Biocatalysis
Ta nj a G ul de r
Enzymes are catalysts evolved in nature to achieve the speed
and coordination of a multitude of chemical reaction necessary
to develop and maintain life.
B ar an Gro u p M ee tin g
0 7 /11 /2 00 9
Induced fit
Model for the enzyme-substrate interaction introduced
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
Mechanism of trans itions stat e st abilization
Isomer ases catalyze isomerization changes within a single
molecule
Catalysis by bond strain
Ligases join two molecules with covalent bonds
Enzyme reaction
affinity of the enzyme to the transition state is greater than to
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
1
B a ra n Gr ou p Me eti ng
07 /1 1/2 0 09
Biocatalysis
Ta nj a G ul de r
pyruvate decarboxylase mechanism
initial step of the serine protease catalytic mechanism:
R'
A sp
As p
H is
O
H
O
O
N
His
H
O
N
Me
N
N
Se r
H
R'
H
O
H
N
Se r
N
R' H
R
O
R
Electrostatic catalysis
stabilization of charged transition states by forming ionic bonds
with residues of the active site
initial step of the carboxypeptidase catalytic mechanism:
O
Glu
R
O
O R
Glu
HN
R'
H
O
H
C O2
C O2
O
HN
Zn
R'
O
O
H
S
N
Me
Me
R
th ia mi n e
py ro ph o sp h ate
(TP P, vita mi n e B 1)
R'
S
Me
N
R
OH
O
Me
OH
OH
O
C O2
O
R'
S
H
Me
N
R
Me
H
OH
H
R'
S
Me
Me
N
R
OH
Advantages of biocatalysts and enzymes
- very high enantioselectivity
- very high regioselectivity
- transformation under mild conditions
- 'green chemistry' e.g. solvent often water
Disadvantages of biocatalysts and enzymes
H
O
2+
Me
N
R
TPP
yl id e
H
O
O
H
R'
O
S
Zn
2+
Covalent catalysis
substrate is forming a transient covalent bond with a residue in
the active site in order to reduce energy of later transitions
states of the reaction
- often low specific activity
- instability at extreme temperatures and pH values
- availability for selected reactions only
- long development time for new enzymes
advances in genomics, directed evolution, gene and genome
shuffling and the exploration of Earth´s biodiversity aided by
bioinformatics and high-throughput screening facilitate the
discovery and optimization of enzymes
It is estimated that biocatalysis and biotransformations
account for 30% of the chemical business by the year 2050
2
B ar an Gro u p M ee tin g
0 7 /11 /2 00 9
Biocatalysis
Ta nj a G ul de r
Literature
Biotransformations on an Industrial Scale
t/a
product
> 1 000 000 high-fructose corn syrup
> 100 000
lactose-free milk
> 10 000
acrylamide
cocoa butter
> 1 000
nicotinamide
D-pantothenic acid
(S)-chloropropionic acid
6-aminopenillanic acid
7-aminocephalosporanic
acid
aspartame
L-aspartate
D-phenylglycine
D-p-OH-phenylglycine
> 100
ampicillin
L-methionine, L-valine
L-carnitine
L-DOPA
L-malic acid
(S)-methoxyisopropylamine
(R)-mandelic acid
L-alanine
enzyme
glucose isomerase
lactase
nitrilase
lipase
nitrilase
aldonolactonase
lipase
penicillin amidase
glutaryl amidase
thermolysin
aspartase
hydantoinase
hydantoinase
penicillin amidase
aminoacylase
dehydrase/
hydroxylase
!-tyrosinase
fumarase
lipase
nitrilase
L-aspartate-!-decarboxylase
further applications:
baby foods, brewing industry, fruit juice, dairy industry, starch,
paper, biofuels, detergents, rubber,....
- K. Drauz, H. Waldmann, Enzy me Cataly sis in Or ganic
Synthesis, Wiley-VCH, 2002
- V. Gotor, I. Alfonso, E. Garcia-Urdiales, Asy mmetric Organic
Synthesis w ith Enzy mes, Wiley-VCH, 2008
- E. Garcia-Junceda, Multi -Step Enzy me Catalysis, WileyVCH, 2008
- D. Enders, K.-E. Jaeger, Asymmetric Synthesi s with
Chemi cal and Bioblogical Methods, Wiley-VCH, 2007
- A.S. Bommarius, B. R. Riebel, Biocataly sis, Wiley-VCH, 2004
- G. Carrea, S. Riva, Organic Sy nthesis with Enzymes in NonAqueous Media, Wiley-VCH, 2008
Desy mmetriza tions
Me O2 C
Pi g l iv er
e ste ra se
Me O2 C
H 2 O/
ac eto n e
O
O
O
H O2 C
R
m es o
Me O2 C
Pi g l iv er
e ste ra se
MeO
C O2 Me
C O2 Me p H 8, 7 d
pr oc hi ra l
O
O
O
by sso c hl am ic
ac id
Wh ite et a l., JAC S .
