Biokatalyse – Schlüssel zur effizienteren organischen Synthese · 1 Wolfgang Kroutil ,...
Transcript of Biokatalyse – Schlüssel zur effizienteren organischen Synthese · 1 Wolfgang Kroutil ,...
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Wolfgang KroutilWolfgang Kroutil, ,
University of Graz, AustriaUniversity of Graz, Austria
[email protected]@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
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„moderne“ Mensch
Energie/Treibstoffe
KleidungNahrungsmittel
Kosmetika Pharmazeutika
Wohnen
Chemische Industrie
Chemische Reaktionen
Beispiele chemischer Umsetzungen
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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??
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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
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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
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… 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
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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
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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
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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
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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
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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
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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
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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
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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
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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).
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From Nature
Biocatalytic
CascadeApplications
Von NaWaRos zu neuen Polymeren
Alcohols to Amines
1st amination 2nd amination
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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%
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Alcohols to Amines
1st amination 2nd amination
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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