B ar an Gro u p M ee tin g

Ta nj a G ul de r
Biocatalysis 0 7 /11 /2 00 9

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
Oxidor eductases catalyze oxidation/reduction reactions
T r ansfer ases transfer a functional group (e.g. methyl group)
Hyd r olases catalyze the hydrolysis of various bonds
Lyases cleave various bonds by means other than hydrolysis and
oxidation
Isomer ases catalyze isomerization changes within a single Mechanism of trans itions stat e st abilization
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 proxim ity and orientation

enzyme-substrate interactions align reactive groups and hold
them close together reduces the overall loss of entropy
Catalysis involving pr oton donor s or acceptors
(acid/base catalysis)
stabilization of developing charges in the transition state
activation of nuceophiles and electrophiles or
stabilization of leaving groups

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 B a ra n Gr ou p Me eti ng
Ta nj a G ul de r
Biocatalysis 07 /1 1/2 0 09

pyruvate decarboxylase mechanism

initial step of the serine protease catalytic mechanism:
R' O R'
S S Me
As p Me OH
A sp His OH
H is N
O O Me N Me OH
O
H H R O
N R
N O
O TPP
N N H yl id e
Se r C O2
Se r R'
H S
H O
O H R' Me
O S
H N R R' Me
Me N Me H H S
N R R' H
R' R N OH
O Me H N OH
O th ia mi n e R Me
py ro ph o sp h ate R
Electrostatic catalysis (TP P, vita mi n e 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
C O2 C O2
O R O R Disadvantages of biocatalysts and enzymes
Glu Glu
O O HN R'
HN R'
H
- often low specific activity
H - instability at extreme temperatures and pH values
O O O O
H 2+ H 2+
- availability for selected reactions only
Zn Zn - long development time for new enzymes

advances in genomics, directed evolution, gene and genome

Covalent catalysis shuffling and the exploration of Earth´s biodiversity aided by
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

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 B ar an Gro u p M ee tin g
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Biocatalysis 0 7 /11 /2 00 9

Literature
Biotransformations on an Industrial Scale
- K. Drauz, H. Waldmann, Enzy me Cataly sis 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, Asy mmetric Organic
Synthesis w ith Enzy mes, Wiley-VCH, 2008
> 100 000 lactose-free milk lactase
- E. Garcia-Junceda, Multi -Step Enzy me Catalysis, Wiley-
> 10 000 acrylamide nitrilase
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, Biocataly sis, Wiley-VCH, 2004
- G. Carrea, S. Riva, Organic Sy nthesis with Enzymes in Non-
6-aminopenillanic acid penicillin amidase
Aqueous Media, Wiley-VCH, 2008
7-aminocephalosporanic glutaryl amidase
acid
aspartame thermolysin Desy mmetriza tions O
O
L-aspartate aspartase O
D-phenylglycine hydantoinase Me O2 C Pi g l iv er
H O2 C
e ste ra se
D-p-OH-phenylglycine hydantoinase
R
> 100 ampicillin penicillin amidase
Me O2 C H 2 O/ Me O2 C
L-methionine, L-valine aminoacylase
m es o ac eto n e
L-carnitine dehydrase/ 9 8 % , >9 9 % e e O O
O
hydroxylase by sso c hl am ic
L-DOPA !-tyrosinase ac id
Wh ite et a l., JAC S .
L-malic acid fumarase 2 0 00 , 86 6 5
(S)-methoxyisopropyl- lipase
amine Pi g l iv er HO 2C Cl
e ste ra se
(R)-mandelic acid nitrilase C O2 Me CO 2H OMe
L-alanine L-aspartate-!-de- MeO
C O2 Me p H 8, 7 d Me O N
S CO 2Me
carboxylase H
pr oc hi ra l 89%, 95% ee ( -)-v ira n tmy cin
further applications:
Wu lff e t al ., A C IE
baby foods, brewing industry, fruit juice, dairy industry, starch, 2 0 04 , 64 9 3
paper, biofuels, detergents, rubber,.... enantiotopos-differentiating hydrolysis

