Structure Elucidation of Bioactive Marine Natural · PDF fileStructure Elucidation of...

Structure Elucidation of Bioactive Marine Natural

Products using Modern Methods of Spectroscopy

(Strukturaufklärung bioaktiver mariner Naturstoffe mit

modernen Methoden der Spektroskopie)

Inaugural-Dissertation

zur

Erlangung des Doktorgrades

der Mathematisch-Naturwissenschaftlichen Fakultät der

Heinrich-Heine-Universität Düsseldorf

vorgelegt

von

Mohamed A.A. Ashour

Aus El-Sharkiya, Ägypten

Düsseldorf, 2006

Gedruckt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf

Eingereicht am : 04.12.2006

Referent : Prof. Dr. Peter Proksch

Koreferent : Dr. Rainer Ebel, Juniorprofessor

Erklärung

Hiermit erkläre ich ehrenwörtlichen, daß ich die vorliegende dissertation

„Strukturaufklärung bioaktiver mariner Naturstoffe mit modernen Methoden der

Spektroskopie“ selbständig angefretigt und keine anderen als die angegebenen Quellen und

Hilfsmittel benutzt habe. Ich habe diese Dissertation in gleicher oder ähnlicher Form in

Keinem anderen Prüfungsverfahren vorgelegt. Außerdem erkläre ich, daß ich bisher noch

keine weiteren akademischen Grade erworben oder zu erworben versucht habe.

Düsseldorf, 04.12.2006

Mohamed A.A. Ashour

In the name of Allah, the Compassionate, the Merciful. Recite! (or read!) in the name of your Lord who created (1) Created man from clots of blood (2) Recite!, your Lord is the most Gracious (3) Who taught by the pen (4) Taught the man what he knew not (5). (The first verses of the holy Qur'an that came dwon from the sky ) To the Almighty God “ALLAH” who has granted me all these graces to fulfil this work and who supported me and blessed me by His power and His mercy in all my life. To Him I extend my heartfelt thanks.

Acknowledgements

Many institutions and individuals were responsible for the crystallisation of this

humble work, whose associations and encouragement have contributed to the accomplishment

of the present thesis, and I would like to pay tribute to all of them.

I specially wish to express my sincere thanks and gratitude to Prof. Dr. Peter

Proksch, chairman of the Department of Pharmaceutical Biology and Biotechnology,

Heinrich-Heine University, Düsseldorf, for his kindness, admirable supervision, direct

guidance, generous considerations and valuable support during my study in his group.

I would also like to express my deep thanks to my sincere teacher Dr. RuAngelie

Edrada for her guidance, fruitful discussions, constructive advises, NMR courses and

particularly for sharing her expertise in NMR data interpretation and revision of this thesis.

I would also wish to thank J. Prof. Rainer Ebel, of the same department for his direct

guidance, valuable comments and suggestions and specially for sharing his expertise in both

NMR spectroscopy and mass spectrometry.

I am deeply indebted to Dr. Victor Wray (Gesellschaft für Biotechnologische

Forschung, Braunschweig), for the measurement of the NMR spectra, HRMS, and his vital

comments in the structure elucidation of the isolated compounds.

I am grateful to Dr. R. van Soest (Zoological Museum, University of Amsterdam) for

the identification of the sponge materials.

I am thankful to both Dr. Steube of DSMZ (Deutsche Sammlung von

Mikroorganismen und Zellkulturen) and Prof. Dr. W.E.G. Müller (Universität

Mainz,Germany) for the cytotoxicity tests.

My deep thanks are also to PD Dr.Wim Wätjen Institut für Toxikologie, Heinrich-

Heine-Universiät Düsseldorf, Universitätsstr. 1, Geb. 22.21, 40225 Düsseldorf, Germany

I am thankful to Dr. Thomas Schmidt (HHU Düsseldorf) for his kind help in the

measurement of some compounds at the GC machine.

I would also like to express my gratefulness to Dr. Peter Tommes, Dr. U. Matthiesen

(HHU Düsseldorf) for the measurement of EIMS, FABMS and high resolution ms data.

I am very thankful to AnagnosTec [Gesellschaft für Analytische Biochemie und

Diagnostik mbH im Biotechnologiepark, TGZ II , D-14943 Luckenwalde, Berlin] for peptide

sequancing by MALDI-TOF-PSD.

I am thankful to the Egyptian Ministry of Higher Education, Missions Office for

providing the financial support for my study in Germany.

My deep thanks are to my friends, Dr. Mustafa abdelgawwad and Dr. Ihab El-

khayat for their kind friendship, valuable help, encouragement and co-operation during the

first monthes of my stay in Germany.

My deep thanks are also to my new and old colleagues at the Department of

Pharmaceutical Biology and Biotechnology, Düsseldorf, Amal Hassan (Egypt), Arnulf Diesel

(Germany), his wife Ine Inderiani (Indonesia), Clécia Freitas (Brasil). Dr. Yasman

(Indonesia), Dr. Franka (Germany), Dr. Carsten (Germany), Dr. Hefni (Indonesia), Dr. Sabrin

(Egypt), Dr. Gamal (Egypt), Dr. Bärbel (Germany), Dr. Tu (Vietnam), Dr. Yosi (Indonesia),

Dr. Haofu (China), Dr Suwigarn (Thailand), Abdessamad (Morocco), Nadine (Germany),

Mirko (Germany), Frank (Germany), Julia (Germany), Sofia (Sweeden), Idi (Indonesia), Yodi

(Indonesia), Triana (Indonesia), Yao (China) and all the others for their help, friendship and

for the good working atmosphere.

My special thanks to my colleague Annika Putz for his help in the revision of the

German summary in my thesis.

My deep thanks to Mareike, Katrin and Mrs Schlag for their kindness and for always

providing me with the materials and glassware which I needed in my work.

I would also like to express my gratefulness to my supervisors for my Master thesis,

Prof. Dr. Hassan Ammar, Prof. Dr. Taha Khalifa and Ass.Prof. Dr. Samir Al-Donditi and

also to Prof. Dr. Hazem kadry and all staff members and colleagues of the Department of

Pharmacognosy, Faculty of Pharmacy, al-Azhar University (Boys), Nasr City, Cairo for

providing me the basic science and for their motivations, recommendations and for teaching

me very precious knowledge about Pharmacognosy and Phytochemistry offering me such a

kind of opportunity to study in Germany and their continuous encouragement and kind

advises.

My special thanks for my wife Dr. Hasnaa Abouseif for her vast understanding,

everlasting, moral support and contineous encouragement and also for my children Yasmin

Ashour and Ahmed Ashour who could relieve any kind of tirdness by their lovely smiles and

nice atmosphere at home.

I

Table of contents

I- Introduction………………………………………………….….………….…….……… 1

1.1- The significance of the study…………………………...……..…………………… 1

1.2 - Natural Products…………………………………………………...………………... 1

1.3- The biological activity of natural products…………...………...…………………... 10

1.4 -The strategies and approaches that infeluence the extraction, isolation

and purification of natural products…………………….…...………………...… 16

1.5-The structural elucidation of the natural products….………...…..……….…..……. 21

1.6 -Marine Natural Products……………………..……....…………...……...………..… 33

1.7- Aim of the work………………………...…….…………….…………...………….. 40

II-Materials and Methods …………………………………….….………….……. ……… 41

2.1- Animal materials………………………………..….……….…………...………….. 41

2.2- Chemicals used……..……………………….…….…..….…..…….…...………….. 48

2.3- Equipments used…………………………….….………….…..………...………….. 49

2.4- Chromatographic methods.…………………….…………..………….…………….. 51

2.5- Procedures of the isolations of the secondary metabolites…..……………………. 57

2.6- Structure elucidation of the isolated compounds………………...………………….. 61

2.7- Bioassays…………………………………………………………………………….. 75

III-Results…………………. …….…………………………….….………….…….……… 78

3.1-Isolated compounds from Elysia rufescence……………………….…………………. 78

3.1.1- Compound 1 (kahalalide F)…………….…………….…………………...……….. 79

3.1.2- Compound 2 (kahalalide E)…………….……………...….……………...……….. 91

3.1.3- Compound 3 (kahalalide D)……………..…………….……...…………...………. 99

3.1.4- Compound 4 (kahalalide B)…………….…………….…….……..……...……….. 104

3.1.5- Compound 5 (kahalalide C)…………….…………….………....………...………. 110

3.1.6- Compound 6 (N,N-dimethyltryptophane metyhl ester)….….…………………….. 113

3.1.7- Compound 7 (β-sitosterol)…………….…….………….……………...………….. 117

3.2-Isolated compounds from Elysia grandifolia…………….……….……….………….. 121

3.2.1- Compound 8 (kahalalide R)…………….……………..…….….………...……….. 122

3.2.2- Compound 9 (kahalalide S)…………….…………….………..…..……...……….. 133

3.2-Isolated compounds from Pachychalina sp..…………….……….……….………….. 144

3.3.1- Compound 10 (5α,8α-epidioxy-24ξ-methylcholesta-6,22-dien-3β-ol)..…….…..… 145

3.3.2- Compound 11 (5α,8α-epidioxy-24ξ-ethylcholesta-6,22-dien-3β-ol)..……..……… 150

II

3.3.3- Compound 12 (8-hydroxy-4-quinolone)…………… …………….……...……….. 155

3.4-Isolated compounds from Hyrtios erectus……………………....….…………………. 160

3.4.1- Compound 13 (hyrtiosine A)…………….…………………..………...………….. 161

3.4.2- Compound 14 (5-hydroxy-1H-indol-3-carbaldehyde)…………….……..………... 164

3.4.3- Compound 15 (indol-3 carbaldehyde)……………………….………………….… 167

3.4.4- Compound 16 (5-deoxyhyrtiosine A)…………..…..…….…………...…..….…… 170

3.4.5- Compound 17 (isohyrtiosine A)………………….…….…………….……….…… 173

3.4.6- Compound 18 (16-hydroxyscalarolide)….………………….……...…….……….. 176

3.4.7- Compound 19 (scalarolide)…………….…….……….………..……...….……….. 182

3.4.8- Compound 20 (12-O-deacetyl-12-epi-scalarin)….…………….………………….. 187

3.4.9- Compound 21 (7-dehydrocholesterol peroxide)………………....……....…..……. 190

3.5-Isolated compounds from Petrosia nigricans..……………….....….…………………. 194

3.5.1- Compound 22 (24ξ-ethyl-cholesta-5-en-3β-ol)…………….…………….……….. 195

3.5.2- Compound 23 (4α-methyl-5α-cholesta-8-en-3β-ol)…………….………..………... 197

3.5.3- Compound 24 (5α,8α-epidioxy-24ξ-ethyl-cholesta-6-en-3β-ol)………………….. 199

3.5.4- Compound 25 (phenylacetic acid)…………..…..…….…….…………….….…… 202

3.5.5- Compound 26 (p-hydroxyphenylacetic acid)…………..…..…….….……….…… 204

3.5.6- Compound 27 (methyl 2-(4-hydroxyphenyl)acetate)……….…………………….. 207

3.5.7- Compound 28 (ethyl 2-(4-hydroxyphenyl)acetate)………….…………………….. 209

3.5.8- Compound 29 (butyl 2-(4-hydroxyphenyl)acetate)……...….…………………….. 211

3.5.9- Compound 30 (adenosine)…………….…………..……………..………..………. 214

3.5.10- Compound 31 (nicotinamide)…………..……….……...…….………….….…… 217

3.5.11- Compound 32 and 33 (petrocerebrosides 1and 2 )…………..….……… ….…… 219

3.5.12- Compound 34 (nigricine 1)…………….…….……..…..………………..………. 228

3.5.13- Compound 35 (nigricine 2)…………..………….……...…….………….….…… 234

3.5.14- Compound 36 (nigricine 3)…………….…….……..…….……….……..………. 238

3.5.15- Compound 37 (nigricine 4)…………..………….……………………….….…… 242

3.5.16- Compound 38 (nigricinol)…………….……...……..……………..……..………. 245

3.6-Isolated compounds from Callyspongia biru..…….…………....….…………………. 251

3.6.1- Compound 39 (Indole-3-acetic acid)….……………………..……..…….……….. 252

3.6.2- Compound 40 (2`-deoxythymidine)…………….………………………..………... 254

IV-Discussion…………..…………………………………………………………………… 256

4.1- Isolation, characterisation, structure elucidation, and biological evaluation of

III

natural products ………………….………………………………………………….. 257

4.1.1- Isolation of natural products …………...……………………………………….. 257

4.1.2- Characterisation of natural products …………...…………………………...…….. 258

4.1.3- Structure elucidation of natural products ……………...………………………….. 261

4.1.4- Biological activity of natural products …………..………………….…………….. 262

4.1.5- Technology of HPLC …………...…………………………………..…………….. 263

4.2- Isolated compounds from genus Elysia ( sacoglossan mollusc) ……...…………...... 267

4.2.1- The responsibility for production of the kahalalides …………..…………….... 268

4.2.2- The possible biosynthetic pathway of the kahalalides .…...…………………….. 270

4.2.3- Structure-activity relationship ………………...….……………………………….. 271

4.3- Isolated compounds from Pachychalina sp………………………………….………. 272

4.3.1- Epidioxy sterols …………..………………………………………..…………….... 272

4.4- Isolated compounds from Hyrtios erectus……………...…….…...………….………. 275

4.4.1- Excessive harvesting of the wild-type living organism ………..……………….... 275

4.4.2- Biosynthetic pathway of the scalaran-typ sesterterpenoids .…………………….. 276

4.4.3- The pharmacological activity of scalaran-typ sesterterpenoids …………...….….. 277

4.4.4- Indole derivatives from Hyrtios erectus.……………………….………………….. 277

4.4.5- Biosynthetic pathway of indole derivatives from Hyrtios erectus …………….….. 278

4.5- Isolated compounds from Petrosia nigricans………......…………………….………. 279

4.5.1- Biosynthetic pathway of aromatic acids …………...……………….…………….. 279

4.5.2- Cerebrosides from Petrosia nigricans …………...……………………………….. 280

4.5.3- Biosynthesis of cerebrosides …………...………………………...……………….. 280

4.5.4- New purine derivatives from Petrosia nigricans …………...…………………….. 281

4.5.5- The possible biosynthetic pathway of nigricines 1-4………..…...……………….. 282

4.6- Isolated compounds from Callyspongia biru ..…………...…………………….……. 284

V-Summary…………..…………………………………………………………………… 286

VI-References ……..…..…………………………………………………………………… 288

List of abbreviation .…………………………………….………………………………… 302

IV

Zusammenfassung Moderne Techniken und überlegene Werkzeuge haben die Arbeit von Naturstoff-Chemikern

erleichtert und ermöglichen im Vergleich zu älteren Methoden eine beträchtlich schnellere

Entdeckung, Isolierung und Strukturaufklärung von bioaktiven Naturstoffen. Naturstoffe

besitzen oft neue oder ungewöhnliche strukturelle Eigenschaften und / oder viel versprechende

biologische Aktivität. Die Anwendung neuer Methoden reduziert Zeit, Aufwand und Kosten

der Entdeckung bioaktiver Naturstoffdrogen. In dieser Studie wurde oben genanntes Ziel mit

Hilfe von modernen Techniken wie NMR, MS und HPLC Systemen als effiziente Werkzeuge

verfolgt. Das in dieser Studie verwendete biologische Material umfasst sechs marine

Invertebraten, die vier verschiedenen geographischen Standorten entstammen. Insgesamt

wurden 40 Substanzen in reiner Form gewonnen und deren Struktur aufgeklärt. Einige von

diesen zeigten sehr viel versprechende biologische Aktivität und stellen somit potentielle

Kandidaten für die Entwicklung zukünftiger Naturstoffdrogen dar.

1. Elysia rufescens

Der Mollusk Elysia rufescens (Sacoglossa) wurde beim schwarzen Punkt „Kahala“ auf der

Oahu Insel (Hawaii) gesammelt. Aus Extrakten dieser Tiere konnten neben fünf bekannten

Kahalaliden (Kahalalid B, C, D, E und F) β-Sitosterol und ein bislang als Fabaceen-

Sekundärmetabolit bekannter N,N-Dimethyltryptophanmethylester gewonnen werden.

2. Elysia grandifolia

Der Mollusk Elysia grandifolia (Sacoglossa) wurde im Golf von Mannar und Palk Bay

(Rameswaram, Indien) gesammelt. Im LCMS-Spektrum des methanolischen Rohextrakts

zeigten sich neben den Peaks der Kahalalide B, C, D, E, F, G, J, K, und O auch zwei neue

Peaks ähnlich den Kahalaliden R und S. Im Fokus des Isolationsprozesses standen diese neuen

Substanzen. Zusätzlich zu diesen konnten noch zwei bereits bekannte Kahalalide isoliert

werden (Kahalalid D und F).

3. Pachychalina sp.

Der unbekannte Schwamm Pachychalina sp. stammte aus Pulau, Baranglompo (Indonesien).

Im methanolischen Rohextrakt fanden sich sowohl zwei 5α, 8α-Epidioxysteroide als auch das

bereits bekannte 8-Hydroxy-4-quinolon, welches als Bestandteil der Tinte des Riesen-Oktopus

Octopus dofleini martini beschrieben wurde.

V

4. Petrosia nigricans

Der Schwamm Petrosia nigricans wurde in Baranglompo (Indonesien) gesammelt. Der

methanolische Extrakt wurde intensiv untersucht, es konnten insgesamt 17 Substanzen isoliert

werden. Davon waren zehn bereits bekannte Naturstoffe, zwei neue Cerebroside, ein bis-

Indolderivat und vier neue Oxopurinderivate.

5. Callyspongia biru

Proben des Schwammes Callyspongia biru stammten aus Taka Bako (Indonesien). Aus dem

methanolischen Rohextrakt konnten die fünf bekannten Substanzen Indol-3-carbaldehyd, Indol-

3-essigsäure, p-Hydroxyphenylessigsäure, p-Hydroxyphenylessigsäuremethylester und 2`-

Deoxythymidin gewonnen werden.

6. Hyrtios erectus

Individuen des Schwammes Hyrtios erectus wurden bei El-Quesir im Roten Meer (Ägypten)

gesammelt. Im methanolischen Rohextrakt befanden sich neun Substanzen, darunter ein

Epidioxycholesterolderivat, drei Sesterterpene vom Scalaran-Typ, vier bekannte Indolderivate

und ein neues 5-Hydroxyindolderivat.

Introduction

1

I. Introduction

1.1- The significance of the study:

Although effective drugs for many diseases that afflict mankind have been discovered,

many health problems remain untreatable. These problems include various types of cancer;

viral infections such as HIV and viral hepatitis, particularly types related to hepatitis C viral

infections; severe fungal infections, especially in immunocompromised patients;

cardiovascular diseases, and inflammatory and allergic disorders (Gullo, 1994). Only,

approximately, one third of all diseases can be treated efficiently (Müller et al. 2000).

Therefore the search for novel therapeutic agents continues, and the need still remains to

uncover the initial structural lead that interacts with therapeutic targets. Natural products give

a good chance for the discovering of an effective medication of the remaining untreatable

diseases either, by direct therapeutic effect, after semisynthetic modification or by new

synthesis of chemicals based on the natural product models (Cragg et al. 1997). The discovery

of natural products with therapeutic potential is affected by many factors comprising all steps

of extraction, isolation, characterisation, structural elucidation, and biological evaluations. In

the last few years these methods were highly advanced in order to save time, and economy of

isolation process, discover, and ensure the proper biological activity of natural products. Now,

using this technological evolution, one is sometimes able to extract, isolate, and elucidate the

natural product in only one day (Steinbeck 2004). This work deals with natural products and

covers many aspects in which they are included.

1.2- Natural Products

Natural products have inspired chemists and physicians for millenia. Their rich

structural diversity and complexity has prompted synthetic chemists to produce them in the

laboratory, often with therapeutic applications in mind, and many drugs used today are natural

products or natural product derived. Recent years have seen considerable advances in our

understanding of natural product biosynthesis coupled with improvements in approaches for

natural product isolation, characterization and synthesis; these could be opening the door to a

new era in the investigation of natural products in academia and industry (Clardy & Walsh,

2004). Nature has continuously provided mankind with a broad and structurally diverse array

of pharmacologically active compounds that have proved to be indispensible for the cure of

Introduction

2

deadly diseases or as lead structures for novel pharmaceuticals (Newmann et al, 2000a). In

the present drug discovery programs, compounds derived from natural products account for

more than 40% of the newly registered drugs (Cragg, et al, 1997)

The discovery of antibiotics gave the study of natural products a great boost in

microbiology departments and ensured that natural products remained central to growing

pharmaceutical companies ( Firn & Jones 2003).

1.2.1- Definitions of „Natural products“:

Most scientists define the natural products as chemical substances that are made by

organisms and are not active participants in primary metabolism. In other words they are

chemicals that are usually found in few families or species and which do not seem to serve a

purpose in minute-to-minute activities of the cells. Indeed another term used for natural

product is the term secondary metabolite or secondary product. The term secondary products

or secondary metabolites is widely used now to distinguish this type from the other one „

primary metabolites“. (Firn 2004)

Bennett (1995) mentioned five different definitions for the term secondary metabolism :

• secondary metabolites are those metabolites which are often produced in a phase

subsequent to growth, have no function in growth (although they have survival function),

are produced by certain restricted taxonomic groups of microorganisms, have unusual

chemical structures, and are often formed as mixtures of closely related members of a

chemical family. (Martin & Demain 1978 )

• the simplest definition of secondary products is that they are not generally included

in standard metabolic charts (Davies, 1985)

• A metabolic intermediate or product, found as a differentiation product in restricted

taxonomic groups, not essential to growth and the life of the producing organism, and

biosynthesized from one or more general metabolites by a wider variety of pathways than

available in general metabolism. (Bennett& Bentley, 1989).

• Secondary metabolites are not essential for growth and tend to be strain specific.They

have a wide range of chemical structures and biological activities. They are derived by

unique biosynthetic pathways from primary metabolites and intermediates (Vining,1992)

Introduction

3

• Secondary metabolism includes biochemical pathways that are not necessary for

growth or reproduction of an organism, but which can be demonstrated genetically,

physiologically or biochemically (Hunter 1992).

1.2.2- Primary and secondary metabolism : Primary metabolism refers to the processes producing the carboxylic acids of krebs

cycle, α-amino acids, carbohydrates, fats, proteins and nucleic acids, all are essential for the

survival and well-being of the organism. All organisms possess the same metabolic pathways

by which these compounds are synthesized and utilized. Secondary metabolites on the other

hand, are non essential to life but contribute to the species´fitness for survival (Torssell 1997).

Other descriptions of primary metabolism refer to all biochemical processes for the

normal anabolic and catabolic pathways which result in assimilation, respiration, transport,

and differentiation. Primary metabolism shared by all cells are virtually identical in most

living organisms in order for production of extremly similar molecules giving the same

biological function. The secondary products, having no role in the basic life process, are

produced by pathways derived from primary metabolic routes. Secondary metabolite products

accounting for the plant colours, flavours and smells, are source of fine chemicals, such as

drugs, insectisides, dyes, flavours, and fragrances, and phyto-medicines found in medicinal

plants. The concept of secondary metabolism was first introduced by Kössel,1891,

(Haslam,1986; Seigler,1998; Turner, 1971).

1.2.3- The role of secondary metabolite in the producing organism „ to every thing there is a season“

In certain scientific circles it is something of a sport to theorize about function, often with

the intent of finding one overriding axiom true for all secondary metabolism. Speculations

range from the notion that they are waste products or laboratory artefacts, to the concept that

they are neutral participants in an evolutionary game, to ideas of chemical weaponry and

signalling, through a number of other creative notions. (Bennett, 1995).

A) Waste product hypothesis

The role of the secondary metabolites has been rather ambiguous, and initially they were

thought to be just waste materials. The relatively large number and amount of secondary

metabolites observed in nature and the notion that these compounds arose from´´errors´´ in

primary metabolisms in plants, led to the idea that secondary compounds arise and

Introduction

4

accumulate as „waste-products“. However, considering their non-motile nature and the lack of

sophosticated immune system, plants have developed their own defense system against

pathogens and predators, and systems to lure motile creatures, for fertilisation and

dissemination (Luckner, 1990; Seigler, 1998)

B) The overflow or excess of the primary metbolism hypothesis:

In instance of unbalanced growth, secondary metabolites have been envisioned by some as

shunt metabolites produced in order to reduce abnormal concentration of normal cellular

constituents. The synthesis of enzymes designed to carry out secondary metabolism permits

primary metabolic enzymes to continue to function until such time as circumstances are

propitious for renewed metabolic activity and growth. This could be linked to the depletion of

nutrients such as phosphorous or nitrogen ( Bu´Lock, 1980; Haslam, 1986).

C) The increased fitness hypothesis:

This hypothesis is based on the fact that many natural products trigger very specific

physiological responses in other organisms and in many cases remarkably bind to its

complementary receptors. In other words, natural products may aid an organism´s survival in

the absence of an immune system. This supports the hypothesis that secondary metabolites

increase the fitness of the individuals that possess them and that those individuals have been

favoured by the process of natural selection. Secondary metabolites have an important

ecological role in the interaction with the environment, and are like the communication

interface between a plant and its friends and enemies in the environment, (Harborne, 1986;

Rosenthal and Janzen,1979; Swain, 1977; Torssell,1997).

D) Secondary metabolism is a defense system :

Some scientists refer the production of the secondary metabolits to be a potent chemical

defense system against herbivorous, deterrants, pathogens….etc. The immobility of plants in

diverse and changing physical environment, along with the danger of attack by herbivores and

pathogens, has led to the development of numerous chemical and biochemical adaptations for

protection and defense (Knox and Dodge 1985, Harborne 1988), plants for example can

produce higly toxic compounds or compounds mimicking substances normally produced by a

herbivore, for example growth hormones or phermones.

It may be sufficient for the plant to produce compounds that are unpleasant, odorous,

or distasteful, or that possess digestibility reducing properties, i.e. compounds that decrease

the uptake of nutrients, thus preventing over-browsing of the plant. One example of a

compound with strong antifeedant activity against numerous insects is azadirachtin (1),

produced by the indian neem tree (Azadirachta indica). The tree has been used for centuries

Introduction

5

to protect other plants and clothes from insects. The structure of the active compound is very

complex and not proven until 1985. Due to its potency and selectivity against insects this

compound has been commercialized as antifeedent (Bratt, 2000).

Indeed many plants do not keep permanent stores of their defense compounds, but

manufacture them in response to predation. One example is the tobacco plant (Nicotiana

sylvestris) that produces nicotine (2), a compound that deters a wide variety of herbivores.

The level of nicotine produced by the plant is regulated by the extent to which it is being

attacked by herbivores and the wild tobacco plant can increase the amount of nicotine

produced by 3-4 times as response to an attack.(Mann, 1994).

E) The screening hypothesis :

This hypothesis was introduced by Richard D. Firn & Clive G. Jones [(Firn and Jones 1996,

1998, 1999, 2000, &2003), (Jones and Firn 1991), (Firn , 2003 and 2004)]

The hypothesis is composed of two parts.

1. The identification of a fundamental constraint that must be a very significant factor in the

evolution of secondary metabolism.

Potent, specific biological activity is a rare property for a molecule to possess – that is

why large screening programmes are needed to find useful biological activity. The low

frequency of potent, specific biological activity is a consequence of the specificity of

ligand/binding site interactions. Some organisms can increase their fitness by making and

exploiting biological active molecules. However, the low frequency of evolving new

molecules places severe constraints on the evolution of the biochemical pathways leading to

such products.

2. A proposal as to how secondary metabolism might have evolved with such a constraint

including a prediction as to the metabolic traits that would be expected to minimize the

effects of these constraints

How do organisms generate sufficient chemical diversity to enhance their chances of

finding the rare beneficial chemical? How do organisms retain the capacity to generate so

much chemical diversity when indivdual compounds or pathways become redundant?. The

Screening Hypothesis proposes that certain metabolic traits (matrix pathways, non-enzymic

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OO H

N

N

(1 ) (2 )

Introduction

6

transformations, branched pathways, shared pathways and enzymes with a broad substrate

tolerance) would all help increase generation and retention of chemical diversity and

evolution of secondary metabolism.

Some consequences of this hypothesis:

• One should not expect all naturally made chemicals to have a role in the organisms

that make them. Many will have no role and will never have had any role in the organisms

in which they are found. Many chemicals will simply have been made because the

metabolic machinery cabable of their production has a benefit for the producer. If only one

product that those pathways can produce has a beneficial biological activity, the pathways

will be sustained if the costs of possessing that capacity is sustainable.

• One should not assume that some biological activity found in a screening trial

conducted by humans has any significance to the role of the chemical in the organism that

produces it. If organisms are producing chemical diversity they must inevitably produce

chemicals with structures that will possess fortuitous biological activity in non-target

organisms.

• The metabolic traits predicted by the screening hypothesis will sometimes make it

hard to precisely genitically manipulate secondary product pathways. For example, it is

proposed that in order to enhance the production and retention of chemical diversity, many

enzymes involved in secondary product biosynthesis will have low substrate specificity.

Consequently, if a new enzyme is introduced into an organism to cause the production of a

new secondary product, there is high probability that existing enzymes in the transformed

organism will further elaborate the new product to produce more novel diversity.

• Some of the metabolic traits predicted (for example low substrate specificity) might

be exploited in biotransformation and bioremedation studies.

• The flux of carbon through secondary metabolite pathways must have been very large

throughout the period of life on earth. The metabolic traits predicted by the screening

hypothesis may have played a part in encouraging the catabolism of this huge amount of

chemical diversity. The world has never been a clean place chemically hence organisms

must have the capacity to survive and thrive in the presence of chemical diversity. Most

synthetic chemicals released into the environment in small amounts will find enzymes that

can transform them.

Introduction

7

1.2.4- The secondary metabolite pathways: Secondary metabolites are produced through other metabolic pathways than that of primary

metabolites. These pathways are more characteristic for the particular family or genus and are

related to the mechanism of evolution of species. In the fact, the specific constituents in a

certain species have been used to help with systemic determination, groups of secondary

metabolites being used as markers for botanical classification (chemotaxonomy)

(Torssell,1997) .

The differentiation between primary and secondary metabolism is not clear and the

two types are linked together because primary metabolism provides the small molecules that

are the starting materials of the secondary metabolic pathways (Figure 1.1).

1.2.5- The chemical diversity of the secondary metbolites: “Organic chemistry just now is enough to drive one mad. it gives the impression of a

primeval tropical forest, full of the most remarkable things, a monstrous and boundless

thicket, with no way to escape, into which one may well dread to enter. „ (Wöhler in a letter

written to Berzelius in 1835).

CO2 + H2O (photosynthesis)

Monosaccharides

CH3COCOOH pyruvic acid

CH3COSCoAAcetyl-SCoA

OOC-CH2COSCoA-

Malonyl-SCoA

PolyketidesPhenolsfatty acids

mevalonic acidHO

HOO

OH OPP

active isoprene

Isoprenoids(Terpenes, Steroids, Carotenoids)

Aliphatic amino acids

shikimic acidHO

HO

HOO

OH

hv

Aromatic amino acids

Polysaccharides Glycosides Nucleic acids

Cinnamic acid derivativesOther aromatic compoundsLignan

PeptidesProteinsAlkaloidsPhenols

Figure 1.1. Primary metabolites and their links to secomdary metabolism

Introduction

8

When Wöhler wrote those words he was expressing his despair at the emerging

complexity of the composition of natural products. Yet within a few decades, this complexity

was tamed by the Kekule` theory of structure. Increasingly, confident chemists took up the

challenge of determining the structures of ever more complicated natural products. There are

more than 5oo,ooo secondary chemical products already estimated in plants alone. The

majority of natural products described in plants are made by a relatively small number of key

pathways and the structural diversity is therefore largely a consequence of diversity of product

within these few broad groups of chemical clases of natural products (Firn & Jones,1996).

„It is widely accepted that the mutation of a gene coding for an enzyme will result in various

outcomes“. (Firn 2004)

This concept leads to :

1) The enzyme activity will be unaltered (the most likely scenario).

2) The kinetic properties of the enzyme will be changed, usually in a detrimental way.

3) The enzyme will change its substrate specificity.

4) Sites necessary for the allosteric control of the enzyme will be altered.

It is not widely believed that a mutation of the gene coding for an enzyme will change the

type of transformation carried out. Thus most biochemical inventivness will arise from

existing catalytic functions being applied to new substrates rather than new types of

transformations being applied to the original substrate. Such biochemical inventivness

requires that multiple copies of a gene must exist if the gene being mutated codes for enzyme

that is involved in primary metabolism, otherwise a loss of an essential part of primary

metabolism would result (Firn & Jones,1996)

Some simple scenarios to illustrate the constraints that might have operated during the

evolution of natural product pathways. (Firn 2004)

A

B C D

B´ C´ D´E1

E1

E2

E2

E3

E3

Fig. 1.2 : relaxed substrate specificity facilitates the generation and retention of chemichal diversity. such predictable characteristics have been found in "secondary metabolism"-carotenoids, phenylpropanoids, terpenoids, etc..

A

B

C

D

E4

E4

E4

Fig .1 3 single product converted to multiple products by one enzyme.

Introduction

9

In (figure 1.2) there is a diagram showing how 3 enzymes (E1-3) convert A to D. Now

if the substrate specificity is relaxed the order in which the substrates are converted in

unimportant pathway, hence the 3 enzymes will generate 6 products not 3. So this helps

generate diversity. Furthermore, if D increases the fitness of the producer then B´,C´ and D´

may still get made even though they play no role currently- this helps retain chemical

diversity.

This phenomenon is evident in the present study where, Kahalalide F, the major

depsipeptide in a saccoglossan molluscs Elysia sp. has antipredatory effect against fish

predators, its biosynthesis was accompanied with the production of other depsipeptides, that

have no antipredatory effects.

Another example, (figure 1.3) illustrates another proposal where one enzyme produces

multiple products instead of the usual single one. Again this helps generate and retain

chemical diversity.

The third example (figure 1.4) of illustrating this principle is to consider a hypothetical

pathway which has three enzymes leading from a precursor. If the enzymes have a very strict

(narrow) substrate specificity then only three products will be formed.

However if the enzymes have a broad substrate tolerence it is possible that they could

each carry out their own little piece of biochemistry on a selected part of any molecule

irrespective of the whole structure of the molecule. Theoretically, many more structures

could be generated using just these three enzymes as shown in ( figure 1.5).

X XA

XA

B

X*A

B

E1 E2 E3

Fig. 1.4 . hypothetical pathway. illustrated three enzymes of very strict substrate spicificity , resulting in production of only three products.

Introduction

10

1.3- The biological activity of natural products.

1.3.1- The history of biological benefits of natural products Man has used natural products since the down of time as remidies for diseases, spices,

narcotics, dyes, and poison for warfare and hunting. Most of these compounds were used in

their crude forms and active components were mostly not isolated until the nineteenth

century. Morphine (3), first isolated from opium (Papaver somniferum and P. setigrum) in

1803 is a well-known example. It is one of the powerful analgesics known, and also possesses

strong narcotic effects (Mann, 1987).

X XA

XA

B

X*A

B

E1 E2 E3

A

XA A

B

E1 E2

E3

XA

X*A

B

E3

A A

E1

E1E3E3

X*X* X

E2

X*

B B

AE2X*

A

Fig. ( 1.5 ) many structures generates from the hypothetical pathway in which enzymes have a broad substrate tolerance

H O

O

N

H O

3

H ON

H

O

N4

Introduction

11

Another example is quinine (4) an anti-malaria agent isolated from the Cinchona tree

as early as in the seventeenth century (Mann 1987). Of course there was a widely held

view in the 19th century that God had created all organisms for man`s benefit. Hence

many considered that the chemicals other organisms contained were for human use. Such

views would have influenced the thoughts of some scientists (Firn and jones 2003, Firn

2004).

Before the 19th century, the only real interest in natural products came from herbalists

and physicians. They were interested in the fact that plants (and sometimes fungi)

contained substances that were thought to be useful in treating patients. For some

considerable time, botanists had been sent from the United Kingdom, and many other

countries, to scout the world for new, exotic plants, and the number of species that were

accessible to herbalists and physicians increased. By the 19th century chemistry had began

to ask questions about the nature of substances and there was a growing scientific interest

in the natural products however this interest was still biased towards their use by humans

rather than their role in the organisms that made them. (Firn 2004)

1.3.2- The importance of biological activity testing of the isolated natural

products: The fact that microbially-derived antibiotics found by screening make up most of a

$16.5 billion per annum antimicrobial pharmaceutical sales (Nisbet 1992), explains why

so many large antibiotic screening programmes have been conducted during the last half

century (Firn 2004).

This antimicrobial screening of the isolated natural products represents only one of

many lines of biological activity testing that made in order to overcome the most recently

encountered problematic diseases for example, viral hepatitis, AIDS, cancers, autoimmune

diseases, parasites,etc.

The biological activity testing should also expand to comprise most toxicological

studies in order to show the most advantages together with the disadvantages of the given

total extract or the purified natural product. At the end of these biological and

toxicological studies we can decide whether the given product is usefull or not.

This problematic equation (effective, cheap, of wide safty margin, and widely

available natural product drugs) is not easy to be accomplished. For example :

Introduction

12

(i) 400,000 microbial cultures were assayed over a 10 year period and only three

utilisable compounds were found (Fleming et al. 1982, Nelson 1961)

(ii) 21,830 isolates screened in one year to give 2 possible compounds (Woodruff et al

1979).

(iii) 10,000 Microorganisms gave only one clinically effective agent (Woodruff and

MacDaniel 1958).

Those sceptical of such evidence leads man to ask, Is biological activity a rare

property for a natural product to possess? in another word, Is it true that, the

majority of secondary metabolites have no biological benefits?

A meaningful answer requires a definition of the term BIOLOGICAL ACTIVITY.

At a time when natural products were mainly of interest only to herbalists, the father

of modern toxicology, Paracelsus (1493-1541) astutely observed that whether one thought

of a substance as a poison or not depend entirely on the dose that was adminstered. it is

therefore obvious that a discussion of the biological activity of any compound is

meaningless unless some comparative standard is adopted with regards to the dose. The

problem is especially acute when the biological activity is evaluated on the basis of an

adverse effect on an organism, because a sufficient dose of many substances will have an

adverse effect on virtually any organism (Rodricks 1992).

For purposes of this discussion it seems sensible that the only realistic bases for

judging the biological activity of a substance is whether the compound would have a

significant effect at a dose that a recipient organism would receive. A biological activity

that is found when the compound is applied to test organisms at unrealistic concentrations

cannot sensibly be regarded as meaningful. The importance of these considerations can be

illustrated by considering model dose-response curves for three hypothetical substances

C1, C2, and C3 (Fig. 1.6 ). It is appearent that all three substances can be regarded as

showing biological activity in that all saturate the response when applied at high

concentrations (>10-5 M). However, suppose that each substance occures in an organism

at a concentration of 10-8 M. In that case, only compound C1 can be considered to possess

meaningful biological activity. Compounds C2 and C3 show no significant activity at their

endogenous concentrations and would therefore confer no selective advantage to the

organism [Firn and Jones 1996] .

Introduction

13

The law of mass action explains why substances C2 and C3 are actively bound to the

protein binding sites producing their effect at high concentrations and inactive at low

concentrations.

Where: Ka = rate of association

Kd = rate of dissociation

The rate of association and the rate of dissociation are properties of the particular

protein and molecule combination if the former is larger than the latter, there is a high

chance of the protein having a molecule bound to it at any moment (the ratio of the two

rate constants is used to assess the strength of any binding process). The law of mass

action tells us that the equilibrium will be driven to the right as the concentration of the

molecule increases. Consequently there is a much greater probability of finding an

interaction between any chemical and any protein if you test the chemical at high

concentration than that at low concentration. This relationship is also helpful when

assessing the safety margin of a tested compound.

Also, it is obvious that the wider the range of organisms for which biological activity

of a compound is assessed, the greater the chance that some effect will be found, and also

it must be borne in mind that the selective pressures that operate on any individual within

a population at any one time will be quite specific and only a limited range of

Chemical + Protein Chemical protein complexKa

Kd

100%

75%

50%

25%

0%10-11 10-510-710-9

C1C2C3

Res

pons

e %

of m

axim

um

Concentration of applied substances (Molar)

Fig. 1.6 „the dose makes the poison“. The dose response curves of three

hypothetical substances C1,C2 and C3 illustrate that whether or not a substance

is considered to be biologically active depends on the dose being considered .

(firn &Jones 1996)

Introduction

14

opportunities will be available where chemical interactions can be beneficially used.[ Firn

and Jones 1996] .

Firn and Jones 1996, mentioned some limitations that affect the biological activity

studies of the secondary metabolites and consequently minimize the chance of detecting

highly biologically active compounds, specially for commercial use, for example:

- The high selectivity required for a biologically active substance means that many

active compounds will be rejected at an early stage because of lack of specificity.

- Some active compounds are unstable and are either lost during isolation or are

unsuitable for use .

- Some compounds are too difficult to synthesise or extract from cultures and are never

developed.

- Some compounds have already been isolated (and possibly patented before).

- The type of biological activity found is not meaningful in terms of the particular

usage sought.

- The screening process is inappropriate.

- Some forms of biological activity are dependent on the presence of other compounds

(synergists) which are lost during the purification processes before the substance is

bioassayed.

Each of the above reasons could be valid in some circumstances but even taken together

they only partially account for the fact that screening natural products has produced relatively

few biologically active compounds with high selective functions.

Although there are several limitations that minimize the chance of detecting the

biologically active natural product, there are many active natural products that extensively

affect the different forms of our life and can be beneficially used as nutritional supplements ,

dyes, insecticides and also there are many natural products that are already used for treatment

of many diseases that counteract our healthy life.

1.3.3- Approaches to the discovery of biologically active natural products: In general, drug discovery strategies can be trivially separated into three categories:

1. Chemically driven, finding biological activities for purified compounds.

2. Biologically driven, bioassay-guided approach beginning with crude extracts.

3. Combination of chemically and biologically driven approaches (vide infra).

Introduction

15

For marine-derived drug discovery, strategies may involve one or more of the

following elements:

1. In vivo screens

2. Mechanism-based screens

3. Functional, whole cell, or tissue-based assays

4. On-site assays versus post collection assays

5. „Dereplication,“ via biological profiles or chemical profiles, e.g., thin layer

chromatography (TLC), nuclear magnetic resonance (NMR), and high pressure liquid

chromatography (HPLC), (McConnell et al. 1994)

Beginning in the 1970´s through today, the majority of the academic-based research

efforts have become essentially „biologically driven“ i.e., the object of the search has shifted

to discover natural products with biological activity. The biological activities include

exploring their potential as agrochemicals (Crawley 1988) and pharmaceuticals, as well as

their possible chemical ecological roles (Bakus et al. 1986; Hay and Fenical 1988)

Drug discovery in industry has evolved to the use of specific assays with target receptors

and enzymes involved in the pathogenesis of disease rather than cellular or tissue assays

(Johnson and Hertzberg 1989), and has benefitted immensely from breakthroughs in receptor

technology (Hall 1989; Reuben and wittcoff 1989). These assays reflect new opportunities

due to the recent identification of previously unrecognized biomolecular targets for therapy

(Larson and Fischer 1989). More specifically, this approach for most disease areas is

characterized in industry by:

1. Essentially exclusive reliance on biological activity of crude extracts in numerous target-

specific assays, i.e., enzyme assays and receptor-binding assays, for selection of crude

extracts and bioassay-guided fractionation of the crude extracts (prioritization criteria

emphasize selectivity and potency).

2. High volume, automated screening, i.e., thousands of samples per year for smaller

companies and thousands per week for larger companies.

3. The use of „functional“ or whole-cell assays to confirm activity in a particular disease

state and to further prioritize samples for fractionation.

4. The use of gentically engineered microorganisms, enzymes, and receptors.

Because of low correlation between cytotoxity and antitumor activity, a number of

programs have utilized in vivo tumor models directly for drug discovery (Johnson and

Hertzberg 1989). From 1958 through 1985, NCI used in vivo L1210 and P-388 murine

Introduction

16

leukemia assays as primary screens (Suffness et al. 1989; Boyd et al. 1988) and was

successful primarily in identifying compounds possessing clinical activity against leukemias

and lymphomas. Unfortunately, they were not very successful in finding compounds active

against slow growing tumours in humans. Further, these in vivo assays were expensive, time

consuming, and relatively insensitive (Suffness et al. 1989). In other disease areas it was

shown that activity in in vitro antiviral assays does not translate well to in vivo activity, e.g.,

using Herpes simplex. In contrast, reasonable correlations exist between in vitro and in vivo

antifungal activity, e. g., using Candida albicans.(McConnell et al., 1994)

Chemically and biologically driven approaches can be combined. The combination

means that selecting extracts for chemical fractionation based on the biological activity

profile of the crude extract. However, instead of using a bioassay-guided approach to purify

the compounds responsible for the activity of the extract, NMR and some chromatographic

techniques are used to isolate the chemically most interesting substances. Ideally the

structurally unusual or novel compounds are also responsible for the activity of the extract.

This approach works well when the active compounds are present in high concentration and

the assay turnaround time is longer than a couple of weeks. This approach is indeed

productive with respect to isolating numerous new compounds, at least some of which usually

express some of the activity observed for the crude extract, but is obviously not the best

method to identify the most active compounds if they are present in low concentrations

(McConnell et al., 1994).

1.4- The strategies and approaches that influence the extraction, isolation

and purification of natural products. Unlike the medicinal chemist, who usually concentrates on a series of compounds of

similar chemical and physical properties and hence is able to master the limited number of

separation techniques applicable to the specific chemotype, the natural product chemist must

be prepared to deal with molecules of the whole spectrum of bioactive metabolites. These can

vary in hydro- and lipophilicity, charge, solubility, and size (McAlpine and Hochlowski1994).

In general, the more hydrophilic metabolites may be candidates for ion exchange

chromatography, reversed phase silica gel chromtography, or size exclusion chromatography

on polysaccharide resins. The more lipophilic metabolites can be further purified by

chromatography on normal phase silica gel, florisil, alumina, or lipophilic size exclusion

resins such as sephadex LH-20. They may be also candidates for a variety of high-speed

Introduction

17

countercurrent techniques or chromatography on polyresins (McAlpine and Hochlowski

1994). Natural product chemistry is one of the oldest branches of the chemical sciences, its

origin dating back to the first decades of the 19th century, or even earlier. Presently after

almost 200 years of study, this is still vibrant and evolving. What are the reasons for this

continuous and continuing interest? Possible answers would have to include the challenges

offered by the detection, isolation and purification procedures; by the permanently improving

methods of structure elucidation; and by the complexities of the biogenetic pathways leading

to these compounds (Shamma 1989).

The modern highly specific and sensitive screens to detect bioactive molecules have

become available and as researchers have looked at ways to concentrate extracts for primary

screening, novel metabolites have been discovered from fermentations in which they were

produced in levels as low as 1µg/l. (McAlpine and Hochlowski1994). High performance

liquid chromatography (HPLC) including both normal and reversed phases (RP) is now a

well-developed and widely used technique for separation of complex mixtures (Exarchou, et

al, 2005). All parts of the process leading to an elucidated structure have experienced an

immense speed-up in the past fifty years. Separation technology, analytical and spectroscopic

methods have improved steadily and with good fortune, a chemist might be able to go from a

crude extract to a full set of 2D NMR spectra in one day (Steinbeck 2004).

The traditional way of studying natural products includes fractionation of a crude

mixture or extract, separation and isolation of the individual components using liquid

chromatography and structure elucidation using various spectroscopic methods (UV, IR,

NMR, MS). (Exarchou, et al, 2005). It is therefore of great importance to be able to gain

information about extract constituents before investment in the preparative isolation process

(Lambert, et al, 2005).

The chemical components of a biological source are isolated one by one, by

chromatography of the respective extracts, and then their structures are elucidated, such a

complete analytical screening is, unfortunately very time-consuming and material-intensive,

since it is a major effort – and a waste of resources – to isolate all of the compounds in a pure

form, even the known ones. Furthermore, to obtain the required milligram quantities of the

sometimes rare biological source – and of expensive adsorbents and eluents, and thirdly,

unstable compounds will decompose already during the preparative separation and thus

escape the analysis (Bringmann and Lang 2003). A solution of this problem is coupling of the

two steps, the isolation and the structural elucidation, by combining them online. This may

help with the facile acquisition of metabolic profiles, for deciding which of the extracts to be

Introduction

18

analyzed should have the highest priority, for avoiding a tedious isolation of known

compounds, and for checking whether a compound preparatively isolated is genuine natural

product or possibly an artifact (Bringmann and Lang 2003).

In order to discover new bioactive compounds from their biological sources, which

could become new leads or new drugs, extracts should be submitted at the same time to a

chemical screening and to various biological or pharmacological targets. The chemical

screening or metabolite profiling is aimed at distinguishing between already known

compounds (dereplication) and new molecules directly in crude extracts. Thus, the tedious

isolation of known compounds can be avoided and a targeted isolation of constituents

presenting novel or unusual spectroscopic features can be undertaken. (Wolfender, et al 2005)

Metabolite profiling in crude extracts is not an easy task to perform since natural

products display a very important structural diversity. For each compound the order of the

atoms and stereochemical orientations have to be elucidated de novo in a complex manner and

the compounds can not simply be sequenced as it is the case for genes or proteins.

Consequently a single analytical technique does not exist, which is capable of profiling all

secondary metabolites in the biological source. The dereplication procedure strongly relies

mainly on hyphenated techniques coupled to HPLC such as LC-UV-photodiode array

detection (LC/UV-DAD), LC-mass spectroscopy (LC-MS, LC-MS-MS) and LC-NMR which

has been successfully and practically achieved in the last decade. (Wolfender, et al 2005, and

Exarchou, et al, 2005). A common step in the purification and analysis of chemicals of

unknown structure is the separation of the individual components from a chemical or

biological mixture. Chromatographic separation techniques have been coupled with large

number of detection methods, including ultraviolet-optical spectroscopy, Raman

spectroscopy, mass spectrometry, conductivity measurements and NMR spectroscopy. Each

technique can be characterised in terms of easy implementation, intrinsic sensitivity, structural

information produced, and non destructive nature (Webb , 2005).

The combination of LC-UV and LC-MS information can be helpful in a first step of

dereplication, especially when this information is combined with taxonomical considerations

for cross search in natural product databases. This approach is, however, limited by the

unavailability of general LC-MS and LC-MS-MS databases. For the on-line de novo structure

determination of natural products, LC-NMR (see figures 1.7 and 1.8) plays a key role and

allows the recording of precious complementary on-line structure information when LC-UV-

MS data are often insufficient for unambiguous peak identification. (Wolfender, et al 2005).

Introduction

19

The online technique of liquid chromatography coupled with solid-phase extraction

and NMR (LC-SPE-NMR) has recently been used to analyze mixtures originating from

natural product extracts, drug metabolites, and pharmaceutical impurities. The growing use of

this technique results largely from the capability of on-line LC-SPE to isolate, enrich and

allow NMR analysis of an individual analyte present in a complex micture (Xu and Alexander

, 2005).

Fig. (1.7) Various LC-NMR modes and their applications in natural product analysis

(Exarchou, et al, 2005)

Fig. (1.8) Schematic diagram of the experimental set-up used for HPLC-NMR coupling; BPSU= Brucker peak

sampler unit; ( ) capillary junctions; (-----) electric junctions. (Albert, 2002)

Other isolation strategies are focused on the biological activities of the constituents of

the given biological source through what is called „bioactivity-guided isolation“ , where the

isolation steps will continue only with regard to the bioactve fractions and subfractions of the

Introduction

20

biological mixture or extract until the isolation of the bioactive pure compound (Gunatilaka et

al , 1994)

An example for bioactivity-guided isolation for natural product-based anticancer

agents is demonstrated in figure 1.9, where the bioactive compounds and their analogues have

been subjected to cytotoxicity assays with a view to selecting candidates for further

development as anticancer agents.

With few exceptions, intense and systematic bioassay-guided studies of extract and

compounds from marine organisms by academic, government, and industerial research groups

using clinically relevant assay to discover naturally occuring substances with therapeutic

potential have only been underway for less than 10 years. Because of the tremendous

advances in understanding of the biology of certain diseases and the concomitant explosion of

new assays, researchers are now in a position to explore fully the potential of natural products.

These biological advances highly complement the devlopment of new chemical technology,

i.e., new separation and structure elucidation techniques. ( McConnell et al., 1994).

Extraction

screening for bioactivity

Bioactivity-guided fractionation

Confirm bioactivity

Structure elucidation

SAR- Studies

Plant material

Hex.Ext. MEK Ext. MeOH Ext.

Bioactive Ext.

Solvent-solvent partition

Sephadex LH-20 gel filtration

Si gel and/or RP CC Si gel and/or

RP PTLC

Si gel and/or RP HPLC Bioactive

compounds

physical and spectroscopic data

Analog synthesis

Cytotoxic assay

Fig (1.9) , Steps in bioactivity-guided isolation,( Gunatilaka et al , 1994)

Introduction

21

1.5- The structural elucidation of the natural products: Tens of thousands of new compounds have to be synthesized or extracted from natural

sources in order to discover a potential drug. Despite the use of rational drug design

techniques, economic success mainly depends on the number of new candidates available for

activity tests. Consequently, research groups have begun to introduce new automation

procedures such as synthesis robots and combinatorial chemistry. These enhancements on the

production side are only part of the whole task. Additional efforts in the subsequent structure

elucidation process are also vital in order to avoid bottlenecks ( Neudert and Penk 1996). In

natural product drug discovery programs, the major bottleneck has always been structure

elucidation (Jaspars 1999). Conventional approaches to the structure elucidation of organic

compounds are based on the use of spectroscopic data from different sources. The

spectroscopist´s task is to interpret the spectra and to derive structure proposals. The

efficiency of this process depends mainly on his or her knowledge of structure-spectrum

correlations, acquired in the course of everyday work (Neudert and Penk 1996).

The usual spectroscopic methods that used in the structural elucidation of natural product

chemistry includes UV, IR, NMR, and MS (Exarchou, et al, 2005). Recently, the 3D

structural determination was available through X-ray spectroscopy even if no other additional

spectral informations exist. An X-ray crystal structure determination is the ultimate analysis.

No other analytical technique currently available can deliver such complete and unambiguous

information about the nature of the substance being investigated, but this technique has some

limitations :

- Good single crystals are required, and these are sometimes difficult or impossible to obtain

without considerable effort.

- Decomposition of the compound during crystallisation attempts can be a difficulty with

reactive compounds.

- The analysis is done on a single crystal, which may not be representative of the bulk

material.

- The conformational results apply to the solid state and may be different to the molecular

conformations present in solution, which is where most reactions take place.

The modern and highly advanced technology applied in NMR spectroscopy and mass

spectrometry provide unequivocal structural information for the individually isolated

compounds (Exarchou, et al, 2005).

Introduction

22

1.5.1- Mass spectrometry :

Mass spectrometry (MS) has been appropriately used for analysis of molar masses of

molecules for the past 50 years (Burlingame,1992). MS remains the method of choice for

determining molecular formulas and identifying known substances. Where applicable,

depending largely upon volatility and fragmentation patterns, MS can be a very powerful tool,

as has been demonstrated in the analysis and sequencing of peptides and carbohydrates. In

parallel with development of NMR, the field has benefited greatly from studies of peptides

and proteins where the problem of volatility has been paramount (Hensens 1994).

The application of MS to large biomolecules and synthetic polymers has been limited

due to low volatility and thermal instability of these materials. These problems have been

overcome to a great extent through the development of soft ionization techniques such as

chemical ionization (CI), (Silverstein, et al ,1991, and Cotter, 1980) , secondary-ion mass

spectrometry (SIMS) (Silverstein, et al ,1991, Cotter, 1980 and Bletsos et al, 1991 ), field

desorption (FD) (Silverstein, et al ,1991, and Cotter, 1980), fast atom bombardment (FAB),

(Silverstein, et al ,1991, and Cotter, 1980), electrospray ionization (ESI) (Fenn J. B.2003, and

Ashcroft, A. E., 1997) and matrix asssted laser desorption ionization mass spectrometry

(MALDI), (Karas, et al, 1988, Karas, and Hillenkamp, 1991, and Tanaka, K., 2003).

Mass spectrometry (fig. 1.10) is an analytical technique that can provide both

qualitative (structure) and quantitative (molecular mass or concentration) information on

analyte molecules after their conversion to ions. The molecules of interest are first introduced

into the ionization source of the mass spectrometer, where they are first ionized to acquire

positive or negative charges. The ions then travel through the mass analyser and arrive at

diffferent parts of the detector according to their mass (m)-to-charge(z) ratio (m/z). After the

ions make contact with the detector, usable signals are generated and recorded by a computer

system. The computer displays the signals graphically as a mass spectrum showing the

relative abundance of the signals according to their m/z ratio ( Ho, et al , 2003).

Fig (1.10) a simple Mass spectrometer diagram

Introduction

23

The analyser and detector of the mass spectrometer, and often the ionisation source

too, are maintained under high vacuum to give the ions a reasonable chance of travelling from

one end of the instrument to the other without any hindrance from air molecules. The entire

operation of the mass spectrometer, and often the sample introduction process also, is under

complete data system control on modern mass spectrometers. (Ashcroft, A. E., 1997)

Methods of sample ionization:

The choice of ionization methods depends on the nature of the sample and type of

information required from the analysis. so-called “soft ionization” methods such as field

desorption and electrospray ionization tend to produce mass spectra with little or no

fragmentation content whereas “hard ionization” methods such as electron ionization (EI)

which are also refered to as electron impact ionization tend to produce mass spectra with large

amount of fragments or daughter ion peaks. There are several methods that are used

effectively to provide ionization in the currently available mass spectrometrs. In most

ionization methods there are the possibility of creating both positively and negatively charged

sample ions, depending on the proton affinity of the sample. Before embarking on an analysis

the user must decide whether to detect the positively or negatively charged ions (Ashcroft, A.

E., 1997).

Collision Induced dissociation ( CID):

Collision induced dissociation (CID) sometimes also called collisionally activated

decomposition (CAD) is one of the most common fragmentation procedures that is applied in

biopolymer (e.g. peptides or polysaccharides) sequencing, structural elucidation, and analyte

identification through finger-printing. The precursor ion enters the collision cell containing a

high pressure of energised, chemically inert collision gas (e.g. Ar, He, N2, CO2 etc.) The

precursor ions undergo repeated collisions with the collision gas, building up potential energy

in the molecule, until eventually the fragmentation threshold is reached and product ions are

formed, see figure (1.11). The types of fragmentaion that occur vary considerably with the

type of product ion and amount of energy involved. At lower energies (close to the threshold),

fragmentation reactions are often limited to neutral losses (H2O, MeOH, CO, CO2, MeCN

etc.) depending on the nature of the precursor ion. These neutral loses are often not cosidered

structurally significant, although they can be used to obtain information about functional

groups. At higher energies, retro-synthetic type reactions are often observed. These are much

more structurally significant, and often result in cleavage of the molecule at characteristic

positions.

Introduction

24

If the energy is too high, C-C bond cleavage can occur leading to uncontrolled

fragmentation; this should be avoided. Usually it is best to work at around the fragmentation

threshold, or just above, to maintain most control over the fragmentation processes. Ion-trap

and FT-MS instruments allow for the most control over CID, but also tend to produce less

energetic reactions. Triple quadrupole and Q-TOF instruments tend to produce more energetic

CID with more fragmentation, but less operator control. Ion-trap and FT-MS allow multistage

fragmentation experiments to be conducted through, which is essential for structural

elucidation studies (Ojima et al 2005 and Gates, 2005b).

1.5.2- Nuclear magnetic resonance spectroscopy of Natural Products

(NMR): Structural elucidation in general involves determining an extended sequence of bond

connectivities. Natural product chemists have to build up a structure from scratch in a logical

manner as they require some biogenetic knowledge and chemical intuition. Indeed they have

to be quite successful in solving structures where initially they had no idea what class of

compound was involved (Rycroft 1988)

Advances in radio frequency and probe technology, in the application of higher

magnetic fields and the ever expanding repertoire of pulse sequences in one-dimentional (1D)

and two-dimensional (2D) NMR and in the biological area of three-dimensional (3D) NMR

and even four-dimensional (4D) NMR, will inevitably be passed down to the structural

organic chemist to allow the resolution of more complex structural problems on increasingly

Collision gas

Fragnent ion

Neutral lost

Collision cell

fragment ions(product ions)

Activated fragment ion(continues to fragment)Fragmenting

ionActivated

ion

Precursor ion

Fig (1.11). A Schematic diagram of CID fragmentation

Introduction

25

smaller sample quantities. This non destructive methodology contrasts sharply with chemical

degradation studies that so typically have dominated natural product structure determination

in the not too distant past (Hensens 1994). The most of the recent advanced NMR techniques

will be disscussed, briefely, through the following structural elucidation strategies:

1.5.2.1- The most applied structral elucidation strategy depends largely on NMR and

MS modern techniques:

1) the establishment of the molecular formula: the determination of the molecular formula

is critical in the structure determination process of natural product. MS remains the

method of choice for determining molecular formulas and identifying known substances

(Hensens 1994). Depending on the particular instrumentation available, the accuracy of

these methods does not always define an emperical formula uniquely but provides a range

of formulas especially in the molecular weight range above 500 Dalton (Da). increasing

number of examples have recently appeared in the litrature where determining emperical

formulas of natural products with molecular wieght 500 Da or more have required an

interplay between MS and NMR methods.

2) Determination of carbon count of the molecule: a proton–decoupled 13C spectrum can in

principle provide a reliable carbon count of the molecule. However, depending on carbon

spin-lattice relaxation times, the flip angle (pulse width) and the acquisition time

employed, quaternary carbons can sometimes appear as week signals that may not be

readily distinguished from impurity peaks that are present. In this case, changing the

solvent pH, temperature, and/or parameter selection may be helpful. Demonstrating its

long-range connectivity to an assigned proton usually ensures that a quaternary carbon

belongs to molecule. It should be taken in mind that the No. of 13C resonances in the

molecule does not immediately infer the correct carbon count and degeneracy in chemical

shift position is often the reason this drowback may be largely overcome by remeasurment

in different solvents. In other cases the degeneracy may be associated with a symmetry

feature of the molecule such as monomeric versus dimeric forms or overlaping of more

than one carbon peaks.

3) Determination of the proton count: The next step in the strategy is the determination of

the proton count, several methods are currently available to determine carbon

multiplicities and consequently the carbon-bound proton count. for example the J-

modulated attached-proton-test (APT), and the more preferred one „distortionless

Introduction

26

enhancement by polarization transfer“ (DEPT). DEPT is more preferred for various

reasons, it has reduced dependence on J, is not very sensitive to misset pulses or in

homogeneities in the rf, has definit sensitivity advantages, require less amount based on

the availabe aparatus. The solvent peak is effectively suppressed in all spectra, which may

thus advantageously detect nonquaternary carbons obscured by the solvent peak. Having

established the No. of carbon-bound protons in the molecule, it remains to determine the

No. of active protons, which is usually less straightforward. Deuterium (2H) exchange

experiments are often used. As far as MS methods are concerned for the determination of

the No. of active protons in a molecule, the formation of trimethylsilyl (TMS) ethers and

esters have enjoyed great popularity. MS data for the derivatives are compared with that

of the corresponding deuterated d9-TMS derivatives.

4) Determination of the No. of possible empirical formulas: With the molecular weight, 1H

and 13C counts in hand, severe restrictions are now placed on the No. of possible empirical

formulas for a given molecule. This is particularly the case if some knowledge of

elements present has been obtained from either a combustion analysis or by inference

from NMR data. The HRMS data can only be regarded as consistent with the calculated

empirical formula as distinct from rigorously establishing it. This should be kept in mind

because journals such as the Journal of the American Chemical Society and Journal of

Organic Chemistry, for example, set widely varying, acceptable limits in 1991 of ± 3 and

± 13 mmu, respectively for mol.weights up to 500 and ± 6 and ± 16 mmu respectively, for

molecular weights 500 to 1000.

5) Determination of partial structures: This can be established from the 1H-1H connectivity

data, obtained from conventional double irradiation or 2D-COSY-Type experiments.

Included in the development of partial fragments is usually some knowledge of

preliminary 13C NMR data as well as one-bond 1H-13C correlations. One-bond 1H-13C

techniques, which by definition are limited to nonquaternary carbons, provide little

connectivity data unless used in conjunction with long-range 1H-13C data where such 13C

NMR assignments are essential. The most important information the 2D 1H-13C

correlation experiment offers is that can provide a clear distinction between methine and

methylene proton positions in crowded regions of a 1H NMR spectrum. This information

cannot always be unambiguously obtained from COSY-type experiments and when

attempted, is solely inferred from the No. and size of the couplings involved, a process

that is not unambiguous and difficult in situations where there is significant overlap.

Introduction

27

5a) H-H connectivities: this may be established from the basic correlation spectroscopy

(COSY) experiment, double-quantum filtered COSY (DQF-COSY), the complementary

relayed COSY (RCOSY), and homonuclear Hartman-Hahn spectroscopy (HOHAHA) or

total correlation spectroscopy (TOCSY) experiments.

5b) 1H-13C one-bond connectivities: The 2D-heteronuclear correlation (HETCOR)

experiment, which correlates a 13C nucleus with its attached protons or the more preferred

and more sensetive experiment, 1H-detected heteronuclear multiple quantum coherence

experiment, (HMQC) or Hetero nuclear single quantum coherence (HSQC) are applicable

in this stage of the strategy.

6) Determination of the total Structure: this stage of the strategy can be performed by

NMR experiments that are used to bridge isolated spin systems in natural product

compound. For example :

6a) 1H-1H through space correlations: this correlations can be obtained from J-coupled

methods e.g. long range correlations in the COSY experiment (LOCOSY), or from dipolar

coupled methods e.g. 1D- (NOE) or 2D- (NOESY) nuclear overhauser effect or Rotating

frame NOESY, (ROESY) which give indications of distance-dependent „through space“

dipolar interactions. 1D- or 2D NOE experiment can give valuable informations not only

about the sequence and attachment of the separated partial structures but also about the

stereochemical/conformational informations.

6b)1H-13C long range connectivities: powerful as this widely applicable methodology of 13C-detected and especially 1H-detected 1H-13C long range correlation experiments has

become, it nevertheless has its shortcomings. By implication, this technique depends on

each carbon being strategically located usually 2- or 3-bonds away from a proton in the

molecule. Many NMR experiments may be used in this stage e.g. long range version of

the HETCOR experiment (LR-HETCOR) or correlation via long range coupling

(COLOC) which are 2D- 13C-detected 13C-1H long range correlation experiments. But the

more applicable and widely used experiment is heteronuclear multiple bond correlation

(HMBC), which inverse 1H-13C long range coupling through 1H-detected 1H-13C long

range correlation experiment. Because of the extreme usefulness of the HMBC

experiment, it is highly recommended that for optimal results, several experiments be run

under different experimental conditions (e.g., solvent or temperature), optimized for

different couplings and that these experimental conditions be reported.

Introduction

28

1.5.2.2- The Computer-Assisted Structural elucidation strategy (CASE):

The most time-consuming process, that of assembling structures using the

substructure information extracted from the spectra can be performed by computers (Neudert

and Penk 1996). The ideal of computer assisted structure elucidation (CASE) is to generate,

exhaustively and without redundancy, all possible structures that are consistent with a

particular set of spectroscopic data. The aim is to achieve this goal with the minimum amount

of human intervention to overcome the major bottleneck in the natural product drug

discovery (Jaspars 1999).

Some approaches without resorting to 2D NMR data, have been tried using 13C NMR

data alone. One example of this type of system is Richert´s Specsolv (Will et al. 1996), which

is a new module of the NMR database SpecInfo. SpecInfo has used data from thousands of

compounds to calculate typical chemical shifts for a carbon with a particular set of neighbours

(a substructure). SpecSolv allows the user to enter the 13C NMR spectrum of the unknown ,

without having to give the molecular formula, and structures matching these chemical shifts

are returned. For 80% all compounds containing only C,H,N,O,S,P and halogens, the correct

structure is derived. The program relies on a subspectrum search, which is then translated to a

collection of substructures. The substructures are assembled to give the greatest degree of

overlap, and the 13C chemical shift is calculated for each generated structure. The structure

which gives the correct 13C NMR spectrum is returned to the user as the most likely candidate

structure. With „exotic unknowns“ such as complex natural products no final structure can be

proposed by SpecSolv due to the lack of spectral matches. In addition, two carbons with the

same neighbouring groups, but in different conformations may have very different chemical

shifts, and this may confound the subspectrum search. Although 13C shift based programs are

likely to find great utility in a synthetic laboratory with a high turnover of compounds, it is

unlikely to fulfil the ideal of CASE (Jaspars 1999).

