B ar an Group M eeting Tanja G ulder Biocatalysis 07/11/2009
Enzymes are catalysts evolved in nature to achieve the speed Induced fit and coordination of a multitude of chemical reaction necessary Model for the enzyme-substrate interaction introduced to develop and maintain life. by Koshland Enzymes are globular proteins which range from 62 (monomer of 4-oxalocrotonate tautomerase) to over 2 500 amino acid residues (animal fatty acid synthase), but only a small portion (~ 3-4 amino acids are directly involved in catalysis)
Classification
Oxidoreductases catalyze oxidation/reduction reactions T ransferases transfer a functional group (e.g. methyl group) Hyd rolases catalyze the hydrolysis of various bonds Lyases cleave various bonds by means other than hydrolysis and oxidation Isomerases catalyze isomerization changes within a single Mechanism of transitions state stabilization molecule Ligases join two molecules with covalent bonds Catalysis by bond strain affinity of the enzyme to the transition state is greater than to Enzyme reaction the substrate itself ground state destabilization effect
Catalysis by proximity and orientation enzyme-substrate interactions align reactive groups and hold them close together reduces the overall loss of entropy Catalysis involving proton donors or acceptors (acid/base catalysis) stabilization of developing charges in the transition state activation of nuceophiles and electrophiles or stabilization of leaving groups
1 Tanja G ulder B aran Gr oup Meeting Biocatalysis 07/11/2009
pyruvate decarboxylase mechanism initial step of the serine protease catalytic mechanism: O R' R' S S Me As p Me OH A sp His OH His N OH O O Me N O M e H R O H N R O N O TPP N Ser N H ylide Ser CO2 H R' S H O O O H R' S Me H N R N R' S Me N R R' H M e Me H H R' R N O H O Me H N OH O thiamine R Me py rophosphate R Electrostatic catalysis (TP P, vitamine B 1) stabilization of charged transition states by forming ionic bonds Advantages of biocatalysts and enzymes with residues of the active site - very high enantioselectivity initial step of the carboxypeptidase catalytic mechanism: - very high regioselectivity - transformation under mild conditions - 'green chemistry' e.g. solvent often water CO2 CO2 O O R Glu R Glu Disadvantages of biocatalysts and enzymes O O HN R' HN R' - often low specific activity H H - instability at extreme temperatures and pH values O O O O - availability for selected reactions only H 2+ H 2+ Zn Zn - long development time for new enzymes
advances in genomics, directed evolution, gene and genome shuffling and the exploration of Earth´s biodiversity aided by Covalent catalysis bioinformatics and high-throughput screening facilitate the substrate is forming a transient covalent bond with a residue in discovery and optimization of enzymes the active site in order to reduce energy of later transitions states of the reaction It is estimated that biocatalysis and biotransformations account for 30% of the chemical business by the year 2050
2 B ar an Group M eeting Tanja G ulder Biocatalysis 07/11/2009