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Biocatalysis 0 7 /11 /2 00 9

Dynamic k inetic resolut ions

NH2 C AL B , OAc Ac
NH 90%
enzyme-metal combination 9 8 % ee
Ph Me
Ph Me
4 , Na 2 C O 3, R
lipase to lu en e , 9 0 °C , 3 d
R-sec-alcohol R-ester
Ac yl- OR

metal Ru-catalysts:
subtilisin
S-sec-alcohol S-ester
Ph O O H
A cyl -OR H Ph
Ph N
Ruthenium-catalyzed reactions
Ph Ph
OAc Ph Ph
H Ph
Ph Ru Ph 2
Ru Ph Cl
OH C A LB , Ph Ru
Cl OAc OC CO
CO OC CO
7 8- 92 % OC
Ph Me > 9 9% e e S ho v' s ca tal ys t 1
1, to lu e ne , 70 °C, Ph R Me
2 4- 72 h O O
Ph R H R
Ph
used for the production of R-phenylethanol by DSM
R R
Ph R R
Ph H
Cl 3 R Ru
Ph Ru Ru R
OH C A LB , OAc 7 8 -9 2% CO O OC
OAc OC CO OC CO
>9 9 % e e
4
Ph Me
Ph R Me [R u ] = 2 , 31 h Ph
[R u ], K OtB u, N a 2C O 3, R = p -M eO -C 6H 4
[R u ] = 3 , 3h O
to lu e ne , rt
Ph
Ph
Ph 5
O Ru Cl Cl
OC Ru 6
OH su b tili si n, P rC O 2 C H 2C F 3 CO
O Pr Cl
95% 2
Ph Me 92 % ee
2 , TH F, r t Ph S Me

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Biocatalysis 0 7 /11 /2 00 9

Tandem-DRK-Diels-Alder reaction Meerwein-Ponndorf-Verley-Oppenauer reaction

O OH
OH O CO 2Et O C AL B, Al Me 3 OAc
C AL B, 9 6% , 9 6% e e
C O2 Et Ph Me Ph Me
EtO O OAc

6 , NE t 3 , M S, Me C N , 3 5 °C , 3 d BIN OL

8 1% , 97 % e e
O CO 2Et to l ue n e, rt, 3 h

O DRK with enzyme-base combination

R
Hydantoinase-carbamylase system

O bo ra te b u ffe r, O
R R
( S)
p H 9, 4 0o C
Palladium-catalyzed reactions NH NH
HN HN
OAc OAc OH or
C A LB , i P rOH O ra ce ma se
S R O
Me Ph Me Ph Me R Ph
TH F, 25 °C , D -hy da n toi n as e
1 .5 d L- hy da n toi na s e
+ Pd (0 ) - P d( 0) 7 1% , 9 8% e e

i P rOAc
R C O2 H R CO 2H
Pd Ph
AcO L H2 N NH HN N H2
O O
Vanadium-catalyzed reactions L -ca r bo -
D -ca rb o -
m yo la se
EtO OAc my ol as e
OAc 10 0 kg sca le p il ot p ro ce ss
OH C AL B,
OH fo r te rt -Le u ci n a t De g us sa
Me R R CO 2H
Me R CO 2H
Me V O(OS iPh 3 )3 ,
a ce ton e , 2 5 °C , 4 .5 d NH2 NH2
9 1% , 9 9% e e

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Biocatalysis 0 7 /11 /2 00 9

Cyanohydrine-mediated DRK Ox idations

Cl Cl OH Cl OH
M an d el o ni tril e
ly as e HCl drawback: co-factor dependence of oxidases/reductases
O R CN R C O2 H
KC N ,
i Pr OH /H2 O qu a n t., 8 3% e e 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 CO 2Me

a n ti p la tel et N de h yd ro g en a se
cl o pi d og re l (P la vi x) su b stra te p ro d uc t
S (re d .) (o x.)