Another approach to CASE, which does incorporate the use of 2D NMR data, is to

compose a new NMR pulse sequence which enables the direct determination of proton spin

systems in a molecule (Eggenberger and Bodenhausen 1989).The generation of proton spin

systems is also possible using a graph theoretical method which determines a C-C

connectivity matrix for protonated carbons by the direct determination of the matrix product

of 1H-1H COSY and 1H-13C COSY (1bond) spectra. These last two methods are useful in

generating spin systems only, but they do not allow the generation of complete structures

without the use of further long range data. This goal can be achieved through the application

Introduction

29

of CASE programs which are able to use routinely available 2D NMR data (e.g. 1H-1H

COSY, HMQC or HSQC, HMBC, NOESY and INADEQUATE) (Jaspars 1999).

The human thought process:

It is important to appreciate firstly, how a spectroscopist will elucidate a structure

from spectroscopic data. The process is summerized in Fig. (1.12). Normally the molecular

formula is derived from a combination of 13C NMR, DEPT and MS data. Using IR, UV and 13C NMR the functional groups can be proposed, and 1H NMR coupling data or 2D NMR

correlations are used to assemble substructures. These are then combined into „working

structures“ which are possible combination of the substructures. These are then checked for

consistency with the 2D NMR data and MS fragmentations etc. The 13C chemical shifts of

the surviving structure(s) are then compared with litrature, database or predicted values to

confirm the 2D structure of the molecule. To determine the relative stereochemistry of the

molecule, 1H coupling constant (J) and NOE data are used. The absolute streochemistry can

then be determind by a variety of methods such as optical rotatory dispersion-circular

dichroism (ORD-CD), derivatisation or degradation. It is important, as early as possible, to

know whether the unknown compound has previously been described, a process known as

dereplication, which can be performed using a combination of molecular formula,

substructures and chemical/structural databases (Corely and Dorley 1994).

Once it has been established that the compound in question has not been reported

before, the process of structure elucidation as depicted in Fig (1.12) can begin (Jaspars 1999).

Pure compound

MS, NMR

NMR, IR

UV

NMR

X-RAY

Molecular formula

Functionalgroups

Substructures

Very secure 3D molecular structure

Unsaturation Number (UN)

Working 2DStructures

List of working 2Dstructures

New 2Dmolecular structure

Known molecular structure

Reasonable 3Dmolecular structure

Dereplicate by MF

Draw all isomers

Dereplicate by structure

NMR, MS, IR, UV

NMRORDmolecular modeling

Total synthesis.

Fig. (1.12 ) Strategy for structure elucidation from spectroscopic data. (Crews et al. 1998)

Introduction

30

Two common strategies are employed when elucidating organic structures, one

involving C-C correlations from a 2D INADEQUATE spectrum, the other using C-C

connectivities inferred from C-H data. The INADEQUATE strategy is summarised in Fig.

(1.13a). The main problem of this approach is the inherent insensitivity of the INADEQUATE

experiment, which dictates that a large amount of sample is needed, which may be not

available in some cases, and that it must be (if available) soluble in a small amount of solvents

(Jaspars 1999).

The alternative strategy involves the use of more 2D NMR experiments, but these can

be obtained in a reasonable time using inverse detected techniques on a multimilligram

sample. This strategy is outlined in Fig. ( 1.13b) (Jaspars 1999).

Structure of the CASE Program:

The CASE system is composed of several steps (Fig.1.14) (Jaspars 1999).

1- The first step is the input of the spectra, or „peak-picking“, to convert the data into a

more computer digestible form. This can rely on the skill of a spectroscopist who

Get 13C NMR spectrumget multiplicities

Get 2D INADEQUATE

Make C-C map

Get 13C-1H correlation spectrum (e.g. HMQC)

Get one bond 13C-1H correlations assign 1H resonances

Bridge heteroatoms using long range 13C-1H correlation spectrum (e.g.

HMBC)or using NOE data

Generate 2D structure

Get 1H, 13CNMR spectra get multiplicities and integrals

Get 1H-1H correlation data (e.g. COSY)

Get 13C-1H correlation spectrum (e.g. HMQC)

Get one bond 13C-1H correlations assign 1H resonances to 13C resonances

Check assignment of diastereopic protons using COSY and HMQC

Assemble substructures using COSY data

Get long range 13C-1H correlation spectrum (e.g. HMBC)

combine structures into all possible working structures

Check all working structures for consistency with 2D NMR data

2D structure

Fig. (1.13a). A possible structure elucidation strategy using

C-Ccorrelation data.Fig.(1.13b). A possible structure elucidation strategy using H-H

and H-C correlation data

Introduction

31

translates the cross peaks of a 2D spectrum into correlations, or ideally on a sophisticated

peak picking program.

2- The next step is to produce a list of possible components (e.g. CH3, CH2-O etc.) present in

the molecule. Generated substructures can be checked during the process of structure

generation (Prospective checking) or after all complete structures have been generated

(retrospective checking). Clearly, prospective checking will be faster, as those

substructures that are not consistent with the 2D NMR data are removed from the

structure generation process. In the case of retrospective checking a combinatorial

explosion occurs for exhaustive structure generation, even for molecules of a moderate

size. These generated components are fed into the most important part of the program, the

structure generator.

3- The next part is „the structure generator“ which will use the components that are

generated by one of the methods mentioned above to generate exhaustive list of all

possible structures without redundancy, and without missing out any plausible structures.

The structure generator is the part of the CASE program that will take the greatest

amount of CPU time, and this is also where the greatest time savings can be made by the

use of efficient algorithms.

Peak picking routine

Component generator

generate all possible

structures

Structure consistent with2D NMR data?

Yes

Possible solution

Generate substructure

Add componentto substructure

All componentsused up?

Yes

Yes

Substructure consistent with 2D NMR data?

No

Structure generation/Checking routine

Retrospective checking Prospective checking

Fig. (1.14). Components of a CASE program.

Introduction

32

4- The generated structures are checked for consistency with the 2D NMR data. An

innovative feature to determine whether the structure generator is heading in the right

direction is by checking the rate at which 2D NMR constraints are being satisfied. In

general as the generation extends towards the correct structure, the number of constraints

satisfied should increase. As long as this rate of constraint satisfaction is above a

predetermined value, the structure generation continues, if it falls below this level

generation using this particular substructure is discontinued. This is a very powerful way

to direct the structure generation process, and greatly reduces the time taken to achieve a

plausible solution.

5- Determination of stereochemistry: For large molecules the determination of three

dimensional structure is performed by using a combination of molecular modelling and

constraints derived from NOE data as well as coupling constant information (Evans,

1995). The absolute stereochemistry will still need to be determined by the use of

degenerative methods, auxiliary reagents, or optical rotatory dispersion-circular dichroism

(ORD-CD) (Jaspars 1999).

Currently, the modification of CASE systems is continued to overcome as great as

possible, the above encountered problems including the CASE programs, structure generators,

and the database projects, in order to make the CASE more applicable.

Interaction between a spectroscopist and the CASE system will remain important in order

to generate the correct structure rapidly. Therefore CASE will complement the skills of the

spectroscopist, not replace them. The use of CASE system is likely to increase in the near

future, and this will enable the bottleneck so often caused by structure elucidation to be

removed from the natural product drug discovery process (Jaspars 1999).

Introduction

33

1.6- Marine Natural Products 1.6.1- Marine organisms are rich biological sources for bioactive natural product

discovery:

Selection of marine organisms as biological materials in this work was largely

attributed to the tremendous level of worldwide interest in marine natural products with

therapeutic potential in industry, academia, and government research labs (McConnell et al.,

1994, Proksch et al 2002). Marine natural products chemistry is essentially a child of the

1970´s that developed rapidly during the 1980´s and matured in the last decade (Faulkner,

2000a). By 1975 there were already three parallel tracks in marine natural products chemistry:

marine toxins, marine biomedicinals and marine chemical ecology. It is the integration of the

three fields of study that has given marine natural products chemistry its unique character and

vigour . Marine organisms have provided a seemingly endless parade of novel structures. New

carbon skeletons were discovered and several functional groups are uniquely or

predominantly marine (Faulkner 2000a).

The first natural products isolated from marine organisms that proved to be valuable

lead structures for the development of new pharmaceuticals were the unusual nucleosides

Picture from : Marine Pharmacology: Potentialities in the Treatment of Infectious Diseases, Osteoporosis and Alzheimer’s Disease. (Bourguet-kondracki and Kornprobst, 2005)

Introduction

34

spongouridine and spongothymidine from the caribbean sponge [Cryptotethia crypta,

Tethydae, (Bregmann and Feeney 1951)] which served as models for the development of

adenine arabinoside (ARA-A), (Vidarabin, Thilo), for treatment of Herpes simplex infection

and cytosine arabinoside (ARA-C), (Cytarabin, Alexan, Udicil), for the treatment of leukemia

respectively (Ireland et al 1993). The discovery of sizeable quantities of prostaglandins, which

had been discovered as important mediators involved in inflammatory disease, fever and pain

in the gorgonian Plexaura homomalla by Weinheimer and Spraggnis in 1969 is considered

as the take-off point of systematic investigation of marine environments as sources of novel

biologically active componds (Newman, et al., 2000a; Proksch et al., 2002).

1.6.2- Marine natural products:

Marine natural products with their unique structural features and pronounced

biological activities continue to produce lead structures in the search for new drugs from

nature. Invertebrates such as sponges, tunicates, shell-less mollusks and others that are either

sessile or slow moving and mostly lack morphological defense structures have so far provided

the largest number of marine-derived secondary constituents including some of the most

interesting drug candidates (Proksch et al. 2003)

It is clear that marine natural products chemistry has had a major impact over the past

30 years, and man can not predict what will happen in the next few years (Faulkner 2000a).

Faulkner 2000a, predict a great impact of chemical and biological researches including

genetic engineering concerning different forms of marine living forms ranging from marine

invertebrates to the marine-derived microorganisms. Faulkner also expects that, in the near

future we able to transfer biosynthetic genes from one marine organism to another and

imagine the marine natural product chemist of 2025 still involved in structural elucidation,

but considerable effort to the genetic engineering required to produce unique metabolites by

fermentation of genetically modified microbes. This will accomplish the goal of having the

marine organisms provide the inspiration for new compounds while avoiding their excessive

harvesting.

The sponges are the source of the greatest diversity of marine natural products. About

one-third of all marine natural products have been isolated from sponges, which make them

currently the most popular source of novel compounds (Whitehead, 1999). The marine

sponges are considered not only as a very important source of new natural products but also a

Introduction

35

source for bioactive compounds. These compounds are interesting candidates for new drugs,

primarily in the area of cancer, anti-inflammatory and analgesic (Proksch et al, 2002).

1.6.3- The biological evaluation of marine natural products

Marine organisms have provided a large proportion of the bioactive natural products

reported over the last 20 years, but non of these compounds have reached the pharmaceutical

marketplace (Faulkner 2000b).

Now, marine natural products are already available in the market as effective drugs.

Ziconotide (Prialt) which is a 25-aminoacid peptide isolated from the venom of the marine

snail Conus magus is now available in the market as a potent analgesic for severe chronic

pain, its analgesic effect is comparable to the opioid analgesics (e.g. Morphine) but its mode

of action not includes binding to the opioid receptors and its actions are not blocked by opioid

antagonists. Ziconotide has a unique mechanism of action, binding to N-type calcium

channels on nerves in the spinal cord and blocking their ability to transmit pain signals to the

brain. Unlike the opioid analgesics, it doesn’t cause tolerance or addiction (Hussar 2006).

However, several marine-derived compounds have generated considerable interest

scientifically, commercially, and from public and health point of view, these include

prostaglandins, palytoxin, ciguatoxin. Further, because of their unique and potent biological

activities, several marine-derived compounds have already found use as biological probes or

biochemical tools and are sold commercially, e.g., palytoxin, brevetoxins, ocadaic,

tetrodotoxin, saxitoxin, calyculin A, manoalide, and kainic acid (McConnell et al., 1994).

Several marine derived natural products have a significant biological activity and many of

them, are currently, in different phases of clinical trials as drug candidates. Some of these

bioactive marine-derived natural products will be mentioned below.

Approximately, half of all marine natural products papers report bioactivity data for

new compounds (Faulkner 2000b). Significant number of marine-drived natural products

have been entered into antitumor preclinical or clinical trials since the early 1980s (Newmann

and Cragg 2004).

Table 1.2 Status of marine-derived natural products in clinical and preclinical

anticancer trials*

Name Source Status comment

Didemnin B Trididemnum solidum Phase II Dropped middle 90s (very toxic) Dolastatin 10 Dolabella auricularia Phase I/II

Introduction

36

Giroline Pseudaxinyssa cantharella Phase I Discontinued (hypertension) Bengamide derivative

Jaspis sp. Phase I Licensed to Novartis, Met-AP1 inhibitor, withdrawn 2002

Cryptophycins (also arenastatin)

Nostoc sp.& Dysidea arenaria

Phase I Licensed to Lilly, but withdrawn 2002.

Bryostatin 1 Bugula neritina Phase II TZT-1027 Synthetic dolastatin Phase II also known as auristatin PE and

soblidotin Cematodin Synthetic dolastatin 15 Phase I/II ILX 651, synthetadin

Synthetic dolastatin 15 Phase I/II

Ecteinascidin 743 Ecteinascidia turbinata Phase II/III

Aplidine Aplidium albicans Phase II Dehydrodidemnin B, made by total synthesis

E7389 Lissodendoryx sp. Phase I Synthetic halichondrin B derivative Discodermolide Discodermia dissoluta Phase I Kahalalide F Elysia rufescens / Bryopsis

sp. Phase II Licensed to PharmaMar, (isolated also

from E. grandifolia together with very similar analogues as bioactive depsipeptides in the present work.)

ES-285 (spisulosine)

Spisula polynyma Phase I Rho-GTP inhibitor

HTI-286 (hemiasterlin derivative)

Cymbastella sp. Phase II Synthetic derivative, licensed to Wyeth.

KRN-7000 Agelas mauritianus Phase I Squalamine Squalus acanthias Phase II also has antiangiogenic activity. Æ-941(Neovastat) shark Phase

II/III Defined mixture of <500 kDa from cartilage, also has antiangiogenic activity.

NVP-LAQ824 Synthetic Phase I Derived from Psammaplin trichostatin, and trapoxin structures.

LU103793 Dolabella auricularia Phase II Semisynthetic pseudopterosin Laulimalide Cacospongia mycofijiensis preclinical synthesized by a variety of investigatorsCuracin A Lyngbya majuscula preclinical synthesized Vitilevuamide Didemnum cucliferum &

Polysyncraton lithostrotumpreclinical

Diazonamide Diazona angulata preclinical synthesized and new structure elucidated.

Eleutherobin Eleutherobia sp. preclinical synthesized and also derivatized, can be produced by aquaculture

Sarcodictyin synthetic derivatives.

Sarcodictyon roseum preclinical

Introduction

37

Peloruside A Mycale hentscheli preclinical Salicylhalimides A

Haliclona sp. preclinical first marine Vo-ATPase inhibitor, synthesized.

Thiocoraline Micromonospora marina preclinical DNA polymerase α inhibitor Ascididemnin preclinical Reductive DNA-cleavage agents Variolins Kirkpatrickia variolosa preclinical Cdk inhibitors Dictyodendrins Dictyodendrilla

verongiformis preclinical Telomerase inhibitors

(*Reported from Proksch et al.2002 and Newmann and Cragg 2004)

There are other marine-derived natural products showing vast pharmacological

activities, many of them are currently evaluated as drug candidates and entered clinical trials.

Table (1.3) shows other marine-derived natural products that are, currently, in clinical trials

for non antitumour therapeutic activities.

Table 1.3. Status of marine-derived natural products in clinical and preclinical

trials* other than antitumors.

Name Source Status (disease) comment GTS-21 (aka DMBX)

Amphiporeus lactifloreus Phase I (Alzheimer´s / schizophrinia)

Modification of a worm toxin : contignasterol 8 IZP-94,005

Manoalide Luffariaella variabilis Phase II (antipsoriatic)

discontinued (formulation problems)

IPL-576,092 (aka HMR-4011A)

Petrosia contignata Phase II (antiasthmatic)

licensed to Aventis

IPL-512,602 derivative of IPL-576092 Phase II (antiasthmatic)

licensed to Aventis

IPL-550,260 derivative of IPL-576092 Phase I (antiasthmatic)

licensed to Aventis

Ziconotide (aka Prialt)

Conus magus Phase III (neuropathic pain)

Licensed by Elan to Warner Lambert (it is on the market now)

CGX-1160 Conus geographus Phase I (pain) Contulakin G CGX-1007 Conus geographus Phase I (pain &

epilepsy) Conantokin G; discontinued

AMM336 Conus catus Preclinical (pain) ω-conotoxin CVID χ-conotoxin Conus sp. Preclinical (pain) Conotoxin MR1A/B CGX-1063 Thr10-contulakin G Preclinical (pain) Modified toxin ACV1 Conus victoriae Preclinical (pain) Α-conotoxin Vc1.1 Methopterosin Pseudopterogorgia

elisabethae Phase I (inflamation / wound)

Semisynthetic pseudopterosin derivative

(*Reported from Proksch et al.2002 and Newmann and Cragg 2004)

Introduction

38

Although during 2000 no new marine natural product was approved for patient care by

the U.S. Food and Drug Administration (FDA), during 2000 preclinical pharmacologic

research with marine chemicals continued to proceed at a very active pace, involving both

natural product chemists and pharmacologists from foreign countries and the United States

(Mayer and Hamann 2004)

In order to reflect the worldwide interest towards the marine derived natural products

as drug candidates, Mayer and Hamann 2004 reported a review for preclinical trials involving

pharmacological and toxicological studies for some bioactive marine natural products that are

published during only one year (2000), 35 marine natural products reported as antibacterial,

anticoagulant, antifungal, antimalarial, antiplatelet, antituberculosis, antiviral and 20 marine

natural products having antiinflammatory, immunosuppressant, cardiovascular activity, and

compounds affecting nervous systems. Some of this report is presented in Tables (1.4 and 1.5

respectively.)

Table 1.4. Marine compounds with antibacterial, anticoagulant, antifungal,

antimalarial, antiplatelet, antituberculosis, and antiviral activities, that are reported in

one year (2000) as drug leads in preclinical trials. Drug class Compound/organism Chemistry Pharmacologic activity Molecular

mechanism of

action (MMOA)

Antibacterial Acetylenic acids/sponge Fatty acid Gram-positive and

nigative inhibition

Undetermined

Antibacterial Discorhabdin R /sponge Alkaloid Gram-positive and

nigative inhibition

Undetermined

Anticoagulant Fucoidan/alga Sulfated

polysaccharide

Inhibition of

microvascular thrombus

No effect on P-

and L-selectin

Anticoagulant Proteoglycan/alga Polysaccharide Anticoagulant Inhibition of

thrombin and

potentiation of

antithrombin III

Anticoagulant Sulfated D-galactan/alga Sulfated

Polysaccharide

Anticoagulant Inhibition of

thrombin and

factor Xa

Antifungal Lyngbyabellin B/bacterium Depsipeptide C. albicans inhibition undetermined

Antifungal Naamine D/sponge Alkaloid C. neoformans inhibition Nitric oxide

Introduction

39

inhibition

Antimalarial Ascosalipyrrolidin 1A/fungus Polyketide P. falciparum inhibition p561ck tyrosine

Antimalarial Homofascaplysin A &

fascaplysin/sponge

Sesterterpene P. falciparum inhibition Undetermined

Antimalarial Manzamine A/sponge Alkaloid In vivo P. berghei

inhibition

Undetermined

Antiplatelet Eryloside F/sponge Sterol

glycoside

Platelet aggregation

inhibition

Thrombin

receptor

antagonist

Anti-

tuberculosis

Axisonitrile-3/sponge Sesquiterpene M. tuberculosis inhibition Undetermined

Anti-

tuberculosis

Elisapterosin B/soft coral Diterpene M. tuberculosis inhibition Undetermined

Antiviral Cyclodidemniserinol

trisulfate/ascidian

Polyketide In vitro HIV infection

inhibition

HIV-1 integrase

inhibition

Antiviral Dragmacidin F/sponge Alkaloid In vitro HSV-1 and HIV-

1 inhibition

Undetermined

Antiviral Lobohedleolide, 17-

dimethylamino/soft coral

Diterpene In vitro HIV infection

inhibition

Undetermined

Antiviral Mololipids /sponge Alkaloid In vitro HIV-1 infection

inhibition

Undetermined

Table.1-5. Marine compounds with anti-inflammatory and immunosuppressant effects and

others affecting cardiovascular and nervous system that reported in one year (2000) as drug

candidates in preclinical trials. Drug class Compound/organism chemistry Pharmacologic activity Molecular mechanism

of action (MMOA)

Anti-

inflammatory

Cavernolide /coral Terpene In vitroTNF-α, NO and

PGE2 inhibition

sPLA2, iNOS and COX2

inhibition

Anti-

inflammatory

Contignasterol/ sponge sterol In vivo allergen-induced

plasma protein exudation

inhibition

Undetermined

Anti-

inflammatory

Cyclolinteinone/sponge Sesterterpene In vitro NO and PGE2

inhibition

NF-kB binding, iNOS

and COX2 expresion

inhibition

Anti-

inflammatory

Oxepinamide A/fungus Alkaloid In vivo neurogenic

inflammation assay

Undetermined

Anti-

inflammatory

Sterol/alga Sterol

glycoside

In vivo inflammation

assay

Undetermined

Introduction

40

Cardiovascular Docosahexanoic acid/fish Fatty acid In vivo vascular reactivity

assays and in vitro

biochemical assays

Undetermined

Immuno-

suppressant

Theonellapeptolides/

sponge

peptide In vitro mixed

lymphocyte reaction assay

Undetermined

Nervous system Conantokin G, T/snail Peptide In vivo parkinson´s

disease model

Alteration of striatal

efferent neurons

function

Nervous system Conantokin-G/snail Peptide In vivo block of

dopamine-enhancing drug

methamphetamine

NMDA receptor

antagonism

Nervous system Conantokin-G/snail Peptide In vitro neuronal and

oocyte whole-cell

electrophysiology

Competitive antagonist

of NR2B NMDA

receptors

Nervous system Conantokin-G/snail Peptide In vivo and in vitro

neuroprotective assays

Decrease Ca++ response

to NMDA

Nervous system Conantokin-R/snail Peptide In vitro NMDA receptor

antagonist and in vivo

anticonvulsant assays

NR2 NMDA receptor

selectivity

Nervous system Conantokin-R/snail Peptide In vitro binding assays Disulfide loop not

essential for receptor

and cation binding.

1.7- Aim of the work: The present work is focused on some of the modern methods that currently included in

the recent discovery of natural products with therapeutic potentials. This study deals with the

modern technical applications ranging from the use of various types of HPLC, the use of

hyphenated analytical techniques during the isolation procedures (e.g. HPLC/UV, HPLC/MS,

HPLC/MS/MS, GC/MS), new methods of chemical modification and different techniques in

molecular weight determination during the structural elucidation, different methods of NMR

spectral analysis and bioactivity guided isolation.

In the present study, the isolation of some new natural product is guided by

dereplication procedure using hyphenated techniques coupled to HPLC such as LC-mass

spectroscopy (LC-MS, LC-MS-MS). Some soft bodied marine organisms are included in this

study as they are attractive biological materials that, attract worlwide interest of natural

product chemists and pharmacologists.

Material and Methods

41

II. Materials and Methods

2.1 Animal materials

All biological materials included in this work are marine animals, and collected from four

different geographical zones and including 2 saccoglussan mollusks, Elysia rufescens which

was collected from the black point Kahala, in the Pacific Ocean (Hawaii), and Elysia

grandifolia which was collected from the Indian Ocean (India), and 4 sponges, unidentified

Pachychalina sp., Petrosia nigricans, and Callyspongia biru which were collected from the

Pacific Ocean (Indonesia), in addition to Hyrtios erectus which was collected from the Red Sea

(Egypt).

2.1.1 Sacoglossan mollusks from the genus Elysia :

Genus Elysia belongs to the herbivorous marine mollusks, sacoglossans, with the

ability to sequester from their algal diet functioning chloroplasts, which then may participate in

the biosynthesis of secondary metabolites. (Hamann and Scheuer 1993)

2.1.1.1. Elysia rufescens

Elysia rufescens Pease, 1871

Order: Sacoglossa

Superfamily: Elysioidae

Family: Elysiidae

Elysia rufescens : "Length: 11 mm up to 60 mm; parapodial height: 3 mm. Parapodia is low but

erect, with two or three undulations; rhinophores are long, slender and directed anteriorly. The

pericardial prominence is a white sac at the anterior end of the parapodia. The color is dark red

maculated with spots of creamy green which impart a reticulated appearance to the animal. The

parapodial margine is orange, while the rhinophores are dark red in color tipped with purple".

(Kay, E.A. 1979; Pease, W.H. 1871; Rudman, W.B., 1999). Elysia rufescens was collected and

provided by D. Horgen (University of Hawaii at Manoa) from Black point, Kahala, Oahu

Island in Hawaii, dried and sent as coarse powder to our Laboratory in April 2003.

2.1.1.2- Elysia grandifolia

Elysia grandifolia Kelaart, 1858

Order: Sacoglossan

Superfamily: Elysioidea

Family: Elysiidae


42

The mollusc E. grandifolia (17 molluscs ; 77 g wet wt) which were observed to be feeding on

the algae Bryopsis plumosa (Hudson), were collected by snorkeling from the Gulf of Mannar

and Palk Bay, Rameswaram, India (9°15' N; 79°15'E) at 1 to 2 meter depth in May, 2003,

immediately preserved in methanol and kept frozen until extraction work. The animal was 10

cm in length and had a translucent green color with a large parapodial margin. The parodial

margin had a very thin, black line, and a submarginal yellow or orange band.

Figure ( 2.4 ) Elysia ornata

Fig. ( 2.5 ) Elysia grandifolia

Fig. ( 2.3 ) Elysia rufescens

Fig. ( 2.2 ) Elysia rufescens 60mm, on Bryopsis pennata (algal diet)

Rhinophore

Parapodia

Parapodial undulationsRhinophores

Figure ( 2.1) Elysia sp


43

There was no white line between the orange and yellow bands (distinct. from E.

ornata). The tip of the rhinophores had a similar color as its orange bands. The body is covered

with numerous black and white dots. It had a long renopericardial ridge with 7 pairs of dorsal

vessels as described by Eliot (1906). The figure of the teeth showed blunt tips and rather broad,

denticulate blades as reported by O’Donoughe (1932) for specimens from the Gulf of Mannar.

The sacoglossan was identified by Dr. Kathe Rose Jensen, National Natural History Museum,

Copenhagen. Voucher specimens (EG-TMMP-3) were deposited in the Centre for Marine

Diversity, University of Kerala, India. Jensen (1992) reported that E. grandifolia feeds on

Bryopsis in both the Caribbean and the Indo West Pacific. (Jensen, 1992 & Rudman, 1999). It

is very similar in colour to Elysia ornata. It is possibly the same species, Rudmann 2001

thought that, there are probably good grounds for considering it distinct. It grows to a very

large size, 10 cm or more. He suspect that Elysia ornata and E. grandifolia are part of a group

of similarly coloured species which need closer examination. (Rudman, 2001 & Kelaart, 1858).

2.1.2- The sponges

"Sponge" is the common name for the Poriferae, who are members of the phylum Porifera. It is

named for the pores with which every sponge is covered, 'porifera' meaning 'pore bearer'.

Sponges come in an incredible variety of colours and an amazing array of shapes. They

probably achieved their greatest diversity during the Cretaceous period and between 5000 and

10000 species live today. They are the simplest form of the multicellular organisms and all are

aquatic, benthic organisms. Most are marine, with only 150 fresh water species known today.

They are known to be present in all seas and in several lakes.

Sponges are divided into three distinct groups, as follows:

Hexactinellida, or glass sponges. These are characterized by siliceous spicules consisting of

six rays intersecting at right angles.

Demospongia. This is by far the most diverse sponge group, although they are not well

represented in the fossil record, as they do not possess skeletons that would easily fossilize.

Demosponge skeletons are composed of 'spongin' fibres and/or siliceous spicules.

Demosponges take on a variety of growth forms from encrusting sheets living beneath stones to

branching stalks upright in the water column. They tend to be large and only exhibit the more

complex 'leucon' grade of organization.


44

Calcarea. Members of the group Calcarea, the calcareous sponges, are the only sponges that

possess spicules composed of calcium carbonate.

Morphology:

Sponges have a cellular grade of organization, lack symmetry, and unlike all other

marine invertebrates, they do not possess any structures that can be considered true tissues or

organs. Sponges do not have stomachs , kidneys. They do not have nerve or muscle cells. The

body of a sponge is hollow, and it is composed of a simple aggregation of cells, between which

there is little nervous coordination. However, these cells perform a variety of bodily functions

and appear to be more independent of each other than are the cells of other animals. This

gelatinous matrix is supported by an internal skeleton of spicules of silica, calcium carbonate,

or fibrous protein known as 'spongin'. The sponge's body encloses a vast network of chambers

and canals that connect to the open pores on their surface. The porous nature of the sponges

makes them ideally suited for habitation by opportunistic crustaceans, various worms, etc. in

addition to these macro-organisms, bacteria, fungi, blue-green algae and dinoflagellates are also

observed in many species. Many of the most common types of cells are described below.

Pinacocytes

These cells are the "skin cells" of sponges. They line the exterior of the sponge body

wall. They are thin, leathery and tightly packed together.

Choanocytes

These distinctive cells line the interior body walls of sponges. These cells have a central

flagellum that is surrounded by a collar of microvilli. It is their striking resemblance to the

single-celled protists called choanoflagellates that make many scientists believe that

choanoflagellates are the sister group to the animals Choanocytes. Their flagella beat to create

the active pumping of water through the sponge, while the collars of the choanocytes are the

primary areas that nutrients are absorbed into the sponge. Furthermore, in some sponges the

choanoflagellates develop into gametes.

Mesenchyme Between the two layers is a thin space called mesenchyme or mesohyl. The

mesenchyme consists of a proteinaceous matrix, some cells, and spicules.

Archaeocytes

Archaeocytes are very important to the functioning of a sponge. These cells are

totipotent, which means that they can change into all other types of sponge cells. Archaeocytes


45

ingest and digest food caught by the choanocyte collars and transport nutrients to the other cells

of the sponge. In some sponges, archaeocytes develop into gametes.

Sclerocytes The secretion of spicules is carried out by sclerocytes. Other cells, called spongocytes,

secrete the spongin skeletat fibres when those are present.

Myocytes and Porocytes Poriferans do not have any muscle cells, so their movement is rather limited. However,

some poriferan cells can contract in a similar fashion as muscle cells. Myocytes and porocytes

which surround canal openings and pores can contract to regulate flow through the sponge.

Taxonomic classification The Porifera are sometimes placed into the Subkingdom Parazoa of Animalia, while all the

other animals are in the Metazoa. As described above, they are divided into three distinct

groups, the Hexactinellida (glass sponges), the Demospongia, and the Calcarea (calcareous

sponges). The classification of sponges once relied on the characters of the spicules and the

fibres, since the outer shape and the colour vary with the habitat. However, the classification is

now more complex. The following extract, from the systematics page of the excellent Berkeley

University description of Sponges [http.//www.ucmp.berkeley.edu/porifera/porifera.html.]

explains the development of the current taxonomic classification of sponges.

At one time, a diagnostic feature of the Porifera was the presence of spicules. As a

result, certain fossil groups whose organization was consistent with that of living sponges were

not placed within the phylum Porifera. In particular, groups with a solid calcareous skeleton

such as the Archaeocyatha, chaetetids, sphinctozoans, stromatoporoids, and receptaculids were

problematic. A great deal of insight into the phylogenetic affinities of these groups was gained

with the discovery of more than 15 extant species of sponges having a solid calcareous

skeleton. These species are diverse in form, and would be classified with the chaetetids,

sphinctozoans and stromatoporoids if found as fossils. However, with the living material in

hand, histological, cytological, and larval characteristics can be observed. This information

suggests that these 15 species can readily be placed within the Calcarea and the Demospongia.

This radically changes our view of poriferan phylogeny.

It is widely accepted among poriferan biologists that the Calcarea and the Demospongia

are more closely related to each other than either is to the Hexactinellida. With the discovery of

living chaetetids, stromatoporoids, and sphinctozoans, a fourth class was erected for these so-

called sclerosponges. However, the Sclerospongia is not a natural monophyletic grouping and

is thus being abandoned. The abundant fossil chaetetids, stromatoporoids, and sphinctozoans

are probably part of the classes Demospongia and Calcarea, though some uncertainty still


46

remains. The Archaeocyatha pose a special case. No living representative of this group has

been discovered. Their organization is consistent with that of living sponges. The one

phylogenetic analysis (carried out by Reitner) that included archaeocyaths with other sponges,

grouped them as sisters to the demosponges. Therefore, although the taxonomic term

Archaeocyatha is often accorded phylum status it is likely a sub-clade of the phylum Porifera,

thereby violating the ranking system. (www.bbm.me.uk/portsdown/PH_321_sponge.htm,

Pechenik 2000, Barnes 1986, Goerge and Goerge, 1979, Gerwick and Bernart 1992, Pearse et

al. 1987, and Hooper and van Soest 2002)

2.1.2.1- Pachychalina sp.

The sponge Pachychalina sp. (Phylum: Porifera, Class: Demospongia, order:

Haploscleridea, suborder: Haplosclerina family: Niphatidae) is columnar, clavate,

subcylindrical growth forms, having a well-developed aquiferous system with large canals

rendering the texture highly compresible. Numerous small rounded auiferous openings not

clearly differentiated into oscules or pores. The whole skeleton is rather confused, with the

choanosomal skeleton irregular, diffuse, and occuring as a radially plumose network of

primary, ramified, multispicular fibers, irregularly connected by no proper secondary

multispicular fibers and no visible spongin. The outer surface is light rose in colour, the inner

side is white to faint yellow, the growing branches are finger-like branches. It was collected in

Pulau Baranglompo (Indonesia) at depth of 27 ft. (25. 07. 1997). A voucher specimen has been

deposited in the Zoological Museum Amsterdam under registration number ZMA POR17545

2.1.2.2- Petrosia nigricans

The sponge Petrosia nigricans Lindgren, 1897 (Phylum: Porifera, class: Demospongia,

order: Haplosclerida, suborder: Petrosina, family: Pterosiidae) is a massive sponge, irrigularly

globular, with a large base that produces several fused lobes. Aquiferous system with a terminal

deep aquiferous cavity connected with a unique volcano-shaped osculumat the end of the

lobes. Surface smooth, fine, compact, covered by a fine ectosomal layer, hispid to the touch.

Texture hard, firm. The collected specimens are brown, hard, rag-like, the outer surface is

rough sometimes with long fistulae, the inner side is white to faint yellow. The specimen

shows small krabs introduced their heads into the osculum of the sponges. It was collected in

Pulau Baranglompo (Indonesia) at depth of 30 ft. (25. 07. 1997). A voucher specimens has

been deposited in the Zoological Museum Amsterdam under registration numbers ZMA

POR17546 and ZMA POR17713


47

Figure (2.9), Petrosia nigricans

Figure (2.8), Callyspongia biru

Figure (2.10), Hyrtios erectus

Figure ( 2.7 ) Pachychalina sp.

Fig (2.6) Microscopic view of a Poriferan wall (www.ucmp.berkeley.edu/porifera/pororg.html)


48

2.1.2.3- Callyspongia biru

The sponge Callyspongia biru. (Phylum: Porifera, Class: Demospongia, order:

Haploscleridea, suborder: Haplosclerina family: Callyspongiidae) is lobate repent encrusting to

massive or short anastomosed upright tubes with apical oscule. Surface smooth. Single

ectosomal non-hispid, layer and regular size of single, rounded to polygonal mesh.

Choanosomal a well- developed network with rectangular empty mesh, primary choanosomal

fibers multispicular, non-fasciculated, non ramified, spongin sheath well-defined. The

specimens are blue, soft , finger-like branched sponge. the inner side is white to faint yellow.

It was collected in Taka Bako (Indonesia) at depth of 30 ft. (31. 07. 1997). A voucher specimen

has been deposited in the Zoological Museum Amsterdam under registration number ZMA

POR18290

2.1.2.4- Hyrtios erectus

The sponge Hyrtios erectus. Keller (Phylum Porifera, class Demospongia, order

Dictyoceratida, family Thorectidae) is cake shaped, with protruding lobes forming large erect

rounded digits, occasionally branched, with a rounded apex. The consistency is very spongy

and difficult to tear. It is very distinctive black, was collected from a depth of 15-20 m from El

Quseir, 120 km south of Hurghada, Egypt, in the Red Sea, on January 22, 2005. The color in

life is deep greenish black, the interior slightly lighter black to brown. The texture is

incompressible, and the sponge easily snapped in half as the sponge fibers are packed solidly

with sand grains. The surface is regularly conulose and quite spiky to the touch. black, hard,

rag-like. A voucher specimen has been deposited in the Zoological Museum Amsterdam under

registration number ZMA POR18348.

2.2 Chemicals used

2.2.1. General laboratory chemicals

Agar-Agar Merck

Anisaldehyde (4-methoxybenzaldehyde) Merck

(-)-2- butanol Merck

Dimethylsulfoxide Merck

Formaldehyde Merck

Hexamethyldisilazane Merck

Chlorotrimethylsilane Merck

Hydrochloric acid Merck

Potassium hydroxide Merck


49

Pyridine Merck

Concentrated sulphuric acid Merck

Trifloroacetic acid (TFA) Merck

Concentrated amonia solution Merck

D-Glucose Sigma

L-Glucose Sigma

D-Galactose Sigma

L-Galactose Sigma

Acetic anhydride . Merck

Ortho-phosphoric acid 85% (p.a.) Merck

N-(5-fluoro-2,4-dinitrophenyl)-L-alaninamide. Merck

Aminoacids standards. Merck

Sodium bicarbonate Merck

2.2.2. Solvents

Acetone

Acetonitrile

Dichloromethane

Ethanol

Ethyl acetate

Hexane

Methanol

Butanol

The solvents were purchased from the institute of chemistry, university of Düsseldorf. They

were distilled before using and special grade were used for spectroscopic measurements.

2.3. Equipments used

Balances : Mettler 200

: Mettler AT 250

: Mettler PE 1600

: Sartorious RC210P

Centrifuge : Kendro D-37520 osterde

Fraction collector : ISCO Cygnet


50

Freeze dryer : LYOVAC GT2; Pump TRIVAC D10E

Hot plate : Camag

Syringe : Hamilton 1701 RSN

Mill : Molinex 354

Magnetic stirrer : Variomag Multipoint HP

Mixer : Braun

PH-Electrode : Inolab

: Behrotest PH 10-Set

Rotary evaporator : Büchi Rotavap RE 111; Buchi Rotavap R -200

Drying Ovens : Heraeus T 5050

Sonicator : Bandelin Sonorex RK 102

UV Lamp : Camag (254 and 366)

Vacuum Exicator : Solvent speed vac SPD 111V

2.3.1. Hyphenated HPLC equipments

I. Dionex Analytical HPLC (LC/UV) : HPLC program : Chromeleon Ver 6.3

Pump : Dionex P580A LPG

Detector : Dionex, Photoiode Array Detector UVD 340S

Autosampler : ASI-100T

Column Thermostat: : STH 585

Mobile Solvents:

Methanol LiChrosolv HPLC Merck

ortho-phosphoric acid 0.15 %, pH 2.0 (prepared from

ortho-phosphoric acid 85% p.a.) Merck

Nanopure water Barnstead

II. Semi-preparative HPLC ( LC/UV) :

Pump: : Gynkotek, M40

HPLC program: : Gynkosoft (V. 5.4)

Detector: : Gynkotek, Photoiode Array Detector UVD 340

Autosampler: : Gynkotek Autosampler GINA 50

Printer: : NEC P60

Mobile Solvents:


51

Methanol LiChrosolv HPLC Merck


III. Analytical HPLC (LC /UV/ MS hyphenated system) :

MS spectrometer : Finnigan LC Q-DECA

HPLC system (Pump,

Detector and autosampler) : Agilent 1100 series

Column Knauer (125mm L, 2 mm ID), prepacked with Eurosphere- 100 C-18 (5 µm)

and with integrated pre-column.

Mobile Solvents:

Acetonitrile or Methanol LiChrosolv HPLC Merck

ortho-phosphoric acid 0.15 %, pH 2.0 (prepared from

ortho-phosphoric acid 85% p.a.) Merck


LC /ESI-MS was carried out using a Finnigan QDECA-7000 mass spectrometry connected to a

UV detector. The sample is dissolved in methanol and injected to HPLC/ESI-MS

hyphenated system. HPLC was run on a Eurospher C-18 reversed phase column. The mass

spectra were generated on a dual octopole ion trap mass spectrometer operated in positive

and negative ion modes and fitted with an atmospheric pressure electrospray-ionization

sample introduction device. Fragmentation experiments were performed by automatic MS

technique.

2.4. Chromatographic methods

2.4.1. Thin layer chromatography (TLC)

Pre-coated TLC Alu Plates ( Silica gel 60 F254, Layer thickness 0.2 mm) Merck

Pre-coated TLC glass Plates ( RP-18, F254 S, Layer thickness 0.25 mm) Merck

Preparative TLC glass plate ( Silica gel G 60, Merck

spread on a clean glass sheets and activated in oven at 120 °C 5 Hours)

The compounds were monitored by the UV absorbance at 254 and 366 nm or by spraying with

Anisaldehyde reagent , Methanol-sulphouric acid, Dragendorf´s spray reagents.

Anisaldehyde /H2SO4 spray reagent (DAB10)

Anisaldehyde :5 parts


52

Glacial Acetic Acid : 100 Parts

Methanol : 85 parts

Conc. H2SO4 : 5 parts (added slowly)

The solution was stored in amber-coloured bottles and kept refrigerated until use. TLC

was used for the fractions and for the pure compounds to identify the fractions and determine

the purity of the isolated compounds. After spraying, the TLC plates were heated at 110 °C in

order to monitor the UV-undetected sample components.

2.4.2. Vacuum liquid chromatography

Vacuum liquid chromatography (VLC) is a useful method as rapid and initial

fractionation procedure for a crude extracts or large amounts of fractions. The apparatus

consists of a sintered glass büchner filter funnels with different lengthes and internal

diameters suitable for different sample quantities. Fractions are collected in Erlenmeyer

flasks. Silica gel 60 was packed to a hard cake at a height of 5-10 cm under applied vacuum.

The sample used was mixed with a small amount of silica gel using a volatile solvent. The

resulting sample mixture was then packed onto the top of the column. Step gradient elution

with non-polar solvent (hexane) then increasing the amount of the polar solvent (EtOAc,

MeOH) is added to each successive fraction. The flow is produced by vacuum and the

column is allowed to run dry after each fraction was collected.

2.4.3. Column chromatography

Fractions obtained from VLC were subjected to series of chromatographic columns using

different stationary phases and solvent systems according to the chemical nature and

quantity of the fraction components.

The following separation systems were used :

a) Normal phase chromatography uses a polar stationary phase, typically silica gel in

conjunction with a non-polar mobile phase (n-Hexane, EtOAc, CH2Cl2,..etc). thus

hydrophobic compounds elute more quickly than do hydrophilic compounds.

b) Reversed phase (RP) chromatography uses a non polar stationary phase and a polar mobile

pase (water, methanol). The stationary phase consists of silica packed with n-alkyl chains

covalently bound. For instance, C-18 signifies an octadecyl ligand in the silica matrix. The

more hydrophobic the ligand on the matrix, the greater the tendency of the stationary

phase to elute the hydrophilic components of the sample and retain the hydrophobic ones.


53

c) Size exclusion chromatography involves separations based on molecular size of the sample

components. The stationary phase consists of porous beads. The larger compounds will be

excluded from the interior of the bead and thus will elute first. The smaller compounds

will be allowed to enter the beads and elute according to their ability to exit from the small

sized pores they were internalised through.

2.4.4. Semi-preparative HPLC

Semi-preparative HPLC ( Merck-HPLC apparatus, using MERCK HITACHI pump L-

7100 and MERCK HITACHI UV Detector L-7400, Hitachi,Ltd. Tokyo Japan,) was used for

the purification of relatively pure compounds obtained from fractions eluted by column

chromatography or in some cases used to separate very closely related compounds (see figure

2.11). Each injection was in concentration of 3 mg of the dried fraction dissolved in 1 ml of

solvent system. The injection volume up to 1 ml was injected into the column and the flow rate

was 5 ml/min. The eluted peaks were detected by online UV detector involved in the apparatus.

Figure (2.11) HPLC chromatogram A, shows sub-fraction of E. grandifolia–methanol extract containing three closely related peptides , their retention times are very close to each others. Chromatograms B, C, and D shows the pure compounds Kahalalide S, F, and R respectively after successful separation using preparative HPLC.

27,75 29,00 30,00 31,00 32,00 33,00 34,00 35,00 36,51-222

0

200

400

600

821 ms030701 #4 egme3-3 UV_VIS_1mAU

min

8 - 30,403

9 - 31,390 WVL:235 nm

0,0 10,0 20,0 30,0 40,0 50,0 60,0-50

0

50

100

150

200

250

300

350 ms030707 #6 egab2e UV_VIS_1mAU

min

1 - 0,5372 - 0,6653 - 0,9224 - 1,0225 - 1,0896 - 1,2117 - 1,2668 - 1,3429 - 1,485

10 - 30,098

WVL:235 nm

0,0 10,0 20,0 30,0 40,0 50,0 60,0-50

100

200

300

400 ms030707 #13 egKah F prep UV_VIS_1mAU

min

1 - 0,3392 - 0,9193 - 1,0204 - 1,1095 - 1,239

6 - 31,775

7 - 47,621

WVL:235 nm

Kah F Ret. Time : 31,77

Kah S Ret. Time : 30,10

Kah R Ret. Time : 32,21

Kah F Ret. Time : 31,77

Kah S Ret. Time : 30,10

Kah R Ret. Time : 32,21

0,0 10,0 20,0 30,0 40,0 50,0 60,0-20

25

50

75

100

125

150

180 ms030707 #8 egab4b UV_VIS_1mAU

min

1 - 0,6132 - 0,9083 - 1,0124 - 1,0875 - 1,2076 - 1,2687 - 1,3338 - 1,499

9 - 32,212

WVL:235 nm

CB

A

D


54

For separation of the above mentioned peptides (kahalalide S, F, and R), the separation

was carried out by using Methanol : H2O gradient elution; [ solvent A: nanopure H2O 100%,

solvent B: MeOH 100%, the gradient commenced with 50% solvent B to 100% in 20 min then

the mobile phase was stabilised at 100% MeOH for 10 min, then MeOH-percentage was

decreased until 50 % for an additional 5 min], at a flow rate of 5 ml/min through a C-18

reversed phase column to yield three well-separated peaks of Kahalalide S (8 mg), F (103mg),

and R (20 mg) at retention times 17, 19, and 20 min. respectively. The semi-preparative RP-

columns were (300 x 8mm) pre-filled with Eurospher-100 , C-18 reversed phase silica gel beds.

2.4.5. Analytical HPLC

Analytical HPLC was used to identify the UV-absorbable compounds in the isolated

fractions or to evaluate the pure componds. The plotting of the peaks was guided by UV-VIS

photodiode array detector operating in four different wave lengthes 235, 254, 280 and 340 nm.

The solvent gradients used started with 10:90 [MeOH 100 % : nanopure H2O (adjusted to pH 2

with o-phosphoric acid)] , the commencing methanol ratio was equilibrated for 5 min at 10 % ,

then gradiently increased up to 100% after 35 min., then washing with 100 % MeOH for

additional 10 min. In some cases, special programs were applied using the same solvent

systems but in different ratios according to the chemical nature and the relative retention times

of the compounds being analysed. The flow rate of the mobile phase was at 1 ml / min. The

analytical HPLC column was a reversed phase column, thus eluting the polar compounds

firstly, then the relatively nonpolar compounds.

Role of ortho-phosphoric acid in the analytical HPLC process:

o-phosphoric acid gives a good compound-separation, allowing the UV-detector to

impart sharp peaks, but it has some disadvantages that, in some cases of poly-nitrogenous

compounds (e.g. Purine derivatives, nucleotides and other similar small biologically active

metabolites), the presence of acid makes the nitrogenous compounds highly protonated (in

other word, highly polar according to the affinity of that compounds to accept protons), which

in turn elute the compounds very rapidly and in some cases can not be detected with the UV-

detector). This problem prompted some analytical chemists (e.g. Nordström 2004) to derivatise

such basic compounds to be more hydrophobic and consequently improve their retentions on

reversed phase materials and enhance their ESI response. The following example will explain

the effect of ortho-phosphoric acid in masking of the peaks for the nitroginous compounds

during HPLC analysis.


55

Detection and isolation of new purine derivatives in sub-fractions of EtOAc-Fraction of

Petrosia nigricans:

Fig (2.12) HPLC chromatogram of EtOAc subfraction (Et-18) of Petrosia nigricans, pH 2

nanopure water was maintained by phosphoric acid.

From the experiment (figure2.13), it is noted that, in case of acidified water, low

concentrated sample showed nothing and the test purine peak was completely undetected, while

in more concentrated sample the corresponding peak was broad with low resolution, and short

retention time. On the other hand, the chromatograms that plotted during the measurment in

which the neutral nanopure water was applied, showed a purine peak with high resolution, good

peak-sharpness, longer retention times, and more descriptive. It is noted also the resulting UV-

spectra were varied according to pH value of the mobile phase, and the compounds of the same

chromophoric functions give the same UV spectra.

0,0 10,0 20,0 30,0 40,0 50,0 60,0-20

50

100

150

200 ms040710 #3 tm7-et-18 UV_VIS_1mAU

min

1 - 0,1972 - 0,930

3 - 1,2714 - 1,325

5 - 3,451

6 - 5,425

7 - 10,3248 - 14,0359 - 15,64210 - 16,278

11 - 17,971

12 - 20,167

13 - 29,99914 - 30,811 15 - 46,93416 - 51,396

WVL:235 nm


56

0,0 10,0 20,0 30,0 40,0 50,0 60,0-50

0

50

100

150

200

250

300 ms050506 #15 et18h UV_VIS_mAU

min

1 - Peak 1 - 0,0462 - 0,4593 - 0,5974 - 0,7785 - 1,1086 - 1,2377 - 1,360 8 - 14,6739 - 15,412

10 - 16,822

11 - 47,527

WVL:235 nm

0,0 10,0 20,0 30,0 40,0 50,0 60,0-10

20

40

60

80

110 ms050503 #15 et18h UV_VIS_1mAU

min

1 - 0,7582 - 0,950

3 - 1,088

4 - 1,117

5 - 1,2106 - 1,2587 - 1,318

8 - 24,1559 - 25,234

10 - 29,507

11 - 30,42612 - 30,899

13 - 31,75014 - 32,109

15 - 32,88216 - 33,737

17 - 34,093

18 - 35,397

19 - 36,233

20 - 36,71521 - 37,16522 - 37,67823 - 38,21324 - 38,651

WVL:235 nm

0,0 10,0 20,0 30,0 40,0 50,0 60,0-20

50

100

150

200 ms050503 #13 et18f UV_VIS_1mAU

min

1 - 0,1882 - 0,5153 - 0,5984 - 0,9645 - 1,0576 - 1,1257 - 1,2148 - 1,367

9 - 4,528

10 - 9,83811 - 12,692

12 - 15,50613 - 24,164 14 - 30,573

15 - 30,910

16 - 47,606

WVL:235 nm

0,0 10,0 20,0 30,0 40,0 50,0 60,0-200

0

200

400

600

800

1.000

1.200 ms050506 #13 et18f UV_VIS_1mAU

min

1 - 0,4782 - 0,7813 - 0,9104 - 1,1175 - 1,2646 - 1,346 7 - 13,800

8 - 14,308

9 - 15,40610 - 16,195 11 - 32,48212 - 36,770 13 - 47,608

WVL:235 nm

0,0 10,0 20,0 30,0 40,0 50,0 60,0-10,0

0,0

12,5

25,0

37,5

50,0

70,0 ms050503 #12 et18e UV_VIS_1mAU

min

1 - Peak 1 - 0,0632 - 0,3753 - 0,592

4 - 1,1185 - 1,210

6 - 1,341

7 - 5,034

8 - 10,394

9 - 13,491

10 - 18,680

11 - 24,172 12 - 30,569

13 - 30,907

14 - 36,729

15 - 47,523

WVL:235 nm

0,0 10,0 20,0 30,0 40,0 50,0 60,0-50

100

200

300

400 ms050506 #12 et18e UV_VIS_1mAU

min

1 - Peak 1 - 0,0632 - 0,4573 - 0,8604 - 0,9305 - 1,0126 - 1,1297 - 1,2048 - 1,2729 - 1,33710 - 2,394 11 - 10,578

12 - 13,230

13 - 32,47914 - 36,762 15 - 47,571

WVL:235 nm

Peak #10 16.90

-10,0

70,0

200 300 400 500 595

%

nm

228.1

209.0281.3

No spectra library hits found!

Peak #8 14.27

-10,0

70,0

200 300 400 500 595

%

nm

228.3

281.2242.5


Peak #12 13.32

-10,0

70,0

200 300 400 500 595

%

nm

227.0202.7

300.5


9.25

-500

1.250

2.500

4.000

200 400 595

%

nm

344.7 530.3551.7


Peak #9 4.68

-10,0

25,0

70,0

200 400 595

%

nm

210.4

289.7

560.3


Peak #7 5.07

-10,0

25,0

70,0

200 400 595

%

nm

214.2315.6


A B

C D

E F

Figure (2.13) shows three HPLC chromatograms A, C & E of three pure new purine

derivatives isolated from Petrosia nigricans subfraction Et-18, measured in HPLC using

standard mobile phase that composed of methanol 100 % and acidified water pH 2 using

phosphoric acid. The peak resolution in HPLC chromatograms was improved when the

same samples were chromatographed again using the same mobile phase and the same

gradient program under the same condition, but the difference was only the use of neutral

water instead of pH 2 water, where chromatograms B, D & F, respectively were obtained.


57

2.4.6. Flash chromatography

Flash chromatography was used for rapid isolation of compounds. A silica gel 60 GF254

pre-packed column was used and the sample was dissolved in a small volume of the initial

solvent used. The resulting mixture was then packed onto the top of the column using special

syringe. The flow rate was maintained by an air pump and 5 ml fractions were collected and

monitored by TLC.

2.4.6. Gas Chromatography :

Gas chromatography (GC) was used to determine the nature of the absolute stereochemistry of

sugar units (D or L forms). The sugar fraction of the test sample was injected in the GC

after hydrolysis and derivatisation using a special program. The samples were analysed by

comparison of the retention times with those observed for the authentic samples.

2.5. Procedures of the isolations of the secondary metabolites:

2.5.1. Isolation of secondary metabolites from Elysia rufescens

Methanol extract

Sephadex columnmethanol 100 %

Kahalalide F 120 mg

Kahalalide B

5mg

Fraction A Fraction B Fraction C

total methanol extract

n-Hexane extract

solvent partition n-Hexane : methanol

ß-sitosterol

prep. HPLC

RP-18, column MeOH:H2O

80:20Prep HPLC

N,N-dimethyl-tryptophanmethyl ester

4.6 mg

Kahalalide D

3.1 mg

Kahalalide E

4 mg

Kahalalide C

1 mg


58

2.5.2. Isolation of secondary metabolites from Elysia grandifolia

2.5.3. Isolation of secondary metabolites from Pachychalina sp.

Methanol extract

Sephadex columnmethanol 100 %

Kahalalide F 50 mg

Kahalalide S8mg

Fraction 2-4Fraction 5-6 Fraction 13-14


n-Hexane extract


(90%)

prep. HPLC

Kahalalide D3.4 mg

Kahalalide F53 mg

Kahalalide R20 mg

Dereplication procedure using LC/MSnew compounds were detected, and

cosequently were targeted for isolation

mixture of known kahalalides

Fraction 7-12

10mg LC/MS dereplication procedure detect a mixture of 2 new kahalalides plus kahalalide F


n-Hexane extract


(90%)

silica gel columnDCM:MeOH 9:1

2) preparative HPLC

solvent partition n-butanol : dist. H2O

butanol fraction

aqueous fraction 1) silica gel columnDCM:MeOH 9:1

n.hexane fractionNo. 6

preparative HPLC

sterol peroxide 15 mg

sterol peroxid 24.5 mg

8-hydroxyquinolin-4-one3.8 mg

Methanol fraction


59

2.5.4. Isolation of secondary metabolites from Callyspongia biru

2.5.5. Isolation of secondary metabolites from Hyrtios erectus


Aqueous fraction

solvent partition EtOAc:H2O

silica gel columnDCM:MeOH,

gradient elution2) preparative HPLC

solvent partition n-butanol : dist. H2O

butanol fraction

aqueous fraction 1) silica gel columnDCM:MeOH 9:1

EtOAc fraction

VLCsubfractions, Et.A------------Et.Q

subfraction Et.j

2-deoxy thymidine

indol-3-carbaldehydeindol-3

-acetic acid

bioassay guided isolation using brine shrimp assay and antimicrobial activity testing

subfractions, But.A------------But.V

only subfraction (But.V) shows brine shrimp ++ve assay, and mild antimicrobial activity

alkylpyridinium compound30mg

10mg 2.5mg 1.5 mg

4-hydroxyphenyl-acetic acid methylester

4-hydroxyphenyl-acetic acid

1.7 mg 1.3 mg

M e th a n o l e x tra c t

so lv e n t p a rtit io nD C M : H 2 O

D C M e x tra c tH 2 O e x trc t-c o n c en tra tio n-m e th an o l e x tra c tio n

p p t(m a rin e sa lts )

M eth an o l e x tra c t

V L C

1 2 fra c tio n s

fra c tio n s 4 -1 0-s ilic a g e l c o lu m n-p re p H P L C

H y r tio s in e A

5 -(O H )-in d o l-3 -c a r b a ld e h y d e

5 -d eo x y -H y r tio s in e

A

Iso h y r t io s in eA

5 m g 2 m g 3 m g2 .5 m g

1 0 f ra c tio n sh b a ---- -h b j

h b j h b f

S i g e l co lu m n

h b ff

1 2 -d e a c e ty l-1 2 -e p i-sc a la r in

1 .5 m g

sc a la r o lid e5 m g

S ter o lp er o x id e

4 m g

in d o l-3 -ca r b a ld e h y d e

2 .3 m g

1 6 -h y d r o x y -sc a la r o lid e

3 .5 m g

h b fc


60

2.5.6. Isolation of secondary metabolites from Petrosia nigricans


Methanol fraction

solvent partitionn-Hexane:Methanol

silica gel columnn-Hexane:EtOAc

gradient elution

solvent partition BuOH: dist. H2O

n-Hexane fraction

VLC

24-ethylcholesterol

sterolperoxidesteroid

A B

VLC

n-Hexane

TM7EtOAc fraction

Methanol

H2O fractionTM7 Butanol fraction

2g 15 mg25 mg

1) Si gel columnn2) Flash chromatography3)Si gel column4)preparative TLC

Petrocerebroside 1Petrocerebroside 2

35 mg

TM7 Butanol fraction

Si gel columnCH2Cl2:MeOH

26 fractions

fraction 17

Adenosine23 mg


TM7 EtOAc fraction


et.6,7&8 et.11 et.13,14 et.16 et.17&18

phenylacetic acid

p-hydroxyphenylaceticacid

p-hydroxyphenylaceticacid butylester

p-hydroxyphenylaceticacid ethylester

Prep. HPLC Prep. HPLC

Nigricinol

p-hydroxyphenylaceticacid methylester

Prep. HPLC

4.4 mg

5.2 mg 3.25 mg

2.5 mg

3.2 mg

6.0 mg Nigricine 1 Nigricine 2

Nigricine 4

Nigricine 3

Nicotinamide

Prep. HPLC Prep.

HPLC

2.3 mg

2.9 mg

1.1 mg

7.5 mg

6.1 mg


61

2.6. Structure elucidation of the isolated compounds

2.6.1. Mass spectrometry (MS)

2.6.1.1. Low resolution MS:

Low resolution mass spectra were measured by ESI, EI, MALDI, and FAB-MS on a

Finnigan MAT 8430 mass spectrometer. Measurements were done in Institute für

Pharmazeutische Biologie and Institute für Anorganische und Strukturchemie, Heinrich-Heine

University, Düsseldorf.

2.6.1.2. HRMS (High resolution mass spectra):

High resolution mass spectra were determined on a micromass Q-Tof 2 mass

spectrometer.

2.6.1.3. LC/ESI-MS (liquid chromatography/Electrospray ionization mass spectrometry):

LC/ESI-MS was carried out using a Finnigan LC QDECA mass spectrometer connected

to a UV detector. The samples were dissolved in methanol and injected into HPLC/ESI-MS set-

up. HPLC was run on a Nucleosil C-18 reversed-phase column. Measurements were done at

Institute für Pharmazeutische Biologie, Heinrich-Heine University, Düsseldorf. Standard

mobile phase system was applied, which was composed of 10:90 Acetonitrile:nanopure H2O

(0.1% HCOOH) – 100 % acetonitrile in 35 minutes in gradient elution system.

Electrospray ionization Mass spectrometer ( ESI-MS) :

Electrospray ionization (ESI) is a gentle method of the atmospheric pressure ionization

(API) technique and is well-suited to the analysis of polar molecules ranging from less than

100 Da to more than 100,000 Da in molecular weight. ESI –MS has been shown to be a

successful and popular tool for the analysis of noncovalently bound protein macromolecules,

(Ashcroft, A. E., 2005).

Droplets containing ions

Droplet evaporating

Ions evaporatingfrom the surface of the droplets

Capillary, 3-4 kV

Fig (2.14 ) The electrospray ionization (ESI) process


62

The mechanism of electrospray ionization spectrometer

The sample is dissolved in a polar, volatile solvent and pumped through a narrow,

stainless steel capillary (75-150 µm i.d.) at a flow rate of between 1µl/min and 1 mL/min. A

high voltage of 3-4 Kv is applied to the tip of the capillary, which is situated within the

ionization source of the mass spectrometer, and as a consequence of this strong electric field,

the sample emerging from the tip is dispersed into an aerosol of highly charged droplets, a

process that is aided by a co-axially introduced nebulising gas flowing around the outside of

the capillary. This gas, usually nitrogen, helps to direct the spray emerging from the capillary

tip towards the mass spectrometer. The charged droplets diminsh in size by solvent evaporation

(see figure 2.14), assisted by a warm flow of nitrogen known as the drying gas which passes

across the front of the ionization source. Eventually charged sample ions, free from solvent,

are released from the droplets, some of which pass through a sampling cone or orifice into an

intermediate vacuum region, and from there through a small aperture into the analyser of the

mass spectrometer, which is held under high vacuum. The lens voltages are optimized

individually for each sample (Ashcroft, A. E., 2005).

The ESI-MS was modified by John Fenn, 1987. Fenn´s modifications include the

replacement of the simple orifice into the vacuum system by glass capillary tube (see fig. 2.15).

That glass tube was metallized at each end to provide an electrical contact. The inlet end of the

capillary would be maintained at the required potential below ground so the ions entering the

tube would be in a potential well. The fairly high-velocity flow of bath gas through the glass

(dielectric) tube into the vacuum system could drag the ions out of that potential well up to

whatever potential might be desired at the metallized exit end of that tube. Indeed, the gas flow

could raise the ions to an exit potential of several tens of kV, sufficient to inject the ions into

the analyzer. At the same time all exposed external parts and surfaces of the instrument could

be maintained at ground potential, thereby posing no hazard to an operator. Thus, Fenn`s

modification brought the proteins and biomacromolecules to fly, or as John Fenn himself said,

to give “wings to molecular elephants“ (Fenn 2003).

Fig. (2.15) Fenn´s modification of ESI-MS


63

2.6.1.4. MALDI-TOF-PSD-MS Post source decay mass spectrum was obtained using Matrix-assisted laser

desorption/ionization technique coupled to Time-of-Flight mass analyser. This PSD-MS is

the method of choice for sequence determination of the amino acids of isolated

depsipeptides (or peptides). MALDI-TOF-PSD spectra were obtained on a Perspective

Biosystems Voyager-DE PRO MALDI-TOF mass spectrometer and Bruker Esquire 2000

LC-MS system equipped with an electrospray source. Sample application for MALDI-TOF-

PSD MS was carried out directly on sample plates with a mixture of 1µL of matrix

(saturated 2,5- dihydroxybenzoic acid in 50 % acetonitrile, 0.3%TFA) and 1µL of a 50%

MeOH solution containing about 0.2µg of the sample.

The analysis was measured through AnagnosTec (Gesellschaft für Analytische

Biochemie und Diagnostik mbH im Biotechnologiepark, TGZ II , D-14943 Luckenwalde,

Berlin). Post source decomposition mass spectrum was recorded as peaks corresponding to

molecular weight [M+], [M+] - 1 mole amino acid, [M+] - 2 moles, [M+] - 3 moles, and so on.,

where the decomposition was predominantly at the amide (peptide) bonds (i.e. retro-

biosynthetic decomposition). The data collection was assisted automatically by specific

computer program, plotted and interpretted using a computer program. This method has an

advantage, that the cyclic peptide can be measured directly without linearisation or

decyclisation and it also needs no terminal NH2 group.

Matrix-assisted laser desorption ionization (MALDI)

The use of Laser desorption/ionisation-mass spectrometry (LDI-MS) was limited to

compounds that could be vaporised without being decomposed, since the laser light is absorbed

directly into the analyte, the molecular bonds may be broken owing to the increased internal

energy. In other words, there is an increased risk that measurement would occur in the

decomposed state. This method was usually inadequate for compounds exceeding 1000 Dalton

(Da) and ionization of compounds having a molecular weight exceeding 10000 Da was

considered by chemists at that time to be impossible (Tanaka, 2003).

In 1987, Koichi Tanaka, proposed the application of suitable matrix (glycerin) together

with a good laser light absorpable material, UFMP, (Ultrafine metal powder). The mixing of

this combination with the sample allowed to provide an efficiently enough heating and release

of solid sample from its crystalline state to dissolve in the liquid, thereby assisting ionization

(see figure 2.16). Thus, he was able to measure an ion cluster having a mass number exceeding

100, 000 Da (Tanaka, 2003).


64

The mechanism of MALDI spectrometer:

The mechanism of MALDI is believed to consist of three basic steps ( Gates, P., 2005a):

(i) Formation of a solid solution: it is essential for the matrix to be in access thus leading

to the analyte molecules being completely separated from each other. This eases the

formation of the homogenous solid solution required to produce a stable desorption of

the analyte.

(ii) Matrix extraction: the laser beam is focused onto the surface of the matrix-analyte

solid solution. The chromophore of the matrix couples with the laser frequency causing

rapid vibrational excitation, bringing about localised disintegration of the solid

soltuion. The clusters ejected from the surface consists of analyte molecules surrounded

by matrix and salt ions. The matrix molecules evaporate away from the clusters to

leave the free analyte in the gas-phase.

(iii) Analyte Ionization : The photo-excited matrix molecules are stabilised through proton

transfer to the analyte. Cation attatchment to the analyte is also encouraged during this

process. It is in this way that the characteristic [M+X]+ ( X= H, Na, K etc.) analyte

ions are formed. This ionization reactions take place in the desorbed matrix-analyte

could just above the surface. The ions are then extracted into the mass spectrometer for

analysis (see figure 2.17).

Fig (2.16) Desorption/ionization of macromolecules by using the UFMP-glycerin mixed matrix.


65

Matrix-assisted laser desorption/ionisation - time-of-flight mass spectrometer (MALDI-

TOF MS):

It is a relatively new mass spectrometer in which matrix assisted laser

desorption/ionization procedure was applied together with the efficient mass detector, Time-of-

Flight (TOF) detector which is available in two modes, linear and refractory modes (Fig 2.18).

Fig. (2.17) Schematic diagram of matrix-assisted laser desorption ionization mechanism

Fig. ( 2.18) basic components of a linear (upper) and reflecting (lower ) TOF mass spectrometer.


