1

Wolfgang KroutilWolfgang Kroutil, ,

University of Graz, AustriaUniversity of Graz, Austria

Wolfgang.Kroutil@uniWolfgang.Kroutil@uni --graz.atgraz.at

Biokatalyse – Schlüssel zur effizienteren organischen Synthese

Eduardo Eduardo BustoBusto Eva Eva FischerederFischereder Christine FuchsChristine FuchsBarbara Barbara GrischekGrischek Anja HolzerAnja Holzer Christina Christina KoflerKoflerHorst LechnerHorst Lechner Desiree Desiree PressnitzPressnitz AashritaAashrita RajagopalanRajagopalanNina RichterNina Richter Johann Sattler Johann Sattler Robert SimonRobert Simon

Acknowledgement

EC: AmBioCasMC-RTN (RevCat), MC-ITN (Biotrains)CASCAT, Sandoz, Evonik

Current group members

Cooperation:Jan PfefferFerdinand Zepeck

Kurt FaberKarl Gruber Peter Macheroux

2

„moderne“ Mensch

Energie/Treibstoffe

KleidungNahrungsmittel

Kosmetika Pharmazeutika

Wohnen

Chemische Industrie

Chemische Reaktionen

Beispiele chemischer Umsetzungen

3

Die ideale chemische Reaktion

Minimum an Abfällen; Minimum an Lösungsmittel. Verwendung von nachwachsenden Rohstoffen,

100% Atomökonomie; Energieeffiziente Prozesse; Kaskaden und Domino-Prozesse

Alles Substrat wird zu Produkt: 100% Ausbeute, Hochselektive Prozesse (chemo-, regio-, stereo-)

Standard Anlagen; hohe Raum/Zeit-Ausbeuten, katalytische Prozesse

Selektivität

Effizienz

Umwelt

Ökonomie

Viele organisch-chemische Reaktionen sind ineffizie nt

Catalysis/catalyst

A catalyst is a substance that increases the rate of areaction without itself being consumed or changed

A catalyst increases the rate of the reaction by loweringthe ∆G‡ of the reaction

How does that happen??

4

A cartoon: How does a catalyst work?

Active site: gets the reagents close together

Working modes of a catalyst

1) Gets the reagents close together

2) Activates substrate

3) Activates reagent

4) Stabilises transition state

5) May provide alternative mechanism

6) Employing metal ions for activation

5

Types of catalysts

Metal catalyst

Organo-Metal catalyst (metal with ligand)

Organo catalyst: small organic compound

Biocatalyst = Enzyme

Enzymesind die Katalysatoren in lebenden Organismen

Bausteine: Amino-Säuren

6

… are naturally degradabel

… do not need toxic transition metals (as needed formany metal catalysts)

… have been optimised for specific reactions of millionof years

Enzymes…

… are chiral, allow chiral recognition

7

Working modes of a catalyst

1) Gets the reagents close together

2) Activates substrate

3) Activates reagent

4) Stabilises transition state

5) May provide alternative mechanism

6) Employing metal ions for activation

7) Chiral recognition

8

Louis PasteurLouis PasteurLouis PasteurLouis PasteurChemist

1822-1895

“ Chance favors only prepared mindChance favors only prepared mindChance favors only prepared mindChance favors only prepared mind” ----Louis PasteurLouis PasteurLouis PasteurLouis Pasteur

Resolution of enantiomersResolution of enantiomersResolution of enantiomersResolution of enantiomers

Pasteur’s Discovery of Enantiomers

NH

O O

N

O

O

thalidomide

(R)

- (R)-(+) enantiomer - popular sedative inthe late 1950's and used as a treatmentfor morning sickness in pregnant women

- (S)-(-) "inactive" enantiomer isteratogenic, i.e. caused severedeformities in babies born to motherswho had taken the drug

9

H2N SH

Me

MeH

HO2C

S-penicilamineANTIARTHRITIC

NH2HS

Me

MeH

CO2H

R-penicilamineTOXIC

Perfect chiral recognition of enzymes

Agrobacterium radiobacterEpoxide Hydrolase Jacobsen Co+2-Salen-Complex

O

O

10

Working modes of a catalyst

1) Gets the reagents close together

2) Activates substrate

3) Activates reagent

4) Stabilises transition state

5) May provide alternative mechanism

6) Employing metal ions for activation

7) Chiral recognition

A catalyst exploiting more/all modes?