Literature Biotransformations on an Industrial Scale - K. Drauz, H. Waldmann, Enzyme Catalysis in Or ganic t/a product enzyme Synthesis, Wiley-VCH, 2002 > 1 000 000 high-fructose corn syrup glucose isomerase - V. Gotor, I. Alfonso, E. Garcia-Urdiales, Asymmetric Organic > 100 000 lactose-free milk lactase Synthesis w ith Enzymes, Wiley-VCH, 2008 > 10 000 acrylamide nitrilase - E. Garcia-Junceda, Multi -Step Enzyme Catalysis, Wiley- cocoa butter lipase VCH, 2008 > 1 000 nicotinamide nitrilase - D. Enders, K.-E. Jaeger, Asymmetric Synthesi s with D-pantothenic acid aldonolactonase Chemi cal and Bioblogical Methods, Wiley-VCH, 2007 (S)-chloropropionic acid lipase - A.S. Bommarius, B. R. Riebel, Biocatalysis, Wiley-VCH, 2004 6-aminopenillanic acid penicillin amidase - G. Carrea, S. Riva, Organic Synthesis with Enzymes in Non- 7-aminocephalosporanic glutaryl amidase Aqueous Media, Wiley-VCH, 2008 acid O aspartame thermolysin Desymmetrizations O L-aspartate aspartase O D-phenylglycine hydantoinase Pig liv er MeO2 C HO2 C D-p-OH-phenylglycine hydantoinase esterase R > 100 ampicillin penicillin amidase MeO C L-methionine, L-valine aminoacylase 2 H 2O/ MeO2 C m es o ac etone O L-carnitine dehydrase/ 98%, >99% ee O O hydroxylase by ssoc hlam ic L-DOPA !-tyrosinase ac id White et al., JACS . L-malic acid fumarase 2000, 8665 (S)-methoxyisopropyl- lipase amine Pig liv er HO2C Cl (R)-mandelic acid nitrilase esterase CO2 Me MeO CO2H OMe L-alanine L-aspartate-!-de- CO Me MeO N carboxylase 2 pH 8, 7d S CO2Me H pr oc hiral 89%, 95% ee further applications: ( -)-v irantmy cin Wulff et al., A CIE baby foods, brewing industry, fruit juice, dairy industry, starch, 2004, 6493 paper, biofuels, detergents, rubber,.... enantiotopos-differentiating hydrolysis
3 B ar an Group M eeting Tanja G ulder Biocatalysis 07/11/2009
Dynamic kinetic resolutions CALB , Ac NH2 OAc NH 90% enzyme-metal combination 98% ee Ph Me Ph Me 4, Na 2CO3, R lipase toluene, 90°C, 3d R-sec-alcohol R-ester Ac yl- OR metal Ru-catalysts: subtilisin S-sec-alcohol S-ester A cyl-OR Ph O H O Ph H N Ruthenium-catalyzed reactions Ph Ph Ph Ph OAc H Ph Ph Ph 2 Ph Ru Ru Ph Cl OH CA LB , OC Ph Ru Cl OAc CO OC CO CO 78- 92% OC Ph Me > 99% ee S hov' s catalys t 1 1, toluene, 70°C, Ph R Me 24- 72h O O Ph Ph R H R used for the production of R-phenylethanol by DSM Ph R R R Ph H R Cl 3 Ph Ru R Ru Ru R OH CA LB , OAc 78-92% CO O OC CO OAc >99% ee OC CO OC Ph Me 4 [Ru], K OtB u, Na CO Ph R Me [Ru] = 2, 31h Ph 2 3, O R = p-M eO -C 6H 4 toluene, rt [Ru] = 3, 3h Ph Ph Ph 5 O Ru Cl Cl OC Ru 6 OH subtilisin, P rCO2CH 2CF3 CO O Pr Cl 95% 2 Ph Me 92% ee 2, THF, r t Ph S Me
4 B ar an Group M eeting Tanja G ulder Biocatalysis 07/11/2009
Tandem-DRK-Diels-Alder reaction Meerwein-Ponndorf-Verley-Oppenauer reaction
O OH OAc OH O CO2Et O CALB, AlMe CALB, 3 Ph Me 96%, 96% ee EtO O CO2 Et OAc Ph Me
BINOL 6, NE t3, M S, MeCN, 35°C, 3d 81%, 97% ee
O CO2Et toluene, rt, 3h O DRK with enzyme-base combination
R Hydantoinase-carbamylase system
O borate buffer, O R ( S) pH 9, 40oC R Palladium-catalyzed reactions NH NH HN HN OH or OAc OAc CA LB , iP rOH O racemase S R O Me Ph Me Ph Me R Ph THF, 25°C, D-hy dantoinas e 1.5d L- hy dantoinas e + Pd(0) - P d( 0) 71%, 98% ee iP rOAc R CO2 H Pd Ph R CO2H AcO L H2 N NH HN NH2 O Vanadium-catalyzed reactions O L-car bo- D-carbo- m yolase EtO OAc my olas e 100 kg scale pilot process CALB, OAc OH OH for tert -Leucin at Degus sa R CO2H Me R R CO2H Me Me V O(OS iPh 3)3, acetone, 25°C, 4.5d NH 91%, 99% ee 2 NH2
5 B ar an Group M eeting Tanja G ulder Biocatalysis 07/11/2009
Cyanohydrine-mediated DRK Oxidations Cl Cl OH Cl OH M andelonitrile O ly as e CN HCl drawback: co-factor dependence of oxidases/reductases R R CO2 H KCN, iPr OH/H2O quant., 83% ee solutions: - closed-loop systems with an additional enzyme for co-factor regeneration produced by DSM Chemie Linz, - electrochemical co-factor recycling Nippon Shokubai, Clariant - application of metals for regeneration - living whole cells Cl CO2Me
antiplatelet N dehydrogenase clopidogrel (P lavix) substrate produc t S (red.) (ox.)