66

Matrix-assisted laser desorption/ionisation-time-of-flight in source decompostion mass

spectrometer (MALDI-TOF-ISD MS):

A recent extension of the MALDI in source design dramatically increased the ion

resolution and mass accuracy. The technique was dubbed “time lag focusing” (TLF), “delayed

extraction” (DE) or “pulsed ion extraction” (PIE). After desorption/ionization, the ions are

kept in the ion source under field-free conditions for a short period of time, like 100-400 ns,

before they are extracted with a high electrical field and accelerated towards the detector.

During the brief time between ion desorption and the extraction pulse, analyte ions can

undergo prompt fragmentaion within the ion source (in source decompostion, ISD). Many

reports of this phenomenon were published in which linear TOF instruments were incorporated

to provide a protein sequencing. Because the fragmentation occurs in the source, product ions

experience some of the effects of pulsed ion extraction, but due to the higher laser power

required to induce fragmentation it is very difficult to obtain high resolution and mass accuracy

for product ions in linear mode. Suckau and Cornett 1998, presented results from ISD

measurements made using an instrument with ion reflector, which provided isotopic resolution

for product ions up to several thousand Da. (Suckau and Cornett 1998).

Matrix-assisted laser desorption/ionisation-time-of-flight post source decay mass

spectrometer (MALDI-TOF-PSD MS):

PSD analysis is an extention of MALDI/MS that allows one to observe and identify

structurally informative fragment ions from decay taking place in the field free region after

leaving the ion source (Spengler, 1997). Mass spectrometric analysis of product ions from post

source decay of precursor ions that were formed by matrix-assisted laser desorption ionization

(MALDI-PSD) has evolved into a powerful method for primary structure analysis of

biopolymers. Especially in the field of peptide sequencing, MALDI-PSD has been widely

applied, mainly because of its high sesitivity for prepared sample amounts in the range 30-100

fmol and because of its high tolerance of sample impurities and sample inhomogenity

(Spengler, 1997).

It was first concluded that ions formed by MALDI must be extremely stable and

internally cool and that MALDI is therefore a very soft ionization technique (i.e. provide highly

stable molecular ion or provide no fragment ions ), the main reason for this assumption was

that these large biopolymers could be desorbed and ionized intact (which was impossible with


67

other ionization techniques) and these molecular ions had obviously survived hundreds of

microseconds during their flight through the instrument until detection (Spengler, 1997).

More recently, fragment ions, which are produced after leaving the ion source during

the flight in the field-free region were used for peptide sequencing, (see figure 2.19). These

ions are called “Post-source decay” or PSD ions and can be observed in instruments equipped

with an ion reflector. In contrast to classical Edman sequencing, PSD-sequence determinations

are possible even from complex mixtures like proteolytic digests if a gated electrostatic ion

selector is used (Suckau and Cornett 1998).

Mechanism of MALDI-PSD:

With reflector TOF-MS, it is in theory possible to obtain structural information on a

selected quasimolecular ion by mass analysis of daughter ions issued from in-flight

fragmentation of the parent ion. Intact molecular ions leaving the ion source and having

acquired sufficient internal energy during the desorption process (photoactivation, low energy

collisions,) can release this energy by undergoing fragmentation while traveling the first field-

free drift path of the instrument called post-source-decay or (PSD). The fragment ions have the

same velocity as their precursor ions but have different energy as a function of their mass.

Fragment ions are then discriminated as a function of their kinetic energy (thus their mass) by

the time dispersions induced by the electrostatic reflector. Large fragment ions (with higher

keinetic energy) will penetrate deeper into the reflectorn than smaller fragment ions and will

appear at later time on the resulting reflectron time-of-flight spectrum (Chaurand et al ,1999).

An important feature of MALDI-PSD instrument is their MS/MS capability, allowing one to

preselect a certain precursor ion in a mixture of multiple components. Precursor selection is

done by electrostatic “beam blanking” or “ion gating”.

All ions passing the beam blanking device are deflected off the ion detector except a

certain mass window which is transmitted without deflection. Deflection is performed by a fast

high voltage drop applied to the device which typically built of small plates, wires or strips.

Positioning of the ion gate within the flight path is always a compromise between position-

dependent dispersion of precursor ions of different masses and high transmission for (low

energy) PSD ions already formed at the position of the ion gate (Spengler, 1997).


68

2.6.1.5. GC/MS (hyphenated Gas chromatography /Mass spectrometry):

GC/MS is a combination of two microanalytical techniques: a separation technique, GC,

and an identification technique, MS. GC/MS combination overcomes certain deficiencies or

limitations caused by using each technique individually and gives a two-diminsional

identification consisting of both GC retention time and a mass spectrum for each component of

the mixture. This combination has several advantages. First, it can separate the components of a

complex mixture so that mass spectra of individual compounds can be obtained for qualitative

U2

U1L2

Detector

Small PSD fragments

Large PSD fragments

Stable ions(precursor)

L1

Ion gate

Sample

d1d2

first field free drift regionUa

DE

neutral fragment+ve fragment

(M+H)+

Fig.(2.19) Principle of PSD analysis in MALDI/MS, using two-stage gridded ion reflector. Mass analysis of these ions is done in a series of consecutive steps by lowering the potentials U1 (decelerating voltage) and U2 (reflecting voltage) of the reflector grid. Total instrument lengths can vary between 1m and several meters.


69

purposes, second , it can provide a quantitative information on these same compounds. GC/MS

can provide complete mass spectrum for as little as 1 pmol of an analyte. It gives direct

evidence for the molecular weight and the characteristic fragmentation pattern or chemical

fingerprint that can be used as the bases for identification. GC/MS is limited to the analysis of

those compounds that can be made volatile without thermal decomposition.

GC/MS consists essentially of three components: the gas chromatograph, the mass

spectrometer and the data system. The sample was dissolved in a volatile solvent (methanol, n-

hexane or EtOAc) and injected manually (10µl) in the GC/MS set-up working in the EI-MS

mode. The sample which eluted from the GC-column, was divided -in the same time- into two

parts, one of them passed through GC-detector (electron capture detector, ECD), and another

part passed through the EI-MS detector involved in the system. GC chromatograms were

obtained as quantitative peak intensities plotted versus retention times. EI-MS of each peak

was recorded depending on the retention times of each compond.

2.6.2. Nuclear magnetic resonance spectroscopy (NMR) NMR measurements were done at the Institut für Anorganische Chemie und

Makromolekulare Chemie of Heinrich-Heine University, Düsseldorf. ID and 2D, 1H and 13C-

NMR spectra were recorded at 300º K on Bruker DPX 300, ARX 400, 500 or GBF 600 NMR

spectrometers. All 1D and 2D spectra were obtained using the standard Bruker software. The

samples were dissolved in deuterated solvents ( DMSO-d6, CDCl3, CD3OD, Pyridine-d5), the

choice of the solvent depends mostly on the solubility of the compound. Residual solvent

signals of (CD3OD at 3.3 ppm ,1H, and 49.0 ppm,13C), (CDCl3 at 7.26 ppm and 77.0 ppm),

(DMSO-d6 at 2.49 ppm and 39.5 ppm) were considered as internal reference signal for

calibration. The observed chemical shift values (δ) were given in ppm. and the coupling

constant (J) in Hz.

2.6.3. The optical activity

Optically active compounds are compounds which have at least one chiral carbon.

Optically active compounds can rotate the plane polarized light. Optical rotation was

determined on a Perkin-Elmer-241 MC Polarimeter. The substance was stored in a 0.5 ml

cuvette with 0.1 dm length. the angle of rotation was measured at a wavelength of 546 and 579

nm of a mercury vapour lamp at room temperature (25ºC). The specific optical rotation was

calculated using the equation:


70

Where [α]20D = the specific rotation at the wavelength of the sodium D-line, 589 nm, at a

temperature of 20ºC.

[α]579 and [α]546 = the optical rotation at the wavelength 579 and 546 nm respectively,

calculated using the formula:

Where:

α = the measured angle of the rotation in degrees

I = the length in dm of the polarimeter tube,

C = the concentration of the substance expressed in g/100 ml of the solution.

2.6.4 Special procedures:

2.6.4.1 LC/MS dereplication procedure for targeting new kahalalides of Elysia grandifolia

Total methanol extract of E. grandifolia was subjected to ESI-MS analysis using

LC/MS set-up available in the Institut für Pharmazeutische Biologie, Heinrich-Heine

University, Düsseldorf. ESI-MS spectrum (Fig 2.20 & 2.21 ) detects many positive molecular

ion peaks, 879.8, 914.9, 596.7, 1478.8, 1494.3, and 1240.0, of known kahalalides, kahalalide B,

kahalalide C, kahalalide D, kahalalide F, kahalalide G and kahalalide J, respectively, that have

been found and were previously reported from E. rufescens extract.

Indeed , it shows two minor new kahalalides (based on mol. wt.) close to the major one

(kahalalide F). The corresponding molecular ion peaks of both peptides appeard in both sides

left and right to the corresponding peak of the major peptide. The mol.wt. of the first one,

kahalalide S (M+1= 1536.6), was 58 mass units more than kahalalide F and slightly more polar,

and the other, kahalalide R (M+1= 1520.5), was 16 mass unit (which equal to one oxygen

atom) less than the first new one, and slightly less polar than kahalalide F (Fig. 2.11)

Both new kahalalides were targeted for isolation based on the obtained data including

the molecular weight and the HPLC retention times, where the total methanol extract was

chromatographed by gel filtration, to separate as pure as possible the targeted peptides.

Complete isolation and purification was carried out by semi-preparative HPLC.

[α] =100 x α

I x Cλ

[α] =[α] 579 x 3.199

[α] 579[α] 546

4.199

20D


71

Figure (2.20) ESI-MS survey for the methanol extract of Elysia grandifolia detect 2 new

depsipeptides, kah.S [m/z 1536.5 (M+H)]+ and kah. R [m/z 1520.5 (M+H)]+ in addition

to the known kah. F [m/z 1478.8 (M+H)]+.


72

2.6.4.2. Determination of amino acid diastereoisomers of isolated kahalalides :

Marfey reagent N-(5-flouro-2,4-dinitrophenyl)-L-alanine amide (FDAA) was used to

prepare diastereoisomers of amino acid derivatives. This diastereoisomers can be very useful in

quantitative determination of D- and L- amino acids in protein hydrolysates by HPLC analysis.

The L- amino acid derivatives were eluted more rapidly than the D-isomers due to the strong

intramolecular hydrogen bonding (more hydrophobic) in the latter diastereoisomers (Marfey,

BG

E

C

D

O

J

K

Fig ( 2.21 ): ESI-MS survey for the methanol extract of Elysia grandifolia detect many of

known depsipeptides. (Kahalalides B, C, D, E, G, J, K, O).


73

1984). Marfey´s procedure was applied for the isolated kahalalides after complete acid

hydrolysis.

Each peptide (0.1 mg) was hydrolysed by heating in a sealed vial with 6N HCl (2 ml)

until complete hydrolysis and liberation of free amino acids. The hydrolysate was cooled, dried

and dissolved in 0.5 ml of 1 % acetone solution of N-(5-fluoro-2,4-dinitrophenyl)L-

alaninamide [FDAA], followed by 20 µmol of NaHCO3, the contents were mixed and heated

over a hot plate at 40 °C for 1 hour with frequent mixing, and after cooling, a 20 µmol HCl was

added, and the mixture was filled to a volume of 1 ml by addition of MeOH. Amino acid

standards were individually derivatised in the same manner using 2.5 µmol amino acid

standard to react with 3.6 µmol FDAA to obtain the amino acid-DAA derivative.

The obtained derivatives were centrifuged and the supernatants were injected in the

HPLC/MS spectrometer, the elution time was 1 hour for each peptide-hydrolysate-DAA

derivative. Standard amino acid-DAA derivatives were used in order to determine the

molecular weight (MW) and the retention time (RT) of each amino acid unit. After comparison

of the MW and RT of the hydrolysate derivatives with that of derivatised standards,

diastereoisomers were detected in each peptide hydrolysate. The test was repeated again using

the same procedure by co-chromatography (peak enrichment technique) to confirm this

determination. The derivatised standards were added separately to the derivatised hydrolysate

of each kahalalide and run again under the same condition in HPLC-MS spectrometer, to show

what diastereoisomer-standard peak will be enriched or completely overlap the unknown peak

of the hydrolysate derivatives. Complete overlapping means complete stereosimilarity between

standard amino acid and the corresponding amino acid in the hydrolysate.

Kahalalide Free aminoacids2 N HCl / 130 °C / 20 Hours

Free aminoacids1 % acetone solution of FDAA

1M NaHCO3 / 40 °C /1 HourAminoacid-DAA derivative

NH

O

H2N

N+

O

-O

N+

O

O-

F

N-(5-Fluoro- 2,4-dinitrophenyl )L-alaninamide

NH2

R

OOH

L-aminoacid

NH2

R

OOH

D-aminoacidNHO

H2N

N+

OO-

N+

O

O-

NH

R

O

O

NHO

H2N

N+

O

-O

N+

O

O-

HN

R

OOH

L-aminoacid- DAA deriv.

D-aminoacid- DAA deriv.

- HF

(A.A.Diastereoisomer-DAA deriv.)

strong intramolecular hydrogen bonding

H

9-membered ring

12-membered ringHPLC ret. time (longer) due to


74

The following reaction products were detected in the new kahalalide hydrolysates ( kahalalide

R and kahalalide S) using LC/MS analysis:

2.6.4.3. Acid hydrolysis and GC analysis :

Petrosia cerebrosides (2.0 mg) were hydrolysed by treatment with 3N HCl and stirred at

80 °C for about 5h. The solution was concentrated using N2. The residue was re-dissolved in (-

)-2-butanol (0.5 mL) and one drop of triflouroacetic acid. The solution was transferred to an

ampoule, sealed and heated at 130 °C in an oven overnight until complete butanolysis. The

solution was concentrated to dryness in a vacuum evaporator and then connected to the freeze

dryer overnight until complete dryness. The residue was then treated with

hexamethyldisilazane-chlorotrimethylsilane-pyridine (0.1 mL, 1:1:5) for 30 min at room

temperature. The solution was then centrifuged and the supernatant (1µL) was analysed by GC

on HP-5 column. The injection port and detector temperatures were 200 °C and 220 °C,

respectively. The temperature gradient was programmed as linear increase from 135 °C to 200

°C at 1 °C/min. Four peaks of hydrolysate were detected at 37.21, 39.91, 41.98, 42.95 minutes,

which was in agreement with the four peaks obtained for authentic D-galactose at 37.49,

40.15, 42.43, 43.32 minutes treated simultaneously in the same manner, [Leontein, et al.1977

and Gerrit, et al., 1977].

NH

O

H2N

F

N+OO-

N+O

O-

N-(2,4-dinitro-5-fluoro-phenyl)-L-alaninamide

C9H9FN4O5Exact Mass: 272,06

Mol. Wt.: 272,19(FDAA)

NH

ONH2

N+OO-

N+O

O-HNCH

CCH

OH3C

H3C

NH

OH2N

N+OO-

N+O

O-HNCH

CH2C

O

H2CC OH

O

DAA- glutamic acid

HO

C14H17N5O9Exact Mass: 399,10

Mol. Wt.: 399,31

HO

DAA- valine


Mol. Wt.: 369,33

NH

OH2N

N+OO-

N+O

O-HNCHC

CH

O

CH3

H2CCH3

DAA- Isoleucine

HN

O

H2N

N+

O

O-N+

O

O-

DAA-phe.ala.

NH

ONH2

N+OO-

N+O

O-

HN

NH2

O

DAA-ornithine

NH

CHCH2C

OHO


Mol. Wt.: 383,36

OH


Mol. Wt.: 417,37

OH


Mol. Wt.: 384,34

HNCHC

CHO OHH3C

NH

O

NH2

N+OO-

N+O

O-

DAA-threonine

HO

C13H17N5O8Exact Mass: 371,11Mol. Wt.: 371,30

N

CO

NH

ONH2

N+OO-

N+O

O-

DAA-proline

OH


Mol. Wt.: 367,31


75

2.7. Bioassays

2.7.1. Antimicrobial activity

Microorganisms The crude extracts and the pure compounds were tested for activity against the following

standard strains: gram positive bacteria Bacillus subtilis DSM2109, Staphylococcus aureus

ATCC 25923 and gram negative bacteria Escherichia coli DSM10290; the yeast

Sacharomyces cerevisiae DSM1333 and two fungal strains Cladosporium herbarum

DSM62121 and C. cucumerinum DSM62122.

Culture preparation The agar diffusion assay was performed according to the Bauer-Kirby-Test (DIN 58940,

Bauer et al, 1966). Prior to testing, a few colonies (3 to 10) of the organism to be tested, were

subcultured in 4 ml of tryptose-soy broth (Sigma, FRG) and incubated for 2 to 5 h to produce a

bacterial suspension of moderate cloudiness. The suspension was diluted with sterile saline

solution to a density visually equivalent to that of a BaSO4 standards. The standards were

prepared by adding 0.5 ml of 1 % BaCL2 to 99.5 ml of 1% H2SO4 (0.36 N). The prepared

bacterial broth is inoculated onto Müller-Hinton-Agar plates (Difco, USA) and dispersed by

means of sterile beads.

Agar diffusion assay For screening, aliquots of the test solution were applied to sterile filter-paper discs (5

mm diameter, Oxoid Ltd) using a final disc loading concentration of 500 µg for the crude

extract and 50 and 100 µg for the pure compounds. The impregnated discs were placed on agar

plates previously seeded with the selected test organisms, along with discs containing solvent

blanks. The plates were incubated at 37°C for 24 hr. and anti-microbial activity was recorded as

the clear zones of inhibition surrounding the discs. The diameter was measured in mm.

2.7.2 Bioactivity guided isolation using brine shrimp lethality test:

This technique is an in vivo lethality test involving the whole body of a tiny crustacean,

the brine shrimp, Artemia salina Leach. It has been previously utilised in various bioassay

systems including the analysis of pesticide residues, mycotoxins, stream pollutants,

anaesthetics, dinoflagellate toxins, morphine-like compounds, toxic oil dispersants,

carcinogenic phorbol esters and toxicants in marine environments (Mayer, et al., 1982).


76

This test takes into account the basic premise that pharmacology is simply toxicology at a

lower dose, and that toxic substances might indeed elicit, at a lower nontoxic dose, interesting

pharmacological effects. The procedure determines LC50 values in µg/mL of active compounds

and extracts in the brine medium.

Sample preparation Crude extract, butanol fraction, EtOAc fraction, and all butanol sub-fractions of

Callyspongia biru were subjected to the brine shrimp lethality test. The test samples were

dissolved in a suitable organic solvent and the appropriate amount was transferred to 10 mL

sample vials. Bioassay was done on 1.0 & 0.5 mg test samples. The samples were then dried

under nitrogen and redissolved in 20 µl DMSO. Negative control vials contain the same

amount of DMSO were also prepared.

Hatching the eggs Brine shrimp eggs (Dohse, Aquaristik GmbH, Bonn, Germany) were hatched in a small

tank filled with artificial sea water which was prepared with a commercial salt mixture (33 g,

Sera Sea-Salt, Aquaristik GmbH, Bonn, Germany) and distilled water (1000 mL). The eggs

hatched and became ready for the bioassay after 48 hours. Twenty nauplii were taken out by

pipette (counted macroscopically in the stem of the pipette against a lighted background) and

transferred into each test sample vial. Artificial sea water was added to render the volume 5 mL

and the vials were maintained under illumination. Survivors were counted, with the aid of a

magnifying glass after 24 hours and the mortality at each dose and control were determined.

The LC50 values were determined using the probit analysis method. The LD50 was derived from

the best fit line obtained by linear regression analysis.

Results The most active fraction was butanol subfraction (But V) the last fraction eluted from

the silica gel column of the butanol fraction of the sponge. Where it shows 80 % and 100 %

lethality effects at concentrations of 0.5 mg and 1.0 mg respectively. Therefore the isolation

was continued for this fraction where alkylpyridinium compound was isolated from this

fraction.


77

2.7.4. Cytotoxicity test

Cytotoxicity Assay

Antiproliferative activity was examined against several cell lines, which included MCF-7,

PC12, HeLa, L1578Y and H4IIE, and was determined by an MTT assay as described earlier

(Kreuter et al 1992, Mosmann 1983).

Cell cultures

L5178Y mouse lymphoma cells were grown in Eagle’s minimal essential medium supplement

with 10% horse serum in roller tube culture. H4IIE-cell line was grown in a DMEM-medium

with 10% fetal bovine serum, while HELA, PC12, and MCF-7 cells were grown in RPMI 1640

medium supplemented with non-essential amino acids, sodium pyruvate, 10 µg/mL insulin and

10% fetal bovine serum. The cell culture media contained 100 units/mL penicillin and

100 µg/mL streptomycin and were changed twice per week. The cells were maintained in a

humidified atmosphere at 37°C with 5% CO2.

Determination of cytotoxicity (MTT colorimetric assay )

MCF-7 cells, PC12, HeLa, L1578Y, and H4IIE were plated on 96-multiwell plates with

50.000 cells/well. The cells were allowed to attach for 24 h and then treated with different

concentrations of kahalalides for 24 h. After this treatment the medium was changed and the

cells were incubated for 3 h under cell culture conditions with 20 µg/ml MTT (3-(4,5-dimethyl-

2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide). The conversion of the tetrazolium salt

MTT to a colored formazan by mitochondrial dehydrogenases was determined as a marker of

cell viability according to Mosman 1983 (Kreuter et al 1992, Mosmann 1983). After this

incubation the cells were fixed on the plate with an aqueous solution containing 1%

formaldehyde and 1% calcium chloride and then lysed with 95% isopropanol-5% formic acid.

The concentration of reduced MTT as a marker for cell viability was measured photometrically

at 560 nm.

Results

78

III. Results

3-1 Natural Products from Elysia rufescens:

Sacoglossan molluscs of the genus Elysia have been intensively investigated for their

biologically active natural products which include diterpenoids (Paul and Van Alstyne 1988),

polypropionates (Gavagnin, et al 1994, Cueto et al 2005), and depsipeptides (Hamann and

Scheuer 1993, Hamann et al 1996, Goetz et al 1997, Horgen et al 2000). Elysia-derived

natural products show significant biological and pharmacological activities. The diterpenoids

were reported to be active against anti-HIV opportunistic infections as well as against Herpes

simplex type II (HSV II) (Paul and Van Alstyne 1988). The polypropionates exhibited

ichthyotoxicity (Gavagnin, et al 1994, Cueto et al 2005), while the depsipeptides were shown

to have fish deterrent activity and are used as a chemical defence by the sacoglossan (Becerro

et al 2001). The peptide derivatives which carry the trivial name kahalalides, were further

described to have anti-malarial, anti-cancer, anti-psoriatic, anti-tuberculosis, and anti-fungal

activities (Hamann and Scheuer 1993, Hamann et al 1996, Goetz et al 1997, Horgen et al

2000, Bourel-Bonnet et al. 2005, El-Sayed et al 2000). The name kahalalide, was derived

from the mollusc’s collection site, at Black Point near the shores of Kahala District of the

Oahu Island in Hawaii. Kahalalides have been isolated from the sacoglossans E. rufescens

(Hamann and Scheuer 1993, Hamann et al 1996, Goetz et al 1997), E. ornata (Horgen et al

2000) (Plakobranchidae) as well as from their green algal diet, Bryopsis sp. (Bryopsidaceae)

(Hamann and Scheuer 1993, Hamann et al 1996, Goetz et al 1997, Horgen et al 2000). The

green algal diet, Bryopsis sp., has been found to yield kahalalides A, B, F, G, K, P and Q.

(Hamann and Scheuer 1993, Hamann et al 1996, Goetz et al 1997, Horgen et al 2000, Kan et

al 1999, Dmitrenok et al 2006). The kahalalide peptides consist of 3 to 13 amino acid units,

ranging from a C31 tripeptide to a C75 tridecapeptide, with cyclic and linear components, the

latter terminating in a saturated fatty acid moiety. Ten of these derivatives are cyclic

depsipeptides, kahalalides A to F, K, O, P and Q, while three analogues, kahalalides G, H, and

J are linear peptides. Kahalalide F and its linear analogue kahalalide G are the only congeners

that feature the atypical amino acid, Z-dehydroaminobutyric acid (Z-Dhb).

Due to their biological importance, kahalalides A, B, and F have been chemically

synthesized. (López-Maciá et al 2001a, López-Maciá et al 2001b, Bourel-Bonnet et al.

2005). To date, kahalalide F is the only derivative reported to have significant activity

towards solid tumor cell lines, including human colon and lung cancers, and against some of

Results

79

pathogenic microorganisms that cause opportunistic infections in HIV/AIDS patients

(Hamann and Scheuer 1993). Kahalalide F displays both in vitro and in vivo anti-tumor

activity in various solid tumor models, which include colon, breast, non-small cell lung, and in

particular prostate carcinoma. In vitro anti-proliferative studies showed activity among certain

prostate cancer cell lines (PC-3, DU-145, T-10, DHM, and RB), but no activity was found

against the hormone-sensitive LnCAP (prostate cancer) cell line. In vivo models also

confirmed selectivity and sensitivity of the prostate tumor xenograft derived from hormone-

independent prostate cancer cell lines, PC-3 and DU-145. Furthermore, in vitro evaluation

exhibited that this activity is selective but not restricted to prostate tumor cells (Rademaker-

Lakhai et al 2005). It has also been revealed that kahalalide F induces cell death via oncosis

preferentially in the tumor cell (Suarez et al 2003). Kahalalide F has attracted most attention

and has been the subject of a patent application. Its mode of action and preclinical toxicity has

also been studied (García-Rocha et al 1996, Sewell et al 2005, Janmaat et al 2005, Suarez et

al 2003) and the compound is currently in phase II clinical trial as a potential anticancer

agent (Brown et al 2002, Ciruelos et al 2002, Rademaker-Lakhai et al 2005).

Chemical investigantion of methanol extract of Elysia rufescens led to the isolation of

five known kahalalides in addition to β-sitosterol and N,N-dimethyltryptophan methyl ester

3.1.1- Kahalalide F (1, Known compound)

Kahalalide F was isolated as a white amorphous powder, with [α]D of – 5° (c 0.35

MeOH). It has UV absorbance at λmax 203 nm. FAB-MS showed pseudomolecular ion peak

m/z 1478 [M+1]+ and 1500 [M+Na]+, suggesting the molecular formula C75H124N14O16. The

molecular weight of 1 was also confirmed by ESI-MS, and MALDI-TOF-MS. The 1HNMR

spectrum of kahalalide F revealed 14 deshielded amide NH resonances in the lower field

region, ranging from 6.76 ppm to 9.69 ppm, suggesting the peptidic nature of the compound.

One NH proton, a sharp singlet at δ 9.69, indicated an α,β-unsaturated amino acid, a broad 2H

singlet at δ 7.69 was shown by a cosy experiment to be the terminal NH2 of ornithine.

Compound 1 was ninhydrin-positive, thus supporting the presence of the free amino group of

ornithine. The remaining 11 NH protons were doublets; those that are part of the ring are

sharp, while the NH protons of the linear portion of the molecule are splitted and broad due to

several conformations of the molecule. COSY spectrum showed 14 different spin systems,

that were also confirmed by TOCSY spectrum, 12 spin systems possessing deshielded amide

Results

80

NH resonances at δ 6.76 Val-1 NH, δ 9.69 Dhb NH, δ 8.79 Phe NH, δ 7.62 Val-2 NH, δ 8.82

Ile-1 NH, δ 8.56 Thr-1 NH, δ 7.90 Ile-2 NH, δ 7.95 Orn NH, δ 8.10 Val-3 NH, δ 7.57 Val-4

NH, δ 7.82 Thr-2 NH, δ 7.88 Val-5 NH (Abbreviations used for amino acids and the

designations of peptides follow the rules of the IUPAC-IUB Commission of Biochemical

Nomenclature in: J. Biol. Chem. 1972, 247, 977-983). The other 2 spin systems show no

correlations to amide NH protons, they were assigned to proline and to the aliphatic acid, 5-

methylhexanoic acid. Each NH proton showed COSY correlation to the next α –proton, then

to the β-proton, then to γ- and δ-proton resonances. The COSY spectrum revealed the

presence of one proline spin system, [δ 4.36 (dd, J= 9.1, 6.6 Hz, H-α), 2.03,1.97 (m,m, 2H-β),

1.86 (m, 2H- γ) and δ 3.76, 3.52 (m,m, 2H- δ)]. The presence of 5-methylhexanoic acid (5-

MeHex) was indicated by the 2D NMR data, sequential COSY correlations were observed

between the α-methylene signals at δ 2.13 and the subsequent methylenes at δ 1.47 (m, 2H-3),

1.11 (m, 2H-4) followed by aliphatic methine proton at δ 1.47 (m, H-5) and two methyl

groups at δ 0.82 (d, J = 7.1 Hz ) and 0.82 (d, J = 7.0 Hz ). As shown by the broad NH signals

for the linear part of compound 1, a second regio-isomer of the aliphatic acid was detected

from the COSY spectrum as exhibited by the signals at δ 2.2 (m, 2H-2), δ 1.6 (m, 2H-3), δ

1.14 (m, 2H-4), δ 1.35 (m, H-5) and two methyl groups at δ 0.80 (d, J = 6.9 Hz, CH3 -6 ) and

0.80 (d, J = 7.0 Hz, CH3-7). TOCSY experiment showed an uncommon spin system which

was assigned as dehydroaminobutyric acid (Dhb) at 9.69 (s, NH), 6.34 (q, J =7.0Hz, 1H),

1.26 (d, J =7.5Hz, CH3). The ROESY correlation between NH and CH3 protons indicated a Z

stereochemistry of this uncommon amino acid. This uncommon amino acid was reported as a

constituent of peptides isolated from the terrestrial blue-green alga (Moore et al 1989), and

from an herbivorous marine mollusk (Pettit et al 1989). The ester linkage of the ring part of

the compound was confirmed by HMBC correlation between the β-Proton (shifted downfield

at δ 4.96) of threonine and the carbonyl at δ 164.4 of valine-1. 13C NMR and DEPT spectra displayed signals for 75 carbons including 14 carbonyls

at δ 169.5 , 164.4, 171.3, 172.9, 170.0, 169.7, 170.6, 173.0, 172.6, 171.3, 169.8, 169.0, 172.2,

and 173.8 of val-1, Dhb, Phe, Val-2, Ile-1, Thr-1, Ile-2, Orn, Pro, Val-3, Val-4, Thr-2, Val-5,

and 5-MeHex, respectively. In addition to 6 sp2 methines and 2 sp2 quaternary carbones, 18

methyl signals, 12 methylenes and 22 sp3 methines including 12 α-methines and two

oxygenated β methines one of them is more deshielded at δ 69.98 for Thr-1 indicating the

ester linkage to Val-1, and another at 67.4 ppm for Thr-2 as mentioned in table 3.1.2.

HMBC and ROESY experiments established the connectivity of the amino acids.

The 5-MeHex—Val-5—Thr-2—Val-4—Val-3 connectivity could be established by HMBC

Results

81

correlations between NH signals of Val-5, Thr-2, Val-4 and Val-3 at δ 7.88, 7.82, 7.57 and

8.10 respectively, to the vicinal carbonyls of 5-MeHex, Val-5, Thr-2, and Val-4 at δ 173.8,

172.2, 169.0, and 169.8, respectively. Connections of the amino acids Thr-1—Ile-2—Orn—

Pro were determined through HMBC correlations of the NH signals at 8.56, 7.90, 7.95 ppm to

the vicinal carbonyls at 170.6, 173.0, 172.6 ppm, respectively. The connectivity of Pro to Val-

3 was established through the HMBC correlation between δ-proton of Pro at δ 3.52 and the

carbonyl of Val-3 at δ 171.3, and through the ROESY correlation between δ-protons of Pro at

δ 3.76, 3.52 and the α-proton of Val-3 at δ 4.26. The cyclo [Val-1—Dhb—Phe—Val-2—Ile-

1—Thr-1] linkage was deduced through HMBC correlations of NH signals at 6.76, 9.69, 8.79,

7.62, 8.82 ppm to the vicinal carbonyls 164.4, 171.3, 172.9, 170.0, 169.7 ppm respectively,

while the connectivity of Val-1 to Thr-1 was confirmed by an HMBC correlation between the

β-proton at δ 4.96 of Thr-1 and the carboxyl at δ 169.5 of Val-1, and also the ROESY

correlation between the β-proton at δ 4.96 of Thr-1 and the α-proton of Val-1 at 3.86 ppm.

The deduced data of compound 1 as obtained by NMR were further confirmed by

ESI-MS/MS experiment, MALDI-TOF-PSD MS (matrix –assisted laser desorption/ionisation

– time-of-flight – post source decay mass spectrometry) and by Marfey´s analysis. The

reflectron mode MALDI-TOF mass spectrum performed with delayed extraction (DE)

showed a positive ion signal at m/z 1499.92 which was identified as sodium-ion associated

monoisotopic peak [M+Na]+ of compound 1, and showed also a positive ion signal at m/z

1515.9, [M+K]+ and at m/z 1478.5, [M+1]+.

MALDI-TOF-PSD spectrum confirmed the sequence of the depsipeptide, kahalalide

F, as shown in table (3.1.3). Similar results were obtained from tandem ESI-MS/MS

spectrum, where the positive protonated fragment ion peaks were evident at m/z 1266.7,

1165.7, 1066.6, 967.8, 836.0, 756.8 , 642.7 and 443.5 corresponding to [M+1]+-[5-MeHex-

Val-5], [M+1]+ -[ 5-MeHex- Val-5- Thr-2], [M+1]+ -[ 5-MeHex- Val-5- Thr-2- Val-4],

[M+1]+-[ 5-MeHex- Val-5- Thr-2- Val-4- Val-3], [(5-MeHex- Val-5- Thr-2- Val-4- Val-3-

Pro- Orn-Ile-2)+1]+, [cyclo (Val-1- Dhb- Phe- Val-2- Ile-1- Thr-1) + Ile-2+1]+, [cyclo (Val-1-

Dhb- Phe- Val-2- Ile-1- Thr-1) +1]+ and [(Dhb- Phe- Val-2- Ile) +1]+, respectively.

The stereochemistry of the amino acids were determined using Marfey analysis

(Marfey, 1984), and the experiment resulted in the presence of D-Pro, L-Orn , D-aIle, L-

aThr, D-aThr, D-Val, L-Val, and L-Phe. The NMR data, amino acid sequences, and

stereochemistry of compound 1 were identical to those of kahalalide F (Hamann and Scheuer

1993).

Results

82

3.1.1- Kahalalide F (1, Known compound)

NH

OCH3

OHN

H3C

H3C

OO

CH3

HN

O

CH3

HN

O

CH3

CH3

CH3

O NH

HN

ONH

CH3

H3C

O

NH

O

NH3C

H3C

HN

H3C

H3C HN

O

HO

H3C

NHO

H3CNH

OCH3

CH3

O

O

H3C

val- 1

val-2

val-3

val-4

val-5

L- Phe.

Z-Dhb

D-Ile

L- Orn.

D- pro

L-Thr

D-Ile

D-Thr

H2N

164.4

60.330.8

14.6

18.8

6.76

3.861.39

0.62

0.58

169.5

130.3

131.3

12.5

9.696.34

1.26

171.3

56.3

36.8

138.2 129.0

8.79

4.42

2.93

7.28

172.9

58.632.4

18.9

18.67.62

4.46217

0.62

0.77170.0

57.5

38.8

26.8

14.6

8.82

4.311.73

1.31

1.02

169.757.4

71.1

17.3

8.564.53

4.96

1.07

127.0

127.6

7.28

7.2

170.6

57.3

38.026.6 14.6

7.90

4.37

1.691.30 1.03

11.70.77

11.60.77

173.052.9

29.624.4

40.1

7.95 4.49

1.481.67

2.74

172.6

60.2

29.6

25.4

48.0

4.36

2.03, 1.97

186

3.76, 3.52

171.3

57.630.5

19.6

18.8

8.10

4.261.94

0.86

0.86

7.69

169.0

58.967.4

19.7

7.82

4.263.97

0.98

4.88

169.8

59.1

31.3

195

1817.57

4.28

1.98

0.80

0.80

173.8

36.3

24.0

39.0

28.12.13

1.47

1.11

1.47

172.2

59.630.719.6

18.4

7.88

4.231.960.84

0.84 22.50.82

22.50.82

0,0 10,0 20,0 30,0 40,0 50,0 60,0-20

0

20

40

60

80

100

120 test #1 Kf UV_VIS_1mAU

min

1 - Peak 1 - 0,0462 - 0,496

3 - 1,041

4 - 1,220

5 - 31,780

6 - 47,005

WVL:235 nm

Peak #5 31.87

-10,0

25,0

50,0

70,0

200 400 595

%

nm

202.8

Fig. ( 3.1.1 ): chemical structure with NMR values (up), HPLC chromatogram (down left and UV spectrum (down right) of compound 1, kahalalide F Yield : 120 mg


Results

83

C = O r e g i o n

C = C r e g io n

P h e - p h e n y l& D h b o l e f e n i c

β - T h r - 1

β - T h r - 2 α - C r e g io n

Fig. (3.1.3) : 13C-NMR and DEPT spectra of compound 1

NH

OCH3

OHN

H3C

H3C

OO

CH3HN

O

CH3HN

OCH3

CH3

CH3

O NH

HN

ONH

CH3

H3C

O

H2N

NH

O

NH3C

H3CHN

H3C

H3C HNO

HO

H3C

NHO

H3CNH

O CH3

CH3

O

O

H3C212

313

412

1066

967

[M+1]+

[M+Na]+[M]+1265

1065

868755

642

512

723

610

836

[M+1]+

ESI-MS/MSESI-MS

FAB-MS

Fig.( 3.1.2 ) : FAB-MS , ESI-MS and ESI-MS/MS of compound 1

Results

84

Fig. (3.1.4) : different reigons of 1HNMR spectrum of compound 1

NHs

α Hs

β Hs

γ Hs, & CH3α/βVal-1

δ/γ Orn

Pro

β/meThr-1

β-Pheα/β Phe

β/meThr-2

β/αThr-1

Thr-1

Fig. (3.1.5) : Total COSY (left), part of COSY (right) spectra of compound 1

Results

85

D h b - C H

P h e . p h e n y l

m e - D h b

α V a l - 1

m e - I l e - 1

δ O r n

β T h r - 1

β T h r - 2

δ − P r o

α T h r - 1

α T h r - 2

α V a l - 2

α V a l - 3

α V a l - 4α V a l - 5

α P r o

α I l e - 1α I l e - 2

α O r n

α P h e

γ − P r oβ V a l - 2

β I l e - 1

β P h e

α5 M e H e x

m e - V a l - 2m e - V a l - 1

Fig. (3.1.6) : Total HMQC spectrum of compound 1

Fig.(3.1.7) : NH-detected spin systems (left), Proline and 5-MeHex (right) spin systems from partial TOCSY spectrum of compound 1

Fig.(3.1.8) : NH-detected ROESY correlations (left), upfield part of ROESY spectrum (right) of compound 1

Results

86

Table (3.1.1) Marfey´s analysis results of kahalalide F hydrolysates : Amino acid-DAA deriv.

D.Ile L.Phe L. Val D. Val D.Pro L.Thr D.Thr L. Orn*

MW (+ve mode)

384.0 418.0 370.0 370.0 368.1 372.0 372.0 385.1

MW (-ve mode)

382.4 416.5 368.3 368.3 366.3 370.3 370.3 383.5

Ret.time in minutes

24.53 22.98 20.97 22.77 18.68 15.93 16.60 13.44

&

14.14

H CO HMBC Corre lations

HMBC CorrelationsH C=C

H C-C HMBC Correlations

NH

OCH3

OHN

H3C

H3C

OO

CH3HN

O

CH3

HN

OCH3

CH3

CH3

O NH

HN

ONH

CH3

H3C

O

H2N

NH

O

NH3C

H3CHN

H3C

H3C HNO

HO

H3C

NHO

H3CNH

O CH3

CH3

O

O

H3C

H

Fig.(3.1.9) : Total HMBC spectrum showing different H-detected HMBC correlations (up), NH-detected correlations to C=O (down left), and aliphatic protons–detected HMBC correlations to C=O (down right) of compound 1.

Results

87

* It was detected that L-ornithine isomer has two different retention times, because ornithine

contains α- and δ- reactive amino groups which could both react with FDAA producing two

ESI-MS-detectable products as shown in figure 3.1.12.

Table (3.1.2) : 1H and 13C NMR data of compound 1 in DMSO-d6 Amino

acid No. 13C,PP

M 1H,PPM multiplicity Amino

acid No. 13C,PPM 1H,PPM multiplicity

Val-1 1 2 3 4 5

169.5 60.3 30.8 14.6 18.8

(NH) 6.76 3.86 1.39 0.62 0.58

(d, J=9.0Hz) ( t,J= 9.0 Hz )

(m) (d, J=7.0Hz) (d, J=6.0Hz)

Pro

1 2 3 4 5

172.6

60.2 29.6 25.4 48.0

-

4.36 2.03,1.97

1.86 3.76,3.52

- (dd,J= 9.1,

6.6 Hz ) (m,m)

(m) (m,m)

Dhb 1 2 3 4

164.4 130.3 131.3 12.50

(NH) 9.69 -

6.34 1.26

(s) -

(q, J=7.0Hz) (d,J=7.5Hz)

Val-3

1 2 3 4 5

171.3 57.6 30.5 19.6 18.8

(NH) 8.10 4.26 1.94 0.86 0.86

(d, J=8.5Hz) ( m ) (m) (m) (m)

Phe. 1 2 3 4

5,5` 6,6`

7

171.3 56.3 36.8 138.2 129.0 127.0 127.6

(NH) 8.79 4.42 2.93

- 7.28 7.28 7.2

(d, J=5.5Hz) (q, J=6.5Hz)

(m) -

(m) (m) (m)

Val-4

1 2 3 4 5

169.8 59.1 31.3 19.5 18.1

(NH) 7.57 4.28 1.98 0.80 0.80

(d, J=8.5Hz) ( m ) (m) (m) (m)

Val-2

1 2 3 4 5

172.9 58.6 32.4 18.9 18.6

(NH) 7.62 4.46 2.17 0.62 0.77

(d, J=8.5Hz) ( m ) (m)

(d, J=7.0Hz) (d, J=6.5Hz)

Thr-2 1 2 3 4 -

169.0 58.9 67.4 19.7

-

(NH) 7.82 4.26 3.97 0.98

(OH) ,4.88

(d, J=8.0Hz) (m) (m)

(d,J=6.5Hz) (d,J=5.0Hz)

Ile-1

1 2 3 4 5 6

170.0 57.5 38.8 26.8 14.6 11.7

(NH) 8.82 4.31 1.73 1.31 1.02 0.77

(d, J=10.0Hz) ( m ) (m) (m)

(t, J= 7.6 Hz) (d)

Val-5

1 2 3 4 5

172.2

59.6 30.7 19.6 18.4

(NH) 7.88 2ndconf. (

7.85) 4.23 1.96 0.84 0.84

(d, J=7.5Hz) (d, J=7.5Hz)

( m ) (m) (m) (m)

Thr-1 1 2 3 4

169.7 57.4 71.1 17.3

(NH) 8.56 4.53 4.96 1.07

(d, J=8.0Hz) (t, J=7.8Hz)

(m) (d,J=6.5Hz)

5-Me-Hex.1st conf

1 2 3 4 5 6 7

173.8 36.3 24.0 39.0 28.1 22.5 22.5

- 2.13 1.47 1.11 1.47 0.82 0.82

- ( m ) (m) (m) (m) (m) (m)

Ile-2

1 2 3 4 5 6

170.6 57.3 38.0 26.6 14.8 11.6

(NH) 7.90 4.37 1.69 1.30 1.03 0.77

(d, J=8.2.0Hz) ( m ) (m) (m)

(t, J= 6.5 Hz) (d)

Orn.

1 2 3 4 5 -

173.0 52.9 29.6 24.4 40.1

-

(NH) 7.95 4.49 1.48 1.67 2.74

(NH2),7.69

(d, J=8.50Hz) ( m ) (m) (m) (m)

b.singlet

5-Me-Hex.

2st conf.

1 2 3 4 5 6 7

174.1 33.9 32.8 38.8 29.5 19.5 11.5

- 2.2 1.6

1.14 1.35 0.80 0.80

- ( m ) (m) (m) (m) (m) (m)

Results

88

R T : 5 . 0 0 - 4 5 . 0 0

5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5T i m e ( m i n )

5

1 0

1 5

2 0

2 5

3 0

3 5

4 0

4 5

5 0

5 5

6 0

6 5

7 0

7 5

8 0

8 5

9 0

9 5

1 0 0

Re

lati

ve

Ab

so

rb

an

ce

2 2 . 7 7

2 4 . 5 32 0 . 9 7

1 8 . 8 8

1 6 . 6 0

1 5 . 9 3

1 4 . 1 4

1 3 . 4 4

7 . 2 9 3 7 . 6 03 6 . 2 02 8 . 2 41 2 . 4 9 3 4 . 4 7 3 8 . 8 18 . 6 5 4 4 . 7 6

N L :2 . 7 8 E 5S p e c t r u m M a x i m u m n m = 2 1 0 . 0 -4 0 0 . 0 - n m = 3 9 9 . 5 -4 0 0 . 5 P D A M o h a m m e d 2 9 7

D-isoleucine

L-PhenylalanineD-Valine

L-ValineD-Proline

D-Threonine

L-Threonine

L-Ornithine

D-isoleucine

D-Valine

L-Valine

L-Phenylalanine

D-Isomer enriched

L-Isomer enriched

L-Isomer enriched

D-Proline

D-ThreonineL-Threonine

L-OrnithineL-Isomer enriched L-Ornithine

L-Isomer enriched

L-Isomer enriched

Fig (3.1.10): Marfey´s analysis results of kahalalide F-amino acids (up), ESI-MS

chromatogram showing base peak search for each individual amino acid

(chromatograms right) and their peak enriched analysis (opposite chromatograms

left) for D-Ile, L-Phe, L-Val& D-Val, D-Pro, D- Thr &L-Thr, and L-Orn.

Results

89

D-ThreonineL-Threonine

D-ProlineL-Prolineenriched

L-Threonineenriched

Kah F hyd.D-Proline

D-Thr

L-ThrD-Thr

D-Pro

L-Thr

L-ThrD-Thr

L-Pro

D-Pro

Fig (3.1.11): Compound 1–hydrolysate ESI-MS (+ve) : comparison of D-Thr and D-Pro [right] against enriched L isomers [left], the retention time was not always constant. Therefore, the peak enrichment technique was applied to unambiguously confirm the stereochemistry of each amino acid.

T w o O rn derivatives resulted from L .O rnith ine

2x L .O rn derivativesD . O rn derivatives

Standard D & L O rn

Standard L .O rn

Fig (3.1.12) : Standard L.Orn vs DL.Orn. , showing two possible products for each stereoisomer.

Results

90

Table (3.1.3): The calculated mass fragments from MALDI-TOF-PSD-MS of compound 1 and sequence determination.

RT: 5.00 - 45.00

5 10 15 20 25 30 35Time (min)

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100R

elat

ive

Abso

rban

ce22.77

24.5320.97

18.88

16.60

15.93

14.14

13.44

7.29 36.228.2412.49 34.478.65

D-isoleucine

L-PhenylalanineD-Valine

L-ValineD-Proline

D-Threonine

L-Threonine

L-Ornithine

Fig (3.1.13) : relative peak area of D-Val(right) to the L-Val (left) of compound 1[ratio = 3:2]

Fig.(3.1.14 ) The stereochemical profile of Amino acid units of compound 1

Results

91

3.1.2- Kahalalide E (2, Known compound)

O

HN

NH

HN

O

O

OHN

HN

OO

NH

N

O

O

0.830.83

0.840.79

1.37

1.45

4.278.12

7.084.41

1.58

1.251.18

4.187.9

1.25

3.998.17

2.65, 2.46

5.1

0.85

4.28

3.47, 3.66

1.82, 1.99

2.01.89,

8.264.38

3.083.11,

7.1

10.8

7.56.95

7.05

7.30

D-Ala-1

D-Ala-2

Leu-2

L-Pro

Leu-1L-Trp

9-Me-3-Decol

1.40

1.15

1.14

1.151.10

0.85

1.50

117.5

118.5

121.5

111

136.3

128.1

124

109.2

170.8

171.6

24.0

25.0

22.1 22.0

171.4

170.4

171.4

16.4

171.9

17.1

170.5

71.5

40.033.1

50.5

47.7

50.2

58.9

53.8

26.8

38.9

28.6

27.0

28.1

25.2

46.328.5

27.4

27.029.0

22.021.5

21.021.5

58.0


Mol. Wt.: 836,071

0,0 10,0 20,0 30,0 40,0 50,0 60,0-50

250

600 ms04052 rp2,7 UV_VImAU

min1 - Peak 1 - 0,0712 - 0,6193 - 1,0374 - 1,1215 - 1,1636 - 1,2547 - 1,3468 - 1,421

9 - 33,231

WVL:280 nm

Peak #1133.26

0,0

20,0

40,0

60,0

200 250 300 350 400 450 500 550 595

%

nm

220.8

281.1288.5


Fig (3.1.15): chemical structure of compound 2 with δC and δH values (up), HPLC-chromatogram (down left) and UV-spectrum (down right). Yield : 4 mg

MALDI-TOF-MS

(M+K) +

(M+H) +

(M+ Na )+

ESI-MS(M+1)+

(2M+H 2O)+

ESI-MS(M-1)+

(M+HCOOH)+

(2M+H 3PO4)+

(2M+HCOOH)+

Fig.( 3.1.16 ) : MALDI-TOF-MS and ESI-MS of compound 2

Results

92

Kahalalide E was isolated as a white amorphous powder, with [α]D of + 5° (c 0.25

MeOH). It has UV absorbance at λmax 221, 281, 288 nm. MALDI-TOF-MS showed

pseudomolecular ion peak at m/z 858.57 [M+Na]+ and 874.55 [M+K]+ and 836.65[M+1]+

suggesting the molecular formula C45H69N7O8. The molecular weight of 2 was also confirmed

by ESI-MS, and MALDI-TOF-PSD-MS. The 1HNMR spectrum of 2 showed 5 deshielded

amide-NH resonances in the lower field region, at 7.08, 7.90, 8.12, 8.17 and 8.26 ppm,

suggesting the peptidic nature of the compound. In addition to the most downfield NH proton

which is a sharp singlet at δ 10.8, an ABCD aromatic spin system as resembled by resonances

at 7.50, 6.95, 7.05 and 7.3 ppm was observed and suggested the presence of tryptophane, Trp.

COSY spectrum together with TOCSY experiment indicated 5 spin systems commencing

from the above mentioned five NH-resonances, in addition to the characteristic indole spin

system of Trp. TOCSY spectrum showed also the presence of characteristic proline spin

system (Hα 4.28, Hβ 1.89 & 2.00, Hγ 1.83 & 1.99 and Hδ 3.47 & 3.66 ppm) and β-hydroxy

aliphatic acid, 9-methyl-3-hydroxydecanoic acid (9-Me-3-Decol) [(H-2) at δ 2.65 & 2.46, (H-

3) at δ 5.1, (H-4) at δ 1.40, (H-5) at δ 1.15, (H-6) at δ 1.14, (H-7) at δ 1.15, (H-8) at δ 1.10,

(H-9) at δ 1.15 and two methyls at δ 0.85 and 0.85 ppm].

13C NMR spectra displayed signals for 45 carbons including 7 carbonyls at δ 170.8,

171.6, 171.4, 170.4, 171.4, 171.9, 170.5 of Trp, Leu-1, Pro, Leu-2, Ala-1, Ala-2 and 9-Me-3-

Decol, respectively, in addition to 8 aromatic carbons, 6 α-CH, one downfield aliphatic

methine carbone at 71.5 (C-3 of 9-Me-3-Decol), 3 highfield methines,12 CH2 and 8 methyls

as mentioned in table 3.1.5.

HMBC and NOESY experiments established the connectivity of the individual spin

systems. H-2 of indole at 7.1 ppm showed HMBC correlation to C-3, C-3a, C-7 and C-7a at δ

109.2, 128.1 111.0 and 136.3 respectively. The connectivity of Trp to Leu-1 was evident

through HMBC correlation between Trp-NH at δ 8.26 to the vicinal carbonyl of Leu-1 at

171.6 ppm. The sequence for Ala-1-Ala-2-9-Me-3-Decol was established also by HMBC

correlations of NH resonances at δ 7.90 and 8.17 to vicinal carbonyls at δ 171.9 and 170.5,

respectively. Connection of 9-Me-3-Decol to Trp was established through HMBC correlation

of Hβ-9-Me-3-Decol to carboxyl at 170.8. Although there are no evident HMBC correlations

between Leu-2-NH and the vicinal carbonyl of Ala-1, due to peak broadness, the connection

of Leu-2 to Ala-1 was determined by the HMBC correlation at δ 4.41 (Hα, Leu-2) to Ala-1

carbonyl at 171.4. HMBC experiment showed a correlation between Hα Pro at δ 4.28 and

Leu-2 carbonyl at 170.4 ppm. The connectivity of Leu-1 to Pro was established through the

NOE between Leu-1 NH at 8.12 and Hα-Pro 4.28 ppm. Also, the connection of the amino

Results

93

acids of Pro- Leu-2 was confirmed by NOE between δ-protons Pro at 3.47, 3.66 ppm and Hα

of Leu-2 at δ 4.41. NOESY experiment showed also a through space correlation between Ala-

1 NH at 7.9 ppm to Ala-2 NH at δ 8.17, while Ala-2 NH showed a through space correlation

to Hβ of 9-Me-3-Decol at 2.65 and 2.46. In addition, further NOEs were observed between the

downfield Hβ of 9-Me-3-Decol at 5.1 ppm and Hα of Trp 4.38 ppm. The NMR data of

compound 2 were identical to those of kahalalide E (Hamann et al 1996).

Indole N HTrp-N H A la-1 N H

Leu 2 N H

Leu -1 N H

A la-2 N H

D ow nfield region

C H -2

C H -4 C H -5C H -6C H -7

H α - region

A la-1

Leu -1

A la-2

Leu -2

H β A liphatic acid Trp Pro

H β Leu -1

H δ - Por H β -Por

M e Leu -1

H α A liphatic acid

H β Trp

M e-A la-1

H γ - Por

H γLeu -1H β Leu -2

H γLeu -2

2x M e Leu -2

H -9 A liphatic acid

M e-A la-2

M e Leu -1

2x M e- A liphatic acid

Fig (3.1.17): 1H NMR spectrum.

Indole NH

Trp-NH

Ala-1 NH

Leu -2 NH

Leu -1 NHAla-2 NH

C H -2

C H -4

C H -5

C H -6C H -7

TrpAla-1Ala-2

Leu -1Leu -2

α

β

γ

α

β

γ

α

β

α

β

α

Trp

Indole NH/ C H -2

ABCD spin system

Fig (3.1.18): parts of TOCSY Spectrum of Compound 2 showing NH-detected spin system .

Results

94

C=OTrp

H-3 Fatty acid/C=O-Trp H-3/CO

Pro

C-2C-3a

H-2 Ala-2/C=O Ala-2

CH3/COAla-1

CH3/COAla-2

HβTrp

Indole carbons

C-3

C-2C-3a

H-2 Pro/C=O Leu-2

H-2 /C=O Ala-1

2x Hβ/C=OFatty acid

HMBC correlations to indole aromatic carbons

HMBC correlations to C=O

NH Ala-1/C=O Ala-2

NH Trp/C=O Leu-2

NH Ala-2β/C=OFatty acid

Fig (3.1.19): Total HMBC spectrum of compound 2 (up), different parts (down) showing important HMBC correlations

Results

95

Deduced data of compound 2 obtained by NMR were confirmed by ESI-MS/MS

experiment and MALDI-TOF-PSD MS.

MALDI-TOF – PSD spectrum confirmed the sequence of a depsipeptide, kahalalide

E, as shown in table (3.1.4). Similar results were obtained from tandem ESI-MS/MS

spectrum, where the positive protonated fragment ion peaks were evident at m/z 808.4, 765.2,

650.1, 599.1, 581.3, 537.1, 464.7, 440.0, 415.1, 395.1, 326.9, 308.9, 282.1 corresponding to

[M+1-CO]+, [M-Ala] +, [M+1-Trp]+, [M+1-(Leu+pro+CO)]+, [M+1-(Ala+9-Me-3-Decol)]+,

[M+1-(Leu+Trp)]+, [M+1-(Trp+9-Me-3-Decol)]+, [9-Me-3-Decol+Ala+Ala+Leu+1]+,

[Trp+9-Me-3-Decol+Ala+1-(CO)]+, [Ala+Leu+Pro+Leu+1]+, [9-Me-3-Decol+Ala+Ala+1]+,

[Ala+Leu+Pro+CO+1]+, and [Ala+Leu+Pro+1]+ respectively.

NH Ala-1/NH Ala-2

NH Ala-1/H-2 Ala-1 & H-2Ala-2

NH Leu-1/H-2 Pro

NH Trp /H-2 Leu-1

Fig (3.1.20) : NH-detected NOESY correlations.

Fig (3.1.21) ESI-MS/MS spectrum of compound 2.

Results

96

[M+1] + = [ 9-Me-3-Decol+Trp+Leu+Pro+Leu+Ala+Ala+1]+ = 836.41

-CO-28

[M+1-CO]+ (m/e 808.4 )

- [Pro+Leu+CO]- m/e 237

[Ala+9-Me-3-Decol+Trp+....+Leu+Ala+1-CO]+

m/e 599.1

-[Leu+Ala-CO]

[Ala+9-Me-3-Decol+Trp+1-CO]+

m/e 415

- m/e 156

OHN N

H

HN

O

OO H

N HN

O O

NH

N

O

OHN N

H

HN

O

OO NH

O

NH

OHN

O NHO

NH

-[Ala]-m/e 71

[9-Me-3-Decol+Trp+Leu+Pro+Leu+Ala+1]+

-[9-Me-3-Decol]-m/e 184

[Trp+Leu+Pro+Leu+Ala+1]+

m/e 765

m/e 581

-[Trp]-m/e 186

[Leu+Pro+Leu+Ala+1]+m/e 395

[Pro+Leu+Ala+1]+

-[Trp]-m/e 186

[Leu+Pro+Leu+Ala+Ala+9-Me-3-Decol+1]+

- [Leu]- m/e113

[Pro+Leu+Ala+Ala+9-Me-3-Decol+1]+

m/e 650

m/e 537

-[9-Me-3-Decol]

-m/e 184

[Leu+Pro+Leu+Ala+Ala+1]+

m/e 650

OHN

NH

HN

O

O

O HN

O N

O

O

HNNH

HN

O

O

HN

O N

O

OO

HN

NH

HN

O

O

O N

O

O

O HNHN

O

O HN H

N

O O

NH

N

O

O

HNHN

OHN H

N

O

O

HN

N

O

O

HNHN

O

HN

O N

O

O

HN

HNO

N

O

Om/e 282

- [Pro]

- m/e 97

OHN

NH

HN

O

O

O

O- [Leu]

- m/e113O

HN

NH

O

O

O

[Leu+Ala+Ala+9-Me-3-Decol+1]+

[Ala+Ala+9-Me-3-Decol+1]+m/e 440 m/e 327

- [Leu]- m/e113

Fig (3.1.22) Possible fragmentation pattern and amino acids-sequencing of compound 2 by interpretation of Tandem ESI-MS spectrum.

Results

97

Table (3.1.4): The calculated mass fragments from MALDI-TOF-PSD-MS of

compound 2

X = 9-Me-3-hydroxy Decanoic acid [9-Me-3-Decol]

Results

98

Table (3.1.5) : 1H and 13C NMR data of compound 2 in DMSO-d6 Amino acid Carbon 13C NMR

ppm, Mult 1H NMR, ppm Multiplicity , J= Hz

1 170.8, s NH,8:26 d, J=7.7 2 53.8, d 4.38 q, J=7.4 3 26.8, t 3.11; 3.08 m; dd, J=14.7, 8.1 4 109.2, s 5 124.0, d 7.1; NH,10.80 s; s 6 136.3, s 7 128.1, s 8 117.5, d 7.50 d, J=8.1 9 118.5, d 6.95 dt, J=7.0, 0.7

10 121.5, d 7.05 dt, J=7.0, 1.1

Tryptophan

11 111.0, d 7.30 d, J=8.1 1 171.6, s NH, 8.12 br d 2 50.2, d 4.27 m 3 24.0, t 1.37 m 4 25.0, d 1.45 m 5 22.0, q 0.84 m

Leucine-1

6 22.1, q 0.79 d, J= 5.9 1 171.4, s 2 58.9, d 4.28 m 3 28.1, t 2.00; 1.89 m; m 4 25.2, t 1.99; 1.83 m; m

Proline

5 46.3, t 3.66; 3.47 m; m 1 170.4, s NH, 7.08 br d 2 58.0, d 4.41 m 3 38.9, t 1.58 m; m 4 28.6, d 1.25 m 5 21.0, q 0.83 m

Leucine-2

6 21.5, q 0.83 m 1 171.4, s NH, 7.90 d, J= 7.7 2 47.7, d 4.18 p, J= 7.3

Alanine-1

3 16.4, q 1.18 d, J= 7.0 1 171.9, s NH, 8.17 d, J= 5.6 2 50.5, d 3.99 p, J= 6.3

Alanine-2

3 17.1, q 1.25 d, J= 7.4 1 170.5, s 2 40.0, t 2.65; 2.46 dd, J= 14.7, 5.6; dd, J= 14.7, 7.0 3 71.5, d 5.10 p, 6.3 4 33.1, t 1.40 m 5 28.5, t 1.15 m; m 6 27.4, t 1.14 m 7 27.0, t 1.15 m 8 29.0, t 1.10 m 9 27.0, d 1.50 m

10 22.1, q 0.85 d, J= 6.7

9-Me-3-Decol

11 22.1, q 0.85 d, J= 6.7

Results

99

3.1.3- Kahalalide D (3, Known compound)

N

N H

O

H N

O

HN

O

OO

C H 3

H 3C

NH

N H 2

N H

L -P ro

D -T rp

L -A rg

7-M e-3 -O c to l

7 .3 4

6 .9 7

7 .0 5

7 .48

1 0 .91

7 .1 9

3 .1 4 , 2 .9 84 .5 5

8 .8 5 4 .1

2 .5 2 , 2 .6 1

2 .7 2 , 3 .7 0

0 .8 4

1 .4 1 , 16 3

0 .8 4

1 .60 , 1 .90

1 .5 2

4 .0 7

1 .1 5

7 .63

1 .2 5

3 .03

1 .67 , 1 57

1 .3 9

5 .05

1 .6 8 ,1 .58

C 31H 45N 7O 5E xact M ass: 595 ,3 48

M ol. W t.: 5 95 ,73 3

0,0 10,0 20,0 30,0 40,0 50,0 60,0-20

25

50

75

100

125

160 ms030627 #2 Egme1-13 UV_VIS_3mAU

min

1

WVL:280 nmPeak #7100%

10,0

12,5

25,0

37,5

60,0

200 250 300 350 400 450 500 550 595

%

nm

219.4

203.1

280.6

Fig (3.1.23 ): chemical structure of compound 3 showing δH values (up) and HPLC chromatogram (down left) and UV spectrum (down right). Yield : 3.1 mg

2 M+3HCOOH-1

2 M+2HCOOH-1

M+HCOOH-1

M+1

2M+1

ESI-MS/MS

Fig.(3.1.24) : ESI-MS and ESI-MS/ MS spectra of compound 3

Results

100

Kahalalide D was isolated as a white amorphous powder, with [α]D of - 40° (c 0.25

MeOH). It has UV absorbance at λmax 203, 219, 280 nm. Positive ESI-MS showed

pseudomolecular ion peak m/z 596.7 [M+1]+ and 1191.0 [2 M+1]+ and negative ESI-MS

showed pseudomolecular ion peak at m/z 641.0 [M+HCOOH-1]- and 1327.8 [2M+3HCOOH-

1]- suggesting the molecular formula C31H45N7O5. The molecular weight and the sequence

N

NH

O

HN

O

HN

O

OO

CH3

H3C

NH

NH2

NH

[Mol. wt. +H]+ = m/z 595

N

NH

O

HN

O

HN

OO

O

CH3

H3C

NH

NH

C31H43N6O5•

Exact Mass: 579,329Mol. Wt.: 579,71

-NH2

(80%)

(16 %)

N

NH

O

HN

O

HN

OOO

CH3

H3C

NH

-CH3N2

(100 %)C30H41N5O5

•

Exact Mass: 551,311Mol. Wt.: 551,677

-NHCNHNH2

N

NH

O

NH

O

HN

OO

O

CH3

H3C


(13 %)

N

NH

O OHN

O

CH3

H3C

-(Arg+H2O)

C25H32N3O3•


(18 %)

N O-

NH

O

HN

O

CH3

H3C

C20H25N2O2•

Exact Mass: 325,192Mol. Wt.: 325,425

(43%)

[- (Trp +Fatty acid)+H]

N

HN

O

O

NH

NH2

NH

+H

( 54 % )

-[Arg + Prol]

NH

OHN OH

O

CH3H3C

C20H27N2O3•


( 3 % )

C11H21N5O2•+

Exact Mass: 255,169Mol. Wt.: 255,316

Fig (3.1.25):Possible fragmentation pattern and sequence determination from ESI-MS/MS spectrum of compound 3

Results

101

determination of 3 was confirmed by ESI-MS/MS fragmentation pattern as shown in figure

3.1.25. The 1HNMR spectrum of 3 showed characteristic proton resonances that belongs to

the simplest known kahalalide, which is kahalalide D. Compound 3 is made up of three amino

acids consisting of tryptophane (Trp), proline (Pro), and arginine (Arg) plus one aliphatic acid

[7-Me-3-Hydroxy-Octanoic acid]. 1HNMR showed 2 deshielded amide-NH resonances in the

lower field region, at 6.78 (d, J = 5.68 Hz, Arg-NH) and 8.85(d, J = 6.9 Hz, Trp-NH) in

addition to downfield proton resonance at 7.63 (br t, J = 5.63 Hz, Arg-NH) and a

characteristic indole protons of Trp resonating at 6.97 (t, J = 7.4 Hz, Trp-CH-10), 7.05 (t, J =

7.3 Hz, Trp-CH-9), 7.34 (d, J = 8.2 Hz, Trp-CH-9), 7.48 (t, J = 8.2 Hz, Trp-CH-11), 7.19 (d, J

= 2.3 Hz, Trp-CH-5) and a downfield resonance at 10.91 (d, J = 1.2 Hz) for an indol-NH.

TOCSY experiment and COSY spectrum showed the rest of arginine-proton resonances as

mentioned in table 3.1.6. 1HNMR showed a characteristic proline spin system at δ 4.07 (dd, J

= 8.6, 4.3 Hz, H-2), 1.60, 1.90 (m,m, H-3),1.41, 1.63 (m,m, H-4), 3.70, 2.72 (m,m, H-5)

which was also confirmed by COSY and TOCSY experiments. The aliphatic acid of 3 was

assigned to be 7Me-3-Octol from the COSY and TOCSY spectra of compound 3 at δ 2.52,

2.61 (dd, J = 15.2, 4.95 Hz, H-2a and dd, J = m, H-2b), 1.67,157 (m,m, H-4), 1.25 (m, H-5),

1.15 (m, H-6), 1.52 (m, H-7), the presence of downfield proton resonance at δ 5.05 (m)

indicated the presence of an oxygenated methine group of the fatty acid (H-3) where its

position was established by direct COSY correlations to H-2. As are most of fatty acid

moieties of known depsipeptides, compound 3-fatty acid is an iso-acid showing a two methyl-

proton resonances at δ 0.84 and 0.84 (d, J 6.3 Hz, H-8 and H-9). The presence of a guanidine

group was established through ESI-MS/MS fragmentation where the presence of molecular

ion fragments at 579.3 [M - NH2, (16 %)], 551.3 [M - NHCHNH2, (100 %)] and 536.5 [M –

guanidine (14 %)] confirmed the presence of guanidine group. The connection of a different

spin systems was confirmed by ROESY and ESI-MS/MS fragmentation data. ROESY

spectrum showed a NOEs between (NH-Trp) at δ 8.85 ppm to Hα of 7Me-3-Octol and Hα-Pro

at δ 2.52, 2.61 and 4.07ppm, respectively. H-3 of the fatty acid at δ 5.05 showed an NOE to

Arg NH at δ 6.78, while Arg-NH at 6.78 showed an NOE to proline-Hα at 4.07, and

additional correlations to Trp-NH and Hα of the fatty acid moiety. ROESY, COSY and

TOCSY correlations as well as 1HNMR data are identical to those of kahalalide D (Hamann

et al 1996).