Types of catalysts

Metal catalyst

Organo-Metal catalyst (metal with ligand)

Organo catalyst: small organic compound

Biocatalyst

not chiral

can be chiral, gets reagents together, activates, stabilising transition state?

can be chiral, activates mostly only onepartner, no transition stabilisation

11

Klassische Biotechnologie: Natürliche Verbindungen

Acetobacter sp.

Ethanol Essigsäure

Hefe

Sucrose Ethanol

Acetobacter sp.

Glucose Sorbose Vitamin CSorbitol

NH2

COOMe HOOCCOOH

HNX

+

NH

CO2Me

COOH

HNX

O

Thermolysin

NH

CO2H

COOH

NH2

O

Aspartam

Nicht nat ürliche Reaktionen: z.B. f ür Aspartam

Seit 1988, 2500 t/a

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Beispiel: Acrylamid

CN

NH2

O

Rhodococcus sp.

> 400 g/LOH

O

H2OH2O

Kupfer-Katalyse

H2O H2O

>30000 t/a

Biokatalyse = die Verwendung von Enzymen (=Biokatalysatoren) für die Umsetzung von vorwiegend nicht natürlichen Substraten

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Binding of substrate to enzyme

Weisse Biotechnologie:

Biotechnologie in der chemischen Produktion

• verringerter Energieaufwand

• saubere Prozesse - weniger Nebenprodukte

• reduzierter chemischer Abfall

• verminderte Umweltgefährdung

• nachwachsende Rohstoffe

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In 2009, European Member States and the European Commission identified Key EnablingTechnologies for

their potential impact in strengthening Europe's industrial and innovation capacity.

Six Key enabling Technologies

� nanotechnology� micro and nanoelectronics� advanced materials� photonics� industrial biotechnology� advanced manufacturing systems

KEY ENANBLING TECHNOLOGY

1960 1970 1980 1990 2002

0

20

40

60

80

100

120

140

Cum

ulat

ive

# of

Pro

cess

esS

tart

ed

Industrial biocatalytic processesCurr. Opinion Biotechnol. 13, 548 (2002)

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Examples performed on ton scale

CNNH2

O

nitrile hydratase

H2O acrylamide 30.000 t/a

OOH

OHOH

OH

OH

Glucoseisomerase

OHO

OH

OH

OH

OH

OO

OHOH

OHHO

O

OHOH

OH

OH

OOH

OHOH

OHHO

O

OHOH

OH

OH

OH+β-Galactosidase

fructose 250.000 t/a

250.000 L/dLactose

N CO2H N CO2H

OH

GrowingAgrobacterium

L-Carnitine

on 50000 L-scale

Herkunft unserer Biokatalysatoren

Bilder: Kroutil, Wikipedia, Winkler

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Production of recombinant Enzyme

• Gene identified• Into plasmid (pET21a)• Plasmid into E.coli• Overexpression• Lyophilisation

Protein Expression

• Cell growth conditions:

Flask volume > 3 x medium

Luria-Bertani medium

37°C

Shake ~ 120 rpm

OD600 0.6 – 0.9

Induction with 0.5 mM IPTG

22 h, 20°C, 120 rpm

History Enzyme catalysed PSR Research Subjects Results Summary Acknowledgment

17

Bild: Andreas Winkler

OH

N

MeO

OH

OMe

(S)

Naturstoff: Scoulerine

Beruhigungsmittel und Mittel zurEntspannung der Muskel

(S)-Scoulerine

Croton/Wolfsmilchgewächse

Fotos: Wikipedia

Korallenbäume

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BBE – preparative scale

207 mg

250 mg

250 mg

500 mg racemate20 g/L, 65 mM

OH

ON

O

BBE

(S)

O2 H2O2

OH

ON

O

(R)

+

e.e.>97%(HPLC)

e.e.>97%(HPLC)

buffer, pH 9, 10 mM MgCl2,70% v/v toluene40°C, darkness, 24 h

Catalase 1/2 O2

+ H2O

(S) OH

ON

O

+

OH

ON

O

(R)

50% conv.