2 0 00 , 86 6 5
9 8 % , >9 9 % e e
HO 2C
Me O
CO 2H
N
H
S CO 2Me
89%, 95% ee
Cl
OMe
( -)-v ira n tmy cin
Wu lff e t al ., A C IE
2 0 04 , 64 9 3
enantiotopos-differentiating hydrolysis
3
B ar an Gro u p M ee tin g
0 7 /11 /2 00 9
Biocatalysis
Ta nj a G ul de r
Dynamic k inetic resolut ions
C AL B ,
NH2
enzyme-metal combination
Ph
Me
Ph
4 , Na 2 C O 3,
to lu en e , 9 0 °C , 3 d
lipase
R-sec-alcohol
Ac
OAc
NH
R
90%
9 8 % ee
Me
R-ester
Ac yl- OR
Ru-catalysts:
metal
subtilisin
S-sec-alcohol
S-ester
O
Ph
A cyl -OR
Ph
Ph
OAc
Ph
OH
Ph
Me
Cl
1, to lu e ne , 70 °C,
2 4- 72 h
Ph R Me
7 8- 92 %
> 9 9% e e
Ph
Me
OAc
CO
[R u ], K OtB u, N a 2C O 3,
to lu e ne , rt
Ph R Me
OC
Ph
Ph
Ph
CO
Ru
Ph
Cl
OC
2
CO
S ho v' s ca tal ys t 1
Ph
Ru
OC
Ph
Cl
O
R
Ph
Ph
7 8 -9 2%
>9 9 % e e
OAc
Ph
Ru
Ph
C A LB ,
Ph
H
Ru
OC
OAc
used for the production of R-phenylethanol by DSM
OH
H
N
Ph
Ph
Ruthenium-catalyzed reactions
C A LB ,
O
H
R
R
3
CO
R
O
O
H
OC
R
R
R
H
Ru
Ru
CO
OC
R
CO
4
[R u ] = 2 , 31 h
[R u ] = 3 , 3h
Ph
R = p -M eO -C 6H 4
O
Ph
Ph
O
OH
Ph
Me
su b tili si n, P rC O 2 C H 2C F 3
2 , TH F, r t
OC
O
Pr
Ph S Me
95%
92 % ee
Ph
Ru Cl
CO
5
Cl
Ru
Cl
6
2
4
B ar an Gro u p M ee tin g
0 7 /11 /2 00 9
Biocatalysis
Ta nj a G ul de r
Tandem-DRK-Diels-Alder reaction
OH
O
C AL B,
EtO
Meerwein-Ponndorf-Verley-Oppenauer reaction
CO 2Et
O
O
OH
C O2 Et
O
Ph
OAc
C AL B, Al Me 3
Me
Ph
OAc
9 6% , 9 6% e e
Me
BIN OL
6 , NE t 3 , M S, Me C N , 3 5 °C , 3 d
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
R
Palladium-catalyzed reactions
OAc
Me
OAc
Ph
Me
- P d( 0)
+ Pd (0 )
OH
TH F, 25 °C ,
1 .5 d
Me R
S
Ph
7 1% , 9 8% e e
i P rOAc
AcO
H2 N
EtO
OH
Me
or
ra ce ma se
HN
D -hy da n toi n as e
C O2 H
NH
R
C AL B,
V O(OS iPh 3 )3 ,
a ce ton e , 2 5 °C , 4 .5 d
9 1% , 9 9% e e
N H2
O
D -ca rb o my ol as e
10 0 kg sca le p il ot p ro ce ss
fo r te rt -Le u ci n a t De g us sa
OAc
Me R
CO 2H
HN
L -ca r bo m yo la se
OAc
NH
R O
O
Vanadium-catalyzed reactions
Me
R
L
OH
O
O
bo ra te b u ffe r,
R
p H 9, 4 0o C
L- hy da n toi na s e
Ph
Pd
NH
HN
C A LB , i P rOH
Ph
O
( S)
R
CO 2H
NH2
R
CO 2H
NH2
5
B ar an Gro u p M ee tin g
0 7 /11 /2 00 9
Biocatalysis
Ta nj a G ul de r
Cyanohydrine-mediated DRK
Ox idations
Cl
Cl
O
M an d el o ni tril e
ly as e
KC N ,
i Pr OH /H2 O
OH
Cl
R CN
OH
HCl
R C O2 H
solutions: - closed-loop systems with an additional enzyme
for co-factor regeneration
- electrochemical co-factor recycling
- application of metals for regeneration
- living whole cells
qu a n t., 8 3% e e
produced by DSM Chemie Linz,
Nippon Shokubai, Clariant
Cl
CO 2Me
N
a n ti p la tel et
cl o pi d og re l (P la vi x)
drawback: co-factor dependence of oxidases/reductases
S
de h yd ro g en a se
su b stra te
(re d .)