N AD (P ) N A D( P)H

OH OH
by pr od u ct
H CN n itr il as e co su b stra te
e .g. la cta te
O CN R CO 2H (r e d.) la cta te d eh yd ro g e na se e .g. p yru va te
pH 7 .2 , re g e ne ra tio n o f e n zym e (o x.)
>9 5 %, >9 9% e e
4 0 oC
Ps eu d o mo n a s
ce p ac ia li p as e
Oxidations of alcohols and amines
applied by Lonza, BASF,
OAc
and Mitsubishi Rayon on
a multiton scale
1 2 -H SD H
OAc
OH
Me R
regioselective oxidations of
S CN >9 6 %, >8 4% e e
bile acid depending on
Me
hydroxysteroid
dehydrogenase used
nonselective nitrile hydratase: Rhodococcus r hodochr ous J1 HO OH
- acrylamide production (Nitto process, > 20 000 t/a) OH 7 -H S DH
- nicotinamide synthesis (Lonza, 3000 t/a) 3 -H SD H

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Biocatalysis 0 7 /11 /2 00 9

Deracemization of secondary alcohols Oxygenation of nonactivated carbon centers

3 al mo s t a ll C a to ms a t th e ste ro id
OH Me R n uc le u s ca n b e hy dr ox yl ate d
N aB H 4
ste re os pe ci fic al ly
Me CO 2H R2 OH
L -l a ctate o xid a se O
L -L ac tate O Me
HO
R1
OH p y ru va te Me H
Me CO 2H
N a BH 4
Me C O2 H H H
R 1 = O , OH pr od u ctio n of
O
D -L a cta te R 2 = H , Me co rti co ster o ne

Deracemization of tertiary amines OH

NaO 2 C OH
H N aO 2C
S- am in e ox id a se HO
R N
Me O HO
va ri an t O
Na B H4 N Me O St re p to my ce s
H Me O
u n na tu ra l Me Me c a rb o ph i l us H
N HMe Me Me
pH 7 en a nti om e r
N
O Me 70%
HO
N
N M L 236B
H p ra va stati n
fro m P en i ci l l i um ci tr i nu m
95 % , >9 5 % ee N (P ra va ch o l)
S Me
N
produced by BMS and Sankyo Pharma
ni co tin e 3.6 billion US Dollar annual market value
A r thr o b ac te r Epoxidation
o x yd a n s P se u d om o na s
s p. D SM 86 5 3
R h o d oc oc cu s O
H O O
S equi
N R
O
Me
R 70 % R
HO N CO 2H OH
R = CH 2 C H 2OM e
H NH O
HO N
m eto pr o lo l
Lo p re so r/To p ro l-X L HN
R
HO N e p ib a ti d in e: a na l ge si c h y pe rte ns io n

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Biocatalysis 0 7 /11 /2 00 9

O
Baeyer-Villiger-Oxidations
O
NH
c ycl op e nta n on e HO B PD O
O O
m on o ox yg en a se
O O OH
Co m am o na s HO OH
O N C IMB 98 7 2 O
S S ( +)-sh o wd o my ci n
OH
7 0% , 9 5% e e Ph H
O
HO
R ud ro ff e t a l . C h em . O O2 model for predicting the regio- and
C om mu n . 20 0 6, 3 21 4 HO O
H stereochemical course for the cis
RS RL
Br (+)-tr a n s-ku m au sy ne selective dihydroxylation reaction
H
O

g on i ofu fu ro n e a n al og s
OAc
Aryl dihydroxylations Br Br O CO 2 Et
TD O

R R R AcHN
b a cte ria l 77%, 99% ee OH
cyto ch ro m e-ty pe
mo n oo xy ge n a se d io x yg en a se OH OH Os el tam iv ir NH2 H3 PO4
O (Tam if lu )
e uk ar yo ts p ro ka ryo ts Fa n g e t al . A C IE 20 0 8 , 5 7 88
OH

o r tho a nd me ta h yd ro xyl ati on o ccu rs us in g to lu e ne (TD O, Ps e ud o m on a s pu ti d a Me Me Me

