Post on 26-Mar-2018
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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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
Material and Methods
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
Material and Methods
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.
Material and Methods
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
Material and Methods
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
Material and Methods
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
Material and Methods
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)
Material and Methods
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
Material and Methods
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
Material and Methods
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:
Material and Methods
51
Methanol LiChrosolv HPLC Merck
Nanopure water Barnstead
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
Nanopure water Barnstead
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
Material and Methods
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.
Material and Methods
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
Material and Methods
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.
Material and Methods
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
Material and Methods
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
No spectra library hits found!
Peak #12 13.32
-10,0
70,0
200 300 400 500 595
%
nm
227.0202.7
300.5
No spectra library hits found!
9.25
-500
1.250
2.500
4.000
200 400 595
%
nm
344.7 530.3551.7
No spectra library hits found!
Peak #9 4.68
-10,0
25,0
70,0
200 400 595
%
nm
210.4
289.7
560.3
No spectra library hits found!
Peak #7 5.07
-10,0
25,0
70,0
200 400 595
%
nm
214.2315.6
No spectra library hits found!
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.
Material and Methods
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
Material and Methods
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
total methanol extract
n-Hexane extract
solvent partition n-Hexane : methanol
(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
total methanol extract
n-Hexane extract
solvent partition n-Hexane : methanol
(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
Material and Methods
59
2.5.4. Isolation of secondary metabolites from Callyspongia biru
2.5.5. Isolation of secondary metabolites from Hyrtios erectus
total methanol extract
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
Material and Methods
60
2.5.6. Isolation of secondary metabolites from Petrosia nigricans
total methanol extract
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
Si gel columnCH2Cl2:MeOH
TM7 EtOAc fraction
Si gel columnCH2Cl2:MeOH
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
Material and Methods
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
Material and Methods
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
Material and Methods
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).
Material and Methods
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.
Material and Methods
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.
Material and Methods
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
Material and Methods
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).
Material and Methods
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.
Material and Methods
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:
Material and Methods
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
Material and Methods
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)]+.
Material and Methods
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).
Material and Methods
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
Material and Methods
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
C14H19N5O7Exact Mass: 369,13
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
C15H21N5O7Exact Mass: 383,14
Mol. Wt.: 383,36
OH
C18H19N5O7Exact Mass: 417,13
Mol. Wt.: 417,37
OH
C14H20N6O7Exact Mass: 384,14
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
C14H17N5O7Exact Mass: 367,11
Mol. Wt.: 367,31
Material and Methods
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).
Material and Methods
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.
Material and Methods
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
C75H124N14O16Exact Mass: 1476,93197Mol. Wt.: 1477,87126
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
C45H69N7O8Exact Mass: 835,521
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
No spectra library hits found!
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
C30H40N4O5Exact Mass: 536,3Mol. Wt.: 536,662
(13 %)
N
NH
O OHN
O
CH3
H3C
-(Arg+H2O)
C25H32N3O3•
Exact Mass: 422,244Mol. Wt.: 422,54
(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•
Exact Mass: 343,202Mol. Wt.: 343,44
( 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
C45H63N7O11Exact Mass: 877,459
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
No spectra library hits found!
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••
Exact M ass: 101,048
C5H7NO ••
Exact Mass: 97,053
C6H11NO••
Exact M ass: 113,084
C9H9NO ••
Exact M ass: 147,068
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
C14H18N2O2Exact Mass: 246,137
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
No spectra library hits found!
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)
1H (δ = ppm), couppling
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
1H (δ = ppm), couppling
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)
1H (δ = ppm), couppling
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
Fig (3.4.18):HPLC chromatogram and UV-spectrum of compound 17. Yield : 3.0 mg
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
C10H9NO3Exact Mass: 191,06
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
No spectra library hits found!
Fig (3.4.23):HPLC chromatogram and UV-spectrum of compound 18. Yield : 3.5 mg
[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
C25H38O4Exact Mass: 402,28Mol. Wt.: 402,57
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):
Fig (3.4.31):FAB-MS spectrum of compound 19.
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
C25H38O3Exact Mass: 386,28
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)] 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
Fig (3.4.35):HPLC chromatogram and UV-spectrum of compound 20. Yield : 1.5 mg
Fig (3.4.36):FAB-MS spectrum of compound 20.