N AD (P )
OH
OH
O
H CN
CN
n itr il as e
pH 7 .2 ,
4 0 oC
Ps eu d o mo n a s
ce p ac ia li p as e
R CO 2H
la cta te d eh yd ro g e na se
re g e ne ra tio n o f e n zym e
co su b stra te
e .g. p yru va te
(o x.)
Oxidations of alcohols and amines
1 2 -H SD H
OAc
S CN
N A D( P)H
>9 5 %, >9 9% e e
applied by Lonza, BASF,
and Mitsubishi Rayon on
a multiton scale
OAc
by pr od u ct
e .g. la cta te
(r e d.)
p ro d uc t
(o x.)
OH
Me R
regioselective oxidations of
bile acid depending on
hydroxysteroid
dehydrogenase used
>9 6 %, >8 4% e e
nonselective nitrile hydratase: Rhodococcus r hodochr ous J1
- acrylamide production (Nitto process, > 20 000 t/a)
- nicotinamide synthesis (Lonza, 3000 t/a)
Me
HO
OH
OH
7 -H S DH
3 -H SD H
6
Oxygenation of nonactivated carbon centers
Deracemization of secondary alcohols
OH
Me
R2
L -l a ctate o xid a se
L -L ac tate
O
OH
Me
N a BH 4
C O2 H
R1
p y ru va te
CO 2H
Me
Deracemization of tertiary amines
NaO 2 C
H
S- am in e ox id a se
va ri an t
Na B H4
N HMe
pH 7
R N
Me
N
u n na tu ra l
en a nti om e r
N
Me
O
H
O
CO 2H
H
NH
HO
HO
N
St re p to my ce s
Me c a rb o ph i l us
Me
N
e p ib a ti d in e: a na l ge si c
O
Me
H
Me
HO
p ra va stati n
(P ra va ch o l)
Epoxidation
O
N
HO
produced by BMS and Sankyo Pharma
3.6 billion US Dollar annual market value
ni co tin e
P se u d om o na s
s p. D SM 86 5 3
S
N
Me
OH
N aO 2C
M L 236B
fro m P en i ci l l i um ci tr i nu m
N
S Me
N
HO
O
pr od u ctio n of
co rti co ster o ne
OH
O
Me
H
95 % , >9 5 % ee
A r thr o b ac te r
o x yd a n s
O
70%
N
N
H
HO
O
Me
H
H
R 1 = O , OH
R 2 = H , Me
D -L a cta te
H
al mo s t a ll C a to ms a t th e ste ro id
n uc le u s ca n b e hy dr ox yl ate d
ste re os pe ci fic al ly
OH
O
Me
HO
3
Me R
N aB H 4
CO 2H
Me
B ar an Gro u p M ee tin g
0 7 /11 /2 00 9
Biocatalysis
Ta nj a G ul de r
R
R h o d oc oc cu s
equi
70 %
O
O
R
R
OH
R = CH 2 C H 2OM e
O
m eto pr o lo l
Lo p re so r/To p ro l-X L
h y pe rte ns io n
R
HN
7
O
Baeyer-Villiger-Oxidations
O
O
O
c ycl op e nta n on e
m on o ox yg en a se
Co m am o na s
N C IMB 98 7 2
O
O
HO
O
S
NH
HO
B PD O
O
OH
OH
( +)-sh o wd o my ci n
S
7 0% , 9 5% e e
Ph
O
OH
H
HO
R ud ro ff e t a l . C h em .