Results

102

H -3 a T r p ,H -3 b T rp

H - 2 T r p

H - 2 P ro & H - 2 A r g

H -5 a P ro

H - 3A r ga & b

H -2 F a t ty a c id

N - 6 H /H - 4 A r g

N H A r g /H - 3 F a t ty a c id

c o r r e la t io n sto H - 2 P ro

N H A r gN H In d o le

N -6 HA rg

N H T r p

Fig (3.1.27): Total COSY spectrum (up) showing different spin

systems and NH-detected ROESY correlations (down) of compound 3

Fig (3.1.26): 1H NMR spectrum of compound 3

Results

103

Table (3.1.6): NMR data of compound 3 in DMSO-d6 Amino acid Carbon 1H NMR,

ppm Multiplicity , J=

Hz ROESY correlations

Tryptophan 1 (NH) 8.85 d, J=6.9 Pro H-2; F.A H-2 2 4.55 q, J=7.4 Trp H-3, H-5 3 3.14; 2.98 m; dd, J=14.7, 7.1 Trp H-5 5 7.19 d, J=1.9 indole NH, Trp H-2, H-8, H-

11; Pro H-5 (NH) 10.91 d, J=1.3 Trp H-5; 8 7.34 d, J=8.2 9 7.05 t, J=7.6 10 6.97 t, J=7.3 11 7.48 d, J=8.2

Proline 2 4.07 m Arg NH, Trp NH 3 1.60; 1.90 m; m

4 1.41; 1.63 m; m 5 3.70; 2.72 m; m Arginine 1 NH, 6.78 d, J=5.68 Arg H-3a &b, NH-6; Pro H-2;

Trp H-2; F.a H-3 2 4.1 m 3 1.68;158 m 4 1.39 m

5 1.03 m 6 7.63 br t, J= 5.6

9-Me-3-Decol (F.A)

2 2.52; 2.61 dd, J= 14.7, 5.6; dd, J= 14.7, 7.0

F.A H-3; Trp NH;

3 5.05 p, 6.3 Arg NH 4 1.57;167 m; m

5 1.25 m; m 6 1.15 m 7 1.52 m 8 0.84 d, J= 6.3 9 0.84 d, J= 6.3

P r o l i n eS p i n s y s t e m

7 - M e - 3 - O c t o l( F a t t y a c i d )

Fig (3.1.28): Total TOCSY spectrum (up), and part of TOCSY spectrum showing proline and Fatty acid spin systems of compound 3 (down).

Results

104

3.1.4- Kahalalide B (4, Known compound)

Kahalalide B was isolated as a white amorphous powder, with [α]20D of + 35° (c 0.25

MeOH). It has UV absorbance at λmax 205, 228sh, 277 nm. ESI-MS showed positive

pseudomolecular ion peak m/z 878.8 [M+1]+ and 895.6 [M+H2O]+, 1774.9 [2M+H2O]+ and

1755.8[2M+1]+ and negative pseudomolecular ion peak m/z 913.1 [M+HCOOH-1]-, 975

[M+H3PO4-1]-, suggesting the molecular formula C45H63N7O11. The 1HNMR spectrum of 4

showed 6 deshielded amide-NH resonances in the lower field region, at δ 8.90 (d, J= 0.9 Hz),

8.32 (d, J= 5.4 Hz), 8.03 (d, J= 9.6 Hz), 7.63 (d, J= 9.4 Hz), 7.37 (t, J= 6.2 Hz), and 7.20 (d,

J= 7.3 Hz) of Leu, Tyr, Ser, Phe, Gly, and Thr, respectively, suggesting the peptidic nature of

the compound. COSY spectrum together with TOCSY experiment indicated 6 spin systems

N

O

HO

HN

O

O

NH

O

HN

O

N

O

NH

O

HO

HN

O

O

H

H

H6.68

7.00

2.78,2.83

4.29

8.32

2.11

1.35

1.10

1.50

0.75 0.78

8.034.4

3.35

3.61,3.91

7.37

5.4 1.25

4.45

4.2

7.2

4.38

2.10, 2.05

2.0, 1.8

3.51,3.96

4.10

1.51, 1.40

1.28

0.81 0.75

8.9

4.8

2.91, 2.93

7.63

7.21

7.167.18

157.7

115.0

129.8 127.3

35.5

56.0171.7

173.5

41.227.1

23.038.0

18.517.2

168.150.4

47.2

167.142.0

170.8

59.1

67.0

12.0

171.560.8

28.5

23.0

47.0

137.2

126.5

129.0

127.5

38.5

53.2

23.5173.1

50.8

38.0

11.011.5


Mol. Wt.: 878,022

0,0 10,0 20,0 30,0 40,0 50,0 60,0-50

0

50

100

150

200

250

300 ms050512 #7 rp2-4e UV_VIS_1mAU

min

123456

7

8

9

10

WVL:235 nm

Peak #8 30.81

-10,0

25,0

50,0

70,0

200 300 400 595

%

nm

204.9

277.2 562.4


Fig (29) : compound 4 (Kahalalide B) Yield : 5 mg

Results

105

commencing from the above mentioned 6 NH resonances as mentioned in table 3.1.8, in

addition to the characteristic proton resonances of p-disubstituted benzene ring (the rest of

Tyrosine protons) at δ 6.68 (d, J= 8.2 Hz) and 7.0 (d, J=8.2 Hz) and proton resonance cluster

at δ 7.16-7.21 ( 5 aromatic proton of phenylalanine). COSY and TOCSY spectra revealed the

presence of two more spin systems which were assigned to proline and 5-methyl hexanoic

acid (see table 3.1.8). As in the case of kahalalide F, kahalalide B showed a second regio-

isomer of 5MeHex. 13C chemical shifts were extracted from HMQC and HMBC spectra as

mentioned in table 3.1.8.

HMBC and ROESY experiments established the connectivity of the individual spin

systems. The NHs of Thr, Gly, Ser, Phe, and Leu at δ 7.20, 7.37, 4.4, 7.63 and 8.90,

respectively showed a ROESY correlation to Pro H-2, Thr H-2, Phe NH, Ser H-2 and Phe H-

2, at δ 4.38, 4.2,7.63, 4.4 and 4.8, respectively, thus the connections of the amino acids Thr-

Gly-Ser-Phe-Leu were determined. The connectivity of Leu to Pro was established by

ROESY through NOEs between Leu H-2 at 4.1 ppm and Pro H-5 at 3.51 ppm. Furthermore,

the sequence [5MeHex-Tyr-Ser] was established through NOEs between Tyr NH and Tyr H-

2 at 8.32 and 4.29, respectively to H-2 of 5MeHex and Ser NH at δ 2.11, and 8.03,

respectively.

Again Ser-Gly connectivity through the ester bond between the terminal C-3 of Ser

and the carboxyl of Gly was confirmed by HMBC correlation of Ser H-3 at δ 3.35 to the

carboxyl at 167.1 ppm. The above deduced connectivities of the different amino acids were

confirmed through additional HMBC correlations. NHs of Leu, Tyr, Ser, Phe, Gly, and Thr

showed HMBC correlations to vicinal carbonyls at 173.1, 173.5, 171.7, 168.1, 170.8, 171.5 of

Phe, 5MeHex, Tyr, Ser, Thr, and Pro respectively. ROESY, COSY, TOCSY and HMBC as

well as 1HNMR and 13C NMR data are identical to those of kahalalide B (Hamann et al

1996).

Results

106

Fig (3.1.30): 1H NMR spectrum, NHs and aromatic region (up), and α-protons and higher field region (down)

Fig ( 3.1.31 ) : Part of COSY spectrum of compound 4, showing Lower field part

Results

107

L e uT y r S e r P h e G ly

T h r

D is u b s t i t u t e d p h e n y l ( T y r )

P h e n y l

Figure (3.1.32): Parts of TOCSY spectrum , NH-detected spin systems (up).

L e u T y r S e r P h e G l y

T h r D i s u b s t i t u t e d p h e n y l ( T y r )P h e n y l

t o H - 3 F . A

t o H - 2 F . A

t o H - 2 P h e

t o H - 2 t y r t o H - 2 S e r

t o H - 2 T h r

N H - T h r / H - 2 P r o

fig (3.1.33): Parts of ROESY spectrum of compound 4, NH/NH correlation (up-left), higher field region (up-right) and NH/ aliphatic proton correlations (down)

Results

108

Table (3.1.7 ) ESI-MS/MS Fragment ions of compound 4 and sequence determination

Ion composition m/z M-[C2H5OH (Thr side chain)]+1 832.1 M-[Fatty acid] +1 765.1 M-[Fatty acid + C2H5OH+ H2O] +1 702.6 M-[Fatty acid + Tyr] +1 603.1 M-[Fatty acid + Tyr + H2O] +1 585.2 M-[Fatty acid + Tyr +2 H2O] +1 567.1

Tyr + Ser + Phe + Leu –[H2O] + 1 492.0

Tyr + Ser + Phe + Leu –[H2O + C=O] + 1 464.1 Ser + Phe + Leu –[H2] + 1 345.0 Pro + Thr + Gly +1 256.0

L e u T y rS e r P h e G l y

T h rD i s u b s t i t u t e d p h e n y l ( T y r )

P h e n y l

C 7 - T y r

P r o C = OT y r C = O

S e r C = OP h e C = O 5 M e H e x

C = OT h r C = O

Fig (3.1.34): Part of HMBC spectrum of compound 4 showing NH-detected correlations to vicinal carbonyls.

N

O

HO

HN

O

O

NH

O

HN

O

N

O

NH

O

HO

HN

O

O

H

HHtyr

Pro

Gly

Thr

Leu

Phe

Ser

5M eHexC7H 13O•

Exact M ass: 113,097

C9H 9NO2••

Exact Mass: 163,063

C2H3NO2••

Exact Mass: 73,016

C4H7NO2••


C5H7NO ••

Exact Mass: 97,053

C6H11NO••


C9H9NO ••


C3H4NO2•••

Exact Mass: 86,024

Fig (3.1.35): ESI-MS/MS of compound 4

Results

109

Table (3.1.8) : 1H and 13C NMR data of compound 4 in DMSO-d6

Amino acid Carbon 13C NMR ppm, Mult

1H NMR, ppm

Multiplicity, J= Hz

1 167.1 s (NH) 7.37 t, J= 6.2 Gly 2 42.0 t 3.61 &3.91 dd, J= 17.4, 5.4;

dd J=17.6, 5.9 1 170.8 s (NH) 7.20 d, J= 7.3 2 59.1 d 4.2 dd, J= 8.8, 1.0 3 67.5 d 4.45 m

OH 5.4 d, J= 9.2

Thr

4 12.0q 1.25 d, J= 7.3 1 171.5 s 2 60.8 d 4.38 m 3 28.5 t 2.10, 2.05 m,m 4 23.0 t 1.80, 2.00 m,m

Pro

5 47.0 t 3.51, 3.96 dd, J= 16.7, 8.8; dd, J= 6.5, 9.5

1 172.5 (NH) 8.90 d, J= 0.9 2 50.8 d 4.1 m 3 23.5 t 1.4 , 1.51 m,m 4 38.0 d 1.28 m 5 11.0 q 0.75 d, J= 7.1

Leu

6 11.5 q 0.81 d, J= 7.1 1 173.1 s (NH) 7.63 d, J= 9.4 2 53.2 d 4.8 dd, J= 15.5, 8.8 3 38.5 t 2.91, 2.93 m;m 4 137.2 s

5,5´ 129.0 d 7.21 m 6,6´ 127.5 d 7.16 m

Phe

7 126.5 d 7.18 m 1 168.1 s (NH) 8.03 d, J= 9.6 2 50.4 d 4.4 m

Ser

3 47.2 t 3.35 m 1 171.7 s (NH) 8.32 d, J= 5.4 2 56.0 d 4.29 dd, J= 13.3,7.3 3 35.5 t 2.78 , 2.83 dd, J= 14.1, 8.5;

dd J=14.8, 7.2 4 127.3 s

5,5´ 129.8 d 7.0 0 d, J=8.2 6,6´ 115.0 d 6.68 d, J= 8.2

Tyr

7 157.7 s 1 173.5 s 2 41.2 t 2.11 t, J= 7.2Hz 3 27.1 t 1.35 m 4 23.0 t 1.10 m 5 38.0 d 1.5 m 6 11.5 q 0.75 d, J= 6.9

5-MeHex

7 12.0 q 0.78 d, J=6.9 H-7

Results

110

3.1.5- Kahalalide C (5, known compound)

Kahalalide C was isolated as a white amorphous powder, with [α]D of + 40° (c 0.11

MeOH). It has UV absorbance at λmax 230, 278 nm. ESI-MS showed pseudomolecular ion

peak m/z 914 [M+1]+ and 937 [M+Na]+, 953 [M+K]+ and 971[M+K+H2O]+ and

H N

HN

OO

NH

H N

N HHN

O

OO

O

O H

O

O H

O

NH

N H 2

N H

T yr

T yr

P he

B u tanoic acid

Ile

A rg

T hr

C 47H 63N 9O 10E xac t M ass: 913 ,4 7M ol. W t.: 91 4 ,057

E S I - M S / M S o f K a h C

Fig(3.1.36):Compound 5 (kahalalide C). Yield : 1 mg

Results

111

pseudomolecular ion peak m/z 913.1 [M-1]-, 1827 [2M-1]-. MALDI-TOF-MS showed

positive pseudomolecular ion peak at m/z 914 [M+1]+ and 937 [M+Na]+, suggesting the

molecular formula C47H63N9O10. The indivdual amino acids and the sequence were

established through interpretation of both ESI-MS/MS [see figure 3.1.36 and table 3.1.9] and

MALDI-TOF-PSD [see figure 3.1.38 and table 3.1.10]. This kahalalide was previously

isolated from the same mollusc (E. rufescens) which was collected from the same

geographical zone (Hamann et al 1996).

Table (3.1.9) ESI-MS/MS Fragment ions of compound 5 and sequence determination

Ion composition m/z M-[OH] +1 897 M-[C=O] +1 886.4 M-[C2H5OH (Thr side chain)]+1 869.4 M-[guanidine] +1 855.5 M-[Ile] +1 801.3 M-[Ile + H2O] +1 783.2 M-[Tyr ] + 1 752.3 M-[ Phe + Fatty acid] + 1 697.3 M-[ Phe + Fatty acid + OH] + 1 680.3 M-[ Phe + Fatty acid + OH + OH ] + 1 662.4 M-[Ile + Tyr + H2O] + 1 620.2 M-[Ile + Tyr + H2O + NH3] + 1 603.3 [Arg + Tyr + Thr + Phe - H2O] +1 549.4 [Tyr + Thr + Phe + Fatty acid - H2O] +1 463.9 Tyr + Thr + Phe + Fatty acid – [C=O + OH] 436.0 [Tyr + Thr + Phe + Fatty acid – (C=O+H2O)] +1 434.2 Arg + Tyr + Ile –[C=O] 404.1 [Arg + Tyr + Thr –(NH2 + OH)] +1 386.0 [Arg + Tyr ] +1 320.7 [Arg + Tyr –( OH)] +1 //also // [Thr + Phe + Fatty acid – (OH)] + 1 302.9

Fig (3.1.37): MALDI-TOF-MS of compound 5

Results

112

Table (3.1.10) MALDI-TOF-PSD Fragment ions of compound 5 and sequence

determination

Ion composition m/z M-[C2H5OH (Thr side chain)]+1 869.8 M-[guanidine] +1 855.4 M-[Ile] +1 801.7 M-[Ile + H2O] +1 783.2 M-[Tyr ] + 1 751.8 M-[Tyr + OH] + 1 734.7 M-[ Phe + Fatty acid] + 1 697.3 M-[ Phe + Fatty acid + OH] + 1 680.5 M-[ Phe + Fatty acid + OH + OH ] + 1 662.7 M-[Ile + Tyr + H2O + NH3] + 1 603.3 M-[Arg + Tyr + ( C=O)] + 1 567.6 [Ile + Tyr + Arg + Tyr - ( C=O)] + 1 567.6 [Arg + Tyr + Thr + Phe - H2O] +1 549.4 [Tyr + Thr + Phe + Fatty acid - H2O] +1 463.9 Tyr + Thr + Phe + Fatty acid – [C=O + OH] 436.6 [Tyr + Thr + Phe + Fatty acid – (C=O+H2O)] +1 434.2 Arg + Tyr + Ile –[C=O] 404.1 [Arg + Tyr + Thr –(NH2 + OH)] +1 386.4 [Tyr + Thr + Phe – (C=O + OH + NH)] +1 350.6 [Arg + Tyr ] +1 320.7 [Thr + Phe + Fatty acid ]+ 1 319.6 [Arg + Tyr –( NH3)] +1 304.0 [Arg + Tyr –(OH)] +1 //also // [Thr + Phe + Fatty acid – (OH)] + 1 302.9 [Thr + Phe + Fatty acid – H2O]+ 1 301.3 Arg + Tyr – [C=O] 292.2 Thr + Tyr – [OH] 247.3 Phe + Fatty acid 218.7 Arg + NH 183.7 Tyr-[C=O] 136.3 Phe – [C=O] 120.2 Ile – [H2] +1 112.2 Phe – [NH + C=O] 104.0 Ile – [C=O] 86.1 Arg-[Guanidine + C=O] 70.1

Fig (3.1.38): MALDI-TOF-PSD-MS of compound 5

Results

113

3.1.6 – N,N-dimethyl Tryptophane metyhl ester (6, new marine natural product)

NH

N

O

O3.163.68

3.18

3.2

7.15

7.60

7.32

7.05

6.98

48.6

167.278.7

51.0

23.2

123.8

109.2127.1

118.4

136.0111.3

118.1

120.8


Mol. Wt.: 246,305

0,0 10,0 20,0 30,0 40,0 50,0 60,0-50

100

200

300 ms040608 #5 rp1c3 UV_VIS_3mAU

min

1 - 1,0412 - 1,1093 - 1,1384 - 1,2125 - 1,283

6 - 11,673

7 - 27,706 8 - 51,602

WVL:280 nm

Peak #711.86

10,0

20,0

40,0

60,0

200 250 300 350 400 450 500 550 595

%

nm

218.9

279.5286.5


Fig (3.1.39):Compound 6, structure with 1H and 13C- chemical shift values (up), HPLC chromatogram (down left), UV-spectrum (down right). Yield : 4.6 mg

Fig (3.1.40): ESI-MS (left), ESI-MS/MS of compound 6 (right).

Results

114

Compound 6 [N,N-dimethyl-tryptophan methyl ester] was isolated as a brownish

white amorphous powder, with [α]D of + 20° (c 0.25 MeOH). It has UV absorbance of typical

indole compounds at λmax 218, 279(sh), 286(sh) nm. ESI-MS showed positive

pseudomolecular ion peak m/z 247 [M+1]+ and 493 [2M+1]+, 515 [2M+Na]+ and negative

pseudomolecular ion peak m/z 245.4 [M-1]-, 291.6 [M + HCOO-]-, 537.8 [2M + HCOO-]-,

suggesting the molecular formula C14H18N2O2. 1H NMR spectrum showed characteristic

proton resonances of tryptophan at δ 3.68 (1H, dd, J=10.1,3.8 Hz, H-2), 3.20 (2H, m, H-3),

7.15 (1H, d, J=1.2 Hz, H-5), 7.32(1H, d, J=8.2 Hz, H-8),7.05(1H, t, J=7.3 Hz, H-9 ), 6.98(1H,

t, J=7.0 Hz, H-10 ), 7.6(1H, d, J=8.2 Hz, H-11), 10.86 (1H, s, NH), in addition to one

methoxy group at δ 3.16 (3H, s) and two N-methyls at δ 3.14 (6H, s). 13C NMR showed the

presence of one carboxyl at δ 167.2 (s. C-1), the presence of methoxy carbon and two N-

methyls were confirmed by resonances at δc 48.6 ppm and 51.0 ppm respectively. The α- and

β-carbon resonances of Trp were evident at 78.7 and 23.2 ppm respectively. COSY spectrum

established the connection of H-2 to H-3 and demonstrated the characteristic ABCD spin

system of indole moiety. The exchangeable NH was confirmed by measuring of the 1HNMR

in deuterated methanol. The connection of one methyl to carboxyl and the substitution of the

two amino protons with two methyls were confirmed through HMBC correlations between δH

at 3.16 and δc 167.2 ppm and between δH at 3.18 and δc 78.7 ppm respectively as shown in

table 3.1.11 and figure 3.1.45. These NMR data and UV-spectrum were identical with those

of the terrestrial natural product [N,N-dimethyl-tryptophan methyl ester] which has been

previously isolated from the Australian fabaceous plant Pultenaea altissima (Fitzgerald

1963). To the best of our knowledge this is the first report of 6 as a marine natural product.

Figure (3.1.41): 1H NMR spectrum of compound 6 meassured in DMSO-d6

8.23

04

4.32

51

Inte

gral

1587

.70

1578

.24

(ppm)3.13.2

0.99

97

1.08

82

1.12

12

1.00

00

1.03

41

1.05

15

1.05

04

1.81

47

1.52

46

8.23

04

4.32

51

Inte

gral

5431

.40

3802

.12

3793

.92

3758

.61

3664

.03

3655

.83

3576

.39

3575

.13

3535

.40

3527

.84

3520

.90

3494

.42

3487

.48

3479

.92

2063

.12

2058

.07

2041

.05

1847

.48

1843

.69

1837

.39

1834

.24

1587

.70

1578

.24

(ppm)0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.0

1.08

82

1.12

12

1.00

00

1.03

41

1.05

15

1.05

04

Inte

gral

3802

.12

3793

.92

3758

.61

3664

.03

3655

.83

3576

.39

3575

.13

3535

.40

3527

.84

3520

.90

3494

.42

3487

.48

3479

.92

(ppm)6.97.07.17.27.37.47.57.67.7

2

3

8 5 9 10 11

Indole NH

O-Me N(CH3)2

Results

115

Figure (3.1.42 ): 1H NMR spectrum of compound 6 measured in CD3OD

167.

2096

136.

0285

127.

0536

123.

8469

120.

8520

118.

4349

118.

1846

111.

2993

109.

2482

78.6

834

50.9

883

48.5

712

23.2

161

(ppm)0102030405060708090100110120130140150160170180190200

DEPT

13C NMR spectrumO-Me

N-(Me)2

CH2α-CH

N CH C

CH2

O

O

HN

12

3

4

5 6

78

9

10

11

45

67

89 1011

Figure (3.1.43): 13C NMR and DEPT spectra of compound 6

(ppm) 10.0 8.0 6.0 4.0

10.0

8.0

6.0

4.0

(ppm)

A B C D s p in s y s te mN H /H -5

C H /C H 2

Fig (3.1.44 ): COSY spectrum of compound 6.

1.17

17

2.26

21

8.26

63

7.02

85

Inte

gral

1950

.21

1944

.85

1941

.07

1935

.71

1716

.29

1711

.87

1706

.83

1646

.30

1640

.62

(ppm)3.43.63.8

1.00

00

1.01

08

0.92

94

1.09

58

0.99

32

1.17

17

2.26

21

8.26

63

7.02

85

Inte

gral

3810

.59

3809

.64

3808

.69

3801

.76

3660

.84

3653

.90

3652

.95

3583

.91

3550

.49

3549

.23

3543

.24

3542

.30

3535

.36

3534

.10

3520

.86

3519

.91

3513

.92

3512

.98

3512

.03

3506

.04

3505

.09

1950

.21

1944

.85

1941

.07

1935

.71

1716

.29

1711

.87

1706

.83

1646

.30

1640

.62

(ppm)0.01.02.03.04.05.06.07.08.09.010.0

2

3 8

5

9 10 11

O-Me

N(CH3)2

Results

116

Table (3.1.11): NMR data of 6 (500 MHz, DMSO-d6), Carbon 13C NMR,

ppm 1H NMR*, ppm (Multiplicity, J=

Hz)

1H NMR, ppm (Multiplicity, J= Hz)

HMBC H -------C

1 167.2 s 2 78.7 d 3.86 (dd, J=9.1; 5.4) 3.68 (dd, J=10.1; 3.8) C-1, C-4, C-3, N(CH3)2 3 23.2 t 3.41 (m) 3.20 (m) C-1, C-2, C-4, C-5, C-6, N(CH3)2 4 109.2 s 5 123.8 d 7.16 (s) 7.15 (d, J=1.2) C-4, C-6, C-7 (NH) 10.86 (s) C-6

6 127.1 s 7 136.0 s 8 111.3 d 7.32 (d, J=7.9) 7.32 (d, J=8.2) C-6, C-10 9 120.8 d 7.08 (dt, J=1.0,6.9) 7.05 (t, J=7.3) C-7, C-11

10 118.1 d 7.03 (dt, J=1.0, 6.9) 6.98 (t, J=7.0) C-6, C-8 11 118.4 d 7.61 (dd, J=7.9, 1.0) 7.6 (d, J=8.2) C-4, C-7, C-9

OMe 48.6 q 3.28 (s) 3.16 (s) N-(CH3)2 51.0 q, q 3.28 (s , s) 3.18 (s , s) C-2, C-3, N-Me

• meassured in (CD3OD)

(ppm) 7.2 6.4 5.6 4.8 4.0 3.2

160

120

80

40

(ppm)

N C O

O

HN

23

2

3

Fig (3.1.45) HMBC of compound 6 [Methanol-d4]

Results

117

3.1.7- β-sitosterol(7, Known compound):

Compound 7 [β-sitosterol] was isolated from the ether fraction of the total exract as a

white amorphous powder, with [α]D of - 37° (c 0.35 CHCl3). EI-MS showed molecular ion

peak m/z 414 [M]+ and fragment ions at 386, 368, 271, 255, 69, 55, 43 suggesting the

molecular formula C29H50O. 1H NMR spectrum showed resonances for six methyl groups at δ

0.68 (3H, s, Me-18), 1.10 (3H, s, Me-19), 0.93 (3H, d, J=6.2 Hz, Me-21), 0.81 (3H, d, J=7.25

Hz, Me-27), 0.83 (3H, d, J=7.57 Hz, Me-26) and 0.86 (3H, t, J=7.11 Hz, Me-29). The

resonances at δ 5.35 (1H, m, H-6), 3.52 (1H, m, H-3) were indicative for ∆5-6 mono

hydroxylated steroidal nucleus (Itoh et al , 1983).13C NMR showed six methyl signals at δ

11.8, 19.4, 18.8, 19.6, 19.0 and 12.3 for the methyl groups 18, 19, 21, 26, 27 and 29,

respectively. 13C NMR spectrum showed one quaternary olefenic carbon resonating at δ 140.7

and one olefenic CH at 121.7 and oxygenated methine at 71.8, in addition to two quaternary

aliphatic carbons, 7 aliphatic methines, and 11 aliphatic methylenes (see table 3.1.12). The

above NMR data were identical with those of β-sitosterol, which was previously isolated from

many other natural sources (Greca et al , 1990).

140.7

121.7

71.8

56.7

56.0

50.1

46.1

42.3

42.2

39.7

37.2

36.5

36.2

33.9

31.931.6

28.9

28.2

26.3

24.3

23.0

21.1 19.6

19.4

19.018.8

12.3

11.8

HO 31.9

1.10

0.68

5.35

3.52

0.93

0.83

0.81

0.91

C29H50OExact Mass: 414,386Mol. Wt.: 414,707

Fig (3.1.46): compound 7 Yield : 15 mg

H O

-- - - - - - -- - - - - - - -- - - - - - -- - - - - - - -- - - - - - -- - - - - - -- - - - - - - - --386

38 6 36 8

2 71

30 3

3 14

2 31

1 43

6 9

28

556 9

43

Fig (3.1.47):EI-MS spectrum of compound 7

Results

118

(ppm)0.60.81.01.21.41.61.82.02.22.42.62.83.03.23.43.63.84.04.24.44.64.85.05.25.4

H O

1

3

56

7

89

1 0

1 1

1 21 3

1 4 1 5

1 61 7

1 8

1 9

2 02 1 2 2

2 3

2 42 5

2 6

2 7

2 8

2 9

3 46

1 81 9

Fig (3.1.48): 1HNMR spectrum of compound 7 ( CDCl3)

(ppm)101520253035404550556065707580859095100105110115120125130135140145

6

5

10

1113

18

19

29

(ppm)102030405060708090100110120130140

12

3

4 7

89

12

14

1516

1720

21

2 2

23

24 252 6 27

28

HO

1

3

56

7

89

1 0

1 1

1 21 3

1 4 1 5

161 7

1 8

19

2 02 1 22

2 3

242 5

26

2 7

2 8

29

Fig (3.1.49): 13CNMR and DEPT spectra of compound 7 (CDCl3)

Results

119

Table (3.1.12): 1H, 13C-NMR data of compound 7 in (CDCl3 , 500, MHz) No. 13C (Multiplicity) 1H (Multiplicity, Hz) 1 37.2 t * 2 31.6 t * 3 71.8 d 3.52 (m) 4 42.2 t * 5 140.7 s - 6 121.7 d 5.35 (m) 7 31.9 t * 8 31.9 d * 9 50.1 d * 10 36.5 s - 11 21.1 t * 12 39.7 t * 13 42.3 s * 14 56.7 d * 15 24.3 t * 16 28.2 t * 17 56.0 d * 18 12.3 q 0.68 ( s) 19 20.4 q 1.10 ( s) 20 36.2 d * 21 18.8 q 0.93 (d, J=6.2 Hz) 22 33.9 t * 23 26.3 t * 24 46.1 d * 25 28.9 d * 26 19.6 q 0.83 (d, J=7.5 Hz) 27 19.0 q 0.81 (d, J=7.3 Hz) 28 23.0 t 0.82 (d, 6.6 Hz) 29 11.8 q 0.86 (t, J=7.1 Hz)

* not confirmed δH, because they are present in the highly overlapping multiplet signals of aliphatic methines

and methylenes in the higher field region between 1.00 and 2.28 ppm.

Bioactivity :

Compound 1 (kahalalide F) was reported as the most active kahalalide. Kahalalide F

was reported to have a significant biological activity against solid tumour cell lines, human

colon and lung cancers and some pathogenic microorganisms that cause the opportunistic

infections of HIV/AIDS. Its mode of action and preclinical toxicity have also been studied. It

is currently in Phase I and II clinical trials. Kahalalide F displays both in vitro and in vivo

antitumor activity in various solid tumor models, including colon, breast, non–small cell lung,

and in particular prostate cancer. In vitro antiproliferative studies showed activity among

certain prostate cancer cell lines (PC-3, DU-145, T-10, DHM, and RB), but no activity was

found against the hormone-sensitive LnCAP. In vivo models also confirmed selectivity and

sensitivity of the prostate tumor xenograft derived from hormone-independent prostate cancer

cell lines, PC-3 and DU-145. Further in vitro evaluation showed selective activity but not

Results

120

restricted to prostate tumor cells. Suares et al, 2003 revealed that kahalalide F induces cell

death via oncosis preferentially in tumor cells. (Hamann et al 1993, Hamann et al 1996,

García-Rocha et al 1996, Sewell et al 2005, Janmaat et al 2005, Suarez et al 2003, Brown et al

2002, Ciruelos, et al 2002, Rademaker-Lakhai et al 2005). Kahalalide A was reported to have

antimalarial and antituberculosis activity (Hamann et al, 1996; Copp 2003 and El-Sayed et al

2000). Furthermore kahalalide E was reported to have selective anti-viral activity against

Herpes simplex (Hamann et al, 1996).

The cytotoxic activity studies of 1 against three different cell lines (L5178Y, Hela, and

PC12) and showed ED50 values of 1 as 6.3µg/ml, 6.7µg/ml and >10 µg/ml respectively. The

cytotoxicity assays were summerized in Figure (3.1.50). Compounds 1 (kahalalide F), 3

(kahalalide D) and 4 (kahalalide B) showed mild antibacterial activity against B. subtilis and

S. cereviisae. Kahalalide B showed significant cytotoxicity (99 % inhibition ) compared to

kahalalide D (65.2 % inhibition), kahalalide E (94.8 %), while compound 6 showed no

significant activity against L5178Y cancer cell line at concentration of 10µg/ml each, see

figure (3.1.51).

0%

20%

40%

60%

80%

100%

120%

Cel

l pro

lifer

atio

n %

L5178Y Hela PC12

0µg/ml1µg/ml3µg/ml10µg/ml

Fig (3.1.50): In vitro cytotoxicity assay of kahalalide F against three different cancer cell lines .

02 04 06 08 0

1 0 0

Cel

l gro

wth

%

0.0

µg/m

l con

trol

10µg

/ml k

ah D

10µg

/ml k

ah B

10µg

/ml k

ah E

10µg

/ml c

omp.

6

Fig (3.1.51): In vitro cytotoxicity assay of compound 2 (kah E), 3 (kah D), 4 (kah B) and 6 ( N,N-dimethyl tryptophan methyl ester) against L5178Y cells .

Results

121

3-2 Natural Products from Elysia grandifolia:

Although many natural products were isolated from the genus Elysia this is the first

report for isolated natural products from E. grandifolia. Our interest with this sacoglossan

mollusc came up by the strong biological activity of its crude MeOH extract. Organic extracts

from the specialist herbivore E. grndifolia and its dietary green alga Bryopsis plumosa

exhibited antimicrobial, cytotoxicity, feeding deterrence and ichthyotoxicity (Padmakumar

1998, Bhosale et al , 1999).

Dereplication procdure using LC-MS was applied for targeting new natural products

from E. grandifolia. Inspection of the LC-MS chromatograms suggested the presence of

kahalalide derivatives giving molecular ion peaks corresponding to kahalalides B, C, D, E, F,

G, J, K, O (see figures 2-15 and 2-16). The presence of two unidentified peaks at m/z 1520.2

and 1536.0 aroused our interest to do further chemical work on the mollusc extract.

Chemical investigation of Indian sacoglossan mollusc E. grandifolia Kelaart 1858,

yielded two known analogues, kahalalide F (53.0 mg) and D (3.4 mg) along with two other

new kahalalide derivatives, which we designated as kahalalide R (20 mg) and S (3.4 mg). The

structural elucidation of the new kahalalides R and S will be discussed in detail.

Results

122

3.2.1 Kahalalide R (8, New natural product)

N H

OC H 3

OH N

H 3C

H 3C

O

O

C H 3

H N

O

C H 3

HN

O

C H 3

H 3C

C H 3

ON H

H N

O

NH

C H 3

H 3C

O

H 2N

NH

O

NH 3C

H 3C

H N

H 3 C

H 3C H N

O

O

O

H 4 .4 3

1 6 3 .5

1 3 0 .4

13 0 .1 9

12 .51 2 .92 ,2 .9 4

8 .78

0 .6 2

0 .6 0

1 .38

6 .7 3

3 .8 5

1 .2 8

6 .36 9 .6 9

7 .28

7 .2 9

7 .2 5

1 6 9 .7

6 0 .2

3 0 .1

1 9 .2

1 6 .5

1 7 1 .3 5 5 .6 5

3 6 .1

1 3 7 .0 2 12 8 .5

1 2 9 .5

1 2 6 .7 5

1 7 2 .6 5 5 .4 33 1 .5

18 .5

19 .5

1 7 1 .2

5 9 .5

3 1 .6

1 9 .3

1 9 .6

7.6 1

4 .4 52 .18

0 .6 2

0 .8 0

7 .89

4 .3 8

1 .8 8

0 .82

0.8 3

1 7 1 .3

5 7 .2 7

3 0 .2

2 6 .0

1 4 .6

1 1 .8 5

7 .8 7

4.3 4

1 .7 31 .0 2 ,1 .2 1

0 .8 4

0 .8 2

1 7 1 .55 1 .1 4

3 0 .9 2

2 8 .538 .3

7 .9 3

4 .4 9

1 .6 9 ,1.8 21 .5 2

2 .7 4

7 .72

1 72 .6

5 5 .9 5

31 .0

19 .0

2 2 .8 5

1 6 8 .7

5 6 .3 7

69 .98

17 .31 70 .03

59 .3

3 1 .5

1 8 .6

1 9 .5

17 2 .0

5 5 .6 52 9 .727 .24

4 7 .0

8 .1 1

4 .26

1 .9 4

0 .8 3

0 .8 4

8 .5 5

4 .5 0

4 .9 6

1 .08 8 .8 24 .1 3

1 .94

0 .81

0 .8 2

4 .3 7

2 .0 1 ,1 .98

1 .7 8 ,1 8 9

3 .78 ,3 .5 2

2 .11 1 .5 1

1 .281.2 5

1 .1 0 , 1 .15

1.5 2

0 .8 0

0 .8 1

1 7 2 .5

3 5.1

2 3 .6

22 .622 .0

2 2 .3

3 0 .6

1 8 .0

1 8 .1

O

N HH O

O

1 7 0 .8 8

5 6 .0 3

2 8 .03 8 .51 69 .45 7 .9 1

4 .411 .59 , 1 .52 .75

O

H 3CH N

H 3C

1 71 .2

5 1 .893 0 .72 2 .2

2 2 .9

7 .86

4 .3 71 .9 50 .8 4

0 .8 6

C 7 7H 12 6N 1 4O 1 7E x ac t M ass: 1 51 8 ,9 4 3

M o l. W t.: 1 5 19 ,9 0 8 Fig ( 3.2.1 ): Compound 8, kahalalide R.

0,0 10,0 20,0 30,0 40,0 50,0 60,0-20

50

100

150

180 ms030707 #8 egab4b UV_VIS_1mAU

min

1 - 0,6132 - 0,9083 - 1,0124 - 1,0875 - 1,2076 - 1,2687 - 1,3338 - 1,499

9 - 32,212

10 - 32,976

WVL:235 nm

Peak #9 50% 100% -50%

-10,0

0,0

12,5

25,0

37,5

50,0

60,0

200 300 400 500 595

%

nm

203.5

993.00 995.52

Fig (3.2.2): HPLC chromatogram and UV spectrum of compound 8. Yield : 20 mg

Results

123

Kahalalide R (compound 8) was obtained as a white amorphous powder. The reflector

mode MALDI-TOF mass spectrum of 8 performed with delayed extraction showed positive

monoisotopic ion peaks at m/z 1520.2, [M+H]+, 1542.2 [M+Na]+, and 1557.2 [M+K]+. In (+)-

ESI-MS, a pseudomolecular ion was detected at m/z 1520.8 [M+H]+ that was compatible with

the molecular formula C77H127N14O17 as established by HRESIMS. The 1H and 13C NMR data

of 8 were comparable to those of kahalalide F (compound 1), but had a higher molecular

weight of 42 mass units. Inspection of the 1H and 13C NMR spectra of 8 revealed that

kahalalide R shared very similar structural features with kahalalide F. As in 1, the region

between 6.50 and 9.70 ppm of the 1H spectrum of 8 accounted for a similar number of 14

deshielded amide NH resonances. Eleven of those are doublets, one is a singlet at δ 9.69 for

the α,β-unsaturated amino acid Z-Dhb, and a broad 2H singlet at δ 7.69 for the terminal NH2

of Orn. Like kahalalide F, compound 8 was also ninhydrin-positive, thus supporting the

presence of a free amino group found in Orn. The aromatic region also disclosed the presence

of Phe, the only aromatic amino acid in the structure of the new analogue as well as in the

known congener (kahalalide F). An apparent difference observed between the 1H spectrum of

1378.8

1250

1151

1052

953

742

629

N H

OCH 3

OH N

H 3C

H3C

OO

CH 3H N

O

CH 3

HN

O

C H 3

H 3C

C H3

O N H

H N

ONH

C H 3

H 3C

O

H2N

NH

O

NH 3C

H3CH N

H3C

H3C H N

O

O

O

H

O

N HH O

O

O

H 3CHN

H3C

856 837

Fig ( 3.2.3 ) : ESI-MS (up) and ESI-MS/MS (down) of compound 8

Results

124

8 and 1, was the absence of a second methyl triplet at δ 1.02 that suggested the loss of one Ile

unit in the structure of the new derivative. Major differences to kahalalide F were more

obvious from the 13C NMR and DEPT spectra of 8. The 13C NMR spectrum displayed 77

carbons signals instead of 75 carbons as in kahalalide F. An additional carbonyl signal was

observed between 163.5 and 174.1 ppm. Again, the DEPT spectrum also revealed the

presence of 12 regular amino acid residues in the new analogue 8, as indicated by the 12 α-

methine carbon signals between 51.0 and 60.0 ppm. The DEPT spectrum showed 15 instead

of 12 methylene carbons and a loss of an oxygenated methine carbon signal in the 65 to 75

ppm region, which further indicated a deficit of one Thr unit that was replaced by another

amino acid compared to kahalalide F. Similar to kahalalide F, the sp2-carbon region of the 13C NMR spectrum of 8 showed evidence for the occurrence of Phe and Dhb. The 13C NMR

and DEPT spectral data were in agreement with the molecular formula as determined by

HRMS.

The COSY spectrum of 8 revealed 14 spin systems, 12 of which commenced with a

deshielded amide NH doublet while two other spin systems showed no correlations to any

amide NH proton and were then allotted to Pro and a saturated fatty acid. The COSY

spectrum indicated the presence of 7-methyloctanoic acid (7-Me-Oct). Kahalalide R contained

a linear n-alkyl side chain that showed an iso- type methyl branching at the terminus of the

alkyl chain similar to that found in kahalalide F, which could be detected in the COSY spectra

of both 8 and 1. Sequential correlations of the iso-fatty acid (7-Me-Oct) observed from the α-

methylene signal at δ 2.11 to the subsequent methylenes at δ 1.51 (H2-3), 1.28 (H2-4), 1.25

(H2-5), and 1.10/1.15 (H2-6), which then terminated with an aliphatic methine proton at δ 1.52

(H-7) and two methyl functions at δ 0.80 and 0.81. In the 13C and DEPT spectra of the latter,

characteristic signals appeared for the terminal carbon atoms, i.e., Cω, δ C-8/9 22.5 (2 × q); Cω-1,

δC-7 27.3 (d); and Cω-2, δC-6 38.3 (t) which were unequivocally assigned by HMQC (see Table

3.2.2).

A TOCSY experiment corroborated the assignments obtained from the COSY

spectrum. The TOCSY spectrum unambiguously resolved the amine- and α-proton resonances

of each of the different 13 amino acid residues in the structure of the new depsipeptide

congener 8. Kahalalide R (8) was thus shown to contain six units of Val, one unit each of Phe,

aIle (allo-Ile), aThr (allo-Thr), Orn, Pro, Glu, also the unusual Dhb, and 7-Me-Oct. In the

new analogue, Val and Glu units replaced Thr and Ile previously found in kahalalide F. The

TOCSY spectrum allowed the overlapping 12 methyl doublets (J = 6.5 Hz) that belong to the

six Val units, which occur between 0.60 and 0.85 ppm, to be explicitly assigned to their

Results

125

respective spin systems. The presence of Glu also further explains the additional carbonyl

signal observed in the 13C NMR spectrum while the extra two methylene carbons could be

accounted for by the replacement of the 7-methyl-hexanoic acid (7-Me-Hex) in kahalalide F

with 7-methyl-octanoic acid (7-Me-Oct) in compound 8. Again, two structural regio-isomers

of the fatty acid moiety could be identified in the TOCSY spectrum of 8.

HMBC and ROESY experiments established the connectivity and sequence of the

amino acids in the peptide structure of 8 (Figure 3.2.4). The sequence 7-Me-Oct–Glu–Val-6–

Val-5–Val-4, which was elucidated as fragment I, was established through the HMBC

correlations of NH signals at δ 7.93, 7.86, 7.89, and 8.11 for Glu, Val-6, Val-5, and Val-4,

respectively, to each of their neighboring vicinal (2J) carbonyls of 7-Me-Oct, Glu, Val-6, and

Val-5 resonating at δ 172.5, 170.9, and 171.2, respectively. Connectivities for fragment II,

Pro–Orn–aIle– aThr–Val-3–Val-2–Phe–Z-Dhb–Val-1, were similarly determined through

HMBC correlations of the NH signals at δ 7.93, 7.87, 8.55, 8.82, 7.61, 8.78, 9.69, 6.73,

respectively, to their neighboring vicinal carbonyls at δ 172.6, 171.5, 171.3, 168.7, 170.0,

172.6, 171.3, and 163.5, respectively. The cyclization of Val-1 to aThr was confirmed by an

HMBC cross peak between the carboxyl signal at δ 169.7 for Val-1 and the β-proton of aThr

at δ 4.96, which arises from a characteristic low field acylation shift. This ring closure was

corroborated by the ROESY correlation of the β-proton of aThr with the α-proton of Val-1 at

δ 3.85. The connectivity of fragment I with II was established through the HMBC correlation

of the δ-proton of Pro at δ 3.52 with the carbonyl of Val-4 at 172.6 ppm. This was in

agreement with the ROESY cross peak between the δ-proton of Pro at 3.78 ppm and the α-

proton of Val-4 at 4.26 ppm.

NH

O

OHN

OO HN

O

HN

O

O NH

HN

ONH

OH2N

NH

O

N

HN

NH

O

O

O

NHHO

O

O

HNVal-6

L- Phe

Z-Dhb

D-aIleL- Orn

D- Pro

D-aThr

Val-1

Val-2

Val-3Val-4

Val-5

D-GluO

7-Me-Oct

Fragment I

Fragment II

Figure 3.2.4. Key HMBC correlations of kahalalide R (8)

Results

126

The ROESY spectrum of compound 8 also indicated the Z stereochemistry of

dehydroaminobutyric acid as shown by the NOE effect of the sharp NH singlet at δ 9.69 on

the methyl doublet at δ1.28 (J = 6.93 Hz). Together with kahalalides F and G, the new

analogues kahalalides R (8) and S (9), are the only derivatives that contain the amino acid Z-

Dhb. This uncommon amino acid, Z-Dhb, was reported as a constituent of peptides isolated

from terrestrial blue-green algae (Moore et al 1989), and from an herbivorous marine mollusc

(Pettit et al 1989).

(ppm)0.81.21.62.02.42.83.23.64.04.44.85.25.66.06.46.87.27.68.08.48.89.29.6

NH

OCH3

OHN

H3C

H3C

OO

CH3HN

O

CH3

HN

O

CH 3

H 3C

CH3

O NH

HN

ONH

CH 3

H3C

O

H 2N

NH

O

NH 3C

H3CHN

H 3C

H 3C HN

O

O

O

H

O

NHHO

O

O

H3CHN

H 3C

NH

OCH3

OHN

H3C

H3C

OO

CH3HN

O

CH3

HN

OCH3

CH3

CH3

O NH

HN

ONH

CH3

H3C

O

NH

O

NH3C

H3 CHN

H3C

H3C HNO

HO

H3C

NHO

H3 CNH

O CH3

CH3

O

O

H3C

val- 1

va l-2

val-3

va l-4

val-5

MeHex

L- Phe.

Z-Dhb

D-Ile

L- Orn.

D- pro

L-Thr

D-Ile

D-Thr

H2 N

Val -1

Pro

Dhb

Ile

Val -6

Val -3

Val -2

Thr

7-MeOct

Phe

Glu

Thr-2OH

Ile -1

Thr-2

Thr-2H-3

Thr-1H-3

Orn-NH2

Val -1

phenyl

Ile-2Val -5

Val -4Val -3 Val -2Thr

PheDhb

DhbOrn

0.9191

0.8770

1.0085

0.8996

0.9839

5.3729

3.5226

1.0137

1.1449

0.9366

0.7921

1.0640

3.3494

1.0703

3.3731

4.0004

1.0596

1.0743

1.1608

1.3544

2.0487

2.4072

3.3920

1.8572

1.6315

4.7918

2.2226

2.6673

3.2653

1.7730

3.8804

5.6907

2.2127

3.5910

3.4979

10.000

0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.0

Orn-NH2

Val -1

phenyl

IleVal -6

Val -5

Val -4Val -3

Val -2ThrPhe

Glu

DhbDhb

ThrH-3

Orn

Kahalalide F

Kahalalide R

(ppm)102030405060708090100110120130140150160170

Thr C-3

Thr -2C-3

Thr -1C-3

Region of carbonyls

13C NMR of kahalalide R Region of aromatic carbons

Region of α-carbons

13C NMR of kahalalide F

Higher field region

Fig ( 3.2.5 ): Comparison between 1H NMR of kahalalide F and R (up) and between 13C NMR of kahalalide R and F (down).

Results

127

Fig ( 3.2.7): Total TOCSY spectrum of compound 8 ( Kahalalide R).

0.9191

0.8770

1.0085

0.8996

0.9839

5.3729

3.5226

1.0137

1.1449

0.9366

0.7921

(ppm)6.26.46.66.87.07.27.47.67.88.08.28.48.68.89.09.29.49.69.8

Orn-NH2

Val -1

phenyl

Ile

Val -6Val -5

Val -4Val -3 Val -2ThrPhe

Glu

Dhb

Orn

Dhb

1 . 06 40

3 . 34 94

1 . 07 03

3 . 37 31

4 . 00 04

1 . 05 96

1 . 07 43

1 . 16 08

1 . 35 44

2 . 04 87

2 . 40 72

3 . 39 20

1 . 85 72

1 . 63 15

4 . 79 18

2 . 22 26

2 . 66 73

3 . 26 53

1 . 77 30

3 . 88 04

5 . 69 07

2 . 21 27

3 . 59 10

3 . 49 79

1 0 .0 00

0 . 51 . 01 . 52 . 02 . 53 . 03 . 54 . 04 . 55 . 0

2x MeVal -11x Me Val -2Thr-Me

PheH-3

GluH-4

Dhb-Me

OrnH-5

1xMe Ile2xMeVal -62xMeVal -52x MeVal -42xMe Val -31xMeVal -22xMe 7-MeOct

ProH-5

Val -1

Val -2H-3

ThrH-3

7-MeOctH-2 1st conf.

7-MeOctH-2 2nd conf.

IlePro

Val -6Val -5

Val -4

Val -3

Val -2 Thr

GluOrn

Hα

Val -1H-3

Hβ, Hγ

Fig ( 3.2.6 ): 1H NMR spectrum of 8, NHs and aromatic region (up) and higher field region of α,β,γ and methyl proton signals(down).

(ppm) 8.0 6.0 4.0 2.0

8.0

6.0

4.0

2.0

(ppm)

NH-detected spin system

Proline spin system

7-Me Oct

Results

128

Fig ( 3.2.8): NH-detected TOCSY correlations of compound 8 ( Kahalalide R).

Fig (3.2.9 ): Part of HMQC spectrum of compound 8 showing H----C direct correlations

of α-carbons.

(ppm) 9.6 8.8 8.0 7.2 6.4

4.8

4.0

3.2

2.4

1.6

0.8

(ppm)

Dhb Phenyl

Orn-

Val -1 Val -4 Val -3

Thr Phe

Dhb

α

Val -2

Orn

Ile Glu Val -6

Val -5

Me

β

α

Me

β

α

Me

Me

β

α

β

α

Me

β

α

Me

β

δ

(ppm) 4.8 4.4 4.0 3.6

68

64

60

56

52

48

(ppm)

Thr C-3

Ile Pro

Val -6 Orn

Phe Glu

Pro C-5

Val -5

Val -4

Val -3

Val -2

Val -1

Thr

Results

129

Fig (3.2.10): significant NOEs deduced from the ROESY spectra of compound 8.

Fig (3.2.11): Total HMBC spectrum of compound 8

(ppm) 8.0 6.0 4.0 2.0

160

120

80

40

(ppm)

NH-detected HMBC correlations to vicinal carbonyls

NH

OCH3

OHN

H3C

H3C

OO

CH3HN

O

CH3

HN

OCH3

H3C

CH3

O NH

HN

ONH

CH3

H3C

O

H2N

NH

O

NH3C

H3CHN

H3C

H3C HN

O

O

O

H

O

NHHO

O

O

H3CHN

H3C

Val -5

Val -4 Val -1

Pro

Dhb

Ile

Val -6

Val -3

Val -2

Thr

7-MeOct

Phe

Glu

Orn

Hα-----C=O

HMBC correlations to Aromatic carbons

C=O

Hα NHs

C=C

(ppm) 8.8 8.4 8.0 7.6 7.2 6.8 6.4

4.8

4.0

3.2

2.4

1.6

0.8

(ppm)

y

Hβ-Val-

Me- Dhb Me

NH

OCH3

OHN

H3C

H3C

OO

CH3HN

O

CH3

HN

OCH3

H3C

O NH

HN

ONH

CH3

H3C

O

H2N

NH

O

NH3C

H3CHN

H3C

H3C HN

O

O

O

H

36.1

59.3

29.7

O

NHHO

O

O

H3CHN

H3C

Orn

Orn-Val -1 Val -Val -3

Phe Dhb Val -2

Ile Glu Val -

Val -

Me-Val -1, Val-2

Phenyl Thr

Hα of most amino acids

Hβ-Thr

Hα- Val-1

Results

130

The deduced data obtained by 2D NMR (Figure 3.2.4) that determine the amino acid

sequence of kahalalide R (8) were further confirmed by mass spectrometric methods

involving both ESI (Spengler 1999) and MALDI–TOF–PSD ( Giorgiani et al 2004, Loo and

Loo 1997, Puapaiboon et al 1999) experiments. Similar results were obtained from the (+)

ESI-MS/MS spectrum, where fragment ion peaks were observed at m/z 1251.6, 1151.6,

1052.6, 952.9, 742.7 and 629.5 corresponding to [M–(7-Me-Oct–Glu)]+, [M–(7-Me-Oct–Glu–

Val-6)]+, [M–(7Me-Oct–Glu–Val-6–Val-5)]+, [M–(7-Me-Oct–Glu–Val-6–Val-5–Val-4)]+,

[M–(7-Me-Oct–Glu–Val-6–Val-5–Val-4–Pro–Orn)]+ and [M–(7-Me-Oct–Glu–Val-6–Val-5–

Val-4–Pro–Orn–aIle)]+, respectively. However, this MS/MS fragmentation of 8 was limited to

confirmation of the linear peptide side chain. In contrast, the MALDI–TOF–PSD spectrum

established unequivocally the sequence of the depsipeptide, kahalalide R, as shown in Table

3.2.1. The amino acid sequence of the cyclic component was indicated by ion composition

peaks at m/z 628.4 [Thr(-H)–Val-1–Dhb–Phe–Val-2–Val-3]+, 330.2 [Val-1–Dhb–Phe]+ or

[Dhb – Phe – Val-2]+, 247.1 [Val-2–Phe]+, and 183.1 [Val-1–Dhb]+. The terminal functional

units 7-Me-Oct–Glu were also shown by peaks at m/z 270.1 [7-Me-Oct–Glu]+ and 142.1 [7-

Me-Oct]+. The stereochemistry of the amino acids was determined by Marfey analysis

(Marfey 1984), which identified one mole unit each of D-Glu, D-Pro, L-Orn , D-aIle, D-aThr,

and L-Phe. For the six Val units, Marfey analysis suggested the presence of both D and L

isomers in compound 8. The stereochemistry of the individual Val units could not, however,

be unambiguously determined as there are three moles each of Val in both the cyclic and

linear fragments. This is not suprising as the stereochemistry of Val-3 and Val-4 in kahalalide

F has been long debated. The overall structure of kahalalide F was initially elucidated by

Scheuer’s group in 1993 (Hamann and Scheuer 1993), where Val-3 and Val-4 were

respectively assigned the L and D stereochemistry (Goetz et al 1999). The originally proposed

structure (Goetz et al 1999) was then synthesized by the groups of Albericio and Giralt

(López-Maciá et al 2001a), and showed differences in chromatographic and spectroscopic

behavior between the synthesized and the natural peptide. They indicated that the

stereochemistry of Val-3 and Val-4 should be reversed. Later, Rinehart’s group (Bonnard et al

2003) confirmed this indication and proved that the stereochemistry of Val-3 and Val-4 plays

an important role in the activity of kahalalide F since the depsipeptide with L-Val-3 and D-

Val-4 in its structure was not active while the molecule with D-Val-3 and L-Val-4 possesses

the activity.

Results

131

Table 3.2.1 Important MALDI-TOF-PSD fragment ions of Kahalalide R Ion composition m/z M–[7-Me-Oct– Glu]+ 1250.6

M–[7-Me-Oct–Glu–Val-6] + 1150.7

M–[7-Me-Oct–Glu–Val-6–Val-5] + 1051.6

M–[7-Me-Oct–Glu–Val-6–Val-5–Val-4] + 952.6

M–[7-Me-Oct –Glu–Val-6–Val-5–Val-4–Pro] + 855.5

M–[7-Me-Oct–Glu–Val-6–Val-5–Val-4–Pro–Orn] + 741.4

Cyclo[Thr(-H)–Val-1–Dhb–Phe–Val-2–Val-3] + 628.4

[Val-1–Dhb–Phe] + or [Dhb–Phe–Val-2] + 330.2

[Val-2–Phe] + 247.1

[Val-1–Dhb] + 183.1

[Pro–Orn] + 212.1

[7-Me-Oct-Glu] + 270.1

[7-Me-Oct] + 142.1

RT: 5.00 - 45.00

5 10 15 20 25 30 35 40 45Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

RelativeAbsorbance

22.26

22.46

20.47

24.00

16.2417.04

13.87

13.2037.1527.58 38.387.25 34.3212.27 33.788.30 44.32

NL:3.06E5SpectrMaximnm=21400.0 nm=39400.5 Moham

D-Pro

D-Thr

D-Glu

L-Val

D-ValL-Phe

D-IleL-Orn

Fig (3.2.12): Marfey analysis result and sterochemical profile of Kahalalide R

Results

132

Table 3.2.2. 1H and 13C NMR Data of Kahalalide R in DMSO-d6.

Amino acid No.

13C (ppm)

1H (ppm) mult. Amino acid

No.

13C (ppm)

1H (ppm) mult.

Val-1 1 2 3 4 5

169.7 60.2 30.1 16.5 19.2

(NH) 6.73 3.85 1.38 0.62 0.60

(d, J=8.5 Hz) ( t, J= 9.0 Hz ) (m) (d, J=6.3 Hz) (d, J=6.3 Hz)

Pro

1 2 3 4 5

172.655.629.727.247.0

4.37

2.01, 1.98 1.78, 1.89 3.78, 3.52

(m) (m, m) (m, m) (m, m)

(Z)-Dhb

1 2 3 4

163.5 130.4 130.2 12.5

(NH) 9.69

6.36 1.28

(s) (q, J=7.0 Hz) (d, J=7.5 Hz)

Val-4

1 2 3 4 5

172.655.931.022.819.0

(NH) 8.11 4.26 1.94 0.84 0.83

(d, J=8.5 Hz) (m) (m) (m) (m)

Phe 1 2 3 4 5,5’ 6,6’ 7

171.3 55.65 36.1

137.0 128.5 129.5 126.7

(NH) 8.78 4.43

2.94, 2.92

7.28 7.29 7.25

(d, J=5.5 Hz) (q, J=6.5 Hz) (m, m) (m) (m) (m)

Val-5

1 2 3 4 5

171.259.531.619.319.6

(NH) 7.89 4.38 1.88 0.82 0.83

(d, J=8.5 Hz) (m) (m) (m) (m)

Val-2

1 2 3 4 5

172.6 55.4 31.5 19.5 18.5

7.61 (NH) 4.45 2.18 0.80 0.62

(d, J=8.5 Hz) (m) (m) (d, J=7.0 Hz) (d, J=6.5 Hz)

Val-6

1 2 3 4 5

171.251.8930.722.222.9

(NH) 7.86 4.37 1.95 0.84 0.86

(d, J=8.5 Hz) (m) (m) (m) (m)

Val-3

1 2 3 4 5

170.0 59.3 31.5 18.6 19.5

(NH) 8.82 4.13 1.94 0.81 0.82

(d, J=8.5 Hz) (m) (m) (d, J=7.0 Hz) (d, J=6.5 Hz)

Glu

1 2 3 4 5

170.92nd regio-

isomer56.028.038.5

169.4

(NH) 7.93 (NH) 7.91

4.41

1.59, 1.50 2.75

(OH) 7.71

(d, J=7.5 Hz) (d, J=7.5 Hz) (m) (m) (m, m) (bs)

aThr

1 2 3 4

168.7 56.4 70.0 17.3

(NH) 8.55 4.50 4.96 1.08

(d, J=8.0Hz) (t, J=7.8 Hz) (m) (d, J=6.5Hz)

aIle

1 2 3 4 5 6

171.3 57.2 30.2 14.6 26.0 11.8

(NH) 7.87 4.34 1.73 1.21 1.02 0.82

(d, J=8.2 Hz) ( m ) (m) (m) (t, J=6.5 Hz) (d, J=6.5 Hz)

Orn

1 2 3 4 5

171.5 51.1 30.9 28.5 38.3

(NH) 7.93 4.49

1.69, 1.82 1.52 2.74

(NH2) 7.72

(d, J=8.5 Hz) (m) (m, m) (m) (m) (bs)

7-Me-Oct

1 2 3 4 5 6 7 8 9

172.535.1 23.6 29.5 29.5 38.3 27.322.5 22.5

2.11 1.51 1.28 1.25

1.15, 1.10 1.52 0.80 0.81

(m) (m) (m) (m) (m) (m) * *

*Resonance is underneath the methyl signals of Val and aIle.

Results

133

3.2.2- Kahalalide S (9, New natural product)

N H

OC H 3

OH N

H 3 C

H 3 C

O

O

C H 3

H N

O

C H 3

HN

O

C H 3

H 3 C

C H 3

ON H

H N

O

NH

C H 3

H 3 C

O

H 2 N

NH

O

NH 3 C

H 3 C

H N

H 3 C

H 3 C H N

O

O

O

H 4 .4 3

1 6 3 .0 1 3 1 .0

1 3 0 .1

2 .9 2 ,2 .9 4

8 .7 8

0 .6 2

0 .6 0 1 .3 8

6 .7 3

3 .8 5

1 .2 8

6 .3 6 9 .6 9

7 .2 8

7 .2 9

7 .2 5

1 6 7 .9

6 0 .2

3 0 .1

1 9 .2

1 6 .5

1 7 1 .35 5 .6 5

3 6 .11 3 7 .0

1 2 8 .5

1 2 9 .5

1 2 6 .7 5

1 7 2 .85 5 .4 3

3 2 .5

1 8 .5

1 9 .5

1 7 1 .3

5 9 .5

3 1 .6

1 9 .3

1 9 .6

7 .6 1

4 .4 52 .1 8

0 .6 2

0 .8 0

7 .8 8

4 .3 21 .8 8

0 .8 2

0 .8 3

1 7 1 .3

5 7 .2 7

3 0 .22 6 .0

1 4 .6

1 1 .8 5

7 .8 7

4 .3 4

1 .7 3

1 .0 2 ,1 .2 10 .8 4

0 .8 2

1 7 1 .55 1 .1 4

3 0 .9 22 8 .53 8 .3

7 .9 3

4 .4 9

1 .6 9 ,1 .8 21 .5

2 .7 4

7 .7 2

1 7 0 .2

5 9 .5

3 1 .0

1 9 .0

1 9 .4

1 6 8 .5

5 7 .16 9 .9 8

1 7 .3

1 7 0 .0

5 9 .3

3 1 .5

1 8 .9

1 9 .1

1 7 2 .0

5 5 .6 52 9 .72 4 .8

4 7 .2

8 .1 1

4 .2 6

1 .9 4

0 .8 3

0 .8 48 .5 5

4 .5 0

4 .9 6

1 .0 8 8 .8 24 .1 3

1 .9 4

0 .8 1

0 .8 2

4 .3 7

2 .0 1 ,1 .9 8

1 .7 8 ,1 8 9

3 .7 8 ,3 .5 2

O H2 .1 1 1 .6 1 ,1 .5 2

1 .2 33 .4 1

4 .2 2

1 .1 21 .7 1

0 .8 0

0 .8 1

1 7 2 .5

3 5 .4

2 3 .6

2 2 .6 6 7 .52 2 .3

3 0 .6

1 8 .0

1 8 .1

O

N HH O

O

1 7 0 .8 85 6 .0 3

2 8 .03 8 .51 6 9 .4 5

7 .9 24 .2 11 .5 9 , 1 .5

2 .7 5 , 3 .2 9

O

H 3 C H N

H 3 C

1 7 1 .2

5 1 .8 9

3 0 .72 2 .2

2 2 .9

7 .8 9

4 .2 41 .9 50 .8 4

0 .8 6

V a l-1

V a l-2

V a l-3

V a l-4

V a l-5

V a l-6

T h r

P h e

D h b

I leO rn

P ro

G lu

7 -M e -5 -O c to l

1 2 .5

7 .7 2

C 7 7 H 1 2 6 N 1 4 O 1 8E x a c t M a s s : 1 5 3 4 ,9 3 7

M o l. W t.: 1 5 3 5 ,9 0 7

Fig (3.2.13): compound 9, Kahalalide S

0 , 1 0 , 2 0 , 3 0 , 4 0 , 5 0 , 6 0 ,-

5

1 0

1 5

1 8e g a b 2 U V _ V I

m A

m i

1 -2 -

3 -4 -5 -6 -

7 -

8 -

9 -

W V L :2 3Peak #7 50% 100% -50%

-10,0

0,0

12,5

25,0

37,5

50,0

60,0

200 300 400 500 595

%

nm

203.0

992.76 994.78

Fig (3.2.14): HPLC chromatogram and the corresponding UV spectrum of

compound 9.

Yield: 8 mg

Results

134

Fig ( 3.2.15 ): ESI-MS of compound 9.

(ppm)0.81.21.62.02.42.83.23.64.04.44.85.25.66.06.46.87.27.68.08.48.89.29.6

NH

OCH3

OHN

H3C

H3C

OO

CH3HN

O

CH3

HN

OCH3

CH3

CH3

O NH

HN

ONH

CH3

H3C

O

NH

O

NH3C

H3CHN

H3C

H3C HNO

HO

H3C

NHO

H3CNH

O CH3

CH3

O

O

H3C

val- 1

val-2

val-3

val-4

val-5

MeHex

L- Phe.

Z-Dhb

D-Ile

L- Orn.

D- pro

L-Thr

D-Ile

D-Thr

H2N

Thr-2OH

Ile -1

Thr-2

Thr-2H-3

Thr-1H-3

Orn-NH2

Val -1

phenyl

Ile-2Val -5

Val -4Val -3 Val -2Thr

PheDhb

Dhb

Orn

0.9191

0.8770

1.0085

0.8996

0.9839

5.3729

3.5226

1.0137

1.1449

0.9366

0.7921

1.0640

3.3494

1.0703

3.3731

4.0004

1.0596

1.0743

1.1608

1.3544

2.0487

2.4072

3.3920

1.8572

1.6315

4.7918

2.2226

2.6673

3.2653

1.7730

3.8804

5.6907

2.2127

3.5910

3.4979

10.000

0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.0

Orn-NH2

Val -1

phenyl

IleVal -6

Val -5

Val -4Val -3

Val -2Thr

Phe

Glu

DhbDhb

ThrH-3

Orn

Kahalalide F

Kahalalide R

Orn-NH2Val -1

phenylIleVal -6Val -5

Val -4Val -3

Val -2ThrPhe

Glu

DhbDhb

ThrH-3

Orn

Kahalalide S 7-Me-5OctolH-5

NH

OCH3

OHN

H3C

H3C

OO

CH3HN

O

CH3

HN

O

CH3

H3C

CH3

O NH

HN

ONH

CH3

H3C

O

H2N

NH

O

NH3C

H3C

HN

H3C

H3C HN

O

O

O

H

O

NHHO

O

O

H3CHN

H3C

OH

Val-1

Val-6

Thr

Phe

Dhb

Ile

Orn

Val-2

Val-3Val-4

Val-5

Pro

7-Me-5-Octol

Glu

NH

OCH3

OHN

H3C

H3C

OO

CH3

HN

O

CH3

HN

O

CH3

H3C

CH3

O NH

HN

ONH

CH3

H3C

O

H2N

NH

O

NH3C

H3C

HN

H3C

H3C HN

O

O

O

H

O

NHHO

O

O

H3CHN

H3C

Val-1

Val-6

Thr

Phe

Dhb

Ile

Orn

Val-2

Val-3Val-4

Val-5

Pro

7-MeOct

Glu

Fig (3.2.16): comparison of 1H NMR spectra of Kahalalides F, R and S.