1. D MP
F3 9 /D ), n a p hth al e ne (N D O, P . pu ti d a 11 9 ), or bi ph e n yl d io xy ge n as e s (BP D O, OH 2. O 3 /D MS O O
TD O
S ph i n go m on a s y an o i ku y a e B8 /3 6) O O
OH
R R

TD O OH Al 2O 3
R = H , Cl , B r, I, M e ,
CN , C O 2 Et, etc O
OH O
CO 2H
OH O
Me
OH
NDO HO O
OH
PGE 2 !
H u dl ic ky e t al . J AC S 1 9 8 8, 4 73 5

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Biocatalysis 0 7 /11 /2 00 9

ipso and ortho dioxygenations possible with Ralstonia and O OH

Pseudomonas mutant strains c ar ro t
C O2 R CO 2R
R
CO 2H A lc a li g e ne s OH O
eu tr o ph y u s HO 2C HO 2C O
OH X X
str ai n B 9.4 .5
X = H , o -C l , p -C l, p -M e; R = M e , E t
>95 % ee OH
9 2 -99 % e e
OH
Formation of the 3,5-dihydroxy side chain in statins
P ar ke r e t a l. S yn l e tt 20 0 4, 2 0 95
O
X = O: to p ira m ate H2 NO 2SO O
an ti- ep i le ps y, an ti- mi gr an e X O
N
Re ductions O H
O
Reduction of aldehydes N
F
O
HO O
h o rse li ve r a lc oh o l produced by Pfizer
Me d e hy dr og e n as e a to rv a stati n
Me Me OH (l ip ito r) 12.4 billion US Dollar 2008
+ HO 2C
N AD , E tOH OH
(OC )3Cr
(OC )3Cr (OC )3Cr
S R 3,5-dihydroxy side chain
33 % , 91 % e e 5 1 % , 8 1 % ee common in all statins
Reduction of ketones
OMe d il tia ze m ke to h a lo h yd rin
h yp er te n si on , an g in a p e cto ri s, O r ed u cta se OH de h yd ro g en a se OH
a n d s om e typ e s o f ar rh yth mi a Cl CO 2Et NC CO 2 Et
H Cl CO 2 Et
S R
S OMe OMe
R O
N H H
H O S
baker's
S
S yeast S
C h a da et a l. O OH O O
S a tor va sta ti n
J . M o l . C a ta l. B N 80 % , CO 2tBu
2 0 0 4, 1 03 N H 2N
H O >9 9% e e H O

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Biocatalysis 0 7 /11 /2 00 9

Ca rbon-Ca rbon coupling reac tions

dihydroxyacetone phosphate (DHAP)-dependend aldolase
stereodivergent product generation possible using
stereocomplementary enzymes -> generation of 2 stereocenters
Aldolreactions
H 1,6-bisphosphate aldolase (FruA)
1 1
R R
R2 A ld o la se
R2 OMe OBn
O OH (L ya se c la ss ) OMe OBn
O O 1 . 1 ,6- bi sp h os ph a te
O
a ld o la se (Fru A )
H R2 HO OPO 3
R2 HO
O 2. p ho sp h ata se OMe
R
1 A ld ol a se / OMe
HO HO Tra n sa ld o la se 42 % O
(Tr an sfe r as e cl a ss) HO
O OH R1 O OH
O OH OH OH OH OH OH
OH
O C5 H11
HO
Thiamine diphosphate dependent conversions OMe O O
Sh i ma g ak i e t a l. C h em .
P ha r m B ul l . 19 9 3, 2 82
OH HO Me
H R1 Ke tol a se
R2 Me p en ta myc in OH
R2 R1 (L ya se cl as s)
O O O
1
R
OH R2
OH
acetaldehyde-dependent aldolase
Ke tol a se /
O
HO R1 Tra ns ke tol a se
HO O O (Tra n sfe ra se cl as s) 2-deoxyribose-5-phosphate aldolase (DERA)
O R2
-> generation of 1 stereocenters OH