249 mg

J. H. Schrittwieser, V. Resch, J. H. Sattler, W.-D. Lienhart, K. Durchschein, A. Winkler, K. Gruber, P. Macheroux, W. Kroutil, Angew. Chem. Int. Ed. 50, 1068-1071 (2011).

OH

N

MeO

OH

OMe

(S)

Naturstoff: Scoulerine

Beruhigungsmittel und Mittel zurEntspannung der Muskel

230 mg hergestellt

(S)-Scoulerine

Erste Herstellungim Labor Croton/Wolfsmilchgewächse

Fotos: Wikipedia

Korallenbäume

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J. H. Schrittwieser, V. Resch, J. H. Sattler, W.-D. Lienhart, K. Durchschein, A. Winkler, K. Gruber, P. Macheroux, W. Kroutil, Angew. Chem. Int. Ed. 50, 1068-1071 (2011).

Reaction conditions

Moderate T: 30-50°C

Normal pressure

Water as (co)solvent

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Biocatalytic pathway to optically pure amines

R R'

NH2H

R

O

R'

Substrate

Substrate = α-keto acid

?

N

NN

N

ONH2

F

FF

CF3Sitagliptin(anti-diabetic drug)

ON

O

NH2

Rivastigmin(for dementia and

Alzheimers disease)

ONH2

Mexiletine(anti-arrhytmeticum)

N

OO

Calvin(natural repellents)

N

Bu

H

Monomorine(trail pheromone)

HO O OH

HN

Morphine(analgesic drug)

Chiral amines

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Combining catalysts

Difficult with organo-metal- and organo-catalysts

Biocatalysts are ideal to be combined

αααα-Chiral primary amines, reaction system

R R'

NH2

R R'

Oω-Transaminase, PLP

L- or D

(S)- or (R)*

CO2H

NH2

CO2H

OLDH

Lactate

NADH NAD+

GlucoseGDH

Glucono-lactone

D. Koszelewski, I. Lavandera, D. Clay, D. Rozzell, W. Kroutil, Adv. Synth. Catal. 350, 2761-2766 (2008).D. Koszelewski, M. Göritzer, D. Clay, B. Seisser, W. Kroutil, ChemCatChem 2, 73-77 (2010).M.D. Truppo, J. D. Rozzell, J. C. Moore, N. J. Turner, OBC 7, 395 (2009).

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ΙΙΙΙdeal process: Reductive amination

R'

O

R

NH4+

R'

NH2

R

Biocatalysts

H2O*

single enantiomer

oxidisedH-donor

H-donor

23

24

Enzymes-Toolbox 1

ω-Transaminases

Amino acidoxidases

NAD(P)Hrecycling:

GDH, FDH…

Lactate-DH

Amino aciddehydrogenase

Cloned and Commercial enzymes

O2H2O2

R

NH2

CO2H R

O

CO2H

H2O

NH4+

NAD(P)+NAD(P)H

H2O

R

O

CO2H R CO2H

NH2

25

αααα-Chiral primary amines

R'

O

R R'

NH2

R

ω-Transaminase

CO2H

NH2

CO2H

O

buffer, pH 7.0/ DMSO

NH4+

NAD(P)+NAD(P)H

L-AADHH2O

FDH or GDH

Formate orGlucose

CO2

orGluconolactone

D. Koszelewski, I. Lavandera, D. Clay, G. M. Guebitz, D. Rozzell, W. Kroutil, ACIE. 47, 9337–9340 (2008).M.D. Truppo, J. D. Rozzell, J. C. Moore, N. J. Turner, OBC 7, 395 (2009).

Results, ( S)-enantiomers, 50 mM, 30°C, 24 h

NH2 NH2

NH2 NH2

ONH2

NH2

O

NH2O

EtO

NH2

NH2

NH2

O

e.e. >99%c 94%

(S) (S) (S)

(S) (S) (S)

(S) (S)

e.e. >99%c >99%

e.e. >99%c 89%

e.e. >99%c 67%

e.e. >99%c 17%

e.e. >99%c >99%

e.e. >99%c 99%

e.e. >99%c 99%

c >99%

(S)

e.e. >99%c 97%

P. fluorescens P. denitrificans

P. fluorescens

D. Koszelewski, I. Lavandera, D. Clay, G. M. Guebitz, D. Rozzell, W. Kroutil, ACIE, 47, 9337 –9340 (2008).D. Koszelewski, M. Göritzer, D. Clay, B. Seisser, W. Kroutil, ChemCatChem 2, 73-77 (2010).F. G. Mutti, C. S. Fuchs, D. Pressnitz, N. G. Turrini, J. H. Sattler, A. Lerchner, A. Skerra, , Eur. J. Org. Chem.1003–1007 (2012).