Results

135

Kahalalide S (9) was obtained as a white amorphous powder. The reflector mode

MALDI-TOF mass spectrum of 9 performed with delayed extraction showed positive

monoisotopic ion peaks at m/z 1536.0, [M+H]+, 1558.0 [M+Na]+, and 1574.0 [M+K]+. In

(+)-ESI-MS, a pseudomolecular ion was detected at m/z 1535.9 [M+H]+ that was compatible

with the molecular formula C77H127N14O18 as established by HRESIMS

Kahalalide S differs from kahalalide R by 16 mass unit (one oxygen atom) in the fatty

acid, 7-methyl-5-hydroxyoctanoic acid (7Me-5-Octol). It consists of all amino acid residues

found in kahalalide R. It is also the fourth kahalalide containing unusual

dehydroaminobutyric acid (Dhb), table 3.2.3 shows the structural differences between the four

largest kahalalides (F, G, R and S) .

(ppm)102030405060708090100110120130140150160170

Thr C-3

Thr -2C-3

Thr -1C-3

Region of carbonyls

13C NMR of kahalalide R Region of aromatic carbons

Region of α-carbons

13C NMR of kahalalide F

Higher field region

C-3Thr

C-57Me-5-Octol

DEPT of kahalalide S

13C NMR of kahalalide S

Fig (3.2.17): comparison of 13C NMR spectra of Kahalalides F, R and S.

Results

136

In comparison with kahalalide R, 9 has 16 mass units more, which implied the

occurrence of an additional oxygen atom in the molecule. Inspection of the 1H and 13C NMR

spectra of 9 revealed that kahalalide S shared very similar structural features with 8 (Table

3.2.5). The amino acid residues of depsipeptide 9 were identical to those of 8. The only

obvious difference was exhibited in the 13C NMR and DEPT spectra of 9 where an extra

oxygenated methine carbon was observed at δ 67.5 and the subsequent loss of one methylene

carbon in the 20 to 25 ppm region. COSY and TOCSY spectra of 9 revealed changes

occurring in the fatty acid residue. Instead of 7-Me-Oct as in 8, the fatty acid residue in 9 was

substituted by 5-hydroxy-7-methyloctanoic acid (7-Me-5-Octol). Sequential COSY

correlations were observed between the α-methylene signal at 2.11 ppm to the subsequent

methylene signals at δ 1.61, 1.52 (CH2-3), 1.25 (CH2-4), then to the oxygenated methine at δH-

5 3.41 and further to the methylene proton at δ 1.12 (CH2-6), which terminates with an

isopropyl unit at δ 1.71 (CH-7), 0.80 (CH3-8) and 0.81 (CH3-9) Table 3.2.5.

HMBC and ROESY spectra of 9 also gave similar results to 8, indicating an identical

amino acid sequence to 8. The ROESY spectra of 9 also implied the occurrence of Z-Dhb in

the depsipeptide. The connectivity of 7-Me-5-Octol to Glu was also afforded by the HMBC

correlation of the Glu–NH resonance at δ 7.92 with the carbonyl signal at δ 172.5, which was

assigned to C-1 in 7-Me-5-Octol.

The amino acid sequence of depsipeptide 9 was further confirmed by ESI-MS/MS

fragmentation and MALDI–TOF–PSD (Table 3.2.4) experiments, which again confirmed the

2D NMR results. The terminal unit, 7-Me-5-Octol–Glu, was evident from the peak at m/z

286.2 in the MALDI–TOF–PSD spectrum. Amino acid analysis using Marfey´s method

revealed the prevalence of a similar stereochemistry for each of the amino acids as found in 8.

The same problem was encountered in 9 as in 8 regarding the assignment of the

stereochemistry of each of the six Val units.

Except for the presence of glutamic acid (Glu) in kahalalides R (8) and S (9), the new

derivatives have a similar set of amino acids to those of kahalalide F (1). The new analogues

further differ from kahalalide F with regard to the occurrence of octanoic acid instead of

hexanoic acid. Kahalalide S (9) was found to contain an identical sequence of amino acids

along with the unusual amino acid, Z-Dhb, as in kahalalide R (8) and differed only in the

terminal fatty acid function, which was 7-Me-5-Octol instead of 7-Me-Oct. (see tables 3.2.3

and 3.2.5 )

Results

137

Table ( 3.2.3 ) Structural differences between the four largest kahalalides, (F, G, R and

S) . Aminoacids Kah Mol.wt Mol.formula

Dhb Glu Ileu Orn Phe Pro The Val Fatty acid Structure

form F 1477.9 C75H124N14O16 1 0 2 1 1 1 2 5 5-MeHex cyclic

peptide G 1495.9 C75H126N14O17 1 0 2 1 1 1 2 5 5-MeHex linear

peptide P 1519.9 C77H126N14O17 1 1 1 1 1 1 1 6 7-MeOct cyclic

peptide Q 1535.9 C77H126N14O18 1 1 1 1 1 1 1 6 7-Me-5-

Octol cyclic peptide

Inte

gral

(ppm)66.46.66.87.07.27.47.67.88.08.28.48.68.89.09.29.49.69.8

Orn-NH2

Val -1

phenylIleVal -6

Val -5

Val -4Val -3

Val -2ThrPhe

Glu

DhbDhb

Orn

(ppm)0.60.81.01.21.41.61.82.02.22.42.62.83.03.23.43.63.84.04.24.44.64.85.0

2x MeVal -11x Me Val -2

Thr-Me

PheH-3

GluH-4

Dhb-Me

OrnH-5

2xMe Ile2xMeVal -62xMeVal -52x MeVal -42xMe Val -31xMeVal -22xMe 7-MeOct

ProH-5Val -1

Val -2H-3

ThrH-3

7-Me-5-OctolH-2 1st conf.

7-Me-5-OctolH-2 2nd conf.

IlePro

Val -6Val -5

Val -4

Val -3

Val -2 Thr

GluOrn

Hα

Val -1H-3

Hβ, Hγ7-Me-5-Octol H-5

7-Me-5-Octol OH

Fig (3.2.18 ): 1H NMR spectrum of compound 9, NH and aromatic region (up), high field region (down).

Dhb PhenylOrn-NH2

Val -1Val -4Val -3

ThrPhe Dhb

α

Val -2

Orn

IleGluVal -6

Val -5

Me

β

α

β

α

MeMe

β

α

β

α

Me

β

α

Me

δ

Me

Fig (3.2.19): Part of TOCSY of compound 9 showing NH-detected correlations.

Results

138

Fig (3.2.21): Part of the ROESY spectrum of compound 9.

(ppm) 7.00 6.00 5.00 4.00 3.00 2.00 1.00

120

100

80

60

40

20

(ppm)

α- carbons

β- carbonThr C-5

7-Me-5-Octol

C-5 Pro

=CH-Dhb

Phenyl CHs

Fig (3.2.20): Total HMQC of compound 9

(ppm) 8.8 8.4 8.0 7.6 7.2

4.8

4.0

3.2

2.4

1.6

0.8

(ppm)

phenyl Ile

Val -6 Val -5

Val -4 Val -3 Val -2 Thr Phe

Glu Orn

Results

139

The sequence of kahalalide S was confirmed using MALDI-TOF-PSD modus, and

LC-MS/MS spectra. The reflector mode MALDI-TOF mass spectrum performed with delayed

extraction (DE) showed a positive ion signal at m/z 1558.0, identified as sodium-ion

associated monoisotopic peak [M+Na]+ of kahalalide S, and showed also a positive ion signal

at m/z 1574.0, [M+K]+ and at m/z 1536.0, [M+1]+. MALDI-TOF– PSD spectrum revealed and

confirmed the sequence of a depsipeptide, kahalalide S, as shown in table (3.2.4). Similar

results were obtained from LC-MS/MS spectrum, where the positive protonated fragment ion

peaks could be seen at m/z 1379.9, 1250.6, 1151.6, 1052.6, 952.9, 742.7 and 629.5

corresponding to M-[ 7Me-5-Octol], M-[ 7Me-5-Octol - Glu], M-[ 7Me-5-Octol - Glu- Val-

6], M-[ 7Me-5-Octol - Glu- Val-6- Val-5], M-[ 7Me-5-Octol - Glu- Val-6- Val-5- Val-4], M-[

7Me-5-Octol - Glu- Val-6- Val-5- Val-4- Pro- Orn] and M-[ 7Me-5-Octol - Glu- Val-6- Val-

5- Val-4- Pro- Orn- Ileu] respectively, See figure (3.2.23).

Amino acid analysis using Marfey´s method revealed the presence of D-Glu, D-pro, L-Orn,

D-aIle, D-athr, L-Phe , D-Val & L-Val,

NH-detected HMBC correlations to vicinal carbonyls

Hα-----C=O

HMBC correlations to Aromatic carbons

C=O

HαNHs

C=C

NH

OC H3

OHN

H 3C

H 3C

O

O

CH 3

HN

O

CH3

HN

O

C H3

H 3C

C H3

O NH

HN

O

NH

CH3

H3C

O

H 2N

NH

O

NH3C

H3C

HN

H 3C

H 3C HN

O

O

O

H

O

NHH O

O

O

H 3CH N

H3C

OH

Fig (3.2.22 ): Total HMBC correlations of compound 9

Results

140

Table 3.2.4. Important MALDI-TOF-PSD fragment ions of Kahalalide S Ion composition m/z

M–[7-Me-5-Octol–Glu] + 1250.6

M–[7-Me-5-Octol–Glu–Val-6] + 1150.7

M–[7-Me-5-Octol–Glu–Val-6–Val-5] + 1051.6

M–[7-Me-5-Octol–Glu–Val-6–Val-5–Val-4] + 952.6

M–[7-Me-5-Octol–Glu–Val-6–Val-5–Val-4–Pro] + 855.5

Ile + Cyclo[Thr(-H)–Val-1–Dhb–Phe–Val-2–Val-3] + 741.4

Cyclo[Thr(-H)–Val-1–Dhb–Phe–Val-2–Val-3] + 628.4

[Val-1–Dhb–Phe] + or [Dhb–Phe–Val-2] + 330.2

[Val-2–Phe] + 247.1

[Val-1–Dhb] + 183.1

[Pro–Orn] + 212.1

[7-Me-5-Octol–Glu] + 286.2

NH

OCH 3

OHN

H3C

H 3C

OO

CH3

HN

O

CH3

HN

O

CH3

H 3C

CH3

O NH

HN

ONH

CH3

H3C

O

H2N

NH

O

NH3C

H3C

HN

H3C

H3C HN

O

O

O

H

O

NHHO

O

O

H3CHN

H3C

OH

Val-1

Val-6

Thr

Phe

D hb

IleOrn

Val-2

Val-3Val-4

Val-5

Pro

7-Me-5 -Octol

Glu

1379

1250

1151

1052

953

742

627

681

583

855

909

Fig (3.2.23): ESI-MS/MS of compound 9

Results

141

Table 3.2.5 1H and 13C NMR data of Kahalalide S in DMSO-d6.

Amino acid No.

13C (ppm)

1H (ppm) mult. Amino acid No.

13C (ppm)

1H (ppm) mult.

Val-1 1 2 3 4 5

167.9 60.2 30.1 16.5 19.2

(NH) 6.73 3.85 1.38 0.62 0.60

(d, J=9.0 Hz) ( t, J=9.0 Hz )(m) (d, J=7.0 Hz) (d, J=6.0 Hz)

Pro

1 2 3 4 5

172.055.629.724.847.2

4.37

2.01, 1.98 1.78, 1.89 3.78, 3.52

(m) (m, m) (m, m) (m, m)

(Z)-Dhb

1 2 3 4

163.0 131.0 130.1 12.5

(NH) 9.69

6.36 1.28

(s) (q, J=7.0 Hz) (d, J=7.5 Hz)

Val-4

1 2 3 4 5

170.259.531.019.419.0

(NH) 8.11 4.26 1.94 0.84 0.83

(d, J=8.5 Hz) ( m ) (m) (m) (m)

Phe 1 2 3 4 5,5’ 6,6’ 7

171.3 55.6 36.1

137.0 128.5 129.5 126.7

(NH) 8.78 4.43 2.94

7.28 7.29 7.25

(d, J=5.5 Hz) (q, J=6.5 Hz) (m) (m) (m) (m)

Val-5

1 2 3 4 5

171.359.531.619.319.6

(NH) 7.88 4.32 1.88 0.82 0.83

(d, J=8.5 Hz) (m) (m) (m) (m)

Val-2

1 2 3 4 5

172.8 55.4 32.5 19.5 18.5

(NH) 7.61 4.45 2.18 0.62 0.80

(d, J=8.5 Hz) (m) (m) (d, J=7.0 Hz) (d, J=6.5 Hz)

Val-6

1 2 3 4 5

171.251.930.722.222.9

(NH) 7.89 4.24 1.95 0.84 0.86

(d, J=8.5 Hz) (m) (m) (m) (m)

Val-3

1 2 3 4 5

170.0 59.3 31.5 18.9 19.1

(NH) 8.82 4.13 1.94 0.81 0.82

(d, J=8.5 Hz) (m) (m) (d, J=7.0 Hz) (d, J=6.5 Hz)

Glu

1 2 3 4 5

170.92nd

regio-isomer

56.028.038.5

169.4

(NH) 7.92 (NH) 7.91

4.21

1.59, 1.50 2.75, 3.29 (OH) 7.72

(d, J=7.5 Hz) (d, J=7.5 Hz) (m) (m) (m) (bs)

aThr 1 2 3 4

168.5 57.1 70.0 17.3

(NH) 8.55 4.50 4.96 1.08

(d, J=8.0 Hz) (t, J=7.8 Hz) (m) (d, J=6.5 Hz)

aIle

1 2 3 4 5 6

171.3 57.27 30.2 14.6 26.0 11.9

(NH) 7.87 4.34 1.73 1.21 1.02 0.82

(d, J=8.2.Hz) ( m ) (m) (m) (t, J=6.5 Hz) (d, J=6.5 Hz)

Orn

1 2 3 4 5

171.5 51.1 30.9 28.5 38.3

(NH) 7.93 4.49

1.69, 1.82 1.5

2.74 (NH2) 7.72

(d, J=8.5 Hz) (m) (m, m) (m) (m) (bs)

7-Me- 5-Octol

1 2 3 4 5 6 7 8 9

172.535.423.622.667.5

38.3 27.318.018.1

2.11

1.61,1.52 1.25 3.41

(OH) 4.22 1.12 1.71 0.80 0.81

(m) (m) (m) (m) (d) (m) (m) * *

*Resonance is underneath the methyl signals of Val and aIle.

Results

142

Table 3.2.6 Marfey´s analysis results of kahalalide F, R and S hydrolysates : Amino acid-DAA

deriv. L. aIle

D. aIle

L. Phe

L. Val

D. Val L. Pro

D. Pro

L. aThr

D. aThr

L. Glu

D. Glu

L. Orn*

D. Orn*

Mol. wt (+ve mode) 384.0 384.0 418.0 370.0 370.0 368.1 368.1 372.0 372.0 400.0 400.0 385.1 385.1 Mol. wt

(-ve mode) 382.4 382.4 416.5 368.3 368.3 366.3 366.3 370.3 370.3 398.3 398.3 383.5 383.5

Ret.time in minutes 23.48 24.53 22.98 20.97 22.77 18.02 18.68 15.93 16.60 16.61 17.45 13.44

&

14.14

13.54 &

16.40

Occcurence in Kah. F

- + + + + - + + + - - + -

Occcurence in Kah. R

- + + + + - + - + - + + -

Occcurence in Kah. S

- + + + + - + - + - + + -

*it was detected that each Ornithine isomer has two different retention times, because ornithine contains α- and δ-

reactive amino groups which called be react with FDAA producing ESI-MS detectable products.

Bioactivity :

Crude aqueous methanol extract obtained from E. grandifolia was reported to exhibit

significant antifungal activity against Aspergillus japonica, A. fresenii and A. niger (Bhosale

et al , 1999). Although many natural products were isolated from genus Elysia, this is the first

report for isolated natural products from E. grandifolia.

The known derivatives, kahalalides B, D, E, F, together with the new congeners,

kahalalides R and S, were assayed for their cytoxicity toward L1578Y, HELA, PC12, H4IIE,

and MCF7 cancer cell lines. Kahalalides F and R were found to be comparably cytotoxic

toward MCF7 cells with IC50-values of 0.22 ± 0.05 µmol/L and 0.14 ± 0.04 µmol/L,

respectively. Kahalalide S and E were less cytotoxic in MCF7 cells with IC50-values of 3.55

± 0.7 µmol/L and 4.5 ± 0.49 µmol/L, respectively (Figure 3.2.25). Kahalalide R was cytotoxic

towards the mouse lymphoma L1578Y cell line at an IC50 of 4.28 ± 0.03 nmol/mL, which is

R T : 5 . 0 0 - 4 5 . 0 0

5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4T i m e ( m i n )

5

1 0

1 5

2 0

2 5

3 0

3 5

4 0

4 5

5 0

5 5

6 0

6 5

7 0

7 5

8 0

8 5

9 0

9 5

1 0 0R

elat

ive

Abso

rban

ce2 2 . 7 1

1 8 . 8 6

2 0 . 9 3

2 2 . 9 8

2 4 . 5 1

1 7 . 4 5

1 4 . 1 5

1 3 . 4 3

7 . 2 7 3 7 . 6 32 8 . 2 3 3 6 . 4 71 2 . 4 9 3 8 . 4 83 4 . 4 51 0 . 9 9 4 3 . 7 7

L - V a l i n e

D - V a l i n e

D - T h r e o n i n e

L - P h e - a l a n i n eD - P r o l i n e

L - O r n i t h i n e

D - G l u t a m i c a c i d

D - I s o l e u c i n e

Fig (3.2.24) Marfey analysis results showing the stereochemical profile of amino acids of compound 9.

Results

143

almost identical to that of kahalalide F with an IC50 of 4.26 ± 0.04 nmol/mL. The kahalalides

including kahalalides F and R were found to be inactive toward HELA, H4IIE and PC12

cancer cell lines. This implied the cytotoxic selectivity and specificity of kahalalides F and R.

Statistics

Data are given as mean ± S.E.M. of 3 independent experiments. The significance of changes in the test responses was assessed using a one-way ANOVA followed by LSD test (Analyse-it, Leeds, UK), differences were considered significant at p<0.05.

Fig (3.2.25): Cytotoxicity of Kah B, D, E, F, R and S in MCF7 cells

MCF7 cells were incubated with different kahalalides (1 µmol/L) for 24 h, and then

MTT reduction as a marker of cell viability was measured. Results are expressed as

absorption of reduced MTT (560 nm) ± S.E.M. (n=3), * p < 0.05 vs. control (DMSO).

Antimicrobial activity

In an agar diffusion assay, kahalalide R at a disc loading concentration of 5 µg,

showed strong antifungal activity against the plant pathogens Cladosporium herbarum and C.

cucumerinum with inhibition zones of 16 and 24 mm, respectively. These results were almost

identical to kahalalide F, which exhibited its fungicidal activity with inhibition zones of 17

and 24 mm, respectively, at the same concentration as the latter compound. Using the same

concentration, the fungicidal activity of kahalalides F and R were also comparable to that of

nystatin showing inhibition zones of 19 and 39 mm, respectively. However, kahalalides F and

R did not show a broad spectrum of antibiotic activity as the derivatives did not exhibit any

antibacterial activity. Kahalalide S exhibited neither antibacterial nor antifungal activities.

0

0,1

0,2

0,3

0,4

cell

viab

ility

(abs

orpt

ion

560

nm)

* *0

0,1

0,2

0,3

0,4

cell

viab

ility

(abs

orpt

ion

560

nm)

* *DMSO Kah B Kah D Kah E Kah F Kah R Kah S

Results

144

3.3-Natural products from Pachychalina sp :

From some of the unidentified marine poriferian Pachychalina sp. from the South

China Sea, 15 steroids with five different nuclei including 7-en-sterol, 8-en-sterols,

Anorsterols, 5-en-sterols and sterols with 4-Me cholestanol nuclei and glycerin-3-heptacosyl

ether (Zeng et al 1996, Zeng 2000), in addition to methyl-p-hydroxyphenylacetate, thymine,

uracil, thimidine and 2´deoxyuridine (Xiao et al 1997) have been previously reported.

An unidentified Indonesian Pachychalina sp. was chemically investigated in the

present study and three compounds were isolated which included two 5α,8α- epidioxysterols

(compounds 10 and 11) and 8-hydroxy-4-quinolone (compound 12).

Compounds 10 and 11 are 5α,8α-epidioxysterols. The difference in molecular weight is

only 14 mass units which seems to be an additional CH2 unit in the side chain as explained

below. Both compounds show the same Rf value on TLC and the same UV-spectrum. HPLC

chromatogram shows slight differences in the relative retention times which were

consequently used as the basis for the isolation using semi-preparative HPLC.

0,0 10, 0 20, 0 30, 0 40, 0 50, 0 60, 0-50

0

100

200

300

400 ms0 309 26 # 3 T M 6-he x6 U V_VIS _1mAU

min

1 - 0, 69 82 - 0, 99 43 - 1, 20 04 - 1, 30 35 - 1, 42 26 - 1, 46 7

7 - 38 ,0 06

8 - 38 ,4 33

9 - 39 ,2 23

WVL :23 5 nm

Pe a k # 8 3 8 .5 1

-1 0 ,0

2 0 ,0

4 0 ,0

6 0 ,0

2 0 0 4 0 0 5 9 5

%

n m

2 05 .4

P eak #9 39.35

-10,0

20,0

40,0

60,0

200 400 595

%

nm

203.9

He xan e : EtA cO 7 : 3

C om p ounds 10 & 11giv in g t he sam e R f value0n T LC ( R f = 0.56)

Co mp o u n d -10 Co mp o u n d -11

Plas t ic is er

Co mp o u n d 10

Co mp o u n d 11

10 11 10 & 11

Fig ( 3.3.1 ): HPLC chromatogram showing the mixture of compounds 10 & 11 before

separation (up-left), TLC of both compound showing the same Rf- value and complete

overlapping before separation (up-right), UV-spectrum of compound 10 (down left) and UV-

spectrum of compound 11 (down right).

Results

145

3.3.1- 5α,8α-epidioxy-24ξ-methylcholesta-6,22-dien-3β-ol (10, known compound)

Fig (3.3.4) EI-MS spectrum of compound 10

HO O

O

30.0934.67

66.44

36.91

82.14135.39

130.73

79.41

51.06

36.94

23.3839.32

44.54

51.68 20.65

28.90

56.12

39.812.82

18.01

18.16135.39

132.39

43.04

20.1433.17

20.89

19.631.68

3.96

2.12,1.85

6.49

6.25

1.52

0.881.21

1.27

1.60

0.81

1.25

0.98

2.03

5.08

5.12

1.850.81

1.47

0.83

0.91

1.5

C28H44O3Exact Mass: 428,329

Mol. Wt.: 428,647

0,0 10,0 20,0 30,0 40,0 50,0 60,0-20

0

20

40

60

80

100 ms031006 #3 tm6hex-6-2 UV_VIS_mAU

min

1 - 0,6582 - 0,9663 - 1,0834 - 1,1475 - 1,1756 - 1,3097 - 1,3428 - 1,4099 - 1,444

10 - 38,407

WVL:235 nm

Peak #11 38.30

-10,0

25,0

70,0

200 300 400 500 595

%

nm

202.6

563.1

Fig (3.3.3) HPLC chromatogram and UV-spectrum of compound 10. Yield : 5mg

Results

146

Compound 10 was isolated as crystalline white needles, with [α]D of - 5° (c 0.35

CHCl3). Compound 10 has UV absorption at λmax 203 nm. EI-MS showed molecular ion peak

at m/z 428 [M]+ and fragment ions at m/z 396 [M-(O2)]+, 378 [M-(O2 + H2O)]+, 363 [M-(O2 +

H2O + CH3)]+, 271 [M-(O2 + side chain)]+, and 253 [M-(O2 + side chain + H2O)]+ suggesting

the molecular formula C29H46O3. 1H NMR spectrum showed resonances for six methyl groups

at δ 0.81 (3H, s, Me-18), 0.88 (3H, s, Me-19), 0.98 (3H, d, J=6.31 Hz, Me-21), 0.81 (3H, d,

J=6.3 Hz, Me-26), 0.83 (3H, d, J=6.93 Hz, Me-27), 0.91 (3H, d, J=6.63 Hz, Me-28). The

resonances at δ 3.96 (1H, m, H-3), 6.25 (1H, d, J=8.83 Hz, H-6) and 6.49 (1H, d, J=8.83 Hz,

H-7) suggested a ∆6, mono hydroxylated 5α,8α-epidioxysteroidal compound (Gauvin et al ,

2000). This was confirmed by the presence of an ion fragment at m/z 396 [M-O2] through the

loss of O2 from the molecular ion, presumably by a retro Diels-Alder fragmentation

(Gunatilaka et al 1981), and by 13C NMR signals at 82.14 and 79.41 of C-5 and C-8,

respectively (Yaoita et al 1998 and Yue et al 2001). The presence of two olefenic protons at δ

5.08 (1H, ddd, J=15.2, 8.83, 2.5 Hz) and 5.12 (1H, ddd, J=15.6, 8.81, 2.3Hz) was indicative

to ∆22 unsaturation and assigned to H-23 and H-22 respectively, which were also confirmed

by 13C NMR resonances at 135.39 and 132.39 ppm for C-22 and C-23, respectively. The β-

configuration of hydroxy group at position 3, δH 3.96 (1H, m, H-3) and δC 66.44 ppm (d, C-3)

was suggested by comparison with the published data of 3α- and 3β- hydroxy steroids (Eggert

et al 1976, Wright et al 1978, and Gauvin et al, 2000 ). Although the NMR data of the

position 24 were compared with those given by (Gunatilaka et al 1981) the sterochemistry of

this position, is still undetected because the chemical shifts of the side chain carbons were not

similar enough to those (meassured in C6D6) given by (Gunatilaka et al 1981) for 24(R) or

24(S). Therefore, it preferred not to assign the stereochemistry at C-24 for compound 10. The

position of the double bonds and also the methyl groups were confirmed with 2D-NMR

experiments of the compound. The NMR data assigned compound 10 as 5α,8α-epidioxy-24ξ-

methylcholesta-6,22-dien-3β-ol, that was previously isolated from some marine sponges (like

Axinella cannabina, Tethya aurantia, and Raphidostila incisa (Gunatilaka et al 1981).

Results

147

Table (3.3.1): 1H, 13C-NMR data of compound 10 in (CDCl3, 500, MHz) No. 13C (Multiplicity) 1H (Multiplicity, Hz) HMBC correlation

H-------C

1 34.67 t 1.68 (m)

2 30.09 t 1.5 (m)

3 66.44 d 3.96 (m) C-5

4 36.91 t 1.85 (m), 2.12 (ddd J=13.87, 5.04, 1.89 Hz) C-3, C-5

5 82.14 s -

6 135.39 d 6.25 (d, J=8.83 Hz) C-4, C-5, C-8

7 130.73 d 6.49 (d, J=8.83 Hz) C-5, C-8

8 79.41 s -

9 51.06 d 1.52 (t) C-8, C-10

10 36.94 s -

11 23.38 t 1.21 (m) C-8, C-13

12 39.32 t 1.27 (m)

13 44.54 s -

14 51.68 d 1.6 (m) C-13, C-8

15 20.65 t

16 28.90 t

17 56.12 d 1.25(m)

18 12.82q 0.81 ( s) C-12, C-13, C-14 ,C-17

19 18.01q 0.88 ( s) C-10, C-9, C-5

20 39.8 d 2.03(m)

21 20.89 0.98 (d, J=6.31 Hz) C-20

22 135.39 d 5.08 (ddd, J=15.2, 8.83, 2.5 Hz) C-20, C-24

23 132.39 d 5.12 (ddd, J=15.6, 8.81, 2.3 Hz) C-20, C-24

24 43.04 d 1.85 (m)

25 33.17 d 1.47 (m)

26 20.14 q 0.81 (d, J=6.3 Hz) C-25, C-24

27 19.63 q 0.83 (d, J=6.93 Hz) C-25

28 18.16 q 0.91 (d, J=6.62 Hz) C-25, C-24

Results

148

Fig (3.3.6): 13C NMR and DEPT spectra of compound 10.

(ppm)1.01.52.02.53.03.54.04.55.05.56.06.57.0

(ppm)6.36.46.5

(ppm)5.20

H-6 H-22, H-23

H-7

H-6

H-3

H-22, H-23

H-7

Me-21

Me-19

Me-18

H-12

H-4

H-20

Me-28

Fig (3.3.5): 1H NMR of compound 10.

135.

3948

132.

3903

130.

7340

82.1

423

79.4

074

66.4

458

56.1

227

51.6

834

51.0

575

44.5

381

43.0

455

39.8

003

39.3

188

36.9

403

36.9

114

34.6

677

33.1

750

30.0

935

28.8

994

23.3

816

20.8

971

20.6

564

20.1

460

19.6

356

18.1

623

18.0

178

12.8

274

1020304050607080901001101201300

HO O

O

12

34

56

7

8

1112

13

1416

15

17

18

19

20

21

22

2324

25 26

27

28

910

1 2 3 4

5

6

7

8

9 10 11 12

13

14

15

16 17 18

19,21

20

22

23

24

25

26 27 28

Results

149

Fig (3.3.9): HMBC spectrum of of compound 10

(ppm) 7.00 6.00 5.00 4.00 3.00 2.00 1.00

7.00

6.00

5.00

4.00

3.00

2.00

1.00

(ppm)

23/24

4a7 6 22&23 3

22/20 3/4a3/4b

3/227/25

21/20

Fig (3.3.8): COSY spectrum of of compound 10

(ppm) 7.00 6.00 5.00 4.00 3.00 2.00 1.00

120

100

80

60

40

20

(ppm)

23/24

4a

7

6

22&23 3

22/20

9&14/8

1/5

2/3

19/5

6/4

2/5

21/20

6/5&8

7/5&8

Results

150

3.3.2- 5α,8α-epidioxy-24ξ-ethylcholesta-6,22-dien-3β-ol (11, known compound)

Fig (3.3.12) EI-MS spectrum of compound 11

HO O

O

30.1134.67

66.46

36.91

82.14135.38

130.74

79.41

51.16

35.32

28.2439.40

44.5

51.67 23.38

39.32

56.1

51.0612.84

18.16

21.01

137.46

129.87

39.96

25.35

18.16

31.85

19.00

18.54

3.96

2.12,1.84

6.51

6.25

0.88

0.81

1.03

2.03

5.04

5.14

1.52

1.19, 1.41

0.80

1.47

0.85

0.80

1.53

C29H46O3Exact Mass: 442,345Mol. Wt.: 442,674

Fig(3.3.10): compound 11.

0,0 10,0 20,0 30,0 40,0 50,0 60,0-40

0

25

50

75

100

120 ms031006 #4 tm6hex-6-3 UV_VIS_1mAU

min

1 - 0,3672 - 1,0783 - 1,1514 - 1,2845 - 1,3586 - 1,4307 - 1,482

8 - 39,181

WVL:235 nm Peak #8 39.12

-10,0

25,0

70,0

200 300 400 500 595

%

nm

204.8

Fig (3.3.11) HPLC chromatogram and UV-spectrum of compound 11. Yield : 4.5 mg

Results

151

Compound 11 was also isolated as crystalline white needles, with [α]D of - 10° (c 0.3

CHCl3). Compound 11 has UV absorption at λmax 204 nm. EI-MS showed molecular ion peak

at m/z 442 [M]+ and fragment ions at m/z 410 [M-O2]+ , 398 [M-(C2H3O)]+, 396 [M-(O2 +

CH2)]+, 377 [M-(O2 + H2O + CH3)]+, 363 [M-(O2 + H2O + C2H5)]+, 271 [M-(O2 + side

chain)]+ and 253 [M-(O2 + side chain + H2O)]+. suggesting the molecular formula C29H46O3. 1H NMR spectrum showed resonances for again six methyl groups at δ 0.81 (3H, s, Me-18),

0.89 (3H, s, Me-19), 1.03 (3H, d, J=6.41 Hz, Me-21), 0.80 (3H, d, J=6.92 Hz, Me-27), 0.85

(3H, d, J=6.91 Hz, Me-26), 0.80 (3H, t, J=6.3 Hz, Me-29) as found in compound 10. The

resonances at δ 3.96 (1H, m, H-3), 6.25 (1H, d, J=8.83 Hz, H-6) and 6.51 (1H, d, J=8.83 Hz,

H-7) suggested a ∆6, mono hydroxylated 5α,8α-epidioxysteroidal compound (Gauvin et al

2000). By comparison between the EIMS spectra of compounds 10 and 11 especially for the

most abundant fragment (M-O2, rel. abundance 100%) for both compounds, compound 11 is

14 mass units larger than 10 which indicated the presence of one additional methylene group

in 11. This CH2 group is assigned at position 28 as evident from its COSY spectrum. The

differences in the NMR data between 10 and 11 are evident only for the side chain as

mentioned in table 3.3.2, while the NMR data of the tetracyclic carbon skeleton are

superimposible. Compound 11 formed two stereoisomers due to the stereochemistry at

position 24 as evident from 1HNMR spectrum where the triplet signal at δ 0.80 of CH3-29

showed another set of resonances at the same position with only 1.2 Hz difference in the

chemical shift value. Therefore compound 11 was assigned as a racemic mixture of 24(R)

and 24(S) at a ratio of 1:1. This was also confirmed by the corresponding δC values of the side

chain which showed signals with small intensities compared to those of the tetracyclic part.

The NMR data assigned compound 11 as [5α,8α-epidioxy-24ξ-ethylcholesta-6,22-dien-3β-ol],

which was also previously isolated from some marine sponges like Luffariella sp. (Gauvin et

al 2000) and Raphidostila incisa (Gunatilaka et al 1981).

Results

152

Table (3.3.2): 1H, 13C-NMR data of compound 11 in (CDCl3 , 500, MHz) No. 13C (Multiplicity) 1H (Multiplicity, Hz) HMBC correlation

H-------C

1 34.67 t

2 30.11 t 1.53 (m)

3 66.46 d 3.96 (m) C-5

4 36.91 t 1.85 (m), 2.12 (ddd J=13.87, 5.04, 1.89 Hz) C-3, C-5

5 82.14 s -

6 135.38 d 6.25 (d, J=8.83 Hz) C-4, C-5, C-8

7 130.74 d 6.51 (d, J=8.83 Hz) C-5, C-8

8 79.41 s -

9 51.16 d

10 35.32 s -

11 28.24 t

12 39.40 t

13 44.5 s -

14 51.67 d

15 23.38 t

16 39.32 t

17 56.10 d

18 12.84 q 0.81 ( s) C-12, C-13, C-14 ,C-17

19 18.16 q 0.89 ( s) C-10, C-9, C-5

20 51.06 d 2.03(m)

21 21.01 q 1.03 (d, J=6.31 Hz) C-20

22 137.46 d 5.14 (ddd, J=15.2, 8.83, 2.3 Hz) C-20, C-24

23 129.87 d 5.04 (ddd, J=15.6, 8.81, 2.3 Hz) C-20, C-24

24 39.96 d 1.52 (m)

25 31.85 d 1.47 (m)

26 19.00 q 0.80 (d, J=6.93 Hz) C-25, C-24

27 18.54 q 0.85 (d, J=6.93 Hz) C-25

28 25.35 t 1.19, 1.41 (m,m)

29 18.16 q 0.80 (m) C-28

Results

153

Fig (3.3.13): 1H NMR spectrum of compound 11.

Fig (3.3.14): 13C NMR spectrum of compound 11.

137.

6481

135.

3851

130.

7436

129.

8769

82.1

423

79.4

074

66.4

651

56.1

035

51.6

738

51.1

538

51.0

575

51.0

382

44.5

285

39.9

640

39.3

188

38.7

796

36.9

114

35.3

225

34.6

677

33.4

736

32.3

854

31.8

461

31.7

884

30.1

032

28.9

861

28.8

802

28.2

350

25.3

557

23.3

912

21.0

897

20.9

068

20.6

949

18.9

904

18.9

230

18.5

475

18.2

393

18.1

623

12.8

370

( )102030405060708090100110120130140150160170

NE

P

22 23

7 6

5 8

3

18

4 2

15 19

9 13

13

( )1.01.52.02.53.03.54.04.55.05.56.06.57.0

(ppm)6.4

(ppm)5.05.1

22 23

7 6

21

3

18

4a

20

19

Results

154

Fig (3.3.15): COSY spectrum of compound 11.

Fig (3.3.16): HMBC spectrum of compound 11.

(ppm) 7.00 6.00 5.00 4.00 3.00 2.00 1.00

7.00

6.00

5.00

4.00

3.00

2.00

1.00

(ppm)

22 23 7 6

3

3/4a

3/2

21

21/20

6/7

23/22

23/24

22/20

3/4b

7 6

29/28

4/3

19/5 6/ 5 & 8

7/ 5 & 8

27/25

Results

155

3.3.3- 8-hydroxy-4-Quinolone (12, known compound)

NH

O

181.5

158

128

132

115

125

115.2

109.1

140

OH

7.93

6.33

7.58

7.2

7.03

C9H7NO2Exact M ass: 161,04

0,0 10,0 20,0 30,0 40,0 50,0 60,0-200

500

1.000

1.400 ms031002 #9 but-a2 UV_VIS_mAU

min

1 - 0,6472 - 0,9713 - 1,1524 - 1,1965 - 1,2736 - 1,3517 - 1,3938 - 1,4609 - 1,50910 - 1,55111 - 1,80212 - 4,229

13 - 12,687

14 - 37,007 15 - 47,32916 - 48,089

WVL:235 nmP e a k # 1 2 1 2 .9 7 5 0 % 1 0 0 % -5 0 %

-1 0 ,0

0 ,0

1 2 ,5

2 5 ,0

3 7 ,5

5 0 ,0

6 0 ,0

2 0 0 3 0 0 4 0 0 5 0 0 5 9 5

%

n m

2 3 2 .5

2 0 0 .5

3 2 3 .2

9 9 9 .2 9 9 9 9 .9 4 9 9 7 .6 0

Fig (3.3.17): HPLC chromatogram and UV-spectrum of compound 12. Yield : 3.8 mg

[ M + H ] +

[ 2 M + H ] +

[ M - H ] +

[ 2 M - H ] +

Fig (3.3.18): ESI-MS of compound 12.

Results

156

Compound 12 [8-hydroxy-4-quinolone] was isolated as a brownish yellow

amorphous powder. Compound 12 has UV absorption at λmax (MeOH) 200, 232, 220, 323

and 335 nm. Positive ESI-MS showed molecular ion peak at m/z 162 [M+H]+ and negative

molecular ion peak at m/z 160 [M-H]- suggesting the molecular formula C9H7NO2. 1H NMR

spectrum showed resonances for five aromatic protons for two spin systems, 1st spin system

composed of two resonances at δ 7.93 (1H, d, J= 6.94 Hz) and 6.33 (1H, d, J= 6.94 Hz), the

2nd spin system composed of three proton resonances at δ 7.58 (1H, dd, J= 7.90, 0.94 Hz), 7.2

(1H, t, J= 7.88 Hz) and 7.03(1H, dd, J= 7.90, 0.94 Hz ). COSY spectrum confirmed the

above spin systems. These data sugested the presence of two aromatic rings, the odd

numbered MW suggested the occurence of one nitrogen atom while the presence of three

nitrogens or more was excluded. The disappearence of the exchangeable protons (NH and

OH) was expected because deuterated methanol was used. By searching in the commercially

available computer program „Dictionary of Natural Product (DNP)“ it was suggested that

compound 12 is 8-hydroxy-4-quinolone which was isolated previously (Siuda 1974). The

NMR data were identical to those of 8-hydroxy-4-quinolone which was previously isolated

from the ink glands of the marine giant octopus, Octopus dofleini martini (Siuda 1974). The

deduced data of compound 12 were confirmed by HMQC and HMBC experiments. The

presence of tautomerisation was evident from the presence of four aromatic spin systems

(two spin systems for each tautomer) as evident from the 1H NMR and COSY spectra of the

same sample in deuterated DMSO as shown in figures (3.3.19 and 3.3.20).

1.18

17

1.47

69

2.45

51

2.08

13

1.00

00

2.14

54

2.36

32

2.38

93

0.62

98

0.88

94

0.46

99

1.79

37

3949

.03

3942

.09

3890

.39

3881

.56

3775

.95

3767

.75

3601

.29

3593

.41

3585

.53

3556

.84

3549

.27

3109

.80

3102

.86

(ppm)2.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.512.0

Fig (3.3.19):1H NMR spectrum of compound 12 (measurred in DMSO)

NH

O

12

34 5

6

78

4a

8a

OHN

OH

1'2'

3'4' 5'

6'

7'8'

4a'

8a'

OH

Results

157

Fig (3.3.20):COSY spectrum of compound 12 (DMSO-d6)

Fig (3.3.21):1H NMR spectrum of compound 12 (methanol-d4)

3960

.06

3953

.12

3830

.80

3829

.85

3822

.60

3821

.65

3613

.58

3605

.70

3597

.50

3538

.23

3537

.29

3530

.35

3529

.40

3166

.22

3158

.97

1.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5

6.57.07.58.0

NH

O

12

34 5

6

78

4a

8a

OH

5 6 7 2 3

(ppm) 8.0 7.2 6.4 5.6 4.88.8

8.0

7.2

6.4

5.6

4.8

(ppm)

NH

O

12

34 5

6

78

4a

8a

OHN

OH

1'2'

3'4' 5'

6'

7'8'

4a'

8a'

OH

2 3 5 6 7 2’ 3’

5’

6’ 7’

2/3

5/6

6’/7’ 2’/3’

Results

158

Fig (3.3.22):COSY spectrum of compound 12 ( showing H/H correlations)

Fig (3.3.23):HMQC spectrum of compound 12 ( showing H/C direct correlations)

(ppm) 8.4 8.0 7.6 7.2 6.8 6.4

8.4

8.0

7.6

7.2

6.8

6.4

(ppm)NH

O

12

34 5

6

78

4a

8a

OH

3 7

6 25

(ppm) 8.00 7.00 6.00 5.00 4.00 3.00 2.00

140

120

100

80

60

40

20

(ppm)

NH

O

12

34 5

6

78

4a

8a

OH

3 7 6

2 5

Results

159

Fig (3.3.24):HMBC spectrum of compound 12 ( showing H/C long range correlations)

Bioactivity:

Many of the reported 5α, 8α- epidioxy sterols showed significant biological activity

including antimycobacterial activity (Cantrell et al 1999), inhibitive activity against the

human T-cell leukemia/lymphotropic virus type I (HTLV-I), cytotoxic activity against the

human breast cancer cell line (MCF7 WT) (Gauvin et al 2000), and antitumor activity against

different tumour cell lines (Bok et al 1999). 8-Hydroxy-4-quinolone (compound 12) was

reported to be one of the ink components that is ejected by the giant octopus Octopus dofleini

martini which was reported to have antipredatory activity (Siuda 1974). Some other

quinolone analogues (e.g. 3,8-dihydroxyquinoline) showed mild cytotoxic activity against the

growth of several human tumour cell lines( Moon et al 1996).

Cytotoxic activity of compounds 10 and 11 showed growth inhibitiory effect of 57.2

% and 38.6 %, respectively at a concentration of 10 µg/mL each against L5178Y cancer cells.

Compound 12 showed neither significant antimicrobial activity nor cytotoxic activity in our

available test systems.

(ppm) 8.0 7.2 6.4 5.6

180

160

140

120

(ppm)

3 7 6

2 5

NH

O

12

34 5

6

78

4a

8a

OH

7/5

5/8a

6/4a

6/83/2

3/4a 2/8a

2/4

2/3

7/8a

Results

160

3.4-Natural products from Hyrtios erectus

The secondary metabolites from the genus Hyrtios especially Hertios erectus (also

called H. erecta) have been investigated extensively [Kobayashi et al 1990; Kobayashi et al

1994a; Miyaoka et al 2000; Pettit et al 1998a; Pettit et al 1998b; Pettit et al 1998c; Youssef

2005; Youssef et al 2005, Youssef et al 2002]. Previous chemical investigations of different

Hyrtios sp. and their associated microorganisms have revealed the presence of numerous

structurally unique natural products including scalarane sesterterpenoids, acyclic

triterpenoids, indole alkaloids, macrolides and usual- and unusual steroids. Many of these

compounds possess important biological activities. The most important metabolites of the

genus Hyrtios discovered to date include the powerful anticancer agents spongistatins (Pettit

et al 1993; Pettit et al 1994). Which in turn prompted the same authers to exhaustively

recollect the same sponge Hyrtios erecta (600 kg wet wt.) for further chemical investigations.

The later chemical investigations resulted in the isolation of antineoplastic sesterstatins in

very minute quantities (Pettit et al 1998b, Pettit et al 1998c, and Pettit et al 2005).

However, in the present study, 210 g. dry weight of Hyrtios erectus collected from the

Red Sea – Egypt were chemically investigated and nine compounds were isolated. The

isolated compounds consisted of 8 known compounds and one new 5-hydroxy-1H-indole

derivative.

Results

161

3.4.1- Hyrtiosine A (13, Known compound):

0,0 10,0 20,0 30,0 40,0 50,0 60,0-50

0

100

200

300

400 ms050811 #3 ih-mixa UV_VIS_1mAU

min

1 - 12,104

2 - 47,192

WVL:235 nm Peak #1 12.24

-10,0

25,0

70,0

200 300 400 595

%

nm

213.6

251.6302.9

Fig (3.4.1):HPLC chromatogram and UV-spectrum of compound 13. Yield : 5 mg

[M+H]+[M-(COCH2OH)]+

[M-CH2OH]+

[M-H2O]+

[M-(CH2OH + OH)]+[M-(CH2OH + OH)]+

Fig (3.4.2):(+ve) ESI-MS spectrum of compound 13 and its MS/MS spectrum.

NH

HO

O

OH

133.4

114.0107.5

155.6

113.6

113.3

195.3

67.1

128.0

132

4.71

8.1

7.63

6.76

7.28

C10H9NO3Exact Mass: 191,06Mol. Wt.: 191,18

Results

162

Compound 13, Hyrtiosine A, [ 5-Hydroxy-3-(hydroxyacetyl)-1H-indole] was isolated

as brownish transluscent amorphous powder, with slightly offensive characteristic odour.

Compound 13 shows positive pseudomolecular ion peak at m/z 192 [M+1]+ and negative

pseudomolecular ion peak at m/z 190 [M-1]- suggesting the molecular formula C10H9NO3.

The UV (MeOH) absorption maxima of 13 at λmax 213, 251, 271 and 302 nm indicative of a

3-acyl indole chromophore (Kobayashi et al 1990). The 1H NMR (CD3OD) showed 5 proton

resonances at δ 8.1 (1H, s ; H-2), 7.63 (1H, d, J = 2.21 Hz; H-4), 6.76 (1H, dd, J = 2.21,8.83

Hz; H-6), 7.28 (1H, d, J = 8.83 Hz; H-7), 4.71 (2H, s ; H-9). The 13C NMR resonances

were obtained indirectly from the long range correlations (HMBC spectrum), see table (3.4.1).

The HMBC spectrum showed long range correlations between CH2-9 and the carbonyl C-8 at

195.3 ppm and C-3 at 114.0 ppm. Indeed, the following HMBC correlations were observed

(H/C): H-2/C-3, H-2/C-3a, H-2/C-7a, H-4/C-5, H-4/C-7a, H-4/C-6, H-4/C-3, H-6/C-7a, H-

4/C-5, H-7/C-3a, H-7/C-5. The NMR data and the UV absorption maxima identified 13 as

Hyrtiosine A that was previously isolated from the same sponge (Kobayashi et al 1990) and

from Dragmacidon sp (Pedpradab et al 2004).

Table (3-4.1): NMR data of compound 13 (Methanol-d4, 500 MHz).

No. 13C, (δ = ppm)

1H (δ = ppm), couppling

constant (J= Hz)

HMBC correlations

H C

2 133.4 δ 8.1 (1H, s ) 3, 3a, 7a

3 114.0

3a 128

4 107.5 7.63 (1H, d, J = 2.21) 5, 7a, 6, 3

5 155.6

6 113.6 6.76 (1H, dd, J = 2.21& 8.83) 5, 7a

7 113.3 7.28 (1H, d, J = 8.83) 5, 3a

7a 132

8 195.3

9 67.1 4.71 (2H, s ) 8, 3

Results

163

Fig (3.4.3):1H NMR spectrum of compound 13 (CD3OD).

Fig (3.4.4): HMBC spectrum of compound 13 (CD3OD).

H-9/C-8

(ppm) 8.0 7.2 6.4 5.6 4.8 4.0 3.2

200

160

120

80

(ppm)

2 6

4 9

7

H-6/C-6 direct correlation

Correlations to C-5

H-6/C-4 H-2/C-3

H-9/C-2

H-2/C-3a

H-2/C-7a

Correlations to C-3a Correlations to C-7a

1.0

122

1.0

000

0.9

974

1.0

521

1.8

726

Inte

gral

4047.7

0

3820.3

9

3818.1

9

3636.9

1

3628.0

8

3389.4

3

3387.2

2

3380.9

1

3378.3

9

2341.4

9

0.61.21.82.43.03.64.24.85.46.06.67.27.88.49.09.610.210.811.412.0

1.0

000

3820.3

9

3818.1

9

(ppm)

0.9

974

3636.9

1

3628.0

8

(ppm)

7.26

1.0

521

3389.4

3

3387.2

2

3380.9

1

3378.3

9

(ppm)

6.78

2

4 6

7 NH

HO

O

1

2

34

5

6

7

8

3a

7a

OH

9

Results

164

3.4.2- 5-Hydroxy-1H-indol-3-carbaldehyde (14, Known compound):

0,0 10,0 20,0 30,0 40,0 50,0 60,0-20

50

100

150

180 ms050811 #4 ih-mixb UV_VIS_1mAU

min

1 - 13,623

2 - 47,171

WVL:235 nm Peak #1 13.67

-10,0

70,0

200 400 595

%

nm

211.9

252.3271.8

Fig (3.4.5):HPLC chromatogram and UV-spectrum of compound 13. Yield : 2.5 mg

[M-(CHO)]+[M-(CHO)]+

[M+H]+

Fig (3.4.6):(+ve) ESI-MS spectrum of compound 14 and its MS/MS spectrum.

NH

HO

O

H

139.0

119.1106.5

154.3

113.6

113.3

186.3

126.5

132.2

7.98

7.55

6.80

7.30

9.83

C9H7NO2Exact Mass: 161,05Mol. Wt.: 161,16

Results

165

Compound 14, [5-Hydroxy-3-formyl-1H-indole] was isolated as a brownish

transluscent amorphous powder with characteristic odour. Compound 14 shows a positive

pseudomolecular ion peak at m/z 162 [M+1]+ and a negative pseudomolecular ion peak at m/z

160 [M-1]- suggesting the molecular formula C9H7NO2. The UV (MeOH) absorption maxima

of 14 at λmax 211, 252, 271 and 302 nm were indicative of a 3-acyl indole chromophore

(Kobayashi et al 1990). The 1H NMR (CD3OD) showed 5 proton resonances at δ 7.98 (1H, s ;

H-2), 7.55 (1H, d, J = 2.52 Hz; H-4), 6.80 (1H, dd, J = 2.52, 8.51 Hz; H-6), 7.3 (1H, d, J =

8.51 Hz; H-7) and 9.83 (1H, s ; H-8). The 13C NMR resonances were obtained indirectly

from HMBC spectrum, see table 3.4.2 . The following proton-detected long range correlations

were observed (H/C): H8/C-3, H-8/C-3a, H-2/C-3, H-2/C-3a, H-2/C-7a, H-4/C-6, H-4/C-7a,

H-6/C-7a, H-7/C-3a, H-7/C-5. The NMR data confirmed 14 as 5-hydroxy-3-formyl indole

which has been previously isolated from the same sponge (Kobayashi et al 1990) and from

Dragmacidon sp (Pedpradab et al 2004).

Table (3.4.2): NMR data of compound 14 (Methanol-d4, 500 MHz).

No. 13C, (δ = ppm)


constant (J= Hz)

HMBC correlations

H C

2 139.0 δ 7.98 (1H, s ) 3, 3a, 7a

3 119.1

3a 126.5

4 106.5 7.63 (1H, d, J = 2.52 Hz) 7a, 6

5 154.3

6 113.6 6.8 (1H, dd, J = 2.52& 8.51 Hz) 7a

7 113.3 7.3 (1H, d, J = 8.51 Hz) 5, 3a

7a 132.2

8 186.3 9.83 (1H, s ) 3, 3a

Results

166

Fig (3.4.7):1H NMR spectrum of compound 14 (CD3OD).

Fig (3.4.8): HMBC spectrum of compound 14 (CD3OD).

(ppm) 9.6 8.8 8.0 7.2 6.4

180

160

140

120

100

(ppm)

NH

HO

O

1

2

34

5

6

7

8

3a

7a

H-4/C-7a

6748 2

H-4/C-6

H-2/C-2 direct

H-7/C-5

H-7/C-3a

H-6/C-7a

H-2/C-3

H-2/C-7a

H-2/C-3aH-8/C-3a

H-8/C-3

H-8/C-8 direct

1.0

00

0

1.1

02

2

1.1

30

9

1.3

62

2

1.2

33

0

3.23.64.04.44.85.25.66.06.46.87.27.68.08.48.89.29.610.0

NH

HO

O

1

2

34

5

6

7

8

3a

7a7

7

6

8 4 6

4

2

Results

167

3.4.3- Indol-3 carbaldehyde (15, Known compound, first isolation from genus Hyrtios):

0,0 10,0 20,0 30,0 40,0 50,0 60,0-20

25

50

75

100

140 ms050811 #5 ih-mixc UV_VIS_1mAU

min

1 - 19,113

2 - 47,277

WVL:235 nm Peak #119.10

0,0

0,0

200 300 400 500 595

%

nm

207.5

243.7298.0

Fig (3.4.9):HPLC chromatogram and UV-spectrum of compound 13.

Yield : 2.3 mg

Fig (3.4.10):ESI-MS spectrum of compound 15 [direct injection, +ve mode, (M+H)+].

NH

O

H

12.13

8.3

8.07

7.1

7.30

7.6

9.95

138.5

139.5

119.1

121.5

121.0

120.0 185.2

126.5

114.1

C9H 7NOExact Mass: 145,05

Mol. W t.: 145,16

Results

168

Compound 15, [indole-3-carbaldehyde] was isolated as a yellowish white amorphous

powder, with slightly offensive characteristic oder. Compound 15 shows positive

pseudomolecular ion peak at m/z 146 [M+1]+ suggesting the molecular formula C9H7NO.

The UV (MeOH) absorption maxima of 15 at λmax 206, 243, 255, and 298 nm were

indicative of a 3-acyl indole chromophore. The 1H NMR (DMSO-d6) showed 7 proton

resonances which include the aromatic ABCD spin system at δ 8.07 (1H, d, J = 7.4 Hz ; H-4),

7.10 (1H, dt, J = 1.21, 7.4 Hz; H-5), 7.30 (1H, dt, J = 1.31, 7.4 Hz; H-6) and 7.60 (1H, d, J =

7.4 Hz; H-7) in addition to 3 proton resonances at δ 8.30 (1H, s ; H-2) and the aldehydic

proton at 9.95 (1H, s; H-8) and the most downfield broad singlet at δ 12.13 (1H, s ; H-1) see

table (3.4.3). The HMBC spectrum showed long rang correlation between H-2/C-3, H-2/C-3a,

H-2/C-7, H-2/C-7a, H-2/C-8, H-6/C-7, H-6/C-7a, H-4/C-6, H-4/C-7a, H-4/C-3a, H-4/C-3, H-

5/C-7, H-5/C-3a, H-7/C-3a, H-7/C-5. The NMR data were identical to those of indole 3-

carbaldehyde ( Aldrich, 1990, Hiort, 2002) but this is the first isolation of 15 from genus

Hyrtios.

Table (3.4.3): NMR data of compound 15 (DMSO-d6).

No. 13C, (δ = ppm), multiplicity


constant (J= Hz)

HMBC correlations

H C

1 - 12.13 (1H, s )

2 139.5 8.30 (1H, s ) 3, 3a, 7, 7a, 8

3 119.1, s -

3a 126.5 , s -

4 120.0, d 8.07 (1H, d, J = 7.4 Hz ; H-4) 3, 3a, 7, 7a, 6

5 121.0 7.10 (1H, dt, J = 1.21, 7.4 Hz) 3a, 7

6 121.5 7.30 (1H, dt, J = 1.31, 7.4 Hz) 4, 7, 7a

7 114.1 7.60 (1H, d, J = 7.4 Hz; H-7) 5, 3a

7a 138.5 -

8 185.2 9.95 (1H, s ) 3, 3a

Results

169

Fig (3.4.11):1H NMR spectrum of compound 15 (DMSO-d6).

Fig (3.4.12):HMBC spectrum of compound 15 (DMSO-d6).

(ppm) 9.6 8.8 8.0 7.2

180

160

140

120

(ppm)

4 7

H-2/C-8

5,6 2

H/C-2 direct correlation

H-2/C-7a

H-6/C-7a

H-8/C-3a

H-8/C-3

H-2/C-7a

0.5

789

0.9

239

0.9

870

1.0

000

1.0

425

2.1

817

Inte

gral

( )1.02.03.04.05.06.07.08.09.010.011.012.013.0

2.1

817

Inte

gral

3632.8

23631.5

63625.5

73624.6

23618.0

03616.4

3

3610.4

43609.1

73602.5

53601.6

13595.6

23594.3

6

(ppm)7.157.207.257.30

4 7

1

5,6

2

8

5,6 NH

O

1

2

34

5

6

7

8

3a

7a

Results

170

3.4.4. 5-Deoxyhyrtiosine A (16, Known compound, first isolation from genus Hyrtios):

Fig (3.4.14): (-ve) ESI-MS spectrum of compound 16 and its ESI-MS/MS spectrum.

0,0 10,0 20,0 30,0 40,0 50,0 60,0-50

100

200

350 ms050811 #7 ih-mixe UV_VIS_1mAU

min

1 - 17,006

2 - 18,542 3 - 47,191

WVL:235 nmPeak #1 17.05

-10,0

70,0

200 300 400 500 595

%

nm

208.9

241.3 299.3

Fig (3.4.13):HPLC chromatogram and UV-spectrum of compound 16. Yield : 2 mg

Fig (3.4.15):ESI-MS spectrum of compound 16 (direct injection).

NH

O

OH

133.6

114.4122.0

123.2

124.2

113.0

196.2

66.3

126.3

138.1

4.73

8.19

7. 44

7.22

8.23

7.19

C10H9NO2Exact Mass: 175,06

Mol. Wt.: 175,18

Results

171

Compound 16, 5-deoxyhyrtiosine A, [3-(hydroxyacetyl)-1H-indole] was isolated as

colourless amorphous powder. Compound 16 shows positive pseudomolecular ion peak at m/z

176 [M+1]+ and negative pseudomolecular ion peak at m/z 174 [M-1]- suggesting the

molecular formula C10H9NO2, which seems to be smaller than hyrtiosine A (compound 13) by

only 16 mass units (one oxygene atom). The UV (MeOH) absorption maxima of 16 at λmax

209, 241, 261 and 299 nm were indicative of a 3-acyl indole chromophore. The 1H NMR

(CD3OD) showed 5 aromatic proton resonances (instead of 4 aromatic protons as in hyrtiosine

A) which included an aromatic ABCD spin system at δ 8.23 (1H, dd, J = 6.94, 1.26 Hz; H-4),

7.19 (1H, dt, J = 6.94, 1.26 Hz; H-5), 7.22 (1H, dt, J = 6.94, 1.26 Hz; H-6) and 7.44 (1H, dd,

J = 6.94, 1.26 Hz; H-7) and one proton resonance at δ 8.19 (1H, s ; H-2) in addition to the

most upfield singlet at δ 4.73 (1H, s ; H-9), see table (3.4.4). The 1HNMR confirmed the loss

of a 5-hydroxyl group (compared to Hyrtiosine A). HMBC spectrum showed long range

correlations between H-2/C-3, H-2/C-3a, H-2/C-7a, H-6/C-7, H-6/C-4, H-6/C-7a, H-4/C-6,

H-5/C-7, H-5/C-3a, H-4/C-7a, H-4/C-3a, H-7/C-5, H-9/C-3, H-9/C-8, see table (3.4.4) and

figure (3.4.17). The NMR data and the UV absorption maxima were identical to those of 3-

(hydroxyacetyl)-1H-indole which has been previously isolated from the Bermudian sponge

Tedania ignis and their associated bacterium, Micrococcus sp. and the red alga ( Dillman and

Cardellina 1991, Stierle 1988, Bernart and Gerwick 1990, respectively), but this is the first

isolation of 16 from genus Hyrtios.

Fig (3.4.16):1H NMR spectrum of compound 16 (CD3OD)..

0.88

07

0.77

90

0.88

00

2.25

92

1.91

71

Inte

gral

4114

.85

4112

.96

4107

.91

4106

.65

4097

.83

3728

.02

3721

.08

3720

.14

3623

.98

3622

.09

3618

.31

3617

.05

3614

.21

3611

.37

3610

.11

3604

.44

3602

.86

3597

.19

2367

.02

2.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.5

0.88

07

0.77

90

Inte

gral

4114

.85

4112

.96

4107

.91

4106

.65

4097

.83

(ppm)8.158.208.25

0.88

00

2.25

92

Inte

gral

3728

.02

3721

.08

3720

.14

3623

.98

3622

.09

3618

.31

3617

.05

3614

.21

3611

.37

3610

.11

3604

.44

3602

.86

3597

.19

(ppm)7.27.37.47.5

2 7 5 6

9

4

Results

172

Fig (3.4.17): HMBC spectrum of compound 16(CD3OD).

Table (3.4.4): NMR data of compound 16 (Methanol-d4).

No. 13C, (δ = ppm)


constant (J= Hz)

HMBC correlations

H C

2 133.6 δ 8.19 (1H, s ) 3, 3a, 7a

3 114.4

3a 126.3

4 122.0 8.23 (1H, dd, J = 6.94, 1.26 Hz) 6, 3a, 7a

5 123.2 7.19 (1H, dt, J = 6.94, 1.26 Hz) 7, 3a

6 124.2 7.22 (1H, dt, J = 6.94, 1.26 Hz) 4, 7a, 7

7 113.0 7.44 (1H, dd, J = 6.94, 1.26 Hz) 5

7a 138.1

8 196.2

9 66.3 4.73(2H, s ) 8, 3

2 7 5 6

2/3

4

(ppm) 8.40 8.20 8.00 7.80 7.60 7.40 7.20 7.00

144

136

128

120

112

104

(ppm)

2/7a

2/3a 4/6

4/3a

4/7a

6/4

6/7a

6/7

5/3a

5/7

7/5

NH

O

1

2

34

5

6

7

8

3a

7a

OH

9

Results

173

3.4.5- Isohyrtiosine A (17, New compound):

0,0 10,0 20,0 30,0 40,0 50,0 60,0-50

100

200

300

400 ms050811 #8 ih-mixf UV_VIS_1mAU

min

1 - 17,284

2 - 47,280

WVL:235 nm Peak #1 17.38

-10,0

70,0

200 400 595

%

nm

213.5

241.5285.9


NH

HO

OO

160

132

m /z 191

Fig (3.4.19): EI-MS of compound 17

NH

HO

O

O

133.4

107.5106.3

153.7

113.6

113.4

168.0

51.3

128.4

132.7

3.81

7.91

7.44

6.74

7.24


Mol. Wt.: 191,18

Results

174

Compound 17, isohyrtiosine A, [5-hydroxy-1H-indole-3-carboxylic acid methyl ester]

was isolated as a transluscent needle crystals. Compound 17 shows positive pseudomolecular

ion peak at m/z 192 [M+1]+ and negative pseudomolecular ion peak at m/z 190 [M-1]+ and EI-

MS of 17 showed molecular ion peak of m/z 191 [M]+ suggesting the molecular formula

C10H9NO3. The UV (MeOH) spectrum of 13 showed an absorption maxima 213, 241 and

286 nm, (slightly deviated from those of 13 „Hyrtiosine A“). The 1H NMR (CD3OD) showed

5 proton resonances at δ 7.91 (1H, s ; H-2), 7.44 (1H, d, J = 2.21 Hz; H-4), 6.74 (1H, dd, J =

2.21,8.83 Hz; H-6), 7.24 (1H, d, J = 8.83 Hz; H-7), 3.81(2H, s ; H-9). Although the

molecular weight is identical to that of hyrtiosine A, the 13C NMR spectrum shows some

differences in resonances from those of hyrtiosine A, including the presence of a carboxyl

resonance at 168.01 ppm instead of the keto-carbonyl at 195.3 ppm and the methoxy carbon

at 51.27 ppm replacing the OCH2 at 67.1 ppm which were confirmed by its DEPT spectrum.

The other 13C NMR resonances seems to be similar to those of hyrtiosine A (compound 13),

see table (3.4.5). The attachment of the methoxy group to the carbonyl is also confirmed by

HMBC correlation between CH3-9 and C-8 at 168.1 ppm. Other HMBC correlations were

presented in table 3.4.5 and explained in figure 3.4.22. The NMR data and the UV absorption

maxima identified 17 as isohyrtiosine A (5-Hydroxy-1H-indole-3-carboxylic acid methyl

ester). To the best of our knowledge this is the first report of 17 as a natural product.

Fig (3.4.20):13C NMR (up) and DEPT (down) spectra of compound 17

(ppm)

10203040506070809010011012013014015016017018019000

NH

HO

O

O

12

345

6

7

89

3a

7a

9

3

4 7a58

2

3a

6,7

5152535455565758595105115125135145155165175185195

9

42

6,7

Results

175

Fig (3.4.21):1H NMR spectrum of compound 17

Fig (3.4.22): HMBC spectrum of compound 17

Table (3.4.5): NMR data of cpompound 17 (Methanol-d4).

No. 13C, (δ = ppm) 1H (δ = ppm), couppling constant (J= Hz)

HMBC correlations (H C)

2 133.41 δ 7.91 (1H, s ) 3, 3a, 7, 8 3 107.5 3a 128.4 4 106.27 7.44 (1H, d, J = 2.21 Hz) 3, 5, 7a, 6 5 153.7 6 113.6 6.74 (1H, dd, J = 2.21& 8.82 Hz) 4, 5, 7a 7 113.4 7.24 (1H, d, J = 8.82 Hz) 5, 3a 7a 132.7 8 168.01 9 51.27 3.81 (3H, s ) 8

1.0

00

0

0.9

91

8

1.1

13

9

1.0

63

5

3.1

13

5

In

teg

ral

39

28

.5

37

22

.3

37

20

.1

36

28

.0

36

19

.2

33

75

.8

33

73

.6

33

67

.3

33

64

.8

19

25

.9

0.01.02.03.04.05.06.07.08.09.010.0

1.0

00

0

0.9

91

8

1.1

13

9

1.0

63

5

In

teg

ral

39

28

37

22

37

20

36

28

36

19

33

75

33

73

33

67

33

64

(ppm)

6.706.806.907.007.107.207.307.407.507.607.707.80

92 4 6 7

NH

HO

O

O

1

2

34

5

6

7

8

93a

7a

(ppm) 8.0 7.6 7.2 6.8 6.4

180

160

140

120

(ppm)

NH

HO

O

O

12

345

6

7

89

3a

7a

H-2/C-3

H-7/C-3a H-2/C-3a

H-6/C-4

H-2/C-7a

2 7 6 4

H-4/C-7a

H-4/C-5 H-7/C-5

H-6/C-5

H-6/C-7a

H-4/C-6 H-4/C-3

H-2/C-8

Results

176

3.4.6- 16-Hydroxyscalarolide (18, Known compound):

0,0 10,0 20,0 30,0 40,0 50,0 60,0-50

0

100

200

300

350 ms051011 #3 ihbffa2 UV_VIS_1mAU

min

1 - 0,3172 - 1,1083 - 1,2154 - 1,3585 - 1,420

6 - 33,371

7 - 34,354

8 - 44,083

WVL:235 nm Peak #10 33.43

-10,0

25,0

50,0

70,0

200 300 400 595

%

nm

216.7

590



[M+H]+

[M+Na]+

[M+H-(2H2O)]+

[M+H-(H2O)]+

Fig (3.4.24):FAB-MS spectrum of compound 18.