C om mu n . 20 0 6, 3 21 4
HO
Br
H
H
O
O2
O
RS
(+)-tr a n s-ku m au sy ne
g on i ofu fu ro n e a n al og s
OAc
Br
R
cyto ch ro m e-ty pe
mo n oo xy ge n a se
RL
model for predicting the regio- and
stereochemical course for the cis
selective dihydroxylation reaction
O
Aryl dihydroxylations
O
B ar an Gro u p M ee tin g
0 7 /11 /2 00 9
Biocatalysis
Ta nj a G ul de r
R
R
b a cte ria l
d io x yg en a se
e uk ar yo ts
p ro ka ryo ts
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
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,
Os el tam iv ir NH2 H3 PO4
(Tam if lu )
OH
Fa n g e t al . A C IE 20 0 8 , 5 7 88
OH
Me
Me
OH
TD O
1. D MP
2. O 3 /D MS
S ph i n go m on a s y an o i ku y a e B8 /3 6)
OH
R
CO 2 Et
AcHN
OH
77%, 99% ee
OH
O
Br
TD O
Me
O
O
O
O
R
OH
TD O
OH
R = H , Cl , B r, I, M e ,
CN , C O 2 Et, etc
Al 2O 3
O
CO 2H
OH
NDO
Me
OH
HO
OH
PGE 2 !
O
O
O
H u dl ic ky e t al . J AC S 1 9 8 8, 4 73 5
8
B ar an Gro u p M ee tin g
0 7 /11 /2 00 9
Biocatalysis
Ta nj a G ul de r
ipso and ortho dioxygenations possible with Ralstonia and
Pseudomonas mutant strains
CO 2H A lc a li g e ne s
eu tr o ph y u s HO 2C
str ai n B 9.4 .5
OH
HO 2C
OH
c ar ro t
C O2 R
O
O
R
CO 2R
X
X
X = H , o -C l , p -C l, p -M e; R = M e , E t
9 2 -99 % e e
OH
>95 % ee
OH
O
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
an ti- ep i le ps y, an ti- mi gr an e
H2 NO 2SO
O
X
O
N
H
O
Re ductions
O
Reduction of aldehydes
N
O
Me
HO
h o rse li ve r a lc oh o l
d e hy dr og e n as e
Me
Me
OH
+
HO 2C
N AD , E tOH
(OC )3Cr
(OC )3Cr
S
33 % , 91 % e e
Reduction of ketones
OMe
S
a to rv a stati n
(l ip ito r)
produced by Pfizer
12.4 billion US Dollar 2008
OH
3,5-dihydroxy side chain
common in all statins
R
5 1 % , 8 1 % ee
OMe
R
N
H
(OC )3Cr
d il tia ze m
h yp er te n si on , an g in a p e cto ri s,
a n d s om e typ e s o f ar rh yth mi a
H
F
O
ke to
r ed u cta se
O
Cl
CO 2 Et
OH
Cl
S
h a lo h yd rin
de h yd ro g en a se
CO 2Et
OH
NC
R
CO 2 Et
OMe
O
O
C h a da et a l.
J . M o l . C a ta l. B
2 0 0 4, 1 03
H
S
S
N
H
O
O
baker's
yeast
80 % ,
>9 9% e e
H
S
S
S
N
H
OH
O
O
a tor va sta ti n
H 2N
O
CO 2tBu
9
Ca rbon-Ca rbon coupling reac tions
dihydroxyacetone phosphate (DHAP)-dependend aldolase
stereodivergent product generation possible using
stereocomplementary enzymes
Aldolreactions
-> generation of 2 stereocenters
1,6-bisphosphate aldolase (FruA)
H
1
R
R2
R
R2
O
O
O
1
A ld o la se
(L ya se c la ss )
OH
HO
R
HO
OH
OMe OBn
OMe OBn
1 . 1 ,6- bi sp h os ph a te
a ld o la se (Fru A )
O
R2
H
O
B ar an Gro u p M ee tin g
0 7 /11 /2 00 9
Biocatalysis
Ta nj a G ul de r
O
R1
R2
1
OH
O
A ld ol a se /
Tra n sa ld o la se
(Tr an sfe r as e cl a ss)
O
HO
OPO 3
2. p ho sp h ata se
42 %
OMe
OH
OH
OH
OH
OH
HO
OH
R1
R2
O
O
HO
O
O
O
HO
Ke tol a se
(L ya se cl as s)