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Results, (R)-enantiomers

NH2 NH2

NH2 NH2

ONH2

NH2

O

NH2O

EtO

NH2NH2

O

e.e. >99%c 98%AT-ωTA

(R) (R) (R)

(R) (R) (R)

(R) (R)

e.e. >99%c 69%

e.e. >99%c 97%

e.e. >99%c 54%

e.e. >99%c 96%

e.e. >99%c >99%

e.e. >99%c >99%

e.e. >99%c >99%

(R)e.e. >99%c >99%

AT-ωTA = Aspergillus terreus ω-transaminase (U.T. Bornscheuer)

AT-ωTA AT-ωTA AT-ωTA

AT-ωTA AT-ωTA AT-ωTA

AT-ωTA AT-ωTA

F. G. Mutti, C. S. Fuchs, D. Pressnitz, J. H. Sattler, W. Kroutil, Adv. Synth. Catal. 2011, 353, 3227

sec-amines: 2,6-dialkyl piperidines

potential antifeedant against the pine weevil Hylobius abietis[=Fichtenrüsselkäfer (ge.)]

NH

N RNH2

R

O

O

R

O

(+)-dihydropinidine

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Regioselectivity

R

OO

R

ONH2

R

NH2O

ω-TAn

n n

n = 1,2,3

ω-TA

Regioselectivity

O O

n-PrMgCl, molecular sieve

-78°C, Et 2O, 16 h,77% yield

O O

N N

(2S)conv. >99%,

>99% ee

(2S,6R)>99% ee, >99% de94% over two steps

Pd/C, H2,4 h, 22°C

H H

Cl

ω-TAChromobacter iumviolaceum

N N

(2R)conv. 97%,

>99% ee

(2R,6S),>99% ee, >99% de85% over two steps

H H

Cl

(R)−ω-TAArthrobacter

Pd/C, H2,4 h, 22°C

2,6-disubsituted chiral piperidines

R. C. Simon, B. Grischek, F. Zepeck, A. Steinreiber, F. Belaj, W. Kroutil, Angew. Chem. Int. Ed. 51, 6713-6716 (2012).

shortestes synthesisto date

28

Regioselectivity

N

(2S,6R)

H H

Cl

natural alkaloid (+)-dihydropinidine,© Wikipedia

potential antifeedant against the pine weevil Hylobius abietis[=Fichtenrüsselkäfer (ge.)]

N

(2S,6R)

H H

Cl

N

(2S,6R)

H H

Cl

Regioselectivity

N RR

O O regio- & stereoselectiveamination

spont. ring closure

NH

R

>99% ee, >99% de

"NH3"

>99% ee

[H]

R. C. Simon, B. Grischek, F. Zepeck, A. Steinreiber, F. Belaj, W. Kroutil, Angew. Chem. Int. Ed. 51, 6713-6716 (2012).

29

From Nature

Biocatalytic

CascadeApplications

Von NaWaRos zu neuen Polymeren

Alcohols to Amines

1st amination 2nd amination

22

J. H. Sattler, M. Fuchs, K. Tauber, F. G. Mutti, K. Faber, J. Pfeffer, T. Haas, W. Kroutil,Angew. Chem. Int. Ed. 51, 9156-9159 (2012).

50 mMpH 8.5, 20-30°C

10% v/v DME

400 mM20 h: 98%

30

Alcohols to Amines

1st amination 2nd amination

22

J. H. Sattler, M. Fuchs, K. Tauber, F. G. Mutti, K. Faber, J. Pfeffer, T. Haas, W. Kroutil,Angew. Chem. Int. Ed. 51, 9156-9159 (2012).

50 mMpH 8.5, 20-30°C

10% v/v DME

400 mM20 h: 98%

Tyrosinase

OH

O

OCuCu

OCu

O

- H+

O

O

R R

OCu

R

OCu(II)

OCu(II)

R

Braune Farbe

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