O

HO

OH

H

H H

O

39.618.5

42.0333.2

56.67

18.1

41.6

37.0

57.95

37.4

25.64

75.54

42.74

54.2528.15

67.9

162.25

136.93

175.6

4.5

4.81, 5.00

2.21, 1.7

1.18

1.15

1.88, 1.49

0.88

3.6

0.9

1.81, 0.92

1.57, 1.410.77

0.840.81

0.84

1.60, 1.44

1.79, 0.83

1.09,1.38

17.4

16.72

15.92

21.27 33.27

70.71


Results

177

Compound 18 [16-hydroxyscalarolide] was isolated as a colourless solid with [α]D of

- 23° (c 0.18 CHCl3). Compound 18 has UV absorption at λmax 216 nm and a molecular

formula of C25H38O4, as established from its FAB MS and 13C NMR data. FABMS of the

compound 18 showed pseudomolecular ion peak m/z 425 [M+ Na]+ and pseudomolecular ion

peak m/z 403 [M+ H]+ suggesting the molecular formula C25H38O4.

1H NMR spectrum (Table 3.4.6) displayed resonances for 38 protons including five

singlets assigned to five methyl groups at δ 0.81, 0.84, 0.9, 0.84, and 1.18, eight methylenes,

and five sp3 methines. The 13C NMR spectrum revealed signals for 25 carbons including five

methyls, eight methylenes, two downfield sp3 methines, three upfield sp3 methines, and four

sp3 quaternary carbons and three sp2 quaternary carbons. Analysis of the 1H,1H-COSY led to

the assembly of the following structural fragments: C-1 to C-3; C-5 to C-7; C-9 to C-12 with

a hydroxyl group at C-12; and C-14 to C-16 with an hydroxyl group at C-16. The proton

signals at 4.81(H-24a) and 5.00 (H-24b) (J24a/b = 17.97 Hz) together with the carbon

resonances at 70.71 (CH2, C-24), 162.25 (qC, C-17), 136.93 (qC, C-18), and 175.6 (qC, C-25)

were representative of an α,β-unsaturated butenolide moiety (Youssef et al 2005, Pettit et al

1998b, Miyaoka et al 2000). This was supported by interpretation of the HMBC spectrum

(Table 3.4.6), which indicated that C-16 and C-24 were connected to the sp2 quaternary

carbon C-17, with these situated, along with C-18 and C-25, in the α,β-unsaturated-γ-lactone

ring (Youssef et al 2005, Pettit et al 1998b, Miyaoka et al 2000). Connectivities of the five

ring systems of 18, as well as the assignments of all quaternary carbons, were supported

unequivocally by HMBC data. In addition, the placements of the OHs at C-12 and C-16, were

secured from HMBC correlations of H-11/C-12, H-16/C-17, and H-16/C-18. Moreover,

COSY coss-peaks of H-12/H-11a, H-12/H-11b, H-16/H-15a, and H-16/H-15b supported these

assignments. The partial structures and the above α,β-unsaturated-γ-lactone (C-17, C-18, C-24

and C-25) were found to be connected through quaternary carbons based on HMBC data; also

observed were the cross-peak: Me-20/C-3, C-5, C-19; Me-19/C-3, C-5, C-20; Me-22/C-1, C-

5, C-9; Me-21/C-7, C-9, C-14; Me-23/C-12, C-14, C-18, and H-16/C-14, C-17, C-18, so that

the tetracarbocyclic scalarane skeleton could be constructed. The planar structure of 18 was

thus determined. All trans junctions of the tetracyclic rings (A, B, C, and D) were

demonstrated from 13C NMR chemical shifts of methyl groups (δC 15.92, 16.7, 17.4, 21.27,

and 33.27), (Miyaoka et al 2000). The NMR spectra of 18 were closely related to those of

known scalarane sesterterpenoids [(scalarolide, seterstatins 1-7, and 16-hydroxyscalarolide),

(Walker et al 1980, Pettit et al 1998b, Pettit et al 1998c and Pettit et al 2005, Youssef et al

2005, Miyaoka et al 2000)]. Compound 18 seems to be identical with the 16-

Results

178

hydroxyscalarolide see the comparative NMR data, table 3.4.6. The planar structure of 18 was

supported by COSY, and HMBC spectra. The equatorial geometry of OH at C-12 was

established from the large coupling constant of H-12 (J = 11.04 Hz), (Youssef et al 2005,

Pettit et al 1998b). Similarly, the β-configuration of the hydroxyl at C-16 was established

from the coupling constant (J = 9.45 Hz) (Youssef et al 2005, Pettit et al 1998b). From the

above discussion, compound 18 was assigned as 16-hydroxyscalarolide and its spectral data is

identical with those of 16-hydroxyscalarolide isolated from the Okinawan sponge H. erectus

(Miyaoka et al 2000).

Table ( 3.4.6 ): NMR data of compound 18 (CDCl3, 500 MHz)

16-Hydroxyscalarolide (Miyaoka et al 2000)

compound 18

No. 13C (mult.)

1H [mult., J (Hz)] 13C (mult.)

1H [mult., J (Hz)] HMBC

1 39.7 (CH2) 0.78 (1H, m), 1.72 (1H, br d, 13.0)

39.6, CH2 1.79, m, 0.83, m C-10

2 18.2 (CH2) 1.42 (1H, m),1.62 (1H, m)

18.5, CH2 1.60, m, 1.44, m C-4, C-10

3 42.0 (CH2) 1.09 (1H, m), 1.38 (1H, m)

42.0, CH2 1.09, m, 1.38, ddd (13.5, 13.5, 4.0)

C-4

4 33.3 (C) 33.2, qC 5 56.7 (CH) 0.75 (1H, m) 56.67, CH 0.77, m C-4 6 18.6 (CH2) 1.40 (1H, m), 1.58

(1H, m) 18.1, CH2 1.57, m, 1.41, m

7 41.7 (CH2) 0.92 (1H, m), 1.80 (1H, td, 3.3, 12.4)

41.6, CH2 1.81, m, 0.92, m C-8

8 37.0 (C) 37.0, qC 9 58.0 (CH) 0.88 (1H, m) 58.0, CH 0.88, m C-10, C-12 10 37.4 (C) 37.4, qC 11 25.7 (CH2) 1.49 (1H, dd, 2.1,

13.2), 1.88 (1H, m) 25.7, CH2 1.49, ddd (11.5, 4.2,

1.8) 1.88, m C-12

12 75.5(CH) 3.64 (1H, dd, 4.5, 10.9)

75.5, CH 3.60, dd (11.5, 4.4) C-13

12-OH 5.75 , s 5.75, s 13 42.8 (C) 42.8, qC 14 54.3 (CH) 1.14 (1H, m) 54.25, CH 1.15, m C-8, C-13, C-16 15 28.2 (CH2) 1.66 (1H, dd, 2.1,

12.4), 2.23 (1H, dd, 6.7, 12.5)

28.2, CH2 1.7, m; 2.21, dd (11.7, 7.1)

C-16

16 67.9 (CH) 4.50 (1H, m) 67.9, CH 4.50, dd (9.45, 6.93) C-17, C-18 17 162.3 (C) 162.25, qC 18 136.93 (C) 136.93, qC 19 21.3 (CH3) 0.81 (3H, s) 21.27, CH3 0.81, s C-4,C-3, C-5, C-

20 20 33.3 (CH3) 0.84 (3H, s) 33.27, CH3 0.84, s C-4, C-3, C-5, C-

19 21 17.4 (CH3) 0.90 (3H, s) 17.4, CH3 0.9, s C-8, C-14, C-9,

C-7 22 15.9 (CH3) 0.84 (3H, s) 15.92, CH3 0.84, s C-1, C-10, C-9 23 16.7 (CH3) 1.19 (3H, s) 16.72, CH3 1.18, s C-12, C-13, C-

18,C-14 24 70.7 (CH2)

4.81 (1H, d, 17.8), 4.99 (1H, d, 17.8)

70.7, CH2 4.81, dd (17.87, 1.27) 5.0, d (17.97)

C-17, C-18, C-25

25 175.6 (C) 175.6, qC

Results

179

Fig(3.4.25): 1H NMR spectrum of compound 18

Fig(3.4.26): 13C NMR and DEPT spectra of compound 18

0.9

11

0

0.9

50

0

0.9

42

6

0.9

41

5

1.0

03

1

1.0

47

1

2.9

78

4

7.7

05

3

5.2

65

0

2.7

87

6

2.9

41

4

6.0

46

2

3.2

82

3

Inte

gra

l

29

03

.63

25

05

.45

24

87

.48

24

82

.44

24

16

.86

23

98

.58

22

62

.07

22

55

.13

22

52

.29

22

45

.67

18

27

.95

18

23

.53

18

17

.23

18

12

.82

11

23

.02

11

16

.71

11

11

.04

11

04

.10

93

4.4

99

25

.03

92

2.5

19

13

.68

90

9.9

09

07

.06

90

0.7

68

97

.60

89

4.7

75

92

.74

45

0.5

64

20

.61

41

5.8

84

05

.48

(ppm)

0.00.40.81.21.62.02.42.83.23.64.04.44.85.25.66.06.46.87.27.6

12-OH 24a

23

24b

12 16

15a

19

22, 20

21

O

HO

OH

H

H H

O

12

3

45

67

8

9

10

1112

13

1415

16

1718

19 20

2122

23

25

24

13

17

5.5

98

1

16

2.2

55

0

13

6.9

38

6

75

.54

25

70

.71

10

67

.89

08

57

.95

08

56

.67

55

54

.24

88

42

.74

94

42

.03

52

41

.65

62

39

.67

41

37

.41

50

37

.01

42

33

.26

85

28

.15

27

25

.65

32

21

.27

35

18

.55

53

18

.19

82

17

.40

39

16

.71

89

15

.92

45

1020304050607080901001101201301401501601701801900

18

19

17

12

24

16

25

15 2,6

23,21,22 9,5,14

11

20

10,8

3,7 1

4

O

HO

OH

H

H H

O

12

3

45

67

8

9

10

1112

13

1415

16

1718

19 20

2122

23

25

24

Results

180

Fig(3.4.27): H-H COSY spectrum of compound 18

Fig(3.4.28): Part of HMBC spectrum of compound 18 showing H/C direct and H/C long

correlations of the methyl groups.

(ppm) 7.00 6.00 5.00 4.00 3.00 2.00 1.00

7.00

6.00

5.00

4.00

3.00

2.00

1.00

(ppm)

16/15a

16

12/11b

12-OH 15a

24b

12 24a

12/11a16/15b

Methyls direct correlations

H-15/C-16

14 direct correlation

H-11a / 12-C

(ppm) 2.0 1.6 1.2 0.8

14

12

10

80

60

40

20

(ppm

20,22

H-14/C-16

19

15a

21

14

23

H-23/C-18

H-23/C-14

H-23/C-12

H-23/C-13

H-21/C-14H-21/C-9

H-22/C-9

H-14/C-8

O

HO

OH

H

H H

O

12

3

45

67

8

9

10

1112

13

1415

16

1718

19 20

2122

23

25

24

Results

181

Fig(3.4.29): Part of HMBC spectrum of compound 18 showing H/C direct and H/C long

correlations of the methylene group (CH2-24).

24b

24a

(ppm) 5.6 5.2 4.8 4.4

160

140

120

100

80

60

40

(ppm)

16

H-24b, H-24a/C-18

H-24a/C-24 direct correlation

H-24b, H-24a/C-17

O

HO

OH

H

H H

O

12

3

45

67

8

9

10

1112

13

1415

16

1718

19 20

2122

23

25

24

Results

182

3.4.7- Scalarolide (19, Known compound):


O

HO

H

H H

O

39.718.5

42.033.2

56.65

18.2

41.7

37.2

57.98

37.4

25.7

75.65

42.2

55.217.2

162.1

135.8

175.9

4.81, 5.00

2.39

1.18

3.6

0.840.81

0.84 17.4

16.7

15.9

21.27 33.2

72.1

25.20.89

5.94


0,0 10,0 20,0 30,0 40,0 50,0 60,0-50

100

200

300

450 ms051004 #7 ihbfcf UV_VIS_1mAU

min

1 - 0,5722 - 0,9393 - 1,0804 - 1,1505 - 1,1846 - 1,2677 - 1,448

8 - 35,654

9 - 36,180

WVL:235 nm Peak #8 35.70

-10,0

20,0

40,0

60,0

200 400 595

%

nm

220.1

342.2

Fig ( 3.4.30 ):HPLC chromatogram and UV-spectrum of compound 19. Yield : 5 mg

[M+Na]+

[M+H]+[M+H-(H2O)]+

Results

183

Compound 19 [scalarolide] was isolated as a brownish white amorphous solid with

[α]D of + 23° (c 0.2 CHCl3). Compound 19 has a UV absorption at λmax 220 nm and a

molecular formula of C25H38O3, as established from its FAB MS and 13C NMR data. FAB MS

of compound 19 showed pseudomolecular ion peak m/z 409 [M+ Na]+ and pseudomolecular

ion peak m/z 387 [M+ H]+ suggesting the molecular formula C25H38O3.

1H NMR spectrum (Table 3.4.7) displayed resonances for five methyls at δ 0.81, 0.85,

0.89, 0.85, and 1.13, one downfield sp3 methine at δ 3.67 (dd, J=11.0, 4.4 Hz) and downfield

methylene at δ 4.69 (1H, d, J=16.8 Hz) and 4.68 (1H, d, J=17.3 Hz) and one OH broad

singlet at δ 5.94. The 13C NMR spectrum revealed signals for 25 carbons including five

methyls, eight upfield methylenes, one downfield methylene at δ 72.1 three upfield sp3

methines, one downfield sp3 methine (oxygenated carbon) at δ 75.6, and four sp3 quaternary

carbons and three sp2 quaternary carbons. The presence of five singlet methyl signals at δ 0.81

(3H), 0.85 (6H), 0.89 (3H), and 1.13 (3H) suggested the pentacyclic scalarin derivative

(Walker et al 1980), the presence of α,β-unsaturated-γ-lactone was evident from the 13C

chemical shift of carbons resonating at δ 72.1, 162.1, 135.8 and 175.9 corresponding to C-24,

C-17, C-18, and C-25 respectively which also confirmed by HMBC correlations between the

methylene protons at δ 4.69 (2H, d, J=16.8 Hz; d, J=17.3 Hz) and three downfield sp2

quaternary carbons resonating at δ 162.1, 135.8 and 175.9. The HMBC correlation (3J

coupling) between the methyl proton at δ 1.13 (3H, s, CH3-23 ) and the sp2 Quaternary

carbon resonating at δ 135.8 and an oxygenated sp3 methine carbon at 75.5 (C-12) further

indicaterd that the compound 19 seems to be very similar to compound 18 except the loss of

one oxygenated methine carbon (C-16) and consequently the presence of one upfield methine

carbon more than those present in compound 18. Compound 19 is smaller than 18 by 16 mass

unit which could be demonstrated by the loss of an oxygen atom from position C-16 as

demonstrated above. The 1H and 13C NMR data of Compound 19 was found to be identical

with those of scalarolide that isolated before (Walker et al 1980) from the sponge Spongia

idia .

Results

184

Table ( 3.4.7 ): NMR data of compound 19 (CDCl3, 500 MHz)

scalarolide

(Walker et al 1980)

compound 19

No. 13C (mult.)


1H [mult., J (Hz)] HMBC

1 39.6 (CH2) 39.7 (CH2)

2 18.5 (CH2) 18.5 (CH2)

3 42.0 (CH2) 42.0 (CH2)

4 33.2 (C) 33.2 (C)

5 56.6 (CH) 56.6 (CH)

6 18.1 (CH2) 18.2 (CH2)

7 41.7 (CH2) 41.7 (CH2)

8 37.2 (C) 37.2 (C)

9 57.9 (CH) 57.9 (CH)

10 37.3 (C) 37.4 (C)

11 25.7 (CH2) 25.7 (CH2)

12 75.5(CH) 3.64 (1H, dd, 4.5, 10.9) 75.6 (CH) 3.64 (1H, dd, 4.5, 10.9) C-13

12-OH 5.75 , s 5.75 , s

13 42.1 (C) 42.2 (C)

14 55.1 (CH) 55.2 (CH)

15 17.1 (CH2) 17.2 (CH2)

16 25.2 (CH2) 2.39 (1H, m) 25.2 (CH2) 2.39 (1H, m) C-18

17 162.0 (C) 162.1 (C)

18 135.8 (C) 135.8 (C)

19 21.2 (CH3) 0.81 (3H, s) 21.2 (CH3) 0.81 (3H, s) C-4,C-3, C-5, C-20

20 33.2 (CH3) 0.85 (3H, s) 33.2 (CH3) 0.85 (3H, s) C-4, C-3, C-5, C-19

21 16.3 (CH3) 0.89 (3H, s) 16.4 (CH3) 0.89 (3H, s) C-8, C-14, C-9, C-7

22 15.9 (CH3) 0.85 (3H, s) 15.9 (CH3) 0.85 (3H, s) C-1, C-10, C-9

23 16.6 (CH3) 1.13 (3H, s) 16.7 (CH3) 1.13 (3H, s) C-12, C-13, C-18,C-14

24 72.0 (CH2)

4.69 m 72.1 (CH2)

4.68 (d, J=17.3 Hz),

4.69 (d, J=16.8 Hz)

C-17, C-18, C-25

25 175.9 (C) 175.9 (C)

Results

185

Fig (3.4.32) 1H NMR spectrum of compound 19

Fig (3.4.33):comparison between 13C NMR spectra of compounds 18 and 19.

C-11, C-16

O

HO

OH

H

H H

O

12

3

45

67

8

9

10

1112

13

1415

16

1718

19 20

2122

23

25

24

Comp. 18

OOH

H

H H

O

12

3

4

56

7

8

9

10

1112

13

1415

16

1718

19 20

2122

23

25

24

Comp. 19

175

162

136

75.5

70.7

67.8

57.9

56.6

54.2

42.7

42.0

41.6

39.6

37.4

37.0

33.2

28.1

25.6

21.2

18.5

18.1

17.4

16.7

15.9

1020304050607080901001101201301401501601701800

175.

9844

162.

1457

135.

8164

75.6

518

72.1

320

57.9

873

56.6

537

55.1

816

42.1

809

42.0

498

41.7

073

39.7

032

37.4

004

37.2

255

33.2

539

25.7

188

25.2

742

21.2

808

18.5

626

18.2

419

17.2

144

16.7

699

16.4

347

15.9

756

(ppm)102030405060708090100110120130140150160170180

C-12

C-16 C-24

C-24 C-12

C-25

C-18 C-17

C-17 C-18

C-25 C-11

2365

.16

2349

.08

2338

.99

2321

.65

1840

.87

1837

.09

1830

.47

1826

.06

0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.5

Me-23 Me-19

2Me- 22, 20

Me-21

CH-12 CH2-24 12-OH

Results

186

Fig (3.4.34):HMBC spectrum of compounds 19.

(ppm) 7.00 6.00 5.00 4.00 3.00 2.00 1.00

160

120

80

40

(ppm)

H-24/C-24 direct correlation

H-12/C-25 W-correlation

H-24/C-17

H-24/C-18

H-24/C-25

H-23/C-13

H-23/C-12

H-23/C-14

H-23/C-18

OOH

H

H H

O

12

3

4

56

7

8

9

10

1112

13

1415

16

1718

19 20

2122

23

25

24

Comp. 19

Results

187

3.4.8- 12-O-deacetyl-12-epi-scalarin (20, Known compound):

0,0 10,0 20,0 30,0 40,0 50,0 60,0-20

50

100

150

220 ms051011 #4 ihbffa3 UV_VIS_1mAU

min

1 - 0,3712 - 0,6353 - 1,0474 - 1,099

5 - 1,2136 - 1,3447 - 1,421

8 - 15,0289 - 15,906

10 - 34,369

11 - 44,10112 - 46,836

WVL:235 nm Peak #10 34.41

-10,0

20,0

40,0

60,0

200 300 400 500 595

%

nm

221.9



O

HO

H

H H

HO

39.918.0

41.533.3

56.5

18.5 42.00

39.9

58.7

37.4

26.1

80.5

37.4

52.7

23.6

127.3

136.3

168.0

6.86

2.38, 2.32

0.93

3.57

0.86

0.810.83

0.85 16.8

9.1

16.6

21.3 33.3

98.6


O58.8

5.67

2.55

Results

188

Compound 20 [12-O-deacetyl-12-epi-scalarin] was isolated as a white solid with

[α]D of - 15° (c 0.1 CHCl3). Compound 20 has a UV absorption at λmax 221 nm and a

molecular formula of C25H38O4, as established from its EIMS and 13C NMR data. EIMS of the

compound 20 showed molecular ion peak m/z 402 [M]+, m/z 384 [M-H2O]+, m/z 356 [M-

CH2O2]+ , and m/z 191 [rings A+B+ 4(CH3)]+suggesting the molecular formula C25H38O4.

The 1H and 13C NMR spectra measured in CDCl3 (Table 3.4.8) suggested the scaralane-type

sesterterpenoid. 1H NMR spectrum displayed five singlets assigned to five methyl groups at δ

0.81, 0.83, 0.85, 0.86 and 0.93. One olefinic proton at δ 6.86 (1H, q, 3.1 Hz, H-16), 2

oxygenated methines at δ 5.67 (1H, d, 6.6Hz, H-19) and 3.57 (1H, d, 4.4, 11.7 Hz, H-12) were

also observed. The carbon signals in the low-field region at 98.6 (d, C-19), 127.3 (s, C-17),

136.3 (d, C-16), and 168.0 (s, C-20) were reminiscent of those of scalarin (Tsukamoto et al

2003). H-12 was assigned as axial on the basis of the coupling constant of 11.7 Hz between

H-11 and H-12. The Mol Wt of 20 is identical with that of 18 (both are 402 mass units),

showing the same formula (C25H38O4), the bresence of one olefenic proton resonating at 6.86

ppm indicated that the presence of double bond in the lactone ring should be different from

that of compound 18 and more likely to be between C-16 and C-17 (instead of C-17 and C-18

as in compound 18). This was confirmed by the coupling constant of H-16 (q, J= 3.1 Hz) due

to the adjacent methylene at δ 2.38 and 2.32. furthremore the assignment at the position 18 as

methine (δ 2.55, br s) was evident from the adjacent doublet (H-19, d, J = 6.6 Hz). The NMR

data of 20 were similar to those of 12-epi-scalarin (Tsukamoto et al 2003 and Cimino et al

1977) ) and suggested that 20 was a deacetyl derivative of 12-epi scalarin and identical with

the previously published 12-O-deacetyl-12-epi-scalarin which isolated from Spongia sp.

(Tsukamoto et al 2003).

Fig (3.4.37):1HNMR spectrum of compound 20.

0.9

99

12

87

3.0

5

28

66

.43

(ppm)

1.0

77

2

17

95

.16

17

91

.06

17

83

.81

17

79

.40

(ppm)

3.553.60

3.1

92

7

3.9

76

6

3.3

61

6

3.2

51

7

4.1

10

3

In

teg

ral

46

5.6

9

43

1.6

4

42

3.7

6

41

5.2

5

40

5.4

8

(ppm)

0.760.800.840.880.920.96

1.0

00

0

34

43

.99

34

40

.52

34

37

.06

34

33

.90

(ppm)

6.88

1.0

00

0

0.9

99

1

1.0

77

2

1.2

10

5

1.6

27

6

3.1

92

7

3.9

76

6

3.3

61

6

3.2

51

7

4.1

10

3

In

teg

ral

34

43

.99

34

40

.52

34

37

.06

34

33

.90

28

73

.05

28

66

.43

17

95

.16

17

91

.06

17

83

.81

17

79

.40

12

75

.60

12

71

.19

12

65

.52

12

61

.73

11

90

.80

11

86

.70

11

81

.66

11

77

.56

11

70

.62

11

66

.52

11

61

.48

11

57

.38

46

5.6

9

43

1.6

4

42

3.7

6

41

5.2

5

40

5.4

8

0.60.81.01.21.41.61.82.02.22.42.62.83.03.23.43.63.84.04.24.44.64.85.05.25.45.65.86.06.26.46.66.87.07.27.4

16 12 19

25 16

12 19

15-a & -b

18

24

23 22

21

Results

189

Fig (3.4.38):13CNMR spectrum of compound 20.

Table ( 3.4.8): NMR data of compound 20 (CDCl3, 500 MHz)

12-O-deacetyl-12-epi-scalarin (Tsukamoto et al 2003)

(DMSO-d6)

compound 21

(CDCl3)

No. 13C (mult.)


1H [mult., J (Hz)]

1 40.2 (CH2) 0.80 (1H, m), 1.62 (1H, m)

39.9 (CH2) *

2 18.2 (CH2) 1.57 (1H, m), 1.63 (1H, m)

18.0 (CH2) *

3 41.3 (CH2) 1.10 (1H, m), 1.33 (1H, m)

41.5 (CH2) *

4 33.5 (C) 33.3 (C) 5 56.5 (CH) 0.79 (1H, m) 56.5 (CH) * 6 18.7 (CH2) 1.38 (1H, m),

1.46 (1H, m) 18.5 (CH2) *

7 42.2 (CH2) 0.88 (1H, m), 1.63 (1H, td, 3.3, 12.4)

42.0 (CH2) *

8 37.5 (C) 39.9 (C) 9 58.7 (CH) 0.80 (1H, m) 58.7 (CH) * 10 37.4 (C) 37.4 (C) 11 27.4 (CH2) 1.30 (1H, dd, 2.1, 13.2),

1.55(1H, m) 26.1 (CH2) *

12 78.6(CH) 3.39 (1H, dd, 4.4, 12.0) 80.5(CH) 3.57 (1H, dd, 4.4, 11.7) 13 37.4 (C) 37.4 (C) 14 52.9 (CH) 1.20 (1H, m) 52.7 (CH) * 15 24.1 (CH2) 2.12 (1H,m),

2.25 (1H, m) 23.6 (CH2) 2.38 (1H,m),

2.32 (1H, m) 16 135.7(CH) 6.67 (1H, q, 2.9) 136.3(CH) 6.86 (1H, q, 3.1) 17 128.9 (C) 127.3 (C) 18 58.1 (CH) 2.40 br s 58.8 (CH) 2.55 br s 19 100.5(CH) 5.72 (1H,t 5.9) 98.6(CH) 5.67 (1H,d, 6.6) 20 167.5 (C) 168.0 (C) 21 33.7 (CH3) 0.63 (3H, s) 33.3 (CH3) 0.81 (3H, s) 22 21.7 (CH3) 0.77 (3H, s) 21.3 (CH3) 0.83 (3H, s) 23 16.4 (CH3) 0.80 (3H, s) 16.6 (CH3) 0.85 (3H, s) 24 16.8 (CH3) 0.81 (3H, s) 16.8 (CH3) 0.86 (3H, s) 25 9.8 (CH3) 0.83 (3H, s) 9.1 (CH3) 0.93 (3H, s)

* chemical shifts can not be precisely characterized from 1HNMR spectrum.

13

6.2

12

7.3

98

.5

9

80

.4

6

58

.7

9

58

.6

9

56

.4

4

52

.6

9

42

.0

0

41

.4

6

39

.9

1

39

.8

7

37

.4

2

37

.3

8

33

.2

6

26

.0

6

23

.5

5

21

.2

8

18

.5

3

18

.0

1

16

.7

6

16

.5

5

9.0

89

0102030405060708090100110120130140

O

HO

H

H H

HO

O

12

34

5

67

8

9

10

1112

13

1415

16

1718

19

20

2122

23 24

25

1, 8

3 5 6, 2

7

10, 13

12

11

14

15 16 17

18, 9

19

21, 4

22 24, 23

25

Results

190

3.4.9- 7-dehydrocholesterol peroxide (21, Known compound):

0,0 10,0 20,0 30,0 40,0 50,0 60,0-10

20

40

60

80

100 ms051004 #12 ihbfck UV_VIS_1mAU

min

1 - 0,1492 - 0,9533 - 1,075

4 - 1,123

5 - 1,2516 - 1,2847 - 1,440

8 - 38,395

WVL:235 nm Peak #8 38.32

-10,0

25,0

50,0

70,0

200 300 400 500 595

%

nm

204.7

243.0

566.7

Fig ( 3.4.39 ):HPLC chromatogram and UV-spectrum of compound 21 Yield : 4 mg

[M+Na]+

[M-(O2)]+[M-(O2+H2O)]+

[M-(O2+CH3+H2O)]+


HO O

O

30.0934.67

66.47

36.92

82.14135.36

130.75

79.45

51.04

36.89

23.3939.41

44.71

51.55 20.59

28.22

56.40

35.212.6

18.14

18.55

35.92

23.78

39.41

27.97

22.52

22.79

3.94

2.12,1.85

6.45

6.22

0.85

0.77

0.87

0.84

0.83


Mol. Wt.: 416,64

Results

191

Compound 21 [5α,8α-epidioxy-cholesta-6-en-3β-ol] was isolated as a white needle

crystals. Compound 21 has a UV absorption at λmax 204 and 243 nm. FAB-MS showed

pseudomolecular ion peak m/z 439 [M+Na]+ and characteristic fragment ions of typical 5α,8α-

epidioxy-cholesta-6-en-3β-ol (Gauvin et al, 2000) suggesting the molecular formula

C27H44O3. 1H NMR spectrum showed resonances for five methyl groups at δ 0.77 (3H, s, Me-

18), 0.85 (3H, s, Me-19), 0.87 (3H, d, J=6.5 Hz, Me-21), 0.83 (3H, d, J=6.6 Hz, Me-26), and

0.84 (3H, d, J=6.6 Hz, Me-27). The resonances at δ 3.94 (1H, m, H-3), 6.22 (1H, d, J=8.5

Hz, H-6) and 6.45 (1H, d, J=8.5 Hz, H-7) suggested ∆6, mono hydroxylated 5α,8α-

epidioxysteroidal compound (Gauvin et al , 2000). This was confirmed by the presence of a

fragment ion at m/z 384 (rel. int., 100 %) [M-O2] through the loss of O2 from the molecular

ion, presumably by a retro Diels-Alder fragmentation (Gunatilaka 1981), and by 13C NMR

signals at 82.14 and 79.41 of C-5 and C-8 respectively (Yaoita et al 1998, and Yue et al

2001). The β-configuration of hydroxy group at position 3, δ 3.96 (1H, m, H-3) and 66.44

ppm (d, C-3) was suggested by comparison with the published data of [5α,8α-epidioxy-

cholesta-6-en-3β-ol] (Gauvin et al, 2000). From the above discussion, compound 21 was

assigned as 5α,8α-epidioxy-cholesta-6-en-3β-ol.

Fig(3.4.41): 1H NMR spectrum of compound 21

1.0

000

1.0

273

1.1

227

0.5

055

1.5

805

5.8

412

2.5

838

2.8

445

2.9

968

9.4

172

9.4

897

3.6

926

3.5

921

3.5

351

3.3

569

2.7

143

3257.6

73249.1

6

3124.6

33116.4

3

2002.2

91997.2

41990.9

41985.8

91980.5

41978.0

11974.5

51969.5

0

1842.1

31832.3

6

1064.6

91062.8

01059.6

51057.7

61050.8

21049.2

41045.7

81044.2

0

452.4

5445.8

3439.8

4435.1

1432.9

0428.4

9426.2

8398.5

4

0.40.60.81.01.21.41.61.82.02.22.42.62.83.03.23.43.63.84.04.24.44.64.85.05.25.45.65.86.06.26.46.6

7

18

6 3

21,19,27,26

HO O

O

12

34

56

7

8

1112

13

1416

15

17

18

19

20

2122

2324

25 26

279

10

Results

192

Fig(3.4.42): 13C NMR and DEPT spectra of compound 21

Table (3.4.9): 1H, 13C-NMR data of compound 21 in (CDCl3 , 500, MHz)

5α,8α-epidioxy-cholesta-6-en-3β-ol.

(Guavin et al , 2000)

Compound 20

No. 13C (Multiplicity) 1H (Multiplicity,

Hz)

13C

(Multiplicity)

1H (Multiplicity, Hz)

1 34.74 t 34.67 t 2 30.16 t 30.09 t 3 66.52 d 3.94 (m) 66.48 d 3.94 (m) 4 37.00 t 36.93 t 5 82.24 s 82.14 s 6 135.46 d 6.22 (d, J=8.5 Hz) 135.36 d 6.22 (d, J=8.51 Hz) 7 130.83 d 6.45 (d, J=8.5 Hz) 130.75 d 6.45 (d, J=8.51 Hz) 8 79.54 s 79.45 s 9 51.10 d 51.04 d 10 37.00 s 36.90 s 11 23.46 t 23.39 t 12 39.49 t 39.41 t 13 44.80 s 44.72 s 14 51.63 d 51.56 d 15 20.68 t 20.59 t 16 28.32 t 28.22 t 17 56.46 d 56.40 d 18 12.69q 0.77 ( s) 12.61q 0.77 ( s) 19 18.24q 0.85 ( s) 18.15q 0.85 ( s) 20 35.29d 35.21d 21 18.64q 0.87 (d, J=6.5 Hz) 18.55 q 0.87 (d, J=6.62 Hz) 22 36.00 t 35.93 t 23 23.86 t 23.78 t 24 39.49 t 39.41 t 25 28.06 d 27.97 d 26 22.62 q 0.83 (d, J=6.6 Hz) 22.53 q 0.83 (d, J=6.62 Hz) 27 22.89 q 0.84 (d, J=6.6 Hz) 22.79 q 0.84 (d, J=6.62Hz)

135.3

646

130.7

589

82.1

376

79.4

486

66.4

770

56.3

986

51.5

598

51.0

424

44.7

169

39.4

117

36.9

267

36.8

976

35.9

284

35.2

069

34.6

749

30.0

912

28.2

256

27.9

706

23.7

803

23.3

941

22.7

892

22.5

269

20.5

957

18.5

553

18.1

472

12.6

088

0102030405060708090100110120130140150160170180190

3 6

8 7

4

5

2, 16

15

14, 9

10

12, 24

17

23,11

25

22,1

20

18

HO O

O

12

34

56

7

8

1112

13

1416

15

17

18

19

20

2122

2324

25 26

279

10

21,19 27, 26

Results

193

Bioactivity:

Many scalarane-type sesterterpenoids have been isolated from marine sponges

belonging to the order Dictyoceratida. Scalarane-type sesterterpenes display a variety of

biological activities such as cytotoxic, antimicrobial, antifeedant, antimycobacterial,

ichthyotoxic, anti-inflammatory, and platelet-aggregation inhibitory effects, as well as nerve

growth factor synthesis-stimulating action (Youssef et al, 2005).

The isolated compounds from the Hyrtios erectus were tested for cytotoxic activity

against L5178Y cells and the results wer summarised in table (3.4.10). Compounds 14, 16,

18 showed mild antimicrobial activity against Bcillus subtilis and Saccharomyces cereviisae.

Table (3.4.10): cytotoxic activity of compounds 13-18 and 21 against L5178Y cells.

Compound Cell growth %

(conc. of 3µg/ml)

Cell growth %

(conc. of 10µg/ml)

Cell growth %

(-ve control)

13 98.1 92.4 100 14 100 96.6 100 15 95.5 80.5 100 16 119 94.5 100 17 95.8 64 100 18 59.5 44.7 100 21 86.4 77 100

Results

194

3.5. Natural products from Petrosia nigricans

Marine sponges of the genus Petrosia are known as a source of diverse bioactive natural

products. Sponge species of genus Petrosia are rich in polyacetylenic compounds (Faulkner

1998, Faulkner 2002, Watanabe et al 2005), unusual bioactive steroids and steroidal sulphates

acting as anti-HIV, antivirals, and anti-inflammatory (Goud et al 2003, Giner et al 1999,

Reddy et al 1999, Shatz et al 2000, Sun et al 1992, Qureshi and Faulkner 1999).

Many alkaloidal natural products were isolated from genus Petrosia belonging to

various subclasses, examples are bioactive manzamines (Crews et al 1994, Yousaf et al

2002), mimosamycins (Kobayashi et al 1994, Kobayashi et al 1992), cardioactive pentacyclic

hydroquinones (Gorshkova et al 1999), dihydroisoquinolines (Ramesh et al 1999),

pentacyclic pyridoacridines (Skyler et al 2002, Molinski et al 1988), isoquinoline quinones

(Venkateswarlu et al 1993), 3-alkylpyridinium polymers (Sepcic et al 1997), bis-quinolizidine

alkaloid (Braekman et al 1982, Braekman et al 1984), and dihydrotubastrines –

phenethylguanidine (Sperry and Crews 1998). Petrosia–derived fungal strain Penicillium

brevicompactum was reported to produce cyclodepsipeptides (Bringmann et al 2004).

However, this is the first report of Petrosia nigricans-derived natural products. An

Indonesian Petrosia sp. (Petrosia nigricans, family Petrosiidae) was chemically investigated

and several compounds were isolated. These compounds can be divided into the following

chemical classes:

1- steroidal compounds (24ξ-ethyl-cholesta-5-en-3β-ol, 24ξ-ethyl-cholesta-8(9)-en-3β-ol,

5α,8α-epidioxy-24ξ-ethyl-cholesta-6-en-3β-ol)

2- phenylacetic acid derivatives (phenylacetic acid, p-hydroxyphenylacetic acid, p-

hydroxyphenylacetic acid methylester, p-hydroxyphenylacetic acid ethylester, p-

hydroxyphenylacetic acid butylester*)

3- primary metabolites ( adenosine, nicotinamide)

4- cerebrosides. (petrocerebroside 1*, petrocerebroside 2*).

5- purine derivatives (Nigricine 1*, Nigricine 2*, Nigricine 3*, Nigricine 4*)

6- indole alkaloid *

(* = new natural products).

Results

195

3.5.1- 24ξ-Ethyl-cholesta-5-en-3β-ol (22, known compound)

Fig (3.5.2): 1HNMR spectrum of compound 22 (in CDCl3)

HO

H

H

H140.7

121.7

71.8

56.7

56.0

50.1

46.0

42.3

42.2

39.7

37.2

36.5

36.2

33.9

31.931.6

28.9

28.2

26.3

24.3

23.0

21.119.6

19.4

19.0

18.8

12.3

11.8

31.9

1.01

0.68

5.35

3.52

0.93

0.83

0.81

0.85

C29H50OExact Mass: 414,39Mol. Wt.: 414,71

-------------------------------------------------------- Yield : 2g

[M]+

[M-H2O]+

[M-(CH2+H2O)]+


H O

1

35

6

7

8

9

1 0

1 1

1 21 3

1 4 1 5

1 61 7

1 8

1 9

2 02 1 2 2

2 3

2 42 5

2 6

2 7

2 8

2 9

1.00

00

0.96

84

2.03

24

2.25

93

3.08

37

1.51

52

9.64

89

6.95

80

7.51

17

3.68

96

4.03

74

8.89

47

2.71

20

Inte

gral

( )1.01.52.02.53.03.54.04.55.05.56.06.57.07.5

5 3 4

18 19

Results

196

Compound 22 [24ξ-ethyl-cholesta-5-en-3β-ol] was isolated as a white amorphous

powder, with [α]D of - 17° (c 0.1.2 CHCl3). EI-MS showed molecular ion peak m/z 414 [M]+

and fragment ions at 396 [M-(H2O)]+, 382 [M-(CH2 + H2O)]+, 368 [M-C2H5OH]+, 273 [M-

side chain]+, 255 [M- (side chain+H2O)]+, suggesting the molecular formula C29H50O. 1H

NMR spectrum showed resonances for six methyl groups at δ 0.68 (3H, s, Me-18), 1.01 (3H,

s, Me-19), 0.93 (3H, d, J=6.94 Hz, Me-21), 0.81 (3H, d, J=6.93 Hz, Me-27), 0.83 (3H, d,

J=6.94 Hz, Me-26), 0.85 (3H, t, J=7.56 Hz, Me-29). The resonances at δ 5.35 (1H, m, H-6),

3.52 (1H, m, H-3) were indicative for ∆5-6 mono hydroxylated steroidal nucleus (Itoh et al ,

1983).13C NMR showed six methyl signals at δ 12.3, 11.8, 19.4, 18.8, 19.6 and 19.0 for the

methyl groups 29,18, 19, 21, 26, and 27, respectively. 13C NMR spectrum showed one sp2

quaternary olefenic carbon resonance at δ 140.7 and one sp2 methine at 121.7, and an

oxygenated methine at 71.8. In addition, 13C NMR spectrum shows 7 sp3 methines, and 11

aliphatic methylenes (see table 3.5.1). The above EI-MS, and NMR data were identical with

that of 24ξ-ethyl-cholesta-5-en-3β-ol (Wright et al 1978, Younus et al 1974). Compound 22

was also isolated from Petrosia (Strongylophora) durissima (Shen and Prakash 2000).

Fig (3.5.3): 13CNMR and DEPT spectra of compound 22 (in CDCl3)

HO

1

3

56

7

89

10

11

1213

14 15

1617

18

19

2021 22

23

2425

26

27

28

29

1

3 6

7,8,2

8

9

5

1,10,20

11

12 13,4

14, 17 15

16

18

20

26, 19, 27, 21

22

22

23

24 25

7

28

29

140.

7393

121.

7205

71.8

096

56.7

390

56.0

072

50.1

138

46.0

404

42.3

040

42.2

848

39.7

522

37.2

388

36.4

973

36.2

566

33.8

876

31.9

039

31.6

535

28.9

091

28.2

254

26.3

379

24.2

964

22.9

868

21.0

705

19.5

875

19.3

853

18.9

519

18.8

171

12.3

170

11.8

452

102030405060708090100110120130140

Results

197

3.5.2- 4α-Methyl-5α-cholesta-8-en-3β-ol (23, known compound)

Fig (3.5.4): ES-MS spectrum of compound 23


HO

1

3 56

7

8

9

10

11

1213

14 15

1617

18

19

2021 22

23

2425

26

27

28

[M]+

[M-(side chain)]+

[M-(CH3)]+

[M-(CH3+H2O)]+

[M-(side chain+H2O)]+

[M-(side chain+H2+C3H6O)]+

1.32

19

1.58

33

2.84

05

1.76

99

4.44

08

2.73

05

3.09

01

1.47

10

10.2

83

1.69

63

4.16

55

1.65

07

1.26

24

4.40

96

3.59

28

3.78

06

5.05

51

3.00

00

Inte

gral

( )0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.5

28

3

19 27, 26

21

18 H2O

CHCl3

HO

1

3 56

7

8

9

10

11

1213

14 15

1617

18

19

2021 22

23

2425

26

27

28

HO

31.235.08

76.5 46.88

39..2

20.8728.79

128.1

135.0

36.25

22.7437.0

42.06

51.84 23.73

27.45

54.88

36.2511.22

18.87

18.72

36.14

23.92

39.5

15.04

28.0

22.54

22.81

3.1

0.95

0.6

0.92

0.86

0.85

0.99

C28H48OExact Mass: 400,37

Mol. Wt.: 400,68

----------------------------------------------------------- Yield : 15 mg

Results

198


Compound 23 [4α-methyl-5α-cholesta-8-en-3β-ol] was isolated as white amorphous

powder, with [α]D of + 44° (c 0.5 CHCl3). EI-MS showed molecular ion peak m/z 400 [M]+

and characteristic fragment ions at 385 [M-CH3], 367 [M- (CH3+H2O)], 287 [M-(side chain)],

269 [M-(side chain+H2O)], 227 [M-(side chain+C3H6O+H2)] suggesting the molecular

formula C28H48O. 1H NMR spectrum showed resonances for six methyl groups at δ 0.6 (3H, s,

Me-18), 0.95 (3H, s, Me-19), 0.92 (3H, d, J=6.31 Hz, Me-21), 0.86 (3H, d, J=6.6 Hz, Me-27),

0.85 (3H, d, J=6.61 Hz, Me-26), 0.99 (3H, t, J=6.31 Hz, Me-28). The presence of the typical

3β- hydroxyl group was indicated by the methine signal at δ 3.1. Furthermore, appearance of

the methine signal as a triple doublet with coupling constant of J=4.7, 9.9, 11.1 Hz indicated

that the methine proton is axial and is coupled to three adjacent hydrogens as in other 4α-

methylsterols (König et al, 1998 & Gunasekera et al, 1989). The two axial–axial couplings of

9.9 and 11.1 Hz confirmed the α-equatorial nature of the 4-methyl group. The 13C-NMR

spectrum revealed only two singlets in the olefenic region (δ128.1 and 135.0) indicating the

presence of tetrasubstituted double bond (Gunasekera et al, 1989). The mass spectrum

showed the characteristic peak at m/z 287 (M-side chain). The above NMR and MS data

confirmed compound 23 as 4α-methyl-5α-cholesta-8-en-3β-ol. The structure of 23 was

confirmed by comparing its spectral data with the literature data reported for the same

compound isolated from Agelas oroides and Agelas flabelliformis (König et al, 1998 &

Gunasekera et al, 1989 respectively).

135.

0098

128.

1342

54.8

804

51.8

470

46.8

877

42.0

631

39.5

016

39.2

031

37.0

075

36.2

564

36.1

408

35.0

815

31.2

007

28.7

933

28.0

037

27.4

548

23.9

206

23.7

377

22.8

132

22.7

458

22.5

436

20.8

776

18.8

746

18.7

205

15.0

420

11.2

286

( )102030405060708090100110120130140

19, 21 8

9

18 23, 15

28

1013

25 27, 26

1

12

3

11 16

6 22

20

2

7

5417

14

24

HO

1

3 56

7

8

9

10

11

1213

14 15

1617

18

19

2021 22

23

2425

26

27

28

Results

199

3.5.3. 5α,8α-Epidioxy-24ξ-ethyl-cholesta-6-en-3β-ol (24, known compound)



HO O

O

30.0934.66

66.45

36.90

82.14135.37

130.75

79.44

51.04

36.90

23.3939.39

44.71

51.55 20.61

28.24

56.27

35.712.60

18.15

18.67

33.66

26.31

45.99

22.96

12.29

28.9

18.94

19.56

3.96

6.49

6.25

0.88

0.79

0.92 0.85

0.80

0.82


Mol. Wt.: 444,69

--------------------------------------------------------------------------- Yield : 25 mg

[M]+

[M-O2]+

[M-(H2O)]+

[M-(H2O+O2+CH3)]+

[M-(O2+ side chain)]+

[M-(O2+C3H7O)]+

[M-(O2+ side chain+H2O)]+

1.00

00

0.95

35

1.12

41

1.23

76

4.56

10

4.03

21

3.11

50

10.3

89

4.47

39

Inte

gral

3258

.30

3249

.79

3125

.26

3116

.75

2001

.97

1996

.93

1990

.62

1985

.58

1980

.22

1978

.01

1973

.92

1968

.87

1064

.38

1062

.49

1059

.33

1057

.44

1050

.51

1048

.93

1045

.78

1043

.89

615.

76

611.

34

605.

35

601.

57

458.

12

451.

50

439.

84

424.

39

416.

19

409.

26

405.

48

398.

86

1.01.52.02.53.03.54.04.55.05.56.06.57.0

H-6 H-3 H-7

Me-21

Me-19

Me-18

Me-29, 26, 27

Results

200

Compound 24 [5α,8α-epidioxy-24ξ-ethyl-cholesta-6-en-3β-ol] was isolated as white

needle crystals with [α]D of + 4° (c 0.70 CHCl3). EI-MS showed molecular ion peak m/z 444

[M]+ and characteristic fragment ion peaks at m/z 426 [M-H2O], 412 [M-O2], 379 [M-(H2O,+

O2+CH3)], 353 [M-(O2+ C3H7O)], 271 [M-(side chain +O2)] and 253 [M-(side chain + O2+

H2O)] suggesting the molecular formula C29H48O3. 1H NMR spectrum showed resonances for

six methyl groups at δ 0.79(3H, s, Me-18), 0.88 (3H, s, Me-19), 0.92 (3H, d, J=6.7 Hz, Me-

21), 0.80 (3H, d, J=6.6 Hz, Me-27), 0.82 (3H, d, J=6.90 Hz, Me-26), 0.85 (3H, t, J=7.5 Hz,

Me-29). The resonances at δ 3.96 (1H, m, H-3), 6.25 (1H, d, J=8.51 Hz, H-6) and 6.49 (1H,

d, J=8.51 Hz, H-7) suggested a ∆6 mono hydroxylated 5α,8α-epidioxysteroidal compound

(Gauvin et al , 2000). This was confirmed by the presence of an ion fragment at m/z 412 [M-

O2] through loss of O2, presumably by a retro Diels-Alder fragmentation (Gunatilaka 1981),

and by 13C NMR signals at 82.14 and 79.41 of C-5 and C-8, respectively (Yaoita et al 1998,

and Yue et al 2001). The β-configuration of hydroxyl group at position 3, δ 3.96 (1H, m, H-3)

and 66.44 ppm (d, C-3) was suggested by comparison with the published data of 3α- and 3β-

hydroxy steroids (Eggert et al 1976, Wright et al 1978, and Gauvin et al, 2000 ). Although the

NMR data of the position 24 were in comparison with those reported by Wright et al 1978

and Gauvin et al, 2000, the sterochemistry at this position is still undetected because the

chemical shifts of the side chain carbons which were not similar enough to those given for

24R or 24S.


101520253035404550556065707580859095100105110115120125130135

135.

3779

130.

7577

66.4

612

56.2

734

51.5

512

51.0

338

45.9

910

39.3

959

36.9

036

35.7

085

34.6

737

33.6

680

30.0

972

28.9

021

28.2

462

26.3

224

23.3

928

22.9

629

20.6

236

19.5

742

18.9

402

18.6

779

18.1

532

12.6

075

12.3

014

12

3

4

5

67

8

9

10,4

11,28

12

1314

1516

17

18,29

20

24

27,26,21,19

12

25

2322

HO

1

3 56

7

89

10

11

1213

14 15

1617

18

19

2021 22

23

2425

26

27

28

29

O

O

Results

201

Table (3.5.1): 1H- and 13C-NMR data of compounds 22, 23 and 24 in (CDCl3,

500, MHz) Compound 22 Compound 23 Compound 24

No. 13C

(Multi-

plicity)

1H (Multiplicity, Hz) 13C

(Multi-

plicity)

1H (Multiplicity, Hz) 13C

(Multi-

plicity)

1H (Multiplicity,

Hz)

1 37.2 t 35.08 t 34.66 t

2 31.6 t 31.2 t 30.09 t

3 71.8 d 3.52 (m) 76.5 d 3.1 (ddd, J=4.7, 9.9, 11.1) 66.45 d 3.96 (m)

4 42.2 t 39.50 d 36.90 t

5 140.7 s 46.88 d 82.14 s

6 121.7 d 5.35 (m) 20.87 t 135.37 d 6.25 (d, J=8.51)

7 31.9 t 28.79 t 130.75 d 6.49 (d, J=8.51)

8 31.9 d 128.1 s 79.44 s

9 50.1 d 135.0 s 51.03 d

10 36.5 s 36.25 s 36.90 s

11 21.1 t 22.74 t 23.39 t

12 39.7 t 37.0 t 39.39 t

13 42.3 s 42.06 s 44.71 s

14 56.7 d 51.84 d 51.55 d

15 24.3 t 23.73 t 20.61 t

16 28.2 t 27.45 t 28.24 t

17 56.0 d 54.88 d 56.27 d

18 11.8 q 0.68 ( s) 11.22 q 0.6 ( s) 12.60q 0.79 ( s)

19 20.4 q 1.01 ( s) 18.87 q 0.95 ( s) 18.15q 0.88 ( s)

20 36.2 d 36.25 d 35.70 d

21 18.8 q 0.93 (d, J=6.94 Hz) 18.72 q 0.92 (d, J=6.31) 18.67 q 0.92 (d, J=6.7 Hz)

22 33.9 t 36.14 t 33.66 t

23 26.3 t 23.92 t 26.31 t

24 46.0 d 39.5 t 45.99 d

25 28.9 d 28.0 d 28.89 d

26 19.6 q 0.83 (d, J=6.94 Hz) 22.54 q 0.85 (d, J=6.61) 18.94 q 0.82 (d, J=6.9 Hz)

27 19.0 q 0.81 (d, J= 6.93Hz) 22.81 q 0.86 (d, J=6.61 Hz) 19.56 q 0.80 (d, J=6.6 Hz)

28 23.0 t 15.04 q 0.99 (d, J=6.31) 22.96 t

29 12.3 q 0.86 (t, J = 6.94 Hz)

12.29 q 0.85 (t, J=7.5Hz)

Results

202

3.5.4- Phenylacetic acid (25, known compound) Fig (3.5.11):EI-MS spectrum of compound 25

0,0 10,0 20,0 30,0 40,0 50,0 60,0-50

0

50

100

150

200

250 ms050920 #8 tm7et6g UV_VIS_1mAU

min

1 - 0,5892 - 0,6683 - 1,0114 - 1,113

5 - 1,203

6 - 1,275

7 - 1,420

8 - 12,529

9 - 16,127

10 - 21,371

11 - 46,517

WVL:235 nm

Peak #10 21.64

-10,0

70,0

200 400 595

%

nm

213.8

244.8

299.0


Fig (3.5.10): HPLC chromatogram and UV spectrum of compound 25

COOH1

23

4

56

δΗ−2 3.65 (2H, s)δΗ(4,5, 6,4', 5') 7.3 (5H, m)

C8H8O2Exact Mass: 136,05Mol. Wt.: 136,154'

5'

-------------------------------------------------------------------- Yield : 6.0 mg

[M-(COOH)]+

[M]+

[M-(CH2COOH)]+

Results

203

Compound 25 [phenylacetic acid] was isolated as a white amorphous powder, UV

absorption at λmax 213, 244, 299 nm. EI-MS showed molecular ion peak at m/z 136 [M]+,

and fragment ion peaks at m/z 91 [M-COOH]+, 77 [M-CH2COOH]+, (see figure 3.5.22)

suggesting the molecular formula C8H8O2. A deceptively simple 1H NMR spectrum

(meassured in CDCl3) showed two sets of protons resonating at δ 3.65 (2H, s) suggesting the

presence of lower field methylene group (i.e., attached to aromatic spin system), and at δ 7.3

(5H, m) suggesting monosubstituted phenyl group. The above NMR data together with the

EI-MS fragments confirmed compound 25 to be phenylacetic acid. 1H-NMR and 13C-NMR of

25 is identical with those reported for phenylacetic acid (Aldrich, 1993, 2/981).

Fig (3.5.12):1HNMR spectrum of compound 25

Fig (3.5.13):13CNMR spectrum of compound 25

5.3

930

2.0

000

Integral

3678.8

6

3670.9

8

3664.0

5

3649.2

3

3644.8

2

3641.6

6

0.00.40.81.21.62.02.42.83.23.64.04.44.85.25.66.06.46.87.27.68.08.48.89.29.610.010.4

5.3

930

Integral

3678.8

6

3670.9

8

3664.0

5

3649.2

3

3644.8

2

3641.6

6

(ppm)

7.247.287.327.367.40

CHCl3 H-4, H-5, H-6, H-5’ and H-4’ H-2

H-4, H-5, H-6, H-4’ and H-5’

COOH1

23

4

56

4'

5'

177.6313

133.2294

129.3525

128.6310

127.3339

41.0004

2535455565758595105115125135145155165175185195205

CDCl3

C-5 & C-5’ C-4 & C-4’

C-6 C-3

C-2

C-1

Results

204

3.5.5- p-Hydroxyphenylacetic acid (26, known compound) Fig (3.5.15):EI-MS spectrum of compound 26


COOH

OH

173.4

39.0

126.1

132.2

113.8

156.6

3.4

7.03

6.72

------------------------------------------------------ Yield : 5.2 mg

0,0 10,0 20,0 30,0 40,0 50,0 60,0-200

0

250

500

750

1.000

1.400 ms041210 #13 et13a2 UV_VIS_1mAU

min

1 - 0,2292 - 0,5743 - 1,0834 - 1,1565 - 1,266

6 - 9,356

7 - 10,2748 - 11,5809 - 12,51310 - 30,569

11 - 47,835

WVL:235 nm

Peak #69.66

10,0

25,0

50,0

70,0

200 300 400 500 595

%

nm

223.0

275.0

562.1


[M]+

[M-(COOH)]+

phenyl

Results

205

Compound 26 [p-hydroxyphenylacetic acid] was isolated as a white amorphous powder, UV

absorption at λmax 201, 223, 275 nm. EI-MS showed molecular ion peak m/z 152 [M]+, and

fragment ion peaks at m/z 107 [M-COOH]+, 77 [M-(CH2COOH+OH)]+, (see figure 3.5.26)

suggesting the molecular formula C8H8O3. The difference between 25 and 26 is only 16 mass

units which indicates the presence of one oxygen atom in 26 more than 25. Comparable to

compound 25, 1H NMR spectrum of 26 in DMSO-d6 showed three proton resonances at δ

3.38 (2H, s) suggesting the presence of lower field methylene group, attached to an aromatic

spin system and at δ 7.03 (2H, d, J=8.52 Hz, H-4& H-4') and δ 6.72 (2H, d, J=8.2 Hz, H-5&

H-5'), suggesting p-disubstituted phenyl group instead of 5 proton resonances overlaping at

δ7.3 in 1HNMR of compound 25. The NMR data together with the ESI-MS fragments

suggested compound 26 to be p-hydroxyphenylacetic acid, which was confirmed by HMBC

correlations (see figure 3.5.17). The CH2 at 3.38 showed HMBC correlations to C-1, C-3, C-4

and C-4` at δ 173.4, 126.1, and 132.2, while H-4 and H-4` at δ 7.03 showed HMBC

correlations to C-2, C-5, and C-6 at δ 39.0, 113.8, and 156.6, respectively. The NMR data of

26 are identical to those of p-hydroxyphenylacetic acid (Aldrich, 1993, 2/1018). p-

Hydroxyphenylacetic has been previously isolated from many terresterial biological sources [

e.g. Mellilotus officinalis and Taraxacum officinale (DNP 2005)].


0.72

81

0.77

33

2.00

00

2.02

70

1.99

69

Inte

gral

6085

.89

4625

.59

3515

.86

3507

.34

3341

.83

3333

.63

1787

.89

1700

.25

1665

.57

-4-3-2-1012345678910111213141516

H-2

H-4, H-4'

H-5, H-5'

6-OH Acidic H

2

34

5

OH

6

O

OH1

5'

4'

Results

206


(ppm) 7.2 6.4 5.6 4.8 4.0 3.2 2.4

160

140

120

100

80

60

40

(ppm)

H-2 H-4, H-4'

H-5, H-5'

H-2/C-3

H-2/C-1

H-4/C-5

H-5&H-5' C-5' /C-5

H-4&H-4' /C-6

H-2/C-4 H-2/C-4'

H-5 & H-5' /C-6

H-4& H-4' /C-4' &C-4

H-4&H-4' /C-2

H-5 & H-5' /C-3

2

34

5

OH

6

O

OH1

5'

4'

Results

207

3.5.6- Methyl 2-(4-hydroxyphenyl)acetate (27, known compound)

Fig (3.5.19):EI-MS spectrum of compound 27 (up) compared with standared p-hydroxy-

phenylacetic acid methyl ester (down).

0,0 10,0 20,0 30,0 40,0 50,0 60,0-50

0

100

200

300

350 ms041210 #7 et13b6 UV_VIS_1mAU

min

1 - 0,5642 - 0,9113 - 1,0854 - 1,152

5 - 14,629

6 - 15,275

7 - 30,121

8 - 47,910

WVL:235 nm

Peak #5 14.66

-10

50

90

200 300 400 500 595

%

nm

223.8

275.5562.2



Mol. Wt.: 166,17

OH

O

O3.51

7.06

6.72

3.65

------------------------------------------ Yield : 4.4 mg

[M-(CH2COOMe+OH)]+ [M]+ [M-COOMe]+

Results

208

Compound 27 [methyl 2-(4-hydroxyphenyl)acetate] was isolated as a white amorphous

powder, UV absorption at λmax 201, 223, 275 nm. EI-MS showed molecular ion peak at m/z

166 [M]+, and fragment ion peaks at m/z 107 [M-COOMe]+, 77 [M-(CH2COOMe+O)]+, (see

figure 3.5.30) suggesting the molecular formula C9H10O3. It is clear that compound 27 is 14

mass units larger than compound 26 which indicates the presence of one CH2 group or

exactly, the replacement of the carboxylic proton with methyl group as evident from the 1H

NMR spectrum. The 1H NMR spectrum in methanol-d4 of 27 showed the same set of proton

resonances as compound 26, in addition to the presence of a methoxy proton at 3.65 ppm

which indicated the methyl ester of compound 26. Compared to the authentic sample of

methyl 2-(4-hydroxyphenyl)acetate, compound 27 showed the same molecular ion peak as

well as the same fragmentation pattern (as shown in figure 3.5.19). From NMR data and

EIMS compound 27 was assigned as methyl 2-(4-hydroxyphenyl)acetate which was

previously isolated from many biological sources [like Fusarium oxysporum (Kachlicki P., et

al . (1997)]


2.0

00

0

2.0

95

9

3.0

41

3

1.9

59

5

(ppm)2.83.23.64.04.44.85.25.66.06.46.87.27.6

H-2

H-5 & H-5' H-4&H-4'

H-7

2

34

5

7

OH

6

O

O1

5'

4'

Results

209

3.5.7 Ethyl 2-(4-hydroxyphenyl)acetate (28, known compound) Fig (3.5.22):EI-MS spectrum of compound 28

0,0 10,0 20,0 30,0 40,0 50,0 60,0-100

200

400

600

900 ms050920 #7 tm7et6f UV_VIS_1mAU

min

1 - 0,2022 - 0,5973 - 0,9464 - 1,0935 - 1,1976 - 1,2577 - 1,3228 - 1,4619 - 13,642

10 - 16,770

11 - 17,020

12 - 17,52513 - 47,114

WVL:235 nm

Peak #10 16.72

-10,0

70,0

200 400 595

%

nm

224.3

202.8

275.6


OH

O

O172.640.1

126.2

130.0

115.2156.8

60.1

3.48

7.04

6.68

4.031.1614.2


Mol. Wt.: 180,2

----------------------------------------- Yield : 3.2 mg

[M]+

[M-(CH2)]+

[M-(C2H4O)]+

[M-(C2H5OCO)]+

Results

210

Compound 28 [ethyl 2-(4-hydroxyphenyl)acetate] was isolated as white amorphous powder,

UV absorption at λmax 202, 224, 275 nm. EI-MS showed molecular ion peak m/z 180 [M]+,

and fragment ion peaks at 136 [M-C2H4O]+, 107 [M-COOC2H5]+, 91 [M-(COOEt+O)]+, (see

figure 3.5.33) suggesting the molecular formula C10H12O3. Compound 28 is 14 mass units

more than compound 27 which indicates the presence of one CH2 group or exactly elongation

of one carbon in the alkoxy group of 27 as evident from the NMR data. The 1H NMR

spectrum in DMSO-d6 of 28 showed proton resonances of ethoxy group at δ 4.03 (2H, q,

J=6.93 Hz, H-7) and 1.16 (3H, t, J=6.93 Hz, H-8) instead of a methoxy proton at 3.65 ppm as

in compound 27. The other proton resonances of 28 are the same as those of 27. The

methylene CH2-7 at δ 4.03 of 28 showed an HMBC correlations to C-8 and C-1 at δ 14.2 and

172.0, respectively. The other HMBC correlations were identical with those of compound 26,

see figure (3.5.24). p-Hydroxyphenylacetic acid ethylester is a synthetic product (Bhawal et al

1991, Meltzer et al 1957). However, according to our knowledge, it is the first report of p-

hydroxyphenylacetic acid ethylester as natural product.



1.9

95

2

2.0

00

0

2.1

64

9

2.1

96

5

3.0

18

5

0.60.81.01.21.41.61.82.02.22.42.62.83.03.23.43.63.84.04.24.44.64.85.05.25.45.65.86.06.26.46.66.87.02

H-8

H-7

H-2

H-5 & H-5'

H-4& H-4'

2

34

5

7

OH

6

O

O1

5'

4'

8

(ppm) 7.00 6.00 5.00 4.00 3.00 2.00 1.00

200

160

120

80

40

(ppm)

H-8/C-7

H-2/C-1 H-7/C-1

H-2/ C-4&C-4'

H-8 H-7

H-2 H-5 & H-5'

H-4& H-4'

H-4&H-4’ /C-2

H-4&H-4’ /C-6

H-5&H-5' /C-3

H-5&H-5' /C-6

H-2/C-3

H-7/C-8

2

34

5

7

OH

6

O

O1

5'

4'

8

Results

211

3.5.8 - Butyl 2-(4-hydroxyphenyl)acetate (29, new compound) Fig (3.5.26):EI-MS spectrum of compound 29

[M-(C2H4)]+ [M]+

[M-(C4H8)]+[M-(C3H6)]+

[M-(C4H9OCO)]+

0,0 10,0 20,0 30,0 40,0 50,0 60,0-100

0

100

200

300

400

500

600 ms051002 #15 tm7et6n UV_VIS_1mAU

min

1 - 0,1392 - 0,4013 - 0,4604 - 0,5935 - 0,7776 - 0,9617 - 1,0808 - 1,1579 - 1,24910 - 1,362

11 - 23,016

12 - 23,754

13 - 24,080

14 - 47,958

WVL:235 nm Peak #12 23.78

-10,0

70,0

200 300 400 595

%

nm

224.8203.3

276.2


OH

O

O172.640.1

124.8

130.5

114.1156.1

63.8

3.49

7.03

6.68

3.99

0.8513.1


Mol. Wt.: 208,25

1.51

1.27

30.2

18.4

--------------------------------------------------- Yield : 2.5 mg

Results

212

Compound 29 [butyl 2-(4-hydroxyphenyl)acetate] was isolated as white amorphous powder,

UV absorption at λmax 203, 225, 276 nm. EI-MS showed molecular ion peak m/z 208 [M]+,

and fragment ion peaks at 180 [M-C2H4]+, 166 [M-C3H6]+, 152 [M-C4H8]+, 136 [M-C4H8O]+,

107 [M-COOC4H9]+, and 91 [M-(COOC4H9+O)]+, (see figure 3.5.37) suggesting the

molecular formula C12H16O3. The difference between compounds 28 and 29 is only 28 mass

units which suggested the elongation of the alkoxy group with C2H4 unit as evident from the

NMR spectra of 29. 1H NMR spectrum in DMSO-d6 showed the presence of four sequentially

correlated proton resonances (integrated as 2:2:2:3) at δ 3.99 (2H, triplet, J=6.63 Hz, H-7),

1.51 (2H, quintet, J=7.23 Hz, H-8), 1.27 (2H, sextet, J=7.56 Hz, H-9) and 0.85 (3H, t, J=6.93

Hz, H-8). This indicated the presence of a butoxy group instead of a methoxy group as in

compound 27 and ethoxy group as in compound 28. The other proton resonances of 29 are the

same as those of 27 and 28. The NMR data together with the ESI-MS fragments suggested

compound 29 to be butyl-2-(4-hydroxyphenyl)acetate, which was further confirmed by

HMBC correlations as shown in figure 3.5.28. To the best of our knowledge this is the first

report of a butyl-2-(4-hydroxyphenyl) acetate as a natural product.


2.00

00

1.93

70

1.83

45

1.54

81

2.05

48

2.03

09

2.94

31

Inte

gral

3516

.80

3508

.29

3344

.35

3335

.84

2003

.85

1997

.23

1990

.92

1745

.33

770.

22

763.

60

756.

35

748.

78

742.

16

656.

41

649.

16

641.

59

634.

03

626.

46

619.

21

431.

94

424.

38

417.