R1
1
R2
OH
R1
HO
O
O
OMe
Me
Me
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
p en ta myc in
O
R
OH
OH
C5 H11
Thiamine diphosphate dependent conversions
R2
OMe
O
HO
OH
O
H
HO
O
Ke tol a se /
Tra ns ke tol a se
(Tra n sfe ra se cl as s)
R2
Enzyme classification dependent on the nucleophile:
1. pyruvate-dependent aldolase
2. dihydroxyacetone phosphate (DHAP)-dependend aldolase
3. acetaldehyde-dependent aldolase
4. glycine-dependent aldolase
acetaldehyde-dependent aldolase
2-deoxyribose-5-phosphate aldolase (DERA)
-> generation of 1 stereocenters
O
Cl
O
Me
OH
O
D ER A
Cl
70 % ,
>9 9.5 % e e, 9 6.6 % d e
4 s te p s
OH
O
ato rv as tati n
NC
O
CO 2tBu
10
O
O
HO
B ar an Gro u p M ee tin g
0 7 /11 /2 00 9
Biocatalysis
Ta nj a G ul de r
O
D ER A
OH
O
Tot al Synthesis of Natural Products
O
i n v itro reconstitution of complete biosynthetic pathways
Me
46%
Me
Me
C O2 H
Me
OH
OH
L-tr yp to p h an :
ph e ny lp yr uv ate
a mi n otra n sfe ra se
Td iD
NH2
O
H
HO
Me
Me
OH
S
Me
N
Me
O
Me
Me
S
Me
CO 2H
C O2 tBu
e p oth il o ne A
HO
O
O
Me
S
Me
N
O
O
O
HN
OH
O
S
NH
HO
HO
O
O
OH
Me
Th D P
S
Me
q u in o ne
re du cta se
Td iC
OH HN
HO
N AD P H
OH
NH
OH
(-) -e ph e dr in e
Me
HO
Me
N
N
O6 P2O
HN
OH
D
id em e thy lO
a ste rri qu i no n e D
NH
NHMe
O
CO 2 H
Me
R - ph e ny la ce tyl c ar bi n ol
> 9 8% e e
-C O 2
O
OH
O
Me
P DC ,
Th D P
H2 N Me ,
H2 , Pt
H o ffm ei ste r
C e l l 2 0 0 7,
635 HO
NH
NH
21 %
PD C ,
Th D P
OH
O
O
Me
3 5%
Pyruvate decarboxylase (PDC, thiamin diphosphate depended)
Me
Td iA
O
OAc
O
bi si nd o ly lqu i no n e
s yn the ta se
Tdi A
AT P
NH 2
O
Td iA
O
D ER A
OH
CO 2 H
OTBS
Me
Me
N
H
Me
Wo ng e t a l. A CI E 2 0 02 , 14 0 4
S
O
N
H
PMPO
O
Me
O
Me
CO 2H
Me
N
H2 N
O
HN
p r en yl tra ns fer as es
Tdi B/Td iE
HN
Wa l sh e t a l.
N a t. C h em .
B i ol . 2 00 7 ,
5 84
O
OPP
te rr eq u in o ne
11
O
a cyl ca rri e r
p ro te in En cC
li ga se En cN
A TP
OH
7x
ke to sy nth a se
E nc A-E nc B,
k eto re du c ta se En cD ,
tra n sa cy la se Fab D
O
O
O
HO
S
EncC
HO
O
Ph
O
O
O
O
C OSEn cC
O
O
O
fe rre d o xin ,
fe rr ed o xin -N A DP +
r ed u cta se
En cR
OH
Ph
O
O
O
O
HO 9
Ph
O
HO
wa i lu pe m yci n F
O
O
COSEncC
O
O
de sm e th yl -5 d e ox ye nte ro ci n
~ 2 5 % o ve ra l l yi el d ;
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
O
OH Ph
OH
HO
O
HO
NA D P
O H
O
Ph
OH
OH
HO
O
5 -d eo xy en te ro ci n
" fav o rsk iia se "
fl av op ro te in
En cM
HO
5
SA M
O
MeO
O
me thy ltra ns fer as e
En cK
HO
9 OH
O
O H O
O
OH Ph
OH
5
HO
Ph
HO
O H
O
EncC S
O
w a il up e myc in G
Ph
O
O
O
HO
OH
O
HO
OH
HO 9
O
EncC
O
OH
Fa v or ski i
re ar ra ng m en t
O
S
O
O
NA D PH
7x
SCoA
O
B ar an Gro u p M ee tin g
0 7 /11 /2 00 9
Biocatalysis
Ta nj a G ul de r
HO
O
MeO
e nte ro ci n
O
M oo re e t a l. N a t. C he m. B io l .
20 0 7, 5 57
12