NAD(P ) NA D( P)H
OH OH by pr oduct HCN nitr ilas e e.g. lactate cosubstrate O CN CO2H R (r ed.) lactate dehydrogenase e.g. pyruvate pH 7.2, regeneration of enzym e (ox.) >95%, >99% ee 40 oC Ps eudomonas cepac ia lipas e Oxidations of alcohols and amines applied by Lonza, BASF, OAc and Mitsubishi Rayon on a multiton scale 12-HSDH OAc OH R Me regioselective oxidations of S CN >96%, >84% ee Me bile acid depending on hydroxysteroid dehydrogenase used nonselective nitrile hydratase: Rhodococcus r hodochr ous J1 HO OH - acrylamide production (Nitto process, > 20 000 t/a) OH 7-HS DH - nicotinamide synthesis (Lonza, 3000 t/a) 3-HSDH
6 B ar an Group M eeting Tanja G ulder Biocatalysis 07/11/2009
Deracemization of secondary alcohols Oxygenation of nonactivated carbon centers almos t all C atoms at the steroid R3 OH NaB H Me nuc leus can be hy dr ox ylated 4 stereos pecific ally 2 Me CO H R OH 2 L-lactateoxidase O L-Lac tate Me O HO R1 OH py ruvate Me H Me CO2H NaBH 4 Me CO2 H H H R 1 = O , OH pr oduction of D-Lactate R 2 = H, Me O corticoster one
Deracemization of tertiary amines OH NaO C OH H 2 NaO2C S- am ine ox idase HO R N O HO variant Me O NaB H 4 N Me O H St reptomy ces Me O unnatural Me Me c arbophilus H NHMe enantiom er Me Me pH 7 N O Me 70% HO N N H M L 236B from P enicillium citr inum pravastatin 95%, >95% ee N (P ravachol) S Me produced by BMS and Sankyo Pharma N nicotine 3.6 billion US Dollar annual market value A r thr obac ter Epoxidation ox ydans P seudom onas s p. DSM 8653 O H O Rhodoc oc cus S equi O N O R Me R 70% R HO N CO2H OH R = CH CH OM e H NH 2 2 O HO N m etopr olol HN Lopresor/Toprol-X L R HO N epibatidine: analgesic hy pertens ion
7 B ar an Group M eeting Tanja G ulder Biocatalysis 07/11/2009
O Baeyer-Villiger-Oxidations O NH c yclopentanone HO B PDO O O m onoox ygenase O O OH Com am onas HO OH NCIMB 9872 O O ( +)-showdomy cin S S OH 70%, 95% ee Ph O H HO Rudroff et al. Chem . O2 O O model for predicting the regio- and Com mun. 2006, 3214 HO H stereochemical course for the cis RS RL Br (+)-tr ans-kum ausy ne selective dihydroxylation reaction H O
goniofufurone analogs OAc Br O CO2 Et Aryl dihydroxylations TDO Br
R R R AcHN cytochrom e-ty pe bacterial 77%, 99% ee OH monooxy genase diox ygenase OH OH Os eltam iv ir NH2 H3 PO4 O (Tam if lu) euk ar yots Fang et al. A CIE 2008, 5788 prokaryots OH
Me or tho and meta hydroxylation occurs us ing toluene (TDO, Ps eudom onas putida Me Me 1. DMP OH F39/D), naphthalene (NDO, P . putida 119), or biphenyl dioxy genas es (BP DO, TDO 2. O3 /DMS O O S phingom onas y anoikuy ae B8/36) O O OH R R OH Al O TDO R = H, Cl, B r, I, M e, 2 3 CN, CO2Et, etc O OH O CO2H O OH Me OH O NDO HO OH PGE 2! Hudlic ky et al. J ACS 1988, 4735
8 B ar an Group M eeting Tanja G ulder Biocatalysis 07/11/2009
ipso and ortho dioxygenations possible with Ralstonia and O OH Pseudomonas mutant strains c ar rot CO R CO R 2 R 2 A lc aligenes OH O CO2H HO C eutr ophy us HO2C 2 O OH X str ain B 9.4.5 X X = H, o-Cl, p-Cl, p-M e; R = M e, E t OH >95% ee 92-99% ee OH Formation of the 3,5-dihydroxy side chain in statins P ar ker et al. S ynlett 2004, 2095 O
X = O: topiram ate H2 NO2SO O anti- epileps y, anti- migr ane X O N Reductions O H O Reduction of aldehydes N F O HO O horse liver alc ohol produced by Pfizer Me dehy dr ogenas e atorv astatin Me Me OH (lipitor) 12.4 billion US Dollar 2008 + HO C NAD , E tOH 2 OH (OC)3Cr (OC)3Cr (OC)3Cr S R 3,5-dihydroxy side chain 33%, 91% ee 51%, 81% ee common in all statins Reduction of ketones