12

0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5

2

34

5

7

OH

6

O

O1

5'

4'

8 10

9

4 & 4' 5 & 5'

8 9

10

7

2

Results

213

Fig (3.5.28):HMBC spectrum of compound 29

Table (3.5.2): NMR data of compounds 25, 26, 27, 28 and 29 Compound 25 Compound 26 Compound

27 Compound 28

Compound 29

No. 13C (ppm)

1H (ppm)

(Multiplicity, Hz)

13C (ppm)

1H (ppm)

(Multipli-city, Hz)

1H (ppm)

(Multiplicity,

Hz)

13C

(ppm)

1H (ppm)

(Multiplicity,

Hz)

13C

(ppm)

1H (ppm)

(Multiplicity,

Hz)

1 177.6 - 173.4 - - 172.0 - 172.6 - 2 41.0 3.65 (2H, s) 39.0 3.38 (s) 3.51 (s) 40.1 3.48 (s) 40.1 3.49 (s) 3 133.2 - 126.1 - - 124.3 - 124.8 -

4, 4' 129.3 132.2 7.03 (d,J=8.52)

7.06 (d, J=8.5) 130.0 7.04(d, J=8.5) 130.5 7.03 (d, J=8.51)

5, 5' 128.6 113.85 6.72 (d,J=8.2)

6.72 (d,J=8.5) 115.1 6.68 (d,J=8.5) 114.1 6.68 (d,J=8.51)

6 127.3

7.3 (5H, m)

156.6 - - 156.8 - 156.1 - 7 3.65 (s) 60.1 4.03 (q,J=6.93) 63.8 3.99 (t,J=6.93) 8 14.2 1.16 (t,J=6.93) 30.2 1.51 (t,J=6.93) 9 18.4 1.27 (t,J=6.93) 10

13.1 0.85 (t,J=6.93)

2

34

5

7

OH

6

O

O1

5'

4'

8 10

9

H-2/C-1 H-7/C-1

H-10 H-7

H-2 H-5 & H-5'

H-4& H-4'

H-4&H-4’ /C-2

H-4&H-4’ /C-6

H-5&H-5' /C-3

H-5&H-5' /C-6

H-2/C-3

H-7/C-8

(ppm) 7.00 6.00 5.00 4.00 3.00 2.00 1.00

160

120

80

40

(ppm)

H-7/C-9 H-8 &H-9 /C-10

H-9 H-8

H-8 /C- 9

H-10 /C- 8 H-9 /C- 8

H-8 &H-9 /C-7

H-2/C-3

Results

214

3.5.9 - Adenosine (30, known compound)

0,0 10,0 20,0 30,0 40,0 50,0 60,0-100

0

100

200

300

400

500

600 ms041017 #10 bu17-9 UV_VIS_1mAU

min

1 - 0,950

2 - 1,198

3 - 1,448

4 - 3,048

5 - 47,901

WVL:235 nm

Peak #2 1.19

-10,0

70,0

200 400 595

%

nm

207.4

257.8

559.1

Fig (3.5.29): HPLC chromatogram and UV spectrum of compound 30 Fig (3.5. 30):EI-MS spectrum of compound 30


N

NN

N

NH2

O

OHOH

HO

8.34

8.12

7.33

5.86

5.43

5.41

5.17

4.604.13

3.95

3.54, 3.66

---------------------------------------------------- Yield : 23.0 mg

[M]+

[M-(CH2O+OH+H2O)]+

[M-CH2O]+

[M-(CH2O+OH)]+

[M-(ribose)]+

[M-(C3H5O3)]+

[M-(C4H7O3)]+

[M-(C4H7O4)]+

[M-(ribose+NH2)]+

N

N

H N

H N

Results

215

Fig (3.5. 31):1HNMR spectrum of compound 30

Fig (3.5.32):COSY spectrum of compound 30

1.00

00

0.88

86

1.88

77

1.00

53

2.09

89

0.97

51

1.02

84

1.01

59

1.02

12

1.07

77

1.07

68

1.23

73

Inte

gral

4168

.14

4061

.89

3667

.50

2934

.82

2928

.52

2717

.61

2711

.30

2706

.26

2703

.74

2699

.01

2586

.77

2582

.36

2308

.08

2302

.09

2297

.05

2290

.74

2071

.00

2066

.27

2063

.12

2058

.39

1979

.89

1976

.42

1973

.27

1969

.80

1839

.91

1835

.81

1831

.72

1827

.93

1823

.83

1819

.74

1780

.96

1777

.17

1773

.39

1769

.61

1765

.19

1761

.41

1757

.94

1660

.53

( )0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5

2 8

NH2 1' 2' 3'

4' 5’b

3’-OH

5’-OH

5’a

N

NN

N

NH2

O

OHOH

HO

1

234

5 678

9

1'2'3'

4'

5'

H2O DMSO

(ppm) 5.6 5.2 4.8 4.4 4.0 3.6 3.2

6.0

5.6

5.2

4.8

4.4

4.0

3.6

3.2

(ppm)

1' 2' 3'

4'

5’b

2'-OH 3’-OH

5’-OH

5’a

2'

3' 4'

5’b 5’a

1’/2’ 2’/2'-OH

1'

2'-OH

3’-OH

5’-OH

2’/3’

3’/3'-OH

3’/4’

5’a & 5’b /5'-OH

5’a & 5’b /4'

H2O

H2O

N

NN

N

NH2

O

OHOH

HO

1

234

5 678

9

1'2'3'

4'

5'

2'-OH

Results

216

Compound 30 was isolated as brownish-white amorphous powder, UV absorption at

λmax 207, 257 nm of a very polar peak in HPLC chromatogram suggested the nucleoside

nature of the compound. EI-MS showed molecular ion peak at m/z 267 [M]+, and fragment

ion peaks at 237 [M-CH2O]+, 220 [M-CH2O+OH]+, 202 [M-CH2O+OH+H2O]+, 178 [M-

C3H5O3]+, 164 [M-C4H7O3]+, 148 [M-C4H7O4]+, 135 [M-(ribose)]+, and 119 [M-

(ribose+NH2)]+, (see figure 3.5.41) suggesting the molecular formula C10H13N5O4. 1H NMR

spectrum which was meassured in DMSO-d6 showed resonances at δ 8.12 (s, H-2), 8.34 (s, H-

8), 7.33 (s, 6-NH2) suggesting the presence of 9H-purin-6-ylamine moiety. The existence of a

ribosyl moiety in this compound was evident by a number of protons resonating between δ

3.54 and 5.86 (table 3.5.4). Furthermore, the presence of hydroxyl functional groups at the

positions 2', 3', 5' were determined by resonances at δ 5.43(d, J=6.3) 2'-OH, 5.17 (d, J=4.41)

3'-OH, 5.41 (dd, J=7.25, 4.73) 5'-OH as shown in 1H-NMR and confirmed by its COSY

spectrum (figure3.5.31 & 3.5.32). The above NMR data together with the EI-MS fragments

confirmed compound 30 to be adenosine.

Table 3.5.3 1H-NMR data of Compound 30:

No. 1H (ppm)

(Multiplicity, Hz) 2 8.12 (s) 8 8.34 (s)

6-NH2 7.33 (s) 1' 5.86 (d, J =6.3) 2' 4.60 (dd, J=11.35, 6.3)

2'-OH 5.43(d, J =6.3) 3' 4.13 (dd, J =7.88, 4.73)

3'-OH 5.17 (d, J =4.41) 4' 3.95 (dd, J =6.62, 3.47) 5'a 3.54 (ddd, J =3.47, 7.25, 11.67) 5'b 3.66 (ddd, J =4.09, 8.19, 11.98)

5'-OH 5.41 (dd, J =7.25, 4.73)

Results

217

3.5.10 Nicotinamide (31, known compound)

Fig (3.5.34):EI-MS spectrum of compound 31 (up) compared to authentic sample of

nicotinamide (down)

N

O NH2

9.03 8.19

7.48

8.7

C6H6N2O

Mol. Wt.: 122,12

------------------------------------------------- Yield : 2.9 mg

0,0 10,0 20,0 30,0 40,0 50,0 60,0-100

200

400

600

800

1.000 ms050503 #2 et17a UV_VIS_1mAU

min

1 - 0,5172 - 0,9333 - 1,123

4 - 1,485

5 - 30,581 6 - 47,553

WVL:235 nm Peak #4 1.51

-10,0

70,0

200 400 595

%

nm

260.9218.6202.6



N

O NH2

1

2

3

4

5

6

7

[M]+ [M-NH2]+ [M-CONH2]+

[M-(CONH2 + HCN)]+

Results

218

Compound 31 was isolated as white amorphous powder. It has UV absorption at λmax

202, 218, 260 nm. EI-MS showed molecular ion peak at m/z 122 [M]+, and showed the

same fragmention pattern of authentic sample of nicotinamide where it displays a fragment

ion peaks at m/z 106 [M-NH2]+, 78 [M-CONH2]+, and 51 [M- CONH2+HCN]+, (see figure

3.5.34) suggesting the molecular formula C6H6N2O. 1H NMR spectrum (meassured in

DMSO-d6) showed resonances at δ 9.03 (br s, H-3), 8.7 (br s, H-5), 7.48 (dd, J=7.88, 4.73),

8.19 (d, J=7.88 H-4). The 1H NMR spectrum of compound 31 is identical with those of

nicotinamide (Aldrich 1993, pp 3/338).


4510

.83

4347

.21

4100

.04

4093

.74

4092

.16

3796

.44

3750

.41

3745

.68

3742

.53

3737

.80

6.46.66.87.07.27.47.67.88.08.28.48.68.89.09.29.4

N

O NH2

1

2

3

4

5

6

7

3 5

7 6

Results

219

3.5.11 and 12 Petrocerebrosides 1 and 2 (32 & 33, new compounds)

Fig (3.5.36):EI-MS spectrum of compounds 32 and 33

Cerebrosides are glycolipids composed of a long chain aminoalcohol, known as a

sphingoid base ( sphingosines), a fatty acid residue linked to its amino group (the resulting

amide is called ceramide), and a carbohydrate chain attached to the primary hydroxyl group of

the ceramide. The scientific interest in cerebrosides has recently increased on account of the

role they play as therapeutical immunomodulating agents (Costantino et al 2004, 2003, 1994).

Sphingolipids have come to be used as functional foodstuffs and sphingomyelin isolated from

C22H45O

HNC12H25

OH

OH

O

H

HHO

H

HOH

H

OHOO

HO

HH

HO

H

HOH

H

OHO

C57H109NO14Exact Mass: 1031,78481

C21H43O

HNC11H23

OH

OH

O

H

HHO

H

HOH

H

OHOO

HO

HH

HO

H

HOH

H

OHO


-------------------------------------------------------------------------------------------- Yield : 35.0 mg

PetrocerebrosPetrocerebros

M-Fatty

Results

220

bovine milk has been found to have significant effects in colon cancer prevention. Sphingoid

bases isolated from plants and fungi have been shown to induce apoptosis in the caco-2 cell-

line. The ingestive cerebrocides from maize and Saccharomyces kluyveri prevent aberrant

crypt foci in mice adminstrated with N,N-dimethylhydrazine (Tanji 2004).

Compound 32 and 33 were obtained as mixture [in a ratio of 5:4, respectively as

evident from the fatty acid ratios (see figure 3.5.4)] and as an amorphous powder. The

molecular formulas were deduced as C57H109NO14 and C56H107NO14 based on a molecular ion

peak [M+H]+ 1032.1 and [M]+ 1031.2 for petrocerebroside 1 and [M+H]+ 1018.1 and [M]+

1017.2 for petrocerebroside 2 as obtained from the ESI-MS of the sample. The difference

between both cerebroside derivatives is only 14 mass unit which suggested an additional

methylene group in petrocerebroside 1 when compared to petrocerebroside 2. The position of

this CH2 group was deduced to be in the long chain fatty acid part of the compound as shown

below. The structure elucidation of petrocerebrosides 1 and 2 were obtained using extensive 1H NMR, 13C NMR, DEPT, and 2D NMR experiments, as well as by acid hydrolysis. GC

analysis determined the absolute configuration of the sugar moieties. GC-MS was done to

analyze the molecular weights both the free fatty acid and sphingosine base after acid

hydrolysis.

The cerebroside nature of the petrocerebrosides 1 and 2 were established from the 1H

NMR which showed a triplet-like signal at δ 0.82 for the terminal methyl units and broad

singlet at δ 1.20 for long chain (CH2)n group for both fatty acids and long chain bases. The

presence of several doublets in the region between 3.35-5.30 ppm indicated the occurrence of

the sugar moieties. Two coupling sp2 methines at δ 5.41 and 5.34 showed the presence of only

one double bond. Two doublets at 7.42 and 7.70 ppm exhibited the presence of two amide

NHs belonging to two different compounds. 13C NMR showed two resonances at δ 172.3 and

170.15 indicating two amide carbonyls for petrocerebroside 1 and 2, respectively. Two

resonances at δ 131.5 and 128.1 were assigned for two sp2 methine carbons, while two sp3

methines resonating at 101.2 and 98.9 indicated the presence of anomeric carbons for two

monosaccharides.

Results

221

Fig (3.5.37):1HNMR spectrum of compound 32 and 33

Fig (3.5.38):COSY spectrum of compound 32 and 33

3857

.05

3848

.52

3715

.01

3706

.31

2722

.35

2713

.65

2707

.12

2700

.26

2684

.70

2678

.34

2671

.65

2662

.95

2656

.26

2334

.02

2331

.17

2148

.80

2140

.61

1853

.67

1849

.82

1842

.96

1838

.61

1833

.59

1794

.94

1793

.10

1789

.09

1783

.23

1032

.34

1028

.82

1022

.47

1014

.77

985.

9997

9.80

965.

5895

9.39

952.

7042

5.67

418.

9741

1.95

1.01.52.02.53.03.54.04.55.05.56.06.57.07.5

Amide NHs H-6, H-7 (E-coupling)

Sugar protons and other sp3-down

field

(CH2)n

Terminal methyls

(ppm) 8.0 6.0 4.0 2.0

8.0

6.0

4.0

2.0

(ppm)

(comp-34) NH

H-6, H-7

H-5/H-6

(comp-35) NH

(comp-34) NH/H-2

(comp-35) NH/H-2

H-7/H-8

H-7/H-6

Results

222

Fig (3.5.39):HMQC spectrum of compound 32 and 33

Fig (3.5.40):HMBC spectrum of compound 32 and 33

(ppm) 7.00 6.00 5.00 4.00 3.00 2.00 1.00

120

100

80

60

40

20

(ppm)

2 epimeric protons

2 epimeric carbons 2 epimeric protons/

(ppm) 8.0 6.0 4.0 2.0

160

120

80

40

(ppm)

(comp-32) NH/C-1'

(comp-33) NH/H-1' Hα/C-1'

H-8/C-6 &C-7

H-2" H-2```/C-1" &C-1```

H-6 &H-7/C-5 and C-8

CH2Cl2

OH CH3 (Methanol peaks)

Sugar part Hs/Cs

Results

223

The sample was hydrolysed using 6N HCl and heated over a hot plate under reflux for

7 hours, then cooled with cold water stream. The hydrolysate were extracted with n-hexane to

obtain the fatty acid part which was then examined by GC-MS (figures 3.5.41, 42a &42b)

where mixture of two long chain fatty acids (pentacosanoic acid C25H50O2, m/z 382 and

tetracosanoic acid C24H48O2, m/z 368) were detected. The aqueous part were purified over a

Sephadex column chromatography and the Molish-positive fraction were separated and

utilised for determination of the absolute streochemistry of the monosaccharide units. The

absolute streochemistry of the monosuccharide units were established through butanolysis,

silylation, then GC analysis in comparison with authentic monosaccharides. This experiment

revealed that both sugar units are D-galactoses.

Fig (3.5.41):GC-chromatogram of the fatty acid-containig fraction of

compound 32 and 33 hydrolysate

Fig (3.5.42a):GC-MS of tetracosanoic acid Fig (3.5.42b):GC-MS of pentacosanoic acid

Results

224

Deduced NMR data of both fatty acid and sphingosine moieties were confirmed after

methanolysis. 1H NMR of the fatty acid mixture showed a singlet resonating at δ 3.66

indicating a methoxy group of the derivatised fatty acid. A triplet at δ 0.88 indicated the

terminal methyl, a triplet at 2.3 ppm was assigned for the α-CH2, and the multiplet at 1.61

ppm represented the β-CH2. This data were confirmed by 13C NMR spectrum where

characteristic signals of the long chain fatty acid methyl esters were obtained as shown below

(figures 3.5.43 and 44).

Fig (3.5.43):1HNMR spectrum of fatty acid mixture of compound 32 and 33 hydrolysates.

Fig (3.5.44):1HNMR spectrum of fatty acid mixture of compound 32 and 33 hydrolysates.

Fig (3.5.45):1HNMR spectrum of sphingosine part of compound 32 and 33

1.60

33

1.31

18

1.61

21

41.6

88

3.00

00

Inte

gral

1832

.36

1157

.38

1149

.81

1142

.25

821.

62

814.

06

807.

12

799.

87

792.

30

625.

53

445.

51

438.

89

431.

96

( )0.01.02.03.04.05.06.07.08.09.010.011.012.013.014.0

Terminal methyls

(CH2)n

α-CH2 β-CH2

O-CH3

174.

3759

51.4

427

34.1

188

31.9

136

29.6

891

29.6

506

29.5

832

29.4

483

29.3

520

29.2

461

29.1

498

24.9

512

22.6

883

14.1

178

(ppm)1020304050607080901001101201301401501601700

C-n`-2 C-3`

C-2`Me-O C-1`

C-n`-1

C-n` (CH3)

n`= 24 ---------------- tetracosanoic acid n`= 25 ---------------- pentacosanoic acid

3032

.90

3025

.97

3017

.77

3010

.52

3003

.58

2862

.03

2855

.09

2848

.47

2839

.96

2833

.34

2470

.47

2462

.59

2147

.01

2126

.20

2125

.57

2120

.53

2110

.13

2013

.02

2005

.77

1998

.21

1994

.42

1986

.86

1979

.61

1764

.60

1758

.92

1521

.53

1516

.48

1514

.28

1509

.55

1502

.30

1363

.58

1356

.33

1349

.08

1342

.14

1334

.58

1025

.93

1019

.00

1012

.06

1005

.44

961.

6295

3.74

951.

5394

2.07

1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5

Solvent peaks (pyridine)

H-6, H-7

sp3-down field region H2-8

H-5a, H-5b

(CH2)n

Terminal methyls

Results

225

Fig (3.5.46):COSY spectrum of sphingosine part of compound 32 and 33

Fig (3.5.47):HMQC spectrum of sphingosine part of compound 32 and 33

(ppm) 6.00 5.00 4.00 3.00 2.00 1.00

6.00

5.00

4.00

3.00

2.00

1.00

(ppm)

H-7/H-8

H-6/H-5a & b

H-2/H-1a,H-1b, H-3

H-3 H-2

H-8/H-9

H-4 /H-3

H-5a /H-5 b H-4/H-5a & b

H-8 H-1a, H-1b, H-4

H-5a

6.0 4.0 2.0 0.0

120

80

40

0(ppm)

H-3 H-2

H-8 H-1a, H-1b, H-4 H-5a H-5 b H-6

H-7

MeOH

Results

226

Table 3.5.4 NMR data of compound 32 and 33, in addition to free fatty acids and sphingosine Compounds 35 &36

(DMSO-d6) Fatty acid (CDCl3)

Sphingosine (Pyridine-d5)

No. 13C (ppm)

1H (ppm) (Multiplicity, Hz)

13C (ppm) 1H (ppm) (Multiplicity, Hz)

13C (ppm)

1H (ppm) (Multiplicity, Hz)

1 63.5 4.20 and 4.35 (m & m) 65.0 4.25 & 4.35 (m & m)

**2 55.2 54.5

3.90 (m) 3.4 (m)

58.0 3.6 (m)

**NHs - -

(d, 9.3 Hz) 7.72 (d, 8.2 Hz)

3 75.0 3.55 (m) 75.0 4.04 (m) 4 74.1 3.43 (m) 74.2 4.25 (m) 5 33.0 2.40 & 1.9 ( m &m) 38.2 3.05 (m) &

2.72 (ddd, 14.8, 8.0, 6.8)

6 128.1 5.45 (ddd, 6.4, 8.2, 15.1 Hz)

128.1 6.05(ddd, 15.13, 6.95, 8.19)

7 131.5 5.71 (ddd, 6.6, 8.5, 15.1 Hz)

132.5 5.71 (ddd, 15.13, 6.5, 6.6)

8 34.3 1.9 (m) 34.3 1.99 (m) 9-19 33.0-

29.0 1.2 (br s) 33.0-

29.0 1.26 (br s)

20 14.29 0.85 (t, 6.9 Hz)

14.2 0.82 (t, 6.9) **1´ 172.2

170.0 - 174.4

2´ 35.8 2.3 (m) 34.1 2.3 (t, 7.6) 3´ 31.7 1.6 (m) 31.9 1.6 (m) *4´-(n-1)´

30.0 – 22.5

1.2 (br s) 30.0 – 22.7 1.3 (br s)

n´ 14.29 0.85 (t, 6.9 Hz) 14.1 0.88 (t, 7.0) 1´´ 102.0 4.3 (d, 8.5 Hz) 2´´ 71.0 3.5 (m) 3´´ 69.5 3.63 (m) 4´´ 83.2 3.7 (m) 5´´ 68.4 3.4(m) 6´´ 63.5 3.32 &3.25 (m &m) 1´´´ 99.0 4.7 (d, 4.2 Hz) 2´´´ 70.5 3.2(m) 3´´´ 69.2 3.60 (m) 4´´´ 80.7 3.28 (m) 5´´´ 68.2 3.4(m) 6´´´ 62.2 3.30 &3.4 (m &m)

* n =25 for petrocerebroside 1 n =24 for petrocerebroside 2 ** these pairs of NMR values were assigned for both compounds and can be interchanged, These differences attributed mainly to the differences in stereochemistry of the three chiral centers in each cerebroside at C-2, C-3 and C-4.

Results

227

New purine derivatives isolated from Petrosia nigricans:

Marine organisms particularly sponges have proven to be an exceptionally rich source

of modified nucleosides. The isolation of spongouridine and spongothymidine from

Cryptotethia crypta (Bregman and Feeney 1950) served as models for the development of

adenine arabinoside (ARA-A) for treatment of Herpes simplex infection and cytosine

arabinoside (ARA-C) for the treatment of leukemia (Lindsay et al 1999). Subsequent

development of antiviral analogues demonstrated the potential medicinal importance of these

compounds such as antifungal phidolopine which was isolated from the bryozoan

Phidolopora pacifica (Ayer et al 1984), the hypotensive doridosine which was obtained from

the sponge Tedania digitata (Cook et al 1980) , and the cytotoxic mycalisines which was

found from the sponge Mycale sp. (Kato et al 1985). Many other purines and nucleosides

isolated from marine organisms particularly sponges, display potant bioactivities, such as the

marine derived 1,3-dimethylisoguanine from Amphimedon viridis which showed activity

against an ovarian cancer cell line (IC50, 2.1 µg/mL) (Mitchell et al 1997) and 3,7-

dimethylisoguanine from a Caribbean sponge Agelas lonigssima, which displayed mild

antibacterial activities (Cafieri et al 1995). Investigation of ethylacetate fraction of the

Indonesian sponge Petrosia nigricans, led to the isolation of four new purine derivatives

nigricines 1 to 4. Their structures were elucidated by extensive spectroscopic analysis, 2D-

NMR experiments, EI/MS, ESI/MS, and HRMS.

Results

228

3.5.13. Nigricine 1 (34 , new compounds)

Fig (3.5.49):ESI-MS spectrum of compound 34

0,0 10,0 20,0 30,0 40,0 50,0 60,0-50

0

100

200

300

350 ms040918 #12 tm7et16-7 UV_VIS_1mAU

min

1 - 0,9542 - 1,0533 - 1,1334 - 1,2145 - 1,3016 - 1,3357 - 1,4258 - 1,566

9 - 16,453

10 - 21,56811 - 23,16512 - 27,77213 - 28,905 14 - 47,406

WVL:235 nm

Peak #9 50% 100% -50%

-10,0

25,0

50,0

70,0

200 400 595

%

nm

210.3

290.2

560.9

999.94 999.95


154.9

N

N

N

N

NH

O

O

O


Mol. Wt.: 307,348

31.0

141.5

115.0

153.6

139.1

34.2

36.3

34.8

171.3

65.3

31.2

20.1

13.3

3.62

7.67

7.57

3.91

3.59

2.62

4.08

1.57

1.310.89

----------------------------------------------- Yield : 2.3 mg

Results

229

Compound 34, is the key structure for this group of purine derivatives. The

HRESIMS+ was in agreement with the molecular formula of C14H21N5O3 (measured, m/z

308.170, [M+H] ) with 7 degrees of unsaturation. The UV spectrum of 34 showed absorption

λmax (MeOH) at 210 and 290 nm. Simple 1H NMR spectrum (figure 3.5.51) showed an

exchangeable triplet signal at δ 7.73 ppm for an NH, in addition to signals at δ 7.61 (1H, s ,

H-8), 4.17 ( 2H, t , CH2), 1.67 ( 2H, m , CH2), 1.4 ( 2H, m , CH2), 0.95 ( 3H, t , CH3), 3.80 (

3H, t , N-3 CH3), 4.05 ( 3H, t , CH3). The basic structure of the purine skeleton was evident

through interpretation of 1H NMR and 13C NMR as well as comparison with those of the

literatures of Lindsay et al 1999, Mitchell et al 1997, Lindsay et al 1999, Capon et al 2000,

Yagi et al 1994.

The NMR measurement of compound 34 in deuterated methanol indicated the

presence of only one exchangeable triplet signal at 7.73 ppm for 6-NH suggesting a 6-

derivatized adenine structure, the methyl signals at δ 31.4 and 34.8 showed a characteristic 13C chemical shifts for NCH3 resonances and excluding those of OCH3, often found between

50-60 ppm, the confirmation of the positions of both methyl groups were obtained from

HMBC correlation of both methyl groups to a quaternary carbon at δ 143.1 (C-4), one of

them, N(3)-CH3, showed further HMBC correlation to carbonyl at 159.0 ppm (C-2), while the

other, N(9)-CH3, showed an additional HMBC correlation to the methine carbon at δ 140.6

(C-8). Furthermore, ROESY experiment showed a correlation through space between both

methyl signals, thus, the existence of N(7)-CH3 was excluded. The methine proton signal, H-

8, showed HMBC correlations to both quaternary carbon at 143.1 (C-4) , and 115.0 (C-5).

The remaining carbon in the purine skeleton, C-6, was established through HMBC correlation

of the NH proton signal at δ 7.73 to a quaternary carbon signal at 157.7 ppm (C-6). The

position of β-propionyl side chain was confirmed by sequential COSY correlation between

the proton signals at δ 7.73, 3.80, and 2.72 of 6-NH, β-CH2, and α-CH2 of propionyl group,

and compared with chemical shifts of the same substructure of the previously described

marine derived purine compound, erinacean (5), which was obtained from the antarctic

sponge Isodictya erinacea (Moon et al 1997). In addition, HMBC experiment showed a

correlation of both α- and β- CH2 groups to the carboxyl at 173.0 ppm. The attachment of this

group to the purine skeleton was evident through HMBC correlation betwwen β-CH2 proton

signal and the quaternary carbon at 157.7 ppm (C-6). The last substructure (alkoxy group)

was confirmed through a sequential COSY correlations between 4 aliphatic proton signals at δ

4.17 ( 2H, t. CH2), 1.67 ( 2H, m. CH2), 1.4 ( 2H, m. CH2) and 0.95 ( 3H, t. CH3), the

Results

230

connection of this substructure to the purine skeleton was established through HMBC

correlation between α-CH2 at δ 4.17 and the carboxyl at 173.0 ppm.

LC/MS of compound 34 showed a positve pseudomolecular ion peak at m/z

308(M+1), and at m/z 615 (19 % , 2M+1) and a characteristic fragment ion at m/z 234 [M-

alkoxy group] and 192 [M-(alkoxy group+NCO)] indicating the loss of NCO fragment due to

retro Diels-Alder cleavage of N-1/C-6 and C-2/N-3 bonds which is a characteristic

fragmentation pattern for 2-oxopurines (Cafieri et al 1995). The presence of a fragment ion

peak at m/z 180 indicated 3,9-dimethyl isoguanine skeleton after loss of the side chain (m/z

138). Both MS fragmentaion pattern and NMR spectra corroborate with the structure proposal

and established the identity of 34 as butyl 3-(3,9- dihydro-3,9-dimethyl –2-oxo-2H-purin-6-

ylamino) propanoate. To the best of my knowledge this is the first report of 34 as a natural

product and also as far as I know this is the first report of an 2-oxo-3,9-dimethylpurin-6-

ylamino derivative, which we assign the trivial name nigricine 1.

Fig (3.5.50):13CNMR and DEPT spectra of compound 34

a

bcdef

hig

10

11

12

1

23 4

5

6

7

8

9

N

N N

N

NH

O

OR

O

1 R = Bu2 R = Et3 R = Me

1 0

1 1

1 2

1

23 4

5

6

7

8

9

N

N N

N

N H

O

OO

4

N

N HN

NH

O OH

NH

O1

12

1110

38

6

5

New compounds, nigricines 1 to 4, as well as the known analogue erinacean (5)

Results

231


0.80

50

1.95

62

2.33

89

3.55

15

2.78

01

1.73

62

2.07

32

2.37

25

3.00

00

Inte

gral

3784

.77

2050

.18

2043

.88

2036

.94

1347

.78

1340

.84

1334

.53

809.

30

802.

37

799.

85

795.

43

787.

87

780.

93

690.

13

682.

57

675.

00

668.

07

461.

25

453.

69

446.

75

(ppm)0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.0

*NE

P*

S*

S

I

da

b

c

ef

4. 05

7.6 1

3. 8

3.82.7 2

4.1 7

1.6 7

1.40.9 5

31 .4 34 .8

14 0.6

37 .234 .4

65. 6

3 1.8

20.114. 0

173

15 7 .7

159. 0

115 .0

1 43. 1

N

N

N

N

N H

O

O

O

h

i

g

ia

b

g

cfd e

h

i

a

b

g

cf

d

e

h

da

bc

e

f

3 .9

7 .6 1

3 .6 3

3 .6 0

2 .6 2

4 .0 9

1 .5 5

1 .30 .8 9

N

N

N

N

N H

O

O

O

h

i

g

7 .6 6

N H

Fig (3.5.51):1HNMR spectrum of compound 34 measured in methanol-d4 (up) and DMSO-d6 (down).

NH/

da

bc

e

f

3.9

7.61

3.63

3.60

2.62

4.09

1.55

1.30.89

N

N

N

N

NH

O

O

O

h

i

g

7.66

i/h long range coupling

g/h long range coupling

b/c

b/a

f/e

Results

232

Fig (3.5.53):total HMBC spectrum of compound 34

Fig (3.5.54):part of HMBC spectrum of compound 34

(ppm) 7.00 6.00 5.00 4.00 3.00 2.00 1.00

160

120

80

40

(ppm)

da

b

c

ef

4.05

7.61

3.8

3.82.72

4.17

1.67

1.40.95

31.4 34.8

140.6

37.234.4

65.6

31.8

20.114.0

173

157.7

159.0

115.0

143.1

N

N

N

N

NH

O

O

O

h

i

g

a b c

d e f i

h g

(ppm) 7.2 6.4 5.6 4.8 4.0 3.2 2.4

180

160

140

120

(ppm)

h/i

h/143.1

g/143.1

i/115.0

f/157.7

i/143.1

g/159.0

f/173 d/173 e/173

da

b

c

ef

4.05

7.61

3.8

3.82.72

4.17

1.67

1.40.95

31.4 34.8

140.6

37.234.4

65.6

31.8

20.114.0

173

157.7

159.0

115.0

143.1

N

N

N

N

NH

O

O

O

h

i

g

Results

233

Table 3.5.5. 1H and 13C NMR Data of compound 34 (Nigricine 1) (500 MHz) Position 13C δ ,m

(DMSO-d6)

1H δ,m, j(Hz)

(DMSO-d6)

13C δ ,m

(CD3OD)

1H δ,m, j(Hz)

(CD3OD)

HMBC

(H--------- C)

2 154.9 - 158.9 -

N(3)-CH3 31.0 3.62 31.4 3.80 (s) C-2, C-4

4 141.5 - 142.5 -

5 115.0 - 115.4 -

6 153.6 - 157.6 -

6-NH - 7.67 (t,5.8) - - C-6

8 139.1 7.57 140.6 7.61 (s) C-4, C-5

N(9)-CH3 34.2 3.91 34.5 4.02 (s) C-8, C-4

10 36.3 3.59 (dt,6.30&5.8) 37.2 3.81 (t,6.30) C-6, C-12, C-11

11 34.8 2.62 (t,6.30) 34.8 2.72 (t,6.31) C-12, C-10

12 171.3 - 173.5 -

13 65.3 4.08 (t,6.30) 65.4 4.17 (t,6.94) C-12, C-15, C-14

14 31.2 1.57 (tt,6.93&6.94) 31.8 1.67, (tt,6.93&6.94) C-13, C-15, C-16

15 20.1 1.31(tt,6.93&6.94) 20.6 1.40 (qt,6.93&6.94) C-13, C-14, C-16

16 13.3 0.89 (t,6.94) 13.9 0.95 (t,6.94) C-14, C-15

Results

234

3.5.14 – Nigricine 2 (35, new compounds)


N

N

N

N

N H

O

O

O

3.92

7.57

3.65

3.552.58

4.08

1.12

29.8433.08

138.27

35.3433.3

59.91

14.07171.3

155.45

155.81

113.1

141.1

7.73

C 12H 17N 5O 3E xact M ass: 279,13

M ol. W t.: 279,3

----------------------------------------------------- Yield : 6.1 mg

0,0 10,0 20,0 30,0 40,0 50,0 60,0-50

0

100

200

300

400 ms050503 #7 et17f UV_VIS_1mAU

min

1 - 0,0792 - 0,5843 - 0,7274 - 0,9575 - 1,1176 - 1,205

7 - 8,892

8 - 15,756 9 - 24,17710 - 25,28111 - 30,56912 - 30,904

13 - 47,630

WVL:235 nm Peak #7 8.54

-10,0

25,0

50,0

70,0

200 400 595

%

nm

209.9

289.7

560.5


B

A C

N

N

N

NO

HN

O

O

N

N

N

NO

HN

O

N

N

N

HN

O

N

N

N

NO

NH2

H+ -OEt

retro Diels-Alderfragmentation

-[NCO]

-side chain

1

2

m/z 280

H+

H+

H+

m/z 234

m/z 180 m/z 193

B

A

C

Results

235

Compound 35, showed molecular formula C12H17N5O3 through HRESIMS-TOF

analysis (measured, m/z 280.139, [M+H]+) with 7 degrees of unsaturation. The UV spectrum

of 35 showed absorption λmax (MeOH) at 210 and 290 nm indicating the same chromophoric

functionalities as 34. The difference in molecular weight between 34 and 35 was only 28

mass units indicating a loss of C2H4 group from the alkoxy group as evident from its 1HNMR

spectrum. ESI/MS spectrum showed pseudomolecular ion peak at m/z 280.1 (M+H) and at

m/z 559 (2M+H) and at m/z 838 (3M+H). Tandem MS fragmentation spectrum showed a retro

Diels-Alder fragment ion peak at m/z 192 [M-(ethoxy group +OCN)]+ which confirmed the

loss of an OCN group suggesting 2-oxopurine derivative (Cafieri et al 1995), and also

exhibited a molecular ion fragment at m/z 234 indicating the loss of an alkoxy group.The 1H

NMR and 13C NMR spectra showed signals at chemical shift the same as those of nigricine 1

with the exception of loss of 2 CH2 chemical shifts from the terminal alkoxy group.

The proton signals for 6-NH exhibited HMBC correlations to the adjacent quaternary

carbon signals at 155.45 and 113.1 ppm and also HMBC correlation to the β-methylene

carbon of propionc acid moiety at 35.34 ppm. The aromatic proton signal, H-8, at 7.57 ppm

showed HMBC correlation to N(9)-Me carbon signal at 33.08, quaternary aromatic carbon

signals at 113.1 and 141.1 ppm of C-5 and C-4 respectively. N(3)-Me proton signal exhibited

HMBC correlation to the carbonyl (C-2) at 155.81 ppm. The connection of the ethoxy group

was established through HMBC correaltion between CH2 proton signal to the carboxyl at δ

171.3. Both MS fragmentaion pattern and NMR spectra confirmed the proposed structure and

established the identity of 35 as ethyl 3-(3,9- dihydro-3,9-dimethyl –2-oxo-2H-purin-6-

ylamino) propanoate and was assigned the trivial name nigricine 2.

Table 3.5.6. NMR Data of compound 38 (Ashourine 2) [500 MHz] Position 13C δ ,m

(DMSO-d6) 1H δ,m, j(Hz) (DMSO-d6)

HMBC (H-------- C)

2 155.8 - N(3)-CH3 29.84 3.65 (s) C-2, C-4 4 141.1 - 5 113.1 - 6 155.45 - 6-NH - 7.73 (t,5.68) C-6, C-5, C-10 8 138.27 7.57 (s) C-4, C-5, N(9)-CH3 N(9)-CH3 33.08 3.91 (s) C-8, C-4 10 35.34 3.56 (dt,6.63&5.8) C-6, C-12, C-11 11 33.56 2.58 (t,7.25) C-12, C-10 12 171.3 - 13 59.91 4.08 (q,7.25) C-12, C-14 14 14.07 1.12 (t,7.25) C-13

Results

236



0.8

06

6

0.8

01

3

2.1

72

0

2.5

62

7

2.5

40

9

2.2

29

1

2.1

17

8

3.0

00

0

38

64

.2

38

58

.5

38

52

.8

37

93

.6

20

36

.0

20

28

.7

20

21

.8

20

14

.5

19

45

.2

17

98

.3

17

91

.0

17

84

.1

17

77

.4

17

70

.8

13

00

.8

12

93

.5

12

86

.3

59

0.2

0

58

2.9

5

57

6.0

2

(ppm)00.81.21.62.02.42.83.23.64.04.44.85.25.66.06.46.87.27.68.0

0.9

23

0

2.1

21

6

2.8

80

5

4.9

73

0

1.9

66

3

3.0

00

0

37

82

20

75

20

68

20

61

20

54

19

91

19

85

18

84

18

77

18

75

18

71

16

53

16

52

16

50

16

48

16

47

13

43

13

36

13

29

62

1.7

61

4.4

60

7.2

60

1.2

(ppm)

0.81.21.62.02.42.83.23.64.04.44.85.25.66.06.46.87.27.68.0

a b c

d

e f

g

a b cd

e f

g NH

d

ab

c

ef

N

N

N

N

NH

O

O

O

g

3.92

7.57

3.65

3.55

2.58

4.08

1.12

29.84

33.08

138.27

35.34

33.3

59.91

14.07171.3

155.45

155.81

113.1

141.1

7.73

17

1.3

06

6

15

5.8

13

7

15

5.4

56

6

14

1.1

07

8

13

8.2

73

0

11

3.1

02

4

59

.9

19

2

35

.6

08

5

33

.5

68

1

33

.3

34

9

29

.8

44

2

14

.0

74

4

0102030405060708090100110120130140150160170180190

g a

b c d

e f

d

ab

c

ef

N

N

N

N

NH

O

O

O

g

3.92

7.57

3.65

3.55

2.58

4.08

1.12

29.84

33.08

138.27

35.34

33.3

59.91

14.07171.3

155.45

155.81

113.1

141.1

7.73

Results

237


Fig (3.5.60):HMBC spectrum of compound 35

(ppm) 7.00 6.00 5.00 4.00 3.00 2.00 1.00

7.00

6.00

5.00

4.00

3.00

2.00

(ppm)

g a

b c

d

e f

g

a

b

c

d f

NH

NH

d

ab

c

ef

N

N

N

N

NH

O

O

O

g

3.92

7.57

3.65

3.55

2.58

4.08

1.12

29.84

33.08

138.27

35.34

33.3

59.91

14.07171.3

155.45

155.81

113.1

141.1

7.73

e

(ppm) 7.00 6.00 5.00 4.00 3.00 2.00 1.00

160

120

80

40

(ppm)

g

a

b

f& c d

e

g a b c d

e f

NH

d

ab

c

e f

N

N

N

N

NH

O

O

O

g

3.92

7.57

3.65

3.55

2.58

4.08

1.12

29.84

33.08

138.27

35.34

33.3

59.91

14.07

171.3

155.81

155.45

113.1

141.1

7.73

Results

238

3.5.15 – Nigricine 3, (36 , new compounds)


N

N

N

N

N H

O

O

O

3 . 9 2

7 .5 7

3 .6 0

3 .5 5

2 .5 8

3 . 6 1

2 9 .8 43 3 .3 3

1 3 8 .2 7

3 5 .6

3 3 . 4

5 1 .3 4

1 7 1 .7 8

1 5 5 .4 5

1 5 5 .8 1

1 1 3 . 1

1 4 1 .1

7 . 7 3

C 1 1 H 1 5 N 5 O 3E x a c t M a s s : 2 6 5 , 1 2

M o l . W t . : 2 6 5 , 2 7

--------------------------------------------- Yield : 7.5 mg

0,0 10,0 20,0 30,0 40,0 50,0 60,0-20

50

100

150

200 ms050503 #13 et18f UV_VIS_1mAU

min

1 - 0,1882 - 0,5153 - 0,5984 - 0,9645 - 1,0576 - 1,1257 - 1,2148 - 1,367

9 - 4,528

10 - 9,83811 - 12,692

12 - 15,50613 - 24,164 14 - 30,573

15 - 30,910

16 - 47,606

WVL:235 nm Peak #9 4.68

-10,0

25,0

50,0

70,0

200 300 400 595

%

nm

210.4

289.7

560.3


[2M+1]+ [M-side chain]+

[M+1]+

[M-OMe]+H+

[M-(OMe+NCO)]+H+

Results

239

Compound 36, has the molecular formula C11H15N5O3 based on HRESIMS-TOF

(measured, m/z 266.123 [M+H]) with 7 degrees of unsaturation. The UV spectrum of 36

showed UV λmax(MeOH) absorption at 210 and 290 nm indicating the same chromophoric

functionalities as 34 and 35. The difference in molecular weight between 34 and 36 was only

32 mass units indicating a loss of C3H6 group from the alkoxy group as evident from its 1H

NMR spectrum. ESI/MS spectrum showed pseudomolecular ion peak at m/z 266.1 (M+H),

531 (2M+H) and 796 (3M+H). Tandem MS fragmentation spectrum showed a retro Diels-

Alder fragment ion peak at m/z 192 which confirmed a loss of OCN group indicating 2-

oxopurine derivative (Cook et al 1980, Mitchell et al 1997, Lin et al 1996), and also

exhibited a molecular ion fragment at m/z 234 indicating the loss of a methoxy group.

The 1H NMR spectrum of 36 ( see figure 3.5.63) showed four singlets at δ 7.57 (8-

CH), 3.60 (N(3)-CH3), 3.92 (N(9)-CH3), and 3.61 (O-CH3 ), in addition to a triplet signal at δ

2.58 for the α-CH2 of the propionic acid moiety, multiplet at 3.55 ppm for the β-CH2 of the

propionic acid and an exchangeable triplet signal at 7.73 ppm for 6-NH. DEPT experiment of

36 deduced a downfield methyl carbon signal at 51.34 ppm and absence of the upfieled

methyl carbon signals which were established for 34 and 35. The attachment of the methyl

group directly to the carboxyl of propionic acid part was confirmed through HMBC

correlation between the methyl proton signal and the carboxyl resonance at 171.78 ppm.

Additional HMBC correlations were assigned in figure 3.5.66 and presented in table 3.5.7.

Compound 36 was then elucidated as methyl-(3,9-dihydro-3,9-dimethyl-2-oxo-2H-purin-6-

ylamino) propanoate and was named nigricine 3.


0.8

08

7

0.8

45

1

2.7

43

4

5.3

49

7

2.2

17

3

2.0

00

0

38

7

38

6

38

5

37

9

19

4

17

9

17

9

17

8

17

7

17

7

13

0

13

0

12

9

(ppm)1.21.62.02.42.83.23.64.04.44.85.25.66.06.46.87.27.68.0

a

b c

d e

f NH

d

a

bc

e

fN

N

N

N

NH

O

O

O

3.92

7.57

3.60

3.55

2.58

3.61

29.84

33.33

138.27

35.6

33.4

51.34

171.78

155.45

155.81

113.1

141.1

7.73

Results

240



(ppm) 8.00 7.00 6.00 5.00 4.00 3.00

8.0

7.2

6.4

5.6

4.8

4.0

3.2

2.4

(ppm)

b

a&c d

e

f

d a

b c

f e

d

a

bc

e

fN

N

N

N

NH

O

O

O

3.92

7.57

3.60

3.55

2.58

3.61

29.84

33.33

138.27

35.6

33.4

51.34

171.78

155.45

155.81

113.1

141.1

7.73

171.

155.

155.

141.

138.

113.

51.3

35.6

33.3

33.3

29.8

(ppm)

0102030405060708090100110120130140150160170180

b c d e

f

a

b c

d e f a

141.1 113.1 171.78

d

a

bc

e

fN

N

N

N

NH

O

O

O

3.92

7.57

3.60

3.55

2.58

3.61

29.84

33.33

138.27

35.6

33.4

51.34

171.78

155.45

155.81

113.1

141.1

7.73

Results

241


Table 3.5.7. NMR Data of compound 36 (Ashourine 3) [500 MHz]

Position 13C δ ,m

(DMSO-d6)

1H δ,m, j(Hz)

(DMSO-d6)

HMBC

2 155.81 -

N(3)-CH3 29.84 3.60 (s) C-2, C-4

4 141.1 -

5 113.1 -

6 155.45 -

6-NH - 7.73 (t,5.36) C-6, C-5, C-10

8 138.27 7.57 (s) C-4, C-5, N(9)-CH3, C-6

N(9)-CH3 33.33 3.93 (s) C-8, C-4

10 35.6 3.57

(dt,6.94&6.0)

C-6, C-12, C-11

11 33.4 2.58 (t,6.94) C-12, C-10

12 171.78 -

13 51.34 3.91 (s) C-12

(ppm) 7.2 6.4 5.6 4.8 4.0 3.2 2.4

160

120

80

40

(ppm)

b c

d

f

e a

e&b c d

f

a d

a

bc

e

fN

N

N

N

NH

O

O

O

3.92

7.57

3.60

3.55

2.58

3.61

29.84

33.33

138.27

35.6

33.4

51.34

171.78

155.45

155.81

113.1

141.1

7.73

NH

Results

242

3.5.16 – Nigricine 4 (37, new compounds)


Compound 37, was assigned molecular formula C12H19N5O3 based on ESIMS analysis

with 6 degrees of unsaturation. ESI/MS spectrum showed positive pseudomolecular ion peaks

at m/z 282.1 (M+H), 280 (M-H) and 563 (2M+H). Similar to the previous congeners, MS fragmentation showed a fragment ion peak at m/z 250.2, [M-(OMe)]+ and a characteristic

0,0 10,0 20,0 30,0 40,0 50,0 60,0-10,0

0,0

12,5

25,0

37,5

50,0

70,0 ms050503 #12 et18e UV_VIS_1mAU

min

1 - Peak 1 - 0,0632 - 0,3753 - 0,592

4 - 1,1185 - 1,210

6 - 1,341

7 - 5,034

8 - 47,523

WVL:235 nm

Peak #7 5.09

-10,0

70,0

200 400 595

%

nm

214.1 315.6



N

N

N

N

NH

O

O

O

3.45

3.58, 3.62

3.62

2.6

6.88


Mol. Wt.: 281,31

3.58

3.58

2.55

------------------------------------------------- Yield : 1.1 mg

[2M+1]+

[M-side chain]+

[M+1]+

[M-OMe]+H+

[M-(OMe+NCO)]+H+

Results

243

fragment ion peak at m/z 208, [M-(OMe+NCO)]+ which confirmed the loss of OCN group

indicating 2-oxopurine derivative (Cook et al 1980, Mitchell et al 1997, Lin et al 1996), and

also exhibited a fragment ion peak at m/z 196.2 (24 %) indicating the loss of the side chain.

This fragmentation pattern is typical as those of 36 (nigricine 3), [figures 3.5.62 & 3.5.68]

with the exception of a loss of one double bond and addition of one methyl group at position

7.

The UV spectrum of 37 showed bands at λmax (MeOH) 214 and 315 nm indicating the same chromophoric functionalities as 34, 35, and 36. The difference in molecular weight

between 36 and 37 was 16 mass units. The additional substituent was deduced to be away

from the alkoxy group, because the methoxy group is still present at δ 3.58 as evident from

its 1H NMR spectra. The additional methyl singlet at δ 2.55 indicated an additional N-methyl

function [N(7)-CH3]. Disappearence of the sharp singlet at 7.57 ppm and presence of two

coupled protons at 3.58 and 3.62 ppm indicated the saturation at position 8. The presence of a

triplet signal at 6.88 ppm (J=5.7Hz) ensures the presence of NH which showed a COSY

correlation to adjacent ethylenes at δ 3.58 (2H, m) and δ 2.6 ( 2H, t, J= 6.6 Hz). The NH

signal was shifted upfield indicating the shielding effect of the new methyl group at position

7. From the above NMR data, MS fragmentation pattern and other spectral data, compound

37 was elucidated as methyl-(3,7,8,9-tetrahydro-3,7,9-trimethyl-2-oxo-2H-purin-6-ylamino)

propanoate and assigned the trivial name nigricine 4.

Fig (3.5.69) total 1HNMR spectrum of compound 37

0.91

3134

39.2

5

(ppm)6.90

0.91

31

3.66

18

5.72

34

2.81

98

2.64

66

Inte

gral

3439

.25

1805

.86

1785

.69

1782

.85

1779

.38

1774

.65

1767

.09

1728

.31

1305

.54

1298

.92

1292

.30

(ppm)0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0

B

C

DA

NH E

N

N

N

N

NH

O

O

O

3.45

3.58, 3.62

3.62

2.6

6.883.58

3.58

2.55

B

C

G2

D

A

F

E

G1

G2

G1

NH

Results

244

Fig (3.5.70): part of 1HNMR spectrum of compound 37

Fig (3.5.71) COSY spectrum of compound 37

N

N

N

N

NH

O

O

O

3.45

3.58, 3.62

3.62

2.6

6.883.58

3.58

2.55B

C

D A F

E

B

C

G2

D

A

F

E

G1

G2

G1

N

N

N

N

NH

O

O

O

3.45

3.58, 3.62

3.62

2.6

6.883.58

3.58

2.55

B

C

G2

D

A

F

E

G1

B/C

NH/C G1/G2

Results

245

3.5.17. Nigricinol [4-((1H-Indol-3-yl)methyl)-2-amino-5-(1H-indol-3-yl)-3H-pyrrol-3-one] (38, new compounds)

Fig (3.5.73):GC-MS spectrum of compound 38

Compound 38 was isolated as brownish white amorphous powder. It has UV

absorbance of typical indole compound at λmax 214 and 280 nm. ESI-MS showed

0,0 10,0 20,0 30,0 40,0 50,0 60,0-50

0

100

200

300

350 ms040918 #6 tm7et11f UV_VIS_1mAU

min

1 - Peak 1 - 0,1002 - 1,0443 - 1,1134 - 1,2675 - 1,3116 - 1,3927 - 1,452

8 - 18,275

9 - 19,08110 - 23,016 11 - 29,545 12 - 47,243

WVL:235 nmPeak #8 18.37

-10,0

25,0

70,0

200 400 595

%

nm

214.8

280.0

558.9


NHN

H

NO

NH2

137.9

109.0

124.5133.5

176.5

1283

119.5

119.9

122.3

112.1137.9

137.9 1125

122.3

123.6

122.0

127.232.3

111.0108.0

3.72

7.0

7.087.15

7.16

7.19

7.33

7.43

7.53

7.94

8.05

C21H16N4OExact Mass: 340,13

137.0

-------------------------------------------------------------------- Yield : 3.2 mg

Results

246

pseudomolecular ion peak m/z 339.1 [M-H] while GC-MS showed a molecular ion peak at

m/z 340 [M]+ and fragment ion peaks at m/z 284 [M-(COCNH2)]+, 207 [M-(indolyl group +

NH2)]+ and 177 [M-(3-indolylmethylene group + O + NH2)]+, suggesting the molecular

formula C14H18N2O2. 1H NMR spectrum showed characteristic spin systems for two indole

groups. The first indole group showed an ABCD spin system at δ 7.55 (d, J=7.56 Hz), 7.00

(dt, J=1.2,6.9 Hz), 7.08 (dt, J=1.2, 6.9 Hz), 7.33 (d, J=8.2 Hz) for H-4', H-5', H-6', and H-7',

respectively. Furthermore, the H-2' resonance at δ 7.15 (s) exhibited a COSY correlation to

an exchangeable NH signal at 10.9 ppm. Another indole group showed an ABCD spin system

as shown by 1H signals at 8.05 (dd, J=6.3, 1.2 Hz), 7.16 (dt, J=1.2, 6.9 Hz), 7.19 (dt, J=1.2,

6.9 Hz), 7.43 (dd, J=6.9, 1.2 Hz) for H-4", H-5", H-6", and H-7", respectively. The H-2''

signal at δ 7.94 (s) exhibited a COSY correlation to another exchangeable NH resonance at

11.8 ppm. The first 3-indolyl group is attached to a methylene group resonating at δ 3.72 (s)

which displays an HMBC correlations to carbons at δ124.5, 109.0, and 128.0 of C-2', C-3',

and C-3a', respectively. The methylene protons also exhibited an additional HMBC

correlations with two carbons resonating at 176.5 ppm (C-3) and 110.0 ppm (C-4). The H-2"

methine singlet showed HMBC correlations to carbons resonating at δ 108.0, 127.2, and

137.9 for C-3", C-3a", and C-7a", respectively. The NMR data and mass fragmentation

pattern confirmed 38 to be 4-((1H-indol-3-yl)methyl)-2-amino-5-(1H-indol-3-yl)-3H-pyrrol-

3-one and was assigned as nigricinol.

Fig (3.5.74):total 1HNMR spectrum of compound 38 (up) and part of 1HNMR showing

an aromatic region (down).

1.0

00

0

0.8

26

6

0.9

88

8

1.0

87

0

0.9

92

3

3.4

50

5

1.2

03

1

0.9

77

6

In

te

gra

l

40

31

.9

3

40

30

.6

7

40

25

.6

3

39

67

.6

2

37

67

.1

1

37

59

.5

5

37

16

.6

7

37

15

.4

1

37

08

.4

7

36

66

.8

6

36

58

.6

6

36

02

.5

4

36

01

.2

8

35

95

.6

1

35

94

.3

5

35

88

.0

4

35

86

.1

5

35

79

.8

5

35

78

.5

8

35

76

.6

9

35

72

.9

1

35

48

.9

5

35

47

.6

9

35

40

.7

5

35

33

.8

2

35

32

.5

6

35

06

.7

0

35

05

.4

4

34

98

.5

1

34

91

.5

7

34

90

.3

1

(ppm)

6.806.856.906.957.007.057.107.157.207.257.307.357.407.457.507.557.607.657.707.757.807.857.907.958.008.058.108.158.208.258.30

*NEP*S*S

7’ 5’ 4’ 6’ 5”

4” 7”

2” 6”

1.0000

0.8266

0.9888

1.0870

0.9923

3.4505

1.2031

0.9776

2.4768

In

teg

ral

4031.93

4030.67

4025.63

3967.62

3767.11

3759.55

3716.67

3715.41

3708.47

3666.86

3658.66

3602.54

3601.28

3595.61

3594.35

3588.04

3586.15

3579.85

3578.58

3576.69

3572.91

3548.95

3547.69

3540.75

3533.82

3532.56

3506.70

3505.44

3498.51

3491.57

3490.31

1856.61

(ppm)

0.81.21.62.02.42.83.23.64.04.44.85.25.66.06.46.87.27.68.08.48.89.29.610.010.410.811.2

8’

2’

12

3

4 5

2'

3'4'5'

6'

7'

3a'

7a' 1''

2''

3''4''

5''

6''

7''

3a''

7a''NHN

H

NO

NH2

8'

Results

247

Fig (3.5.75):Part of COSY spectrum of compound 38

Fig (3.5.76):Total HMBC spectrum of compound 38

12

3

4 5

1'

2'

3'4'5'

6'

7'

3a'

7a' 1''

2''

3''4''

5''

6''

7''

3a''

7a''NHN

H

NO

NH2

8'

(ppm) 8 0 7 2 6 4 5 6 4 8 4 0 3 2

200

160

120

80

40

(ppm)

2'

4'6',5'7'

8'6'',5''2''

4'' 7''

H-8'/C-3' & C-4

H-8'/C-3a',C-2

H-8'/C-3

(ppm) 8.0 7.6 7.2 6.8

8.4

8.0

7.6

7.2

6.8

(ppm)

7’ 5’ 4’ 6’ 5” 4” 7” 2” 6” 2’

12

3

4 5

1'

2'

3'4'5'

6'

7'

3a'

7a' 1''

2''

3''4''

5''

6''

7''

3a''

7a''NHN

H

NO

NH2

8'

Results

248


Fig (3.5.78):1HNMR spectrum of compound 38 (measured in DMSO-d6)

(ppm) 8.00 7.80 7.60 7.40 7.20 7.00

136

128

120

112

(ppm)

H-2"/C-3" H-2'/C-3'

H-7'/C-5'

H-5'/C-7'

H-5"/C-7"

H-2"/C-7a"

2'

4' 5'

6' 7' 2''

4''

5'' 6''

7''

H-6'/C-4'

H-4'/C-7a'

H-6"/C-4"

H-6'/C-7a'

H-7"/C-5"

H-5'/C-3a'

H-4"/C-7a"

H-2'/C-7a'

H-4"/C-6"

H-2"/C-3a"

H-2'/C-3a'

H-5"/C-3a"

H-4'/C-6'

H-7'/C-3a'

0.97

06

0.57

24

2.21

51

1.19

69

1.28

27

1.16

10

1.03

57

3.29

53

1.25

23

1.00

00

1.63

45

Inte

gral

5895

.47

5444

.01

3997

.90

3990

.96

3989

.07

3744

.11

3736

.23

3726

.77

3719

.20

3677

.90

3671

.28

3663

.40

3605

.39

3591

.20

3583

.95

3576

.07

3574

.81

3567

.24

3560

.94

3536

.98

3529

.73

3521

.85

3490

.64

3483

.07

3475

.82

1809

.96

(ppm)2.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.512.012.513.013.514.014.5

1.19

69

1.28

27

1.16

10

1.03

57

3.29

53

1.25

23

1.00

00

Inte

gral

3744

.11

3736

.23

3726

.77

3719

.20

3677

.90

3671

.28

3663

.40

3605

.39

3591

.20

3583

.95

3576

.07

3574

.81

3567

.24

3560

.94

3536

.98

3529

.73

3521

.85

3490

.64

3483

.07

3475

.82

(ppm)7.007.107.207.307.407.50

2'

NH2

4' 5' 6'

7'

8'

1' 1''

2''

4''

5'' 6''

7'' 1

23

4 5

1'

2'

3'4'5'

6'

7'

3a'

7a' 1''

2''

3''4''

5''

6''

7''

3a''

7a''NHN

H

NO

NH2

8'

Results

249


(ppm) 8.0 7.6 7.2 6.8

136

128

120

112

104

(ppm)

H-2"/C-3"

H-2'/C-3'

H-7'/C-5'

H-5'/C-7'

H-5"/C-7"

H-2"/C-2” direct

2'

4' 5' 6' 7'

2''

4''

5'' 6'' 7''

H-6'/C-4' H-6"/C-4"

H-6'/C-7a'

H-7"/C-5"

H-5'/C-3a'

H-4"/C-4” direct

H-4"/C-6"

H-2"/C-3a"

H-2'/C-3a'

H-5"/C-3a"

H-7'/C-3a'

NH2/C-5

H-2'/C-7a'

H-7"/C-3a"

H-6"/C-7a"

NH2/C-4

H-2"/C-7a"

NHN

H

NO

NH2

136.5

107.5

124.5132.5

173.4

127.5118.5

118.5

121.3

111.5136.3

136.1 1125

121.2

122.5

122.5

126.0108.5106.5

3.62

6.97

7.057.21

7.15

7.14

7.33

7.44

7.48

7.98

7.99

C21H16N4OExact Mass: 340,13

136.5

10.911.8

7.16

Results

250

Table (3.5.9): NMR data of 38 (500 MHz, Methanol-d4),

Carbon 13C NMR,

ppm

1H NMR, ppm

(Multiplicity, J= Hz)

1H NMR*, ppm

(Multiplicity, J= Hz)

2 137.0 (NH2), 7.15 br s

3 176.5

4 111.0

5 137.9

1' 10.9 (s)

2' 124.5 d 7.15 (s) 7.15 (s)

3' 109.0 s

3a' 128.3 s

4' 119.5 d 7.55 (d, J=7.56) 7.48 (d, J=7.9)

5' 119.9 d 7.00 (dt, J=1.2,6.9) 6.97 (t, J=8.2)

6' 122.3 d 7.08 (dt, J=1.2, 6.9) 7.05 (t, J=7.9)

7' 112.0 d 7.33 (d, J=8.2) 7.33 (d, J=7.8)

7a' 137.9 s

8' 32.3 t 3.72 (s) 3.62 (s)

1" 11.8 (s)

2" 133.5 d 7.94 (s) 7.98 (s)

3" 108.0 s

3a" 127.2 s

4" 122.0 d 8.05 (dd, J=6.3, 1.2) 7.99 (dd, J=7.00)

5" 122.3 d 7.16 (dt, J=1.2, 6.9) 7.15 (t, J=7.3)

6" 123.6 d 7.19 (dt, J=1.2, 6.9) 7.14 (t, J=7.00)

7" 112.5 d 7.43 (dd, J=6.9, 1.2) 7.44 (dd, J=7.5)

7a" 137.9 s

* meassured in DMSO-d6

Biological activity :

None of the isolated compounds from Petrosia nigricans showed significant

antimicrobial activity against B.subtilis, S. cerevisae, C. herbarum, and C. cucumerinum.

All purine derivatives (nigricines 1 to 4) showed no cytotoxic activity against L5178Y

cell line, while nigricinol (compound 38) showed 68.1 % cytotoxic activity at a concentration

of 10 µg/ml.

Results

251

3-6- Natural Products from Callyspongia biru Sponges belonging to the genus Callyspongia are extensively investigated for

bioactive natural products. Many new bioactive natural products have been isolated from

different Callyspongia sp., these natural products includes unusual steroids (Theobald et al

1978, Agrawal and Garg 1997); acetylene derivatives ( Youssef et al 2003, Nakao et al 2002

and Youssef et al 2000); uncommon fatty acids (Carballeira and Pagan 2001, Toth and

Schmitz 1994); diterpenes (Garg and Agrawal 1995) and triterpenes (Fukami et al 1997).

Cyclic peptides have also been described from the genus Callyspongia which includes

phoriospongins A and B from C. bilamellata (Capon et al 2002), callynormine A from C.

abnormis (Berer et al 2004) and callyaerins from C. aerizusa (Min, et al., 2001). Many

natural product-producing fungi have been isolated from some Callyspongia sp., and

cultivated on artificial media for production of natural products [spiciferone derivatives,

macrolides, anthraquinones, and benzofuran have been isolated from C. aerizusa–derived

fungi (Edrada et al 2000, Jadulco et al 2001, Jadulco et al 2002)]. The most attractive

compound, that was isolated from Callyspongia sp., is callystatin A. Callystatin A is a potant

cytotoxic polyketide isolated from Nagasakian marine sponge, Callyspongia truncata

(Kobayashi et al 1997a), and due to the biological importance many studies have been carried

out to synthesize this natural product (Murakami et al 1997, Lautens and Stammers 2002,

Enders et al 2002, Vicario et al 2002).

Methanol extract of an Indonesian sponge Callyspongia biru was investigated in the

present study and five compounds were elucidated. These compounds includes indole-3-

carbaldehyde, p- hydroxyphenylacetic acid, p-hydroxyphenylacetic acid methylester, which

have been described earlier (see compounds 15, 26, and 27) in the present study, in addition

to indole-3-acetic acid, and 2`-deoxythymidine.

Results

252

3.6.1- Indole-3-acetic acid (39, known compound)

0,0 10,0 20,0 30,0 40,0 50,0 60,0-100

200

400

600

900 ms050524 #5 58etj3 UV_VIS_1mAU

min

1 - 0,1852 - 0,4323 - 0,9614 - 1,0465 - 1,1146 - 1,2367 - 1,3098 - 1,390 9 - 17,52210 - 18,190

11 - 19,246

12 - 20,077 13 - 32,831 14 - 47,714

WVL:235 nm Peak #11 19.21

-10,0

70,0

200 400 595

%

nm

224.6

279.6286.6


Fig (3.6.1) HPLC chromatogram and UV spectrum of compound 39

M-1

2M+1

2M-1

3M-1

M+1 M-COOH

Fig (3.6.2) ESI-MS spectrum of compound 39

NH

COOH

6.94

7.05

7.20

7.32

7.48

10.8

3.12

C10H9NO2Mol. Wt.: 175,18

---------------------------------------------------------- Yield : 2.5 mg

Results

253

Compound 39, [indole-3-acetic acid] was isolated as yellowish white amorphous

powder. Compound 39 shows pseudomolecular ion peak at m/z 176 [M+H]+ and in the

negative mode at m/z 174 [M-H], 349 [2M-H], and 524 [3M-H] suggesting the molecular

formula C10H9NO2. The UV λmax (MeOH) absorption of 39 was at 224, 279, 286 nm and

were indicative of an indole chromophore. The 1H NMR (measured in DMSO-d6) showed 7

proton resonances that included an aromatic ABCD spin system at δ 7.48 (1H, d, J = 7.5 Hz,

H-4), 6.94 (1H, t, J = 7.3 Hz, H-5), 7.05 (1H, t, J = 7.4 Hz, H-6), 7.32 (1H, d, J = 8.2 Hz, H-

7). In addition to three proton resonances at δ 7.20 (1H, s , H-2), a most downfield broad

singlet at δ 10.8 (1H, s , H-1) and singlet proton signal at δ 3.12 (2H, s , CH2-8) were also

observed. The NMR data and the UV absorption maxima were identical to those of indole-3-

acetic acid ( Aldrich, 1990 and Hiort, 2002).

Fig (3.6.3) 1HNMR spectrum of compound 39

1.0

00

0

1.1

96

2

1.1

53

3

1.1

93

7

1.2

37

2

1.0

45

2

2.2

45

8

In

te

gra

l

54

18

.7

9

37

47

.8

9

37

40

.3

2

36

64

.3

5

36

56

.1

5

35

95

.3

0

35

29

.4

1

35

22

.1

6

35

14

.2

8

34

80

.8

6

34

73

.6

1

34

66

.3

6

15

77

.9

3

( )1.02.03.04.05.06.07.08.09.010.011.012.0

**

N

E

P**

S

**S

1.1

96

2

1.1

53

3

1.1

93

7

1.2

37

2

1.0

45

2

37

47

.8

9

37

40

.3

2

36

64

.3

5

36

56

.1

5

35

95

.3

0

35

29

.4

1

35

22

.1

6

35

14

.2

8

34

80

.8

6

34

73

.6

1

34

66

.3

6

(ppm)7.007.107.207.307.407.50

NH

4

8 7

6 5 2

NH

COOH

12

45

6

7

8

Results

254

3.6.1- 2'-Deoxythymidine (40, Known compound)

Compound 40, [2'-deoxythymidine] was isolated as white amorphous powder. It

showed pseudomolecular ion peaks at m/z 243 [M+H], 486 [2M+H], 241 [M-H], 287

[M+formic acid-H], 483 [2M-H], 529 [2M+formic acid -H], and 725 [3M-H] suggesting the

0,0 10,0 20,0 30,0 40,0 50,0 60,0-50

0

100

200

300

350 ms050727 #2 58etl8c UV_VIS_1mAU

min

1 - 0,3292 - 0,9573 - 1,0424 - 1,1005 - 1,2266 - 1,2797 - 1,395

8 - 5,774

9 - 32,699 10 - 47,215

WVL:235 nm Peak #8 5.87

-10,0

25,0

70,0

200 300 400 595

%

nm

267.0208.8

557.0


Fig (3.6.4) HPLC chromatogram and UV spectrum of compound 43

C10H14N2O5Mol. Wt.: 242,23

O

HOH

HH

HH

HO N

NH

O

O

11.28

7.7

6.18

5.22

5.03

4.223.75

3.55

2.08

1.77

--------------------------------------------------------

Yield : 10 mg

[M-1]

[2M+1]

[2M-1]

[M+ formic acid-1]

[M+1]

[2M+ formic acid-1]

[3M-1]

[M-(CH2CHOHCHCH2OH+H)]

[M-(CHCH2OH+H)]

[M-(CHOHCHOCH2OH+H)]

[M-(deoxyribose+H)]

[M+H-(deoxyribose)]

Fig (3.6.5) ESI-MS spectrum of compound 40

Results

255

molecular formula C10H14N2O5. The UV λmax (MeOH) of 40 at 208, and 267 nm was deduced

for a nucleoside nature of the compound. Molecular ion fragmentation pattern confirmed the

presence of thymine-2‘-deoxyribose (see figure 3.6.5 ). 1H NMR spectrum showed a broad

singlet at 11.28 (1H, s, 3-NH), 7.7 (1H, s, H-6), 6.18 (1H, t, J = 7.0 Hz, H-1'), 2.08 (2H, m, H-

2'), 4.22 (1H, br s, H-3'), 3.75 (1H, dd, J = 6.6& 3.1 Hz, H-4'), 3.55 (2H, m, H-5'), 1.77 (3H, s,

H3-7). The 1H NMR data were identical with those of thymine-2‘-deoxyribose (Aldrich 1992

). From the above NMR data, mass fragmentation patterns, and UV spectrum, compound 40

was concluded to be thymine 2´-deoxyribose.