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
C25H38O4Exact Mass: 402,28Mol. Wt.: 402,57
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)] 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)]+
Fig (3.4.40):FAB-MS spectrum of compound 21.
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
C27H44O3Exact Mass: 416,33
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)]+
Fig (3.5.1):EI-MS spectrum of compound 22
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
Fig (3.5.5): 1HNMR spectrum of compound 23 (in CDCl3)
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
Fig (3.5.6): 13CNMR and DEPT spectra of compound 23 (in CDCl3)
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)
Fig (3.5.7):EI-MS spectrum of compound 24
Fig (3.5.8): 1HNMR spectrum of compound 24 (in CDCl3)
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
C29H48O3Exact Mass: 444,36
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.
Fig (3.5.9): 13CNMR and DEPT spectra of compound 24 (in CDCl3)
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
No spectra library hits found!
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
C8H8O3Exact Mass: 152,05Mol. Wt.: 152,15
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
Fig (3.5.14): HPLC chromatogram and UV spectrum of compound 26
[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)].
Fig (3.5.16):1HNMR spectrum of compound 26
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
Fig (3.5.17): HMBC spectrum of compound 26
(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
Fig (3.5.18): HPLC chromatogram and UV spectrum of compound 27
C9H10O3Exact Mass: 166,06
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)]
Fig (3.5.20):1HNMR spectrum of compound 27
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
Fig (3.5.21): HPLC chromatogram and UV spectrum of compound 31
OH
O
O172.640.1
126.2
130.0
115.2156.8
60.1
3.48
7.04
6.68
4.031.1614.2
C10H12O3Exact Mass: 180,08
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.
Fig (3.5.23):1HNMR spectrum of compound 28
Fig (3.5.24): HMBC spectrum of compound 28
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
Fig (3.5.25): HPLC chromatogram and UV spectrum of compound 29
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
C12H16O3Exact Mass: 208,11
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.
Fig (3.5.27):1HNMR spectrum of compound 29
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
C10H13N5O4Exact Mass: 267,1Mol. Wt.: 267,24
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
No spectra library hits found!
Fig (3.5.33): HPLC chromatogram and UV spectrum of compound 31
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).
Fig (3.5.35):1HNMR spectrum of compound 31
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
C56H107NO14Exact Mass: 1017,77
-------------------------------------------------------------------------------------------- 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
Fig (3.5.48): HPLC chromatogram and UV spectrum of compound 34
154.9
N
N
N
N
NH
O
O
O
C14H21N5O3Exact Mass: 307,165
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
Fig (3.5.52):COSY spectrum of compound 34
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)
Fig (3.5.56):ESI-MS spectrum of compound 35
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
Fig (3.5.55): HPLC chromatogram and UV spectrum of compound 35
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
Fig (3.5.57):1HNMR spectrum of compound 35
Fig (3.5.58):13CNMR and DEPT spectra of compound 35
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 (3.5.59):COSY spectrum of compound 35
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)
Fig (3.5.62):ESI-MS spectrum of compound 36
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
Fig (3.5.61): HPLC chromatogram and UV spectrum of compound 36
[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.
Fig (3.5.63):1HNMR spectrum of compound 36
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
Fig (3.5.64):13CNMR and DEPT spectra of compound 36
Fig (3.5.65):COSY spectrum of compound 36
(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
Fig (3.5.66): HMBC spectrum of compound 36
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)
Fig (3.5.68):ESI-MS spectrum of compound 37
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
No spectra library hits found!
Fig (3.5.67): HPLC chromatogram and UV spectrum of compound 37
N
N
N
N
NH
O
O
O
3.45
3.58, 3.62
3.62
2.6
6.88
C12H19N5O3Exact Mass: 281,15
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
Fig (3.5.72): HPLC chromatogram and UV spectrum of compound 38
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 (3.5.77):part of HMBC spectrum of compound 38
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
Fig (3.5.79):part of HMBC spectrum of compound 38
(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
No spectra library hits found!
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
No spectra library hits found!
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
No spectra library hits found!
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
No spectra library hits found!
9.25
-500
1.250
2.500
4.000
200 400 595
%
nm
344.7 530.3551.7
No spectra library hits found!
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.