OMe diltiazem keto halohydrin hyper tension, angina pectoris, O r eductase OH dehydrogenase OH and s om e types of ar rhythmia Cl CO Et Cl CO2Et NC CO2 Et H 2 S R S OMe OMe R O N H H H O S baker's yeast S S O S O O Chada et al. S OH ator vastatin J . M ol. Catal. B 80%, CO2tBu N N H N 2004, 103 H O >99% ee H O 2
9 B ar an Group M eeting Tanja G ulder Biocatalysis 07/11/2009
Carbon-Carbon coupling reactions dihydroxyacetone phosphate (DHAP)-dependend aldolase stereodivergent product generation possible using stereocomplementary enzymes -> generation of 2 stereocenters Aldolreactions H 1,6-bisphosphate aldolase (FruA) 1 R 1 R R2 R 2 A ldolase OMe OBn O OH (Lyase c lass ) OMe OBn O O 1. 1,6- bisphos phate O aldolase (FruA ) R 2 H 2 HO OPO3 HO R O 2. phosphatase OMe R 1 A ldolase/ OMe HO HO Transaldolase 42% O HO O OH 1 O OH (Tr ansfer as e class) R O OH OH OH OH OH OH OH O C5 H11 Thiamine diphosphate dependent conversions HO OMe O O Shimagak i et al. Chem . P har m B ull. 1993, 282 OH HO Me 1 H R Ketolase 2 2 1 Me pentamyc in OH R R R (Lyase clas s) O O O
1 R 2 OH R acetaldehyde-dependent aldolase OH O Ketolase/ HO R1 Trans ketolase 2-deoxyribose-5-phosphate aldolase (DERA) HO O O (Transferase clas s) 2 O R -> generation of 1 stereocenters OH
Enzyme classification dependent on the nucleophile: O O DERA O 1. pyruvate-dependent aldolase Cl Cl 4 s teps Me OH 2. dihydroxyacetone phosphate (DHAP)-dependend aldolase 70%, >99.5%ee, 96.6% de 3. acetaldehyde-dependent aldolase 4. glycine-dependent aldolase O O
atorv as tatin NC CO2tBu
10 B ar an Group M eeting Tanja G ulder Biocatalysis 07/11/2009
O O Total Synthesis of Natural Products DERA O OH O O HO Me i n vitro reconstitution of complete biosynthetic pathways
Me 46% Me CO2 H Me L-tr yptophan: CO2H OH OH pheny lpyr uv ate NH 2 aminotransferase O O TdiD Me S H N N HO Me H H Me N PMPO Me CO H CO H Me Me Me Me 2 2 bisindoly l- O quinone Me Me CO tBu 2 O NH 2 s ynthetase O OH O epothilone A O OTBS TdiA AT P TdiA Wong et al. A CI E 2002, 1404 O TdiA O O HO O O O S OH S S Me DERA O HN O Me S Me O Me 35% Me NH N OH OH HO HO O NH OAc NH 21%
Pyruvate decarboxylase (PDC, thiamin diphosphate depended) Hoffm eister Cell 2007, O HN quinone OH HN 635 reductase HO HO OH H NMe, OH TdiC O PDC, 2 Me H , Pt ThDP 2 Me NADP H O OH OH O NHMe NH O Didem ethy l- Me asterriquinone D NH OH R- pheny lacetyl c ar binol (-) -ephedr ine > 98% ee Me O HN P DC, HO Walsh et al. ThDP -CO Me pr enyltrans fer as es Nat. Chem . 2 S N TdiB/TdiE B iol. 2007, ThDP Me HN O 584 N N O6 P2O O Me CO2 H Me H2 N OPP terr equinone
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ketosy nthase O O O O E nc A-E nc B, k etoreduc tase EncD, Fav or skii O Ph acyl carrier OH OH S EncC transacy lase FabD rear rangm ent protein EncC HO O ligase EncN A TP NA DPH O O O O O O COSEncC 7x 7x O HO SCoA HO S EncC
O O O H O OH O O Ph H OH O O O O methy l- 5 O OH Ph HO OH 5 trans fer as e EncC S HO OH EncK HO 9 Ph HO O O O HO SA M O O O Ph 9 OH HO O HO O MeO O desm ethyl-5- wailupemyc in G 5-deoxy enterocin deox yenterocin " fav orsk iiase" flav oprotein EncM ferredoxin, + OH OH ferr edoxin-NA DP NA DP r eductase EncR ~ 25% overall yield; O O for mation of 10 C-C, 5 C-O, and 7 stereo c enters in one pot Ph O O O O H O O Ph O Ph HO 9 HO OH HO O HO OH wailupem ycin F O HO COSEncC O O M oore et al. Nat. Chem. B iol. 2007, 557 MeO O enterocin
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