Fig (3.6.6) 1HNMR spectrum of compound 40

Biological activity:

Total methanol extract, ethylacetate- and butanol-fractions along with n.butanol

subfractions were tested for cytotoxic activity using the brine shrimp assay. The butanol

subfraction (but-V) showed strong cytotoxic activity against the brine shrimp (Artemia salina

Leach). This fraction was further chromatographed and purified to gave (But-Ve3) which

seems to be alkylpyridinium compound. Howevere the structure elucidation of the compound

is still under revision.

Deoxythymidine (compound 40) showed cytotoxic activity 76.2 % and 41.7 % against

L5178Y mouse lymphoma cell line at concentrations 3 and 10 µg/ml respectively.

0.7

653

0.9

736

0.9

741

0.8

594

0.8

270

0.9

703

1.0

000

2.0

458

1.8

595

2.8

184

Inte

gra

l

5629.3

9

3843.1

0

3084.2

6

3077.3

2

3070.3

9

2611.9

9

2608.2

1

2510.7

9

2506.3

8

2110.7

2

1876.8

0

1873.6

5

1870.1

8

1867.0

2

1795.4

6

1787.8

9

1783.4

8

1773.7

1

1769.9

2

1758.2

6

1053.0

1

1046.3

9

1039.7

7

1033.1

5

1027.1

6

1022.1

2

1018.6

5

1014.8

7

1011.4

0

1008.5

6

879.6

2

0 40 81 21 62 02 42 83 23 64 04 44 85 25 66 06 46 87 27 68 08 48 89 29 610 010 410 811 211 612 0

O

HOH

HH

HH

HO N

NH

O

O1 2

345

6

1'2'3'4'

5'

7 7

6

NH 5’-

5' 4'

3' 2'

1'

3’-

Discussion

256

IV- Discussion

The main target of the medicinal chemist is concerned about the exploration of new

effective and less toxic medicaments. However, the main objective of the pharmacognosist or

phytochemist is the presentation of this effective medicament from the natural sources. The

essential topics that the phytochemist will be dealing with through the achievement of this

task includes isolation, chracterisation, structure elucidation, and biological evaluation of the

targeted natural products (Exarchou, et al, 2005; Lambert, et al, 2005).

Concerning the above mentioned

topics many problems will be faced.

However, the employment of the new

technology in the field of natural product

chemistry solved many of these problems.

All parts of the process leading to an

elucidated structure have experienced an

immense speed-up in the past fifty years.

Separation technology, analytical and

spectroscopic methods have improved

steadily and with good fortune, a chemist

might be able to go from a crude extract to

a full set of 2D NMR spectra in one day

(Steinbeck 2004). It should be borne in

mind that the above mentioned tasks

(isolation, characterisation, structure

elucidation, and biological evaluation) are

only a part of the whole process of new

drug discovery. Scheme 1 (Giersiefen et

al., 2003), summarizes the whole drug

discovery process.

Target identification

Target validation

Lead identification

Lead optim ization

- Knock-in and knock-out anim al models

- Antisense nucleic acid and antibodies- Proteom ics- Structural biology/ structural genom ics

- High throughput screening- Natural products screening- NM R-based screening- Virtual screening

- Com binatorial chemistry- Compound library design- Structure-based design

- M edicinal chem istry- Parallel synthesis

- Design of focus compound libraries - M olecular modeling, QSAR

- in vivo pharm acology - Pharmacokinetic and toxicology

- Genom ics- Proteom ics- Bioinform atics

- Cellular and molecular biology

Preclinical and clinical developm ents

Effective Drug

Scheme (1) phase of drug discovery process

(Giersiefen et al., 2003)

Discussion

257

4-1 Isolation, characterisation, structure elucidation, and biological evaluation of natural products:

4.1.1 Isolation of natural products:

Unlike the medicinal chemist, who usually concentrates on a series of synthetic

compounds of known chemical and physical properties and hence is able to master the limited

number of separation techniques applicable to the specific chemotype, the natural product

chemist must be prepared to deal with molecules of the whole spectrum of bioactive

metabolites. These can vary in hydro- and lipophilicity, charge, solubility, and size.

(McAlpine and Hochlowski 1994). In general, the more hydrophilic metabolites may be

candidates for ion exchange chromatography, reversed phase silica gel chromtography, or size

exclusion chromatography on polysaccharide resins. The more lipophilic metabolites can be

further purified by chromatography on normal phase silica gel, florisil, alumina, or lipophilic

size exclusion resins such as sephadex LH-20. They may be also candidates for a variety of

high-speed countercurrent techniques or chromatography on polyresins (McAlpine and

Hochlowski1994). However, this branch of the main task of the natural product chemist was

advanced to large extent. The production of many new packing materials, new isolation

instruments with high degree of resolution and detection facilitated the chemist´s duty in this

branch of the task. Some problems are still unresolved, for example, separation of individual

pure compounds from mixture of cerebrosides, many trials were applied for achievement of

this goal but unfortunately, all failed. The sphingolipids (cerebrosides) are easy to be

separated as a mixture of closely similar chemical constituents from natural sources.

However, they are very difficult to be separated from each other or in another word they are

very difficult to be separated and purified to analytical purity. The difficulty arises probably

from their chromatographic properties being overshadowed by the polar nature of the

glycoside, thus making these metabolites difficult to separate to analytical purity (Jenkins

1999). Furthermore, the fact that, these metabolites are present oftenly as a series of very

similar chemical nature, where the difference in most cases is one or two methylene groups,

thus many of the published cerebrosides were reported as a groups of mixed compounds (e.g.,

Jenkins 1999& Inagaki 2003 ). However, HPLC in reversed-phase mode is now the standard

method for separation of molecular species of cerebroside mixture, often after benzoylation so

that the lipids can be detected by sensitive UV spectroscopy. Mass spectrometric methods are

now being used increasingly for charaterization purposes of such sphingolipids (Christie,

2003)

Discussion

258

4.1.2 Characterisation of natural products:

This branch of the task is the first step in structure elucidation. Therefore it is very

important for the natural product chemist, he has to spend his effort and time to drive the

structure proposal in the right direction. In contrast, the medicinal chemist, who is dealing

with the synthetic chemicals, has no need to spend his time and effort here because he knew

previously what type of chemicals he is dealing with. In the fact, there are many simple and

general chemical tests that are widely available and more applicable since a long time, for

example, Molish’s test for carbohydrates, Mayer`s and Dragendorf`s reagents for alkaloids,

alkali solution (e.g.NaOH, and KOH) for both anthraquinones and phenolic compounds.

However, these reagents were intended to dissolve a small part of the problem. The

phytochemist can not decide precisely and also confirm the structure using these simple

reagents. These reagents tells us about the general chemical class to which the isolated

compound may be related. But in most cases they give no idea about the subchemical class

rather than the smallest details of the structure. Metabolite profiling is not an easy task to

perform since natural products display a very important structural diversity. For each

compound, the order of the atoms and stereochemical orientations have to be elucidated de

novo in a complex manner and the compounds can not simply be sequenced as it is the case

for genes or proteins. Consequently a single analytical technique does not exist, which is

capable of profiling all secondary metabolites in the biological source (Wolfender, et al

2005). However, the employment of advanced analytical and spectroscopic methods like UV-

and IR- spectroscopy solved such problems to a large extent, where they can give a good idea

about the different substructures and/or functional groups of the structure. The hyphenated

techniques coupled to HPLC give not only ideas about the details of functional groups, but

also play an important role in what is called „Dereplication process“ in order to avoid the

tedious isolation of known compounds, and directed the chemist´s effort toward the targted

isolation of constituents presenting novel or unusual spectroscopic features (Wolfender, et al

2005).

In the present study, a lot of chemical compounds that share the same chromophoric functions

were examined by the hyphenated technique HPLC-UV-photodiode array detection (LC/UV-

DAD). This examination showed that, all chemicals having the same chromophoric functions

will show the same UV spectrum with the same absorption maxima even though they are

different in their additional non-chromophoric functions and their molecular weights.

Discussion

259

- Kahalalide F, (compound 1, m/z 1478), R (8, m/z 1520) and S (9, m/z1536), all having the

same chromophoric function and therefore showing the same UV spectrum and having

approximately the same absorption maximum at ~ 203 nm.

- Kahalalide E (2, m/z 836), Kahalalide D (2, m/z 596), N,N-dimethyl tryptophan

methylester. (6, m/z 246), indole-3-acetic acid (39, m/z 175), all having the same

chromophoric function (tryptophan amino acid) and therefore showing the same UV

spectrum and having approximately the same absorption maxima at ~ 220, 279 sh and 285

sh nm.

- A group of 3-acyl indole derivatives, e.g., hyrtiosine A (13, m/z 191), 5-hydroxy-1H-

indole-3-carbaldehyde (14, m/z 161), ), indole-3-carbaldehyde (15, m/z 145) and 5-

deoxyhyrtiosine A (16, m/z 175) all having the same chromophoric function (3-acyl

indole) and therefore showing the same UV spectrum and having approximately the same

absorption maxima at ~ 210, 250, 275 and 300 nm. The same UV spectrum was obtained

for the new indole derivative, isohyrtiosine A (17, m/z 191) with a little deviation in the

UV-absorption maxima. Isohyrtiosine A showed an absorption maxima at 214, 241 and

286 nm due to the presence of carboxyl group instead of a ketonic group.

- New purine derivatives, nigricines 1-3 (34, m/z 307), (35, m/z 279), (36, m/z 265), all

having the same chromophoric function (2-oxo-purine) and therefore showing the same

UV spectrum and having approximately the same absorption maxima at ~ 210 and 290

nm. The same UV spectrum was obtained for both nigricine 4 (37, m/z 281), and

adenosine (30, m/z 267), with a little deviations in the UV-absorption maxima. Ashourine

4 showed an absorption maxima at 214 and 315 nm due to N(7)-methylation and loss of

one double bond at position 8, while adenosine showed an absorption maxima at 207 and

257 nm because it has no 2-oxo- group.

- p-Hydroxyphenyl acetic acid, and its methyl-, ethyl- and butylesters, [(26, m/z 152) (27,

m/z 166) (28, m/z 180) and (29, m/z 208)] all having the same chromophoric function (4-

hydroxyphenyl) and therefore showing the same UV spectrum and having approximately

the same absorption maxima at ~ 202, 224 and 275 nm.

However, the compounds that have no chromophoric functions can not be characterised by

LC-UV hyphenated technique.

Also, NMR spectroscopy play a significant role in characterisation of natural products:

The peptide nature of isolated kahalalides (compounds 1-5, 8& 9) was suggested by:

Discussion

260

1) From 1HNMR, the presense of amide NHs resonating in the downfield region between

7.0-9.0 ppm, α-protons resonating arround 4.0 ppm and the CH3 groups resonating in the

higher field region arround 1.0 ppm.

2) From 13C NMR, The presence of amide carbonyls resonating arround 170 ppm, α- carbons

(sp3 methines) resonating between 50-60 ppm and methyl carbons in the higher region.

3) From a COSY, each amino acid could be distinguished by a sequential correlations

beginning from the amide NH, through α-, β-, γ- to the methyl protons which could be

confirmed by the total correlation spectroscopy (TOCSY).

- The cerebroside nature of compounds 32 and 33 (petrocerebrosides 1 and 2) were

established from the 1HNMR which showed a triplet-like signal at δ 0.82 (terminal

methyls) and broad singlet at δ 1.2 ( long chain (CH2)n groups for both fatty acids and

long chain bases), the presence of several doublets in the region between 4.2-5.3 ppm

indicated the presence of many OHs (of the sugar parts), the presence of multiplet signals

between 3.35 and 5.0 ppm indicated the presence of sugar CHs. In addition to one amide

NH in the lower field region rather than the sp2 methines in aromatic region between 5.0 –

9.0 ppm region. This suggestion cauld be supported by the presence of amide carbonyl

signal in 13CNMR at approximately 170.0 ppm.

- The steroidal nature of the isolated steroidal compounds and also the scalarane type

sesterterpenoids could be suggested from 1HNMR by the presence of a characteristic

methyl around 1.0 ppm and presence of overlaping methylenes and sp3 methines in the

higher field region between 1.0 and 2.0 ppm, which was supported by a characteristic 13C-

NMR spectrum.

- p-Disubstituted phenyl group could be distinguished from 1HNMR by the presence of two

doublets inter-correlated at approximately 6.8 and 7.2 ppm (especially for 4-hydroxy-1-

substituted phenyl group), for example, tyrosine-containig natural products (kahalalide B,

compound 4), and p-hydroxyphenylacetic acid and its derivatives (compounds 26, 27, 28

and 29).

- 3-Substituted-indole could be easily distinguished from 1HNMR by the presence of

ABCD spin system in the aromatic reagion (approximately between 7.00 and 8.2 ppm) for

proton resonances of H-4, H-5, H-6 and H-7, in addition to a relatively sharp singlet or in

rare cases doublet with small coupling constant of H-2 between 7.0 and 8.0 ppm.

Furthermore the sharp singlet in the down field region between 10.0 and 12.5 ppm

indicating the indole NH. The present examples are compounds 2, 3, 6, 15, 16, 38, and

39. In contrast 5-hydroxy-3-substituted indole displays an ABM spin system

Discussion

261

(approximately between 6.80 and 8.0 ppm) for proton resonances of H-4, H-6 and H-7

instead of the above ABCD spin system (see 1H NMR of compounds 13, 14 and 17).

Mass fragmentation pattern, could play a significant role in characterisation of natural

products:

- Comparison of the MS fragmentation of the isolated compound with those of the known

authentic samples may be enough not only for characterisation of substructures but also

for full structure elucidation of the compounds (e.g. compounds 27 and 31).

- MS fragmentation pattern may be helpful in characterisation of some substructures which

seem difficult to be distinguished by other means, rather than they are also helpful in

establishing the relation between the closely related natural products, for example, the

characteristic loss of 42 mass unit through retro Diels-Alder fragmentation from the new

purines, nigricines 1-4, (compounds 34-37) indicated the presence of 2-oxo-purine

derivatives and consequently the loss of NCO fragment, thus, the closed relation between

them could be established.

- MS fragmentation mechanisms (e.g. tandem ESI/MS and MALDI-TOF-PSD) are

described as the methods of choice in characterisation and confirmation of the amino acid

sequence of the isolated peptides, for example, kahalalides F, E, D, B, C, R and S

(compounds 1, 2, 3, 4, 5, 8 and 9 respectively).

4.1.3 Structure Elucidation of Natural Products:

After the characterisation of the isolated natural product and determination of the

subchemical class to which the compound is related, the phytochemist has to demonstrate

unambigiously the small details of the substructures and consequently the elucidation of the

complete structure. Structure elucidation of the isolated new natural product is still the

bottleneck of the objective achievement. Actually, the new technology in the field of NMR

spectroscopy (including 1D- and 2D- NMR experiments) and mass spectrometry facilitated

the chemist´s effort in this part of the whole task. Furthermore, the presence of commercial

and non commercial computer-assisted structure elucidation (CASE) programs even though

they are not widely available and less applicable nowadays but may be the method of choice

in the near future. It is important to say that the interaction between a spectroscopist and a

CASE system will remain important in order to generate the correct structure rapidly.

Therefore CASE will complement the skills of the spectroscopist, not replace them. The use

of CASE system is likely to increase in the near future, and this will enable the bottleneck so

often caused by structure elucidation to be removed from the natural product drug discovery

process (Jaspers 1999).

Discussion

262

4.1.4 Biological evaluation of natural products:

In general, drug discovery strategies can be trivially separated into three categories:

1. Chemically driven, finding biological activities for purified compounds.

2. Biologically driven, bioassay-guided approach begining with crude extracts.

3. Combination of chemically and biologically driven approaches (vide infra).

(McConnell et al. 1994)

Beginning in the 1970´s through today, the majority of the academic-based research

efforts has become essentially „biologically driven“ i.e., the object of the search has shifted to

discover natural products with biological activity. The biological activities include exploring

their potential as agrochemicals (Crawley 1988) and pharmaceuticals, as well as their possible

chemical ecological roles (Bakus et al. 1986; Hay and Fenical 1988).

Drug discovery in industry has evolved to the use of specific assays with target receptors

and enzymes involved in the pathogenesis of disease rather than cellular or tissue assays

(Johnson and Hertzberg 1989), and has benefitted immensely from breakthroughs in receptor

technology (Hall 1989; Reuben and Wittcoff 1989). These assays reflect new opportunities

due to the recent identification of previously unrecognized biomolecular targets for therapy

(Larson and Fischer 1989). More specifically, this approach for most disease areas is

characterized in industry by:

1. Essentially exclusive reliance on biological activity of crude extracts in numerous target-

specific assays, i.e., enzyme assays and receptor-binding assays, for selection of crude

extracts and bioassay-guided fractionation of the crude extracts (prioritization criteria

emphasize selectivity and potency).

2. High volume, automated screening, i.e., thousands of samples per year for smaller

companies and thousands per week for larger companies.

3. The use of „functional“ or whole-cell assays to confirm activity in a particular disease state

and to further prioritize samples for fractionation.

4. The use of genetically engineered microorganisms, enzymes, and receptors.

Because of low correlation between cytotoxity and antitumor activity, a number of

programs have utilized in vivo tumor models directly for drug discovery (Johnson and

Hertzberg 1989). From 1958 through 1985, National Cancer Institute (NCI) used in vivo

L1210 and P-388 murine leukemia assays as primary screens (Suffness et al. 1989; Boyd et

al. 1988) and was successful primarily in identifying compounds possessing clinical activity

against leukemias and lymphomas. Unfortunately, they were not very successful in finding

Discussion

263

compounds active against slow growing tumours in humans. Further, these in vivo assays

were expensive, time consuming, and relatively insensitive (Suffness et al. 1989). In other

disease areas it was shown that activity in in vitro antiviral assays does not translate well to in

vivo activity, e.g., using Herpes simplex. In contrast, reasonable correlations exist between in

vitro and in vivo antifungal activity, e. g., using Candida albicans.(McConnell et al., 1994).

General or non-specific cytotoxic assays could be insufficient to prove or disprove the

activity. For example, the known antitumour drug candidate (kahalalide F, compound 1)

shows no cytotoxic activity against Artemia salina using brine shrimp assay even though it

has potent antitumour activity.

Using bioactivity guided isolation, it should be borne in mind that the active substance

may be present at very low concentration in the total extract and/or subfractions otherwise

many active compounds will be rejected at an early stage because of lack of significant

activity. Firn and Jones 1996, mentioned some limitations that affect the biological

activity studies of the secondary metabolites and consequently minimize the chance of

detecting high biologically active compounds, specially for commercial use, for example:

- The high selectivity required for a biologically active substance means that many

active compounds will be rejected at an early stage because of lack of specificity.

- Some active compounds are unstable and are either lost during isolation or are

unsuitable for use .

- Some compounds have already been isolated (and possibly patented before).

- The type of biological activity found is not meaningful in terms of the particular

usage sought.

- The screening process is inappropriate.

- Some forms of biological activity are dependent on the presence of other compounds

(synergists) which are lost during the purification processes before the substance is

bioassayed.

4.1.5 Technology of HPLC:

High performance liquid chromatography (HPLC) including both normal and reversed

phases (RP) is now a well-developed and widely used technique for separation of complex

mixtures (Exarchou, et al, 2005). HPLC is now available in many forms of hyphenated

systems such as LC-UV, LC-mass spectroscopy (LC-MS, LC-MS-MS) and LC-NMR which

has been successfully and practically achieved in the last decade. (Wolfender, et al 2005, and

Exarchou, et al, 2005). With the aid of the modern HPLC-coupled spectroscopic techniques

Discussion

264

such as HPLC-UV, HPLC-MS as well as HPLC-NMR, the main constituents of plants that

are frequently used in traditional Chinese medicine have been characterized and identified. As

well as compared with reference compounds (Zschocke et al 2005; Albert 2002; Wilson, et

al 1999).

Analytical HPLC that is coupled to a UV detector is ideal for detection of most natural

products that possess chromophoric functions in their structures. However, in some cases,

HPLC-UV hyphenated systems fail to detect the important biological substances such as

purine derivatives, nucleotides and other similar small biologically active metabolites, the

presence of acidified water as a part of the mobile phase make the nitrogenous compounds

protonated (in other word, highly polar according to the affinity of that compounds to accept

protons), which in turn elute the compounds very rapidly and in some cases can not be

detected with the UV-detector). This problem prompted some analytical chemists (e.g.

Nordström et al 2004) to derivatise such basic compounds to be more hydrophobic,

consequently improve their retentions on reversed phase materials and enhance their ESI

response in the case of LC-MS analysis. The use of ortho-phosphoric acid in maintainence of

the mobile phase-pH around 2 give a good compound-separation, allowing the UV-detector to

impart sharp peaks with high resolution, but it is not compatible with all possible analytes. In

some cases of poly-nitrogenous compounds (new purine derivatives and known compound

adenosine in the present study), phosphorylated H2O (pH 2) rendered the compounds very

polar, providing very short retention time and consequently masking their detection by UV

detector. The following example will explain the effect of o-phosphoric acid in masking peaks

of purine derivatives during HPLC analysis.

From the above example, it was clear that, low concentrated analyte couldn`t be

detected by elution of the sample with eluant composed of pH 2 aqueous solution as in fig

4.1.A. In contrast, the same sample concentration could be easily detected (fig 4.1.B) through

elution with mobile phase composed of neutral H2O (free of phosphoric acid). Furthermore,

high concentrated sample (fig 4.1.C) provides a broad peak with relatively low resolution and

short retention time by elution with mobile phase that composed of pH 2 water. while sharp

peak with high resolution separation and relatively delayed retention time could be obtained

through elution with mobile phase composed of neutral water (fig 4.1.D).

Discussion

265

This problem during analysis by HPLC hyphenated systems confirmed that some of

the important bioactive natural products may be undetected even though they have adequate

chromophoric functionalities especially when present at low concentrations (as the case

during an early stage of their isolation from biological sources).

The same problem would be faced during LC-MS analysis of such compounds. Two

major problems are associated with LC-MS analysis. First, no universal LC column packing

material can be used for all possible kinds of analytes. Second, no eluent system is compatible

with both all possible analytes and ESI. A compromise has to be made at some level

0,0 10,0 20,0 30,0 40,0 50,0 60,0-200

500

1.000

1.500

2.000 ms050506 # et17f UV_VIS_mAU

min

1 - 0,4722 - 0,7953 - 0,9774 - 1,1175 - 1,2366 - 1,345 7 - 15,490

8 - 16,660

9 - 17,84910 - 18,311 11 - 32,50312 - 36,766 13 - 47,629

WVL:235 nm

Peak #8 16.58

-10,0

70,0

200 400 595

%

nm

228.3

210.1281.3


0,0 10,0 20,0 30,0 40,0 50,0 60,0-50

0

100

200

300

400 ms050503 # et17f UV_VIS_mAU

min

1 - 0,0792 - 0,5843 - 0,7274 - 0,9575 - 1,1176 - 1,205

7 - 8,892

8 - 15,756 9 - 24,17710 - 25,28111 - 30,56912 - 30,904

13 - 47,630

WVL:235 nm

Peak #7 8.54

-10,0

25,0

50,0

70,0

200 400 595

%

nm

209.9

289.7

560.5

C D

0,0 10,0 20,0 30,0 40,0 50,0 60,0-50

0

50

100

150

200

250

300 ms050506 #15 et18h UV_VIS_mAU

min

1 - Peak 1 - 0,0462 - 0,4593 - 0,5974 - 0,7785 - 1,1086 - 1,2377 - 1,360 8 - 14,6739 - 15,412

10 - 16,822

11 - 47,527

WVL:235 nm

0,0 10,0 20,0 30,0 40,0 50,0 60,0-10

20

40

60

80

110 ms050503 #15 et18h UV_VIS_1mAU

min

1 - 0,7582 - 0,950

3 - 1,088

4 - 1,117

5 - 1,2106 - 1,2587 - 1,318

8 - 24,1559 - 25,234

10 - 29,507

11 - 30,42612 - 30,899

13 - 31,75014 - 32,109

15 - 32,88216 - 33,737

17 - 34,093

18 - 35,397

19 - 36,233

20 - 36,71521 - 37,16522 - 37,67823 - 38,21324 - 38,651

WVL:235 nm

Peak #10 16.90

-10,0

70,0

200 300 400 500 595

%

nm

228.1

209.0281.3


9.25

-500

1.250

2.500

4.000

200 400 595

%

nm

344.7 530.3551.7


A B

Fig(4.1) different HPLC chromatograms of a new purine derivative (compound 35,

ashourine 2); A: diluted solution, the chromatogram was obtained after running in HPLC

using acidic aqueous solvent (pH 2); B: the same sample of A, the chromatogram was

obtained after running in HPLC using neutral water (pH 7); C: concentrated solution, the

chromatogram was obtained after running in HPLC using acidic aqueous solvent (pH 2);

D: the same sample of C, the chromatogram was obtained after running in HPLC using

neutral water (pH 7).

Discussion

266

regarding the packing material, eluent system, or analyte response. Positive ESI is an

excellent interface for reversed-phase chromatography. The solvents used, acidic

acetonitrile/MeOH-H2O mixtures, are suitable for the desolvation process. However, many

biologically important compounds do not separate readily on reversed-phase packing material

due to their high polarity. The need for acetate or formate buffers or ion pairing reagents often

hampers the ESI response (Nordström et al 2004). During the LC-MS analysis of the new

purine derivatives (compounds nigricines 1-4), adenosine (compound 30) and nicotinamide

(compound 31), it was noted that, only the more hydrophobic compound, nigricine 1, could

be measured by LC-MS while the sensitivity was decreased as the side chain (alkoxy group)

was decreased. Adenosine and nicotinamide couldn`t be analysed in the same condition.

Hyphenated HPLC systems play a significant role in comparison the components of

biological extracts or mixtures of substances and a previously known compounds. Thus, the

tedious isolation of known compounds can be avoided and a targeted isolation of constituents

presenting novel or unusual spectroscopic features can be undertaken. (Wolfender, et al

2005).

Furthermore, HPLC-UV-MS hyphenated system is the method of choice for detection

the absolute configuration of amino acids and thus helps in the unambiguous structure

elucidation of new peptides not only by indication of certain amino acids of which the peptide

composed but also by detection and confirmation of stereochemistry of the amino acid. HPLC

analysis of the kahalalide hydrolysates after derivatisation by Marfey`s reagent, N-(5-flouro-

2,4-dinitrophenyl)-L-alanine amide (FDAA), and comparison of their retention times and

molecular weights with those of authentic diastereoisomer amino acids which were treated in

the same manner confirmed the stereochemistry of the kahalalide amino acids (see figures

3.1.11-3.1.15). Due to some of an expected negative effects of factors such as concentration

of the sample, or minute changes in the solvent gradient or in the pH of the mobile phase

that could affect the retention times, peak enrichment technique was applied to give

unambiguous streochemistry determinations. This procedure was made by co-elution of the

kahalalide hydrolysates together with the expected standard amino acids at the same time.

Complete overlapping of both standard and test amino acid peaks indicated complete

stereoidentity (see figures 3.1.11).

LC-ESI/MS/MS hyphenated systems supported online amino acid sequencing of a

mixture of peptides without separation and purification which in turn avoid the cost of

Discussion

267

expensive adsorbents and eluents, and save the time and effort that may be lost during

isolation of known peptides, and for cheking whether a compound that is intended to be

isolated is a genuine new peptide (Bringmann and Lang 2003). The same advantages could

be obtained by online LC-NMR analysis (Wolfender, et al 2005).

4.2. Isolated compounds from genus Elysia ( sacoglossan mollusc):

Genus Elysia is known as a rich source of bioactive natural products such as

diterpenoids (Paul and Van Alstyne 1988) polypropionates (Gavagnin, et al 1994, Cueto et

al 2005) and depsipeptides (Hamann and Scheuer 1993, Hamann et al 1996, Goetz et al 1997,

Horgen et al 2000, Dmitrenok et al 2006). Elysia-derived depsipeptides known as kahalalides

consist of 3 to 13 amino acid units, ranging from a C31 tripeptide to a C75 tridecapeptide, with

cyclic and linear components, the latter terminating in a saturated fatty acid moiety. Ten of

these derivatives are cyclic depsipeptides, kahalalides A to F, K, O, P,and Q while three

analogues, kahalalides G, H, and J are linear peptides. Kahalalide F and its linear analogue

kahalalide G as well as the two new kahalalides R and S are the only congeners that feature

the unusual amino acid, Z-dehydroaminobutyric acid (Z-Dhb). The methanol extract of Elysia

grandifolia showed a promising biological activity such as antimicrobial, cytotoxicity,

feeding deterrence and icthyotoxicity (Padmakumar 1998, Bhosale et al, 1999). These

bioactivities attracted our attention to investigate the methanol extract of the mollusc. The

investigation which was guided by online LC-MS analysis resulted in the detection of many

known kahalalides [kahalalides B, C, D, E, F, G, J, K, O (see figures 2-15 and 2-16)] with

regard to their molecular weights and amino acid sequences through interpretation of ESI-MS

fragmentation patterns. The presence of two unidentified peaks at m/z 1520.2 and 1536.0

aroused our interest to do further chemical work on the mollusc extract. A steady continuous

effort led to the isolation characterisation, and full structure elucidation of two new

depsipeptides, kahalalides R and S.

Discussion

268

4.2.1 Which of the following organism is actually responsible for kahalalides

production? The animal sacoglossan mollusc (Elysia sp.), the plant Bryopsis sp (algal diet

of the mollusc), or the procaryotic symbiont bacterium (Vibrio sp.) ?

The green algal diet, Bryopsis sp., has been found to yield kahalalides A, B, F, G, K,

P, and Q. Kahalalides A-K were isolated from the molluscan E. rufescens, while kahalalide O

was isolated from E. ornata. In the present study, the corresponding molecular ion peaks of

kahalalides B, C, D, E, F, G, J, K, O as well as the new congeners R and S have been

characterized from online LC-ESI-MS analysis of the methanol extract. Furthermore ,

kahalalide F was produced by a symbiont bacterium Vibrio sp. associated with both the

specialist herbivore mollusc Elysia sp and their algal diet Bryopsis sp (Ashour et al 2006).

The existence of the kahalalides in the extracts of these taxonomic unrelated groups

of organisms (animal, plant and procaryote) led the question: which of these organisms is

actually responsible for kahalalides production?

- kahalalides, especially the major one (kahalalide F) play an important ecological role,

since it was reported that they show significant antipredatory effects against fish

predation (Becerro et al 2001). Thus, these compounds increase the fitness of the

mollusc even though they play no role in primary metabolism.

Vibrio sp.

Bryopsis pennataElysia sp.

NH

OCH3

OHN

H3C

H3C

OO

CH3HN

O

CH3HN

OCH3

CH3

CH3

O NH

HN

ONH

CH3H3C

O

H2N

NH

O

NH3C

H3CHN

H3CH3C HN

O

HO

H3C

NHO

H3CNH

O CH3

CH3

O

O

H3C

H

Kahalalides

?

? ?

Fig (4.2) Sources for kahalalides production.

Discussion

269

- Hamann and Scheuer 1993, stated that the herbivorous sacoglossan mollusc E.

rufescens has the ability to sequester from their algal diet „Bryopsis sp“ functioning

chloroblasts, which then perhaps may participate in the biosynthesis of secondary

metabolites (Hamann and Scheuer 1993).

- It was hypothesized that E. rufescens acquires Kahalalide F–producing Vibrio from

the surface of the algal diet Bryopsis sp and maintain these microbes as symbionts

(Ashour et al 2006).

- According to the natural product definitions (Bennett 1995 and Firn 2004) it is

considered that kahalalides are actually secondary metabolites or true natural products

since they are not active participants in primary metabolism and have no function in

growth. Consequently their production is restricted to certain closely related

taxonomic groups of living organisms. Therefore it is not accepted that the above

mentioned organisms of completely different taxonomic groups (animal, plant and

procaryotic members) are responsible for production of one chemical class of closely

related secondary metabolites.

This discussion led to the following conclusion:

1- Kahalalides may be procaryotic products, produced by Vibrio bacterium and

accomulated in both algae and mollusc. (the most likely scenario, but needs further

confirmation).

2- Kahalalides may be plant products, since their production by Vibrio sp is still

unconfirmed (there is no true publication that describe the natural production of the

kahalalides by procaryotic organisms)

3- Kahalalides may be procaryotic products, produced by Vibrio bacterium, and their

production by plant, Bryopsis sp, may be attributed to a possible spontaneous

transplantation of a responsible gene from the bacterium to the plant

4- It is not accepted that kahalalides are actually animal products because of their

production by another completely unrelated organisms, and the presence of

kahalalides in the animal extract may be attributed to accomulation of the secondary

metabolites by contineous intake of the algal diet Bryopsis sp.

5- It is possible that kahalalides are plant or bacterial products liable to modification

through the molluscan metabolism.

6- The isolation of β-sitosterol (compound 7) and a previously known plant secondary

metabolite (N,N-dimethyltryptophan methylester, compound 6) from the animal

extract may confirm the plant responsibility for the production of the kahalalides.

Discussion

270

However, this interesting point needs further study and explanation from the biosynthetically

oriented chemists.

4.2.2 The possible biosynthetic pathway of kahalalides:

In spite of the long debating discussion about the producing organisms, the

kahalalides are considered as non-ribosomal depsipeptides (Marahiel et al 1997). The possible

biosynthetic pathway of kahalalides (as depispeptide natural products) was obtained from the

references (Marahiel et al 1997 and Schwarzer et al 2003). The kahalalide biosynthesis may

be supported by large multifunctional enzymes called non-ribosomal peptide synthetases. The

depsipeptides are assembled from diverse groups of precursors including pseudo-, non

proteinogenic, hydroxy, N-methylated, and D-amino acids. These peptides then undergo

further modifications that lead to additional structural diversity. A typical enzyme module -

concerning the depsipeptide biosynthesis – consists mainly of three steps as shown in figure

4.2

a) Adenylation domain (A): responsible for amino acid activation .

b) Thiolation domain (T) or peptide carrier protein (PCP): responsible for thioestrification of

the activated amino acids by the attachment to the cofactor, phosphopantotheiene.

c) Condensation domain (C): responsible for the peptide bond formation to elongate the

growing peptide chain.

The modification (e.g. epimerization, cyclization, N-methylation, reduction and/or

oxidation) of the peptidyl substrate during this process takes place according to special

considerations regarding the essential properties of the individual species.

Discussion

271

4.2.3 Structure-activity relationship:

In an agar diffusion assay, kahalalide R at a disc loading concentration of 5 µg,

showed strong antifungal activity against the plant pathogens Cladosporium herbarum and C.

cucumerinum with inhibition zones of 16 and 24 mm, respectively. These results were almost

identical to those of kahalalide F, which exhibited its fungicidal activity with inhibition zones

of 17 and 24 mm, respectively, at the same concentration as kahalalide R. Using the same

concentration, the fungicidal activity of kahalalides F and R were also comparable to that of

nystatin showing inhibition zones of 19 and 39 mm, respectively. other kahalalides exhibited

neither antibacterial nor antifungal activities.

The known derivatives, kahalalides B, D, E, F, together with the new congeners,

kahalalides R and S were assayed for their cytoxicity toward L1578Y, HELA, PC12, H4IIE,

OH 3C

H 3C O+ ATP

PP

M g2+

n

OH 3C

H 3C O -AM Pn

SH

PCPOH 3C

H 3C Sn

PCP

OH 3C

H 3C Sn

PCP

O

S

PCP

H2NH 2O

SH

PCP

O

H 3C

H 3C n

O

S

PCP

HN

R R

A-D om ain (T) PCP -D om ain

C -D omain

HNO

CH3O

N H

H3CC H3

O

HOCH3

NH

OH 3C

HNO

CH3

CH3CH3

O

HN

NH

O

NH

CH3H 3C

O

H 2N

NH

O

NH 3C

H 3CHN

H 3C

H 3C HNO

HO

H3C

NHO

H3C NH

O CH3

CH3

O

O

H 3C

H

NH

OC H3

OHN

H3C

H3C

OO

CH3HN

O

CH3HN

OCH3

CH3

CH 3

O NH

HN

ONH

CH3

H 3C

O

H 2N

NH

O

NH3C

H 3CHN

H3C

H3C H NO

HO

H3C

NHO

H3CNH

O CH 3

CH3

O

O

H 3C

H

S

PCP

- repeated condensation (C-Domain)- repeated Epim erisation (E-Domain)

(Cy-D omain)

Cyclization

K ahalalide G

K ahalalide F

SH

Termination

(Te-Domain)PCP

SH

PCP Scheme (4.2) Possible biosynthetic pathway of kahalalide F (modified from

Schwarzer et al 2003)

Discussion

272

and MCF7 cancer cell lines. Kahalalides F and R were found to be comparably cytotoxic

toward MCF7 cells with IC50-values of 0.22 ± 0.05 µmol/L and 0.14 ± 0.04 µmol/L,

respectively. Kahalalide S and E were less cytotoxic in MCF7 cells with IC50-values of 3.55 ±

0.7 µmol/L and 4.5 ± 0.49 µmol/L, respectively. Kahalalide R was cytotoxic towards the

mouse lymphoma L1578Y cell line at an IC50 of 4.28 ± 0.03 nmol/mL, which is almost

identical to that of kahalalide F with an IC50 of 4.26 ± 0.04 nmol/mL. The kahalalides

including kahalalides F and R were found to be inactive toward HELA, H4IIE and PC12

cancer cell lines. This implied the cytotoxic selectivity and specificity of kahalalides F and R.

Furthermore the depsipeptide, kahalalide G, (linear analogue of kahalalide F) lack the

antitumour activity (Hamann et al 1996). This overview indicated that :

1) the presence of the sequential fragment [Val-1-Dhb-Phe-Val-2-Ile-1-Thr-1] with lactone

bond is essential for the antitumour activity.

2) The activity is not affected by substitution of Ile-1 with Valine (as in kahalalide R)

3) Opening of the lactone ring results in loss of the activity (as in kahalalide G)

4) The presence of terminal fatty acid (5-MeHex or 7-MeHex) is also responsible for the

activity

5) Hydroxylation at position 5 of the fatty acid (7-MeHex) is responsible for decreasing the

activity (as in kahalalide S)

6) The activity is not affected by substitution of The -2 and Val-5 of kahalalide F with Val

and Glu respectively (as in kahalalide R).

7) The streochemistry of individual valines is responsible for the biological activity.

4.3 Isolated compounds from unknown Pachychalina sp:

4.3.1 Epidioxy Sterols

Chemical investigation of unknown Indonesian sponge Pachychalina sp led to

isolation and structure elucidation of two 5α, 8α- epidioxy sterols (compounds 10 and 11), as

well as of the previously known Octopus derived natural product, 8-hydroxy-4-quinolone

(compound 12)

Many of the reported 5α, 8α- epidioxy sterols showed significant biological activity

including antimycobacterial activity (Cantrell et al 1999), inhibitory activity against the

human T-cell leukemia/lymphotropic virus type I (HTLV-I) and cytotoxic activity against the

human breast cancer cell line (MCF7 WT) (Gauvin et al 2000), and antitumor activity against

different tumour cell lines (Bok et al 1999). 8-Hydroxy-4-quinolone (compound 12) was

reported to be one of the ink components that is ejected by the giant octopus Octopus dofleini

Discussion

273

martini which was reported to have antipreditory activity (Siuda 1974). Some other quinolone

analogues (e.g. 3,8-dihydroxyquinoline) showed mild cytotoxic activity against the growth of

several human tumour cell lines( Moon et al 1996).

Cytotoxicity study of compounds 10 and 11 showed activity with growth inhibitory activity of

57.2 % and 38.6 % for compounds 10 and 11 respectively at concentration of 10µg/ml each

against L5178Y cancer cells. Compound 12 showed no significant antimicrobial activity.

Biosynthetic pathway of 5α, 8α- epidioxy sterols:

Steroids are formed biosynthetically from active isoprene, isopentenyl pyrophosphate, (see

figure 4.3) which involve the same pathway as those for terpenoid biocynthesis. The

triterpenoid squalene is an intermediate in the steroid biosynthesis (Dewick, 1997). Oxidation

of ∆5,7 steroid results in formation of 5-, 8- epidioxysterol, this oxidation can be produced

spontaneously or enzymatically (Gunatilaka et al 1981)

Until recently, reservation has been expressed that naturally isolated ergosterol

peroxides may be an artefact (Gunatilaka et al 1981). However White and co-workers (Bates

et al 1976) investigated the conversion of ergosterol into its epidioxide in two unrelated fungi

and demonstrated that both chemical (photooxidation) and enzymatic pathways are operative.

Gunatilaka et al 1981 suggested that the peroxidation of ergosterol and semilar ∆5,7 sterols is a

biological process and is aided by peroxidases. This suggestion was confirmed by the recent

isolation of polyketide peroxides from a number of marine sponges (Stierle and Faulkner

1980). Gauvin et al 2000 considered that these peroxides may be biosynthetic precursors for

∆4,7-3,6-diketones where both chemical groups were isolated together from the marine sponge

Raphidostila incisa. Furtheremore, Serebryakov et al 1970 isolated such epidioxy sterols from

fungi in the absence of oxygen. However, these epidioxysterols and their analogues were

previously isolated from many unrelated sponge species for example Axinella cannabina

(Fattorusso et al 1974), Tethya aurantia (Sheikh and Djerassi 1974), Raphidostila incisa

(Malorni et al 1978), Thalysias juniperina (Gunatilaka et al 1981), Haliclona rubens

(Calderon et al 1982), Hyrtios sp.(Koch et al 1983), Axinissa sp.(Iguchi et al 1993),and

Dysidea. fragilis. (Elenkov et al 1994).

Discussion

274

The possible biosynthetic pathway of 8-hydroxyquinolin-4-one from Pachychalina

sp. may include acetylation and cyclization of o-aminobenzoyl CoA as explained in (scheme

4.4)

O PP

O

squalene

oxidosqualene

HO

HO

HO

R

HO O

O

Lanosterol

5α,8α−epidioxysteroids4α -m ethyl-cholesta-8en-3β-ol

24-ethyl-cholesta-5en-3β-ol(e.g. com pound 23)

(e.g. com pound 22) (e.g. compounds 10, 11, 20 and 24)

R

HO

O 2

Scheme (4.3) The biosynthetic pathway of steroids

NH2

O

CoA

NH2

O

CoAO

NH

O

NH

O

OH

Cyclization

Ac-CoA

Scheme (4.4) Biosynthesis of 8-hydroxy-quinolin-4-one (modified from Torssell 1997)

Discussion

275

4.4 Isolated compounds from Hyrtios erectus :

Marine sponges of the genus Hyrtios (family Thorectidae, order Dictyoceratida) have

proven to be a rich source of secondary metabolites, including terpenoids, macrolides, and

tryptamine-derived alkaloids. The most important metabolites of the genus Hyrtios discovered

to date include the powerful anticancer agents spongiastatins 1-3 that were discovered by the

Pettit group in 1993 and 1994, (Youssef 2005).

In the present study, 210 g dry weight of Hyrtios erectus collected from the Red Sea –

Egypt were chemically investigated and nine compounds were structurally elucidated

including 8 known compounds and one new 5-hydroxy-1H-indole derivative.

4.4.1 Exessive harvesting of the wild-type living organisms: The potent antitumour activity of spongiastatins 1-3 from H. erectus (Pettit et al 1993;

Pettit et al 1994) prompted the same authers to exhaustively recollect the wild-type of the

same sponge Hyrtios erecta (600 kg wet wt.) for further chemical investigations. The later

chemical investigations resulted in the isolation of antineoplastic sesterstatins 1, 2 and 3 in

very minute concentrations 3 x 10-7 %; 3 x 10-7 % and 5 x 10-7 % respectively (Pettit et al

1998b), sesterstatins 4 and 5 in concentrations of 1 x 10-6 % and 4 x 10-5 % respectively

(Pettit et al 1998c) and sesterstatin 6 in concentration of 8.3 x 10-7 % (Pettit et al 2005).

Concerning this point (excessive harvesting of a wild–type living organisms), I think

that, we should be aware of the ecological problems that might occur as a result of such

irresponsible collections. Otherwise a big environmental problem takes place, in this case, we

have to balance the advantages against the disadvantages of such exhaustive re-collections. In

order to overcome this problem, I predict the same as Prof. Dr. Faulkner (Faulkner 2000a) he

also predict a great impact of chemical and biological researches including genetic

engineering concerning to different forms of marine living forms ranging from marine

invertebrates to the marine-derived microorganisms, Faulkner also expect that, in the near

future we will be able to transfer biosynthetic genes from one marine organism to another and

imagine the marine natural product chemist of 2025 still involved in structural elucidation, but

considerable effort to the genetic engineering required to produce unique metabolites by

fermentation of genetically modified microbes. This will accomplish the goal of having the

marine organisms provide the inspiration for new compounds while avoiding their excessive

harvesting.

Discussion

276

4.4.2. Biosynthesis of scalaran-type sesterterpenoids:

Tetracyclic sesterterpenes with a scalarane skeleton occur frequently as metabolites of

sponges of the family Thorectidae (order dictyoceratida) which includes the genera Hyrtios,

Cacospongia and Ircinia (Faulkner, 1997). Sesterterpenoids are composed of a C25 backbone.

Sesterterpenoid is built-up of 5 isoprene units. Their biocynthetic pathway involves enzymatic

cyclization of all-trans pentaprenyl methyl ether (which is a detoxification product of

isoprenic alcohol) as illustrated in (scheme 4.5) to form scalarane derivative

(tetracyclopentaprenyl methyl ether). The conversion is aided by cyclase enzymes (Renoux

and Rohmer, 1986)

Although the origin of scalarane-type sesterterpenes is still highly obscure, and since

in most of sponges up to one third of the volume is occupied by symbiotic microbes and

various detritus filtered off by invertebrate Renoux and Rohmer 1986, suggested that the

scalarane-type sesterterpenes which are restricted to a few sponges, (dictyoceratidae family)

might be in fact microbial metabolites. The authors (Renoux and Rohmer 1986) exhibited the

conversion of all-trans pentaprenyl methyl ether to tetracyclopentaprenyl methyl ether

through incubation of the former with cell-free extract of the protozoan ciliate (Tetrahymena

pyriformis).

OPP

OCH3

OCH3O

O

all-trans pentaprenyl methyl ether

tetracyclopentaprenyl methyl etherpentacyclic scalarane derivative

(scalarane derivative)

isopentyl pyrophosphate

Scheme (4.5) Scalarane biosynthesis. (Renoux and Rohmer, 1986)

Discussion

277

4.4.3. The pharmacological activity of scalaran-type sesterterpenoids:

Many scalarane-type sesterterpenoids have been isolated from marine sponges

belonging to the order Dictyoceratida. Scalarane-type sesterterpenes display a variety of

biological activities such as cytotoxic, antimicrobial, antifeedant, antimycobacterial,

ichthyotoxic, anti-inflammatory, and platelet-aggregation inhibitory effects, as well as nerve

growth factor synthesis-stimulating action (Youssef et al, 2005). The most important

scalarane derivatives are spongiastatins1-3 which displayed a potent antineoplastic effect.

(Youssef 2005). However compound 18 (16-hydroxyscalarolide) showed cytotoxic activity

59.5 and 44.7 % at concentrations 3.0 µg and 10.0 µg respectively against L5178Y cells.

4.4.4. Indole derivatives from Hyrtios erectus :

During the 1930’s the essential aminoacid tryptophan was discovered as well as the

plant growth hormone indole acetic acid (Kogl 1933). Tryptophan is a constituent of most

proteins, and serves in man and animal as a biosynthetic precursor for a wide variety of

tryptamine and other indole derived metabolites, several of them of paramount physiological

importance. Tryptophan is ingested during the animal–diet or vegetarian-protein-diet where it

is converted to biologically active derivatives e.g. serotonin and melatonin. Melatonin is

thought to control the day and night rhythms, while serotonin is an important neurotransmitter

acting at the nerve cells to promote feeling of well being, calm, personal security, relaxation,

confidence and concentration. Serotonin neural circuits also help counterbalance the tendency

of brain dopamine and noradrenaline circuits to encourage over-arousal, fear, anger, tension,

aggression, violence, obsessive-compulsive actions, over-eating (especially carbohydrates),

anxiety and sleeping. Serotonin itself can not penetrate the blood brain barrier, but its

precursor tryptophan can penetrate it. The typical diet provide about 1-1.5 g. tryptophan daily,

this amount is sufficient to maintain the brain essential requirement of serotonin.

NHCH2

CH2

NH

CH2

HN

H2N CHCOOH

CH2

HN

H2N CH2CH2

HN

Tryptophan Indol-3-acetic acid Serotonin Melatonin

COOH

OHOMe

COMe

Discussion

278

The sponge Hyrtios erectus is known as rich source of tryptamine derived natural

products. Hyrtiosine A, hyrtiosine B, 5-hydroxy-3-formyl-1H-indole (Kobayashi et al 1990),

hyrtiosulawesine, 1,6-dihydroxy-1,2,3,4-tetrahydro-β-carboline, serotonin, 6-hydroxy-1-

methyl-1,2,3,4-tetrahydro-β-carboline, 5-hydroxy-3-(2-hydroxyethyl)-indole, 6-hydroxy-3,4-

dihydro-1-oxo-β-carboline (Salmoun et al 2002), and hyrtioerectines A-C (Youssef 2005)

were previously isolated from Hyrtios sp.

In the present study, five indole derivatives were isolated (compounds 13-17).

Isohertiosine A, compound 17, is new, its molecular formula is the same as hyrtiosine A. The

difference has been assigned as a methoxy group attached to the carbonyl in 17 instead of

hydroxymethylene group in hyrtiosine A ( compound 13). The known compounds 15 and 16

were not previously reported for H. erectus. Some of 5-hydroxy indole derivatives have been

reported to exert cytotoxic activity against tumour cell models (Youssef 2005). However, the

indole derivative of such basic structure are known to exert their biological effects on CNS.

Compound 17 (isohyrtiosine A) showed growth inhibition activity 64 % at concentrations

10.0 µg/ml against L5178Y cells while other compound has no effect at concentrations up to

10 µg/ml.

4.4.5 Biosynthetic pathway of indole derivatives from Hyrtios erectus

The possible biosynthetic pathway of Hyrtios-derived indole includes hydroxylation

at position 5, then decarboxylation leading to the formation of the intermediate 5-

hydroxtryptamin (serotonin) which has been already isolated before from the same sponge

(Salmoun et al 2002).

Discussion

279

4.5 Isolated compounds from Petrosia nigricans :

The total methanol extract of Petrosia nigricans yielded 17 compounds, (compounds

22-38) belonging to different chemical classes which included steroids, aromatic acid

derivatives, new cerebrosides, new purine derivatives and new indole derivative, in additon to

adenosine and nicotinamide. Although there are a lot of new active metabolites isolated from

genus Petrosia, this is the first report of isolated compounds from Petrosia nigricans. None

of the isolated compounds from Petrosia nigricans showed significant antimicrobial activity

against B. subtilis, S. cerevisae, C. herbarum and C. cucumerinum. All purine derivatives

(nigricines 1 to 4) showed no cytotoxic activity against L5178Y cell line, while nigricinol

(compound 38) showed 68.1 % cytotoxic activity at conc. 10 µg/ml.

4.5.1 Biosynthetic pathway of aromatic acids: Although the isolated aromatic acids are described as catabolic products (waste) of

aromatic amines and amino acids metabolism (O’connor et al 2001), they exhibit a significant

ecological role. For example, p-hydroxybenzaldehyde, which is common in marine sponges,

exhibited a significant antipredetory activity against the major predator Perknaster fuscus of

antarctic sponges (Moon et al 1998). However, the following scheme demonstrates the

possible biosynthetic pathway of aromatic acids.

H2N CH C

CH2

OH

O

HN

H2N CH C

CH2

OH

O

HN

NADH

NAD

Dihydropteridine reductase

Tetrahydrobiopterin (BH4)

Dihydrobiopterin (BH2)

Hydroxylase

OHCO2

NH2

NH

HO

5-Hydroxytryptamine

CH2

NH

HO

OOH

CH2

NH

OOH

NH

HO

O

O

NH

HO

OCH3

NH

O

Hyrtiosine A

5-Deoxyhyrtiosine A

Isohyrtiosine A

Indol-3-carboxaldehyde5-Hydroxy-indol-3-carboxaldehyde

HH

(Serotonin)

Scheme (4.6) Possible biosynthetic pathway of 3-acyl-indole derivatives

Discussion

280

4.5.2 Cerebrosides from Petrosia nigricans: Cerebroside is the principal glycosphingolipid in the brain tissue, hence the trivial

name „cerebroside“, which was first conferred on it in 1874, although it was much later

before it was properly characterized. In fact, glycosphingolipids are found in all nervous

tissues, but they can amount to 2% of the dry weight of grey matter and 12% of white matter.

Cerebroside are found at low levels in animal tissues, such as spleen and erythrocytes, skin

lipids as well as in nervous tissues. Cerebrosides containing α-D-galactose are not found in

humans, but are known to occur in a marine sponge, they are potent stimulators of

mammalian immune systems (Christie, 2003). In the present study cerebroside mixture were

separated from total methanol extract of the sponge Petrosia nigricans, after many trials using

different methods of separation, a mixture of two cerebrosides were separated and elucidated

as petrocerebrosides1 and 2.

4.5.3 Biosynthesis of cerebrosides

The basic mechanism for the biosynthesis of sphinganine involves condensation of

acyl-coenzyme A with serine, catalysed by the enzyme serine acyltransferase as illustrated, to

H2N CH C

CH2

OH

O

H2N CH C

CH2

OH

O

OH

CO2

CO2

NH2

NH2

OH

O2

4-Monooxygenase

tetrahydro-biopterin

dihydro-biopterin

Dehydrogenase

Dehydrogenase

CHO

CHO

OH

Dehydrogenase

Dehydrogenase

COOH

COOH

OH

3-HydroxylaseCOOH

OH

OH

OH

O R2

CHO

OH

OH

COOR1

OHR2 =H, OHR1= H, Me, Et or Bu

Scheme (4.7) Biosynthetic pathway of aromatic acids and aldehydes. (chosen from O’connor et al 2001, Cuskey and Olsen 1988 )

Discussion

281

form 3-keto-sphinganine. The keto group is then reduced to hydroxyl by a specific reductase.

The free sphinganine is rapidly acylated to form a dihydroceramide, by a dihydroceramide

synthase, which can utilize a range of acyl-CoAs. Insertion of the trans-double bond in

position 4 to produce sphingosine occurs only after the sphinganine has been esterified in this

way to form a ceramide. The biosynthesis of cerebrosides takes place through direct transfer

of the carbohydrate moiety from a sugar-nucleotide, eg uridine-5-diphosphate (UDP)-

galactose, UDP-glucose, etc, to the ceramide unit. During the transfer, which is catalysed by a

glycosyl-transferase, inversion of the glycosidic bond occurs (from α to β).

4.5.4 New purine derivatives from Petrosia nigricans:

Marine organisms, particularely sponges, have proven to be an exceptionally rich

source of modified nucleosides. The isolation of spongouridine and spongothymidine from

Cryptotethia crypta (Bregmann, and Feeney 1950) which served as models for the

development of adenine arabinoside (ARA-A) for treatment of Herpes simplex infection and

cytosine arabinoside (ARA-C) for the treatment of leukemia (Lindsay et al 1999), and

subsequent development of antiviral analogues demonstrated the potential medicinal

importance of these compounds such as antifungal phidolopine which was isolated from the

bryozoan Phidolopora pacifica (Ayer et al 1984), the hypotensive doridosine which was

isolated from the sponge Tedania digitata (Cook et al 1980), and the cytotoxic mycalisines

which were obtained from the sponge Mycale sp. (Kato et al 1985). Many other purines and

R SCoA

OCOOH

NH2

CH2OHH

R

O

NH2

CH2OH

Long chain acyl-CoA

Serine

NADPH+H

NADP

3-keto-sphing. reductase

R

OH

NH2

CH2OH

R1

R

OH

NH

CH2OH

OR1

O

OH

R

OH

NH

CH2OH

OR1

Sphingosine

DihydroceramidesynthaseFatty acid

Sphinganine

Dihydroceramide

Cerebroside

Ceramide

R

OH

NH

CH2OH

OR1

OH

R

OH

NH

CH2O

OR1

OH Gal-Gal

Desaturase

Scheme (4.8) Biosynthesis of cerebrosides (Christie, 2003)

Discussion

282

nucleosides isolated from marine organisms particularly sponges display potant bioactivities,

such as the marine derived 1,3-dimethylisoguanine from Amphimedon viridis which showed

activity against an ovarian cancer cell line (IC50, 2.1 µg/mL) (Mitchell et al 1997); 3,7-

dimethylisoguanine from a Caribbean sponge Agelas longssima, which displayed mild

antibacterial activities (Cafieri et al 1995). Figure 4.3 shows the structure of some purine and

pyrimidine derivatives that used as antiviral agents (Baba 2005).

In the present study, investigation of ethylacetate fraction of the Indonesian sponge

Petrosia nigricans, which was collected from Pulau Baranglompo - Indonesia led to the

isolation of four new purine derivatives nigricines 1-4. Their structures were elucidated by

extensive spectroscopic analysis, 2D-NMR experiments, EI/MS, ESI/MS and HRMS.

Erinacean is a related purine derivatives with unusual 6-β-alanine amino acid was isolated

before from the antarctic sponge Isodictya erinacea (Moon et al 1997). To the best of our

knowledge, Purine derivatives with alkyl 3-(3,9-dihydro-3,9-dimethyl–2-oxo-2H-purin-6-

ylamino) propanoate were not reported neither as a synthetic nor as a natural product.

N

NN

N

NH2

OP

OH

OH

O

N

NN

N

NH2

OP

OH

OH

O

Tenofovir (PMPA)Adefovir (PMEA)

N

N

N

N

NH2

O

HO

OH

HO

Vidarabine (Ara-A)

HN

N

N

N

O

OHO

Ganciclovir (GCV)

HO

HN

N

N

N

O

OHO

Acyclovir (ACV)

H2NH2N

HN

NO

O

O

OH

HO

CF3

Trifluridine (TFT)

HN

NO

O

O

OH

HO

I

Idoxuridine (IDU)

HN

NO

O

O

OH

HO

HO

Br

Sorivudine (BVaraU)

HN

NO

O

O

N3

HO

CH3

Zidovudine (AZT)

N

NO

NH2

Cidofovir (HPMPC)

OP

OH

OH

OHO

Fig (4.3) Purine and pyrimidine derivatives used as antiviral agents (Baba 2005).

Discussion

283

4.5.5 The possible biosynthetic pathway of nigricines 1-4:

Synthesis of the purine ring is a central metabolic function of all cells. The products,

AMP and GMP, provide purine bases for DNA and RNA, as well as for a number of essential

coenzymes (NAD, NADP, FAD and Coenzyme A) and signaling molecules (e.g. cAMP). The

purine pathway also functions in pathways that are different and distinct from these

„housekeeping roles.“ It is employed in specialized tissues to assimilate and detoxify NH3

(Smith and Atkins 2002). The proposed biosynthetic pathway of nigricines 1-4 seems to

follow the same biosynthetic pathway of the normal adenylated nucleotides until the

formation of adenylsuccinate then the later substance (adenylsuccinate) is more likely to be

used in de novo nigricines biosynthesis via a lateral pathway as explained in scheme (4.9).

12

4

5 7

9

N

N N

N

NH

O

OO

N

N N

N

NH

O

OR

O

1 R = Bu2 R = Et3 R = Me

N

N

NH

HN

8 O

NH10

11

12 OHO

Erinaceannigricine-4

nigricines:

Fig ( 4.4 ) Nigricines 1-4 as well as the related compound erinacean

Discussion

284

4.6 Isolated compounds from Callyspongia biru:

Sponges belonging to the genus Callyspongia are extensively investigated for

bioactive natural products. Many new bioactive natural products have been isolated from

different Callyspongia sp., these natural products include unusual steroids (Agrawal and Garg

1997); acetylene derivatives ( Youssef et al 2003); uncommon fatty acids (Carballeira and

Pagan 2001); diterpenes (Garg and Agrawal 1995); triterpenes (Fukami et al 1997); many

cyclic peptides have been isolated from the genus Callyspongia [ phoriospongins A and B

from C. bilamellata (Capon et al 2002), Callynormine A from C. abnormis (Berer et al 2004)

and callyaerins from C. aerizusa (Min, et al., 2001)]. Many natural product-producing fungi

N

N NCH

NHN

1

23

4

5 7

8

9

HN

NN

CH

N6

CO2

Aspartate

N10-formyl-THFGlutamine (amide N)

N10-formyl-THF

C-8

N-1

N-9N-3

C-2

C-6

GlycineNH2

COOH

N

N NCH2

N

O

NH

OO

N

N NCH2

N

O

NH

OOH

N

N NCH

N

O

NH

OO R

Nigricine 4

Nigricines 1-3

O

COOHHOOCAspartate

CO2(O)

Ribose P

Ribose P

N

HN N

CHN

HN

COOH

Ribose P

O

N

HN H

NCH

N

HN

COOH

O -Ribose

nucleosidase

Adenylsuccinate

N

N NCH

N

O

NH

OOH

Fumarate

N

N NCH

NNH2 Ribose P

AMP

-P

nucleotidase

Scheme (4.9) Possible biosynthetic pathway of new purine derivatives

from Petrosia nigricans (modified from Smith and Atkins 2002)

Discussion

285

have been isolated from some Callyspongia sp., and cultivated on artificial media for

production of natural products [spiciferone derivatives, macrolides, anthraquinones and

benzofuran have been isolated from C. aerizusa –derived fungi (Edrada et al 2000, Jadulco et

al 2001, Jadulco et al 2002)]. The most attractive compound, that was isolated from

Callyspongia sp., is callystatin A. Callystatin A is a potant cytotoxic polyketide isolated from

Nagasakian marine sponge, Callyspongia truncata (Kobayashi et al 1997). Methanol extract

of Indonesian sponge Callyspongia biru was investigated and p-hydroxyphenylacetic acid, p-

hydroxyphenylacetic acid methylester, indole-3-carbaldehyde, indole-3-acetic acid, and 2‘-

deoxythymidine were isolated. Deoxythymidine (compound 40) showed cytotoxic activity

76.2 % and 41.7 % against L5178Y mouse lymphoma cell line at concentrations 10 and 3

µg/ml respectively.

286

V- Summary

The modern techniques as well as the advanced instruments that employed in the

discovery programms of bioactive natural product facilitated the task of the natural product

chemists and permitted them to detect, target, isolate and elucidate the structure of the

pharmacologically active natural product in significantly short times compared with that done

in the not too distant past. The targeted natural product may show novel or unusual

spectroscopic features and/or promising biological activities. These methodologies are now

applicable to achieve the task without the need of tedious isolation procedures of known

compounds. The application of these new methodolgies resulted in saving the time, efforts

and economy during the processing of the bioactive natural product drug discovery programs.

The present study deals with the application of the modern techniques including NMR, MS,

and hyphenated HPLC systems as very efficient and applicable tools in the achievement of

the above mentioned aim. The biological materials that were employed in this study included

six different soft bodied marine animals which were collected from four different

geographical zones. The study resulted in isolation, purification, and structure elucidation of

40 compounds. Some of them showed a very promising biological activity and recommended

as drug candidates in the near future.

1- The mollusc Elysia rufescens

The sacoglossan molluscs Elysia rufescens were collected from the black point „Kahala“ in

Oahu Island in Hawaii. The animal extract provided 5 known kahalalides (kahalalides B, C,

D, E and F) in addition to β-sitosterol and a previously known fabaceous secondary

metabolite, N,N-dimethyl-tryptophan methylester.

2- The mollusc Elysia grandifolia

The sacoglossan molluscs Elysia grandifolia were collected from the Gulf of Mannar and

Palk Bay, Rameswaram, India. The hyphenated system (LCMS) detected the molecular ion

peaks that corresponding to kahalalides B, C, D, E, F, G, J, K, O as well as two new

molecular ion peaks (corresponding to kahalalides R and S) in the total methanol extract. The

isolation process was directed to target the new ones, and resulted in structure elucidation of

them in addition to another two known kahalalides (kahlalaides D and F).

287

3- The Indonesian sponge Pachychalina sp.

The hithertho new sponge species, Pachychalina sp, was collected from Pulau

Baranglompo, Indonesia. Chemical investigation of the methanol extract of the sponge led to

isolation of two 5α, 8α-epidioxysteroids, as well as the previously known compound, 8-

hydroxy-4-quinolone, which was reported as an ink component that is ejected by the giant

octopus „Octopus dofleini martini“.

4- The Indonesian sponge Petrosia nigricans

The Indonesian sponge Petrosia nigricans was collected from Pulau Baranglompo,

Indonesia. Seventeen compounds were isolated from the total methanol extract. These

compounds included 10 known natural products, 2 new cerebrosides, one bis-indole

derivative and four new 2-oxo-purine derivatives.

5- The Indonesian sponge Callyspongia biru

The Indonesian sponge Callyspongia biru was collected from Taka Bako, Indonesia. The total

methanol extract of the sponge provided five known compounds (indol-3-carbaldehyde,

indol-3-acetic acid, p- hydroxyphenylacetic acid, p-hydroxyphenylacetic acid methylester,

and 2`-deoxythymidine).

6- The Red Sea sponge Hyrtios erectus

The Red Sea sponge Hyrtios erectus was collected from El-Quseir, in the Red sea , Egypt.

The total methanol extract of the sponge provided nine compounds including one

epidioxycholesterol derivative, three scalaran-type sesterterpenes, four known indole

derivatives and one new 5-hydroxy-indole derivative.

288

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List of Abbreviations [α]D : specific rotation at the sodium D-line br : broad signal CI : chemical ionization COSY : correlation spectroscopy d : doublet dd : double of doublets ddd : double double of doublets DEPT : distortionless enhancement by polarization transfer ED : effective dose EI : electron impact ESI : electro spray ionization eV : electronvolt FAB : fast atom bombardment HMBC : heteronuclear multiple bond connectivity HMQC : heteronuclear multiple quantum coherence HPLC : high performance liquid chromatography Hz : herz LC : lethal concentration LC-MS: Liquid chromatography-mass spectrometer m : multiplett MALDI-TOF-PSDMS:

Matrix-assisted laser dessorption ionization-time of flight-post source decay mass spectrometer

MeOD : deuterated methanol MeOH : methanol mg : milligram mL : millilitre MS : mass spectroscopy m/z : mass per charge µg : microgram µL : microliter NMR : nuclear magnetic resonance ppm : part per million Prep. HPLC : preparative HPLC q : quartet ROESY : rotating frame overhauser enhancement spectroscopy RP-18 : reversed phase C-18 s : singlett t : triplett TFA : trifluoroacetic acid TLC : thin layer chromatography UV : ultra-violet VLC : vacuum liquid chromatography

303

Publication

Ashour M., Edrada R, Ebel R., Wray V., Wätjen W., Padmakumar K., Müller W. E. G., Lin

W. H. and Proksch P. (2006): „Kahalalide Derivatives from the Indian Sacoglossan Mollusc

Elysia grandifolia“ , J. Nat. Prod., 69, 1547.

304

Biographic data

Name: Mohamed Abdelghaffar Ali Ashour

Date of birth: 18. December 1969

Place of birth: El-Sharkiya, Egypt

Nationality: Egyptian

Civil status: Married, two children

Address: Universitätsstrasse 1, 40225 Düsseldorf

Home Address: Al-azhar University, Cairo, Egypt

Educational Background:

1975 – 1987: Grade School - Higher School, Belbies Schools for Al-Azhar

Sciences, Belbies, Egypt.

1987 – 1992: Bachelor of Science degree in Pharmacy, Al-Azhar University,

Cairo, Egypt.

1994 – 1999: Master of Science degree in Pharmacy, Al-Azhar University,

Egypt

Thesis: Pharmacognostical Studies of some Sophora sp., belonging to

family Fabaceae

2002-2002: ZMP certificate of German language, Goethe Institut, Cairo,

Egypt.

2003 – present: Ph.D. candidate, Institute of Pharmaceutical Biology, HHU,

Düsseldorf, Germany.

Employment Record:

1994 – 1999: Demonstrator, Faculty of Pharmacy, Al-Azhar Univeristy,

Cairo, Egypt.

1999 – present: Assistant Lecturer, Pharmacognosy Department, Faculty of

Pharmacy, Al-Azhar University, Cairo, Egypt.

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