Enzyme classification dependent on the nucleophile: O O D ER A O

1. pyruvate-dependent aldolase Cl Cl 4 s te p s
Me OH
2. dihydroxyacetone phosphate (DHAP)-dependend aldolase 70 % ,
>9 9.5 % e e, 9 6.6 % d e
3. acetaldehyde-dependent aldolase
4. glycine-dependent aldolase O O
ato rv as tati n NC CO 2tBu

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Biocatalysis 0 7 /11 /2 00 9

O O
D ER A O OH O O
Tot al Synthesis of Natural Products
HO Me i n v itro reconstitution of complete biosynthetic pathways
Me 46% Me Me C O2 H
L-tr yp to p h an : CO 2H
OH OH ph e ny lp yr uv ate
NH2 a mi n otra n sfe ra se O
O Td iD
Me S N
H Me H N
HO PMPO H
Me N Me CO 2H CO 2 H
Me Me Me Me bi si nd o ly l-
O qu i no n e
Me Me C O2 tBu NH 2
O s yn the ta se
O OH O e p oth il o ne A O OTBS Tdi A
AT P
Td iA Td iA
Wo ng e t a l. A CI E 2 0 02 , 14 0 4 O O
O OH
HO O Me D ER A O O O S
S S HN O
Me S Me O
Me 3 5% Me NH
N OH OH HO HO
O NH
OAc NH
21 %

H o ffm ei ste r
Pyruvate decarboxylase (PDC, thiamin diphosphate depended) C e l l 2 0 0 7, O HN q u in o ne OH HN
635 HO re du cta se
OH Td iC HO
O H2 N Me , OH
PD C , H2 , Pt
Me Me
O Th D P OH N AD P H
OH
O NHMe NH O D id em e thy l-
Me a ste rri qu i no n e D NH OH
R - ph e ny la ce tyl c ar bi n ol (-) -e ph e dr in e
> 9 8% e e Me O
P DC , HO HN
Th D P Wa l sh e t a l.
Me p r en yl tra ns fer as es N a t. C h em .
-C O 2
Th D P S N Me Tdi B/Td iE B i ol . 2 00 7 ,
HN 5 84
O N
O6 P2O N O
Me CO 2 H Me H2 N OPP
te rr eq u in o ne

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ke to sy nth a se O O
O O E nc A-E nc B,
k eto re du c ta se En cD , Fa v or ski i O Ph
a cyl ca rri e r OH
OH tra n sa cy la se Fab D re ar ra ng m en t
p ro te in En cC S EncC
HO O
li ga se En cN
A TP NA D PH
O
O
O O O O C OSEn cC
7x 7x O
HO SCoA HO S EncC

O H O
O OH O
O H O Ph
OH
O O O O O me thy l- 5
OH Ph tra ns fer as e
HO OH
5
EncC S HO OH En cK HO
HO 9 Ph
O O O HO SA M O
O O
Ph 9 OH HO O
HO O MeO O
de sm e th yl -5 -
w a il up e myc in G 5 -d eo xy en te ro ci n d e ox ye nte ro ci n
" fav o rsk iia se "
fl av op ro te in
En cM fe rre d o xin ,
fe rr ed o xin -N A DP +
OH OH NA D P
r ed u cta se
~ 2 5 % o ve ra l l yi el d ;
En cR
O O for ma tio n o f 1 0 C -C , 5 C -O,
a n d 7 ste re o c en te rs i n o n e p ot
Ph O
O O H
O
O O Ph O
HO 9 HO OH Ph
HO O HO OH
O
wa i lu pe m yci n F HO
COSEncC
O O
M oo re e t a l. N a t. C he m. B io l .
20 0 7, 5 57
MeO O
e nte ro ci n

12