aff inity of the enzyme to the transition stat e is greater than to

t he substrate itself ground state destabilization eff ect

Biocatalysis
Tanj a Gul der
Bar an Group Meeting
07/11/2009
1
Enzymes are catalysts evolved in nature to achieve the speed
and coordination of a multitude of chemical reaction necessary
to develop and maintain lif e.
Enzymes
are globular proteins which range from 62 (monomer
of 4-oxalocrotonate tautomerase) t o over 2 500 amino acid
residues (animal fatty acid synthase), but only a small portion
(~ 3-4 amino acids are direct ly involved in catalysis)
Classification
Oxi doreductases
catalyze oxidation/reduction reactions
Transferases t ransf er a funct ional group (e. g. met hyl group)
Hydrolases catalyze the hydrolysis of various bonds
Lyases
cleave various bonds by means other than hydrolysis and
oxidation
I somerases catalyze isomerization changes within a single
molecule
Li gases join two molecules with covalent bonds
Induced fit
Model f or the enzyme-substrat e int eract ion introduced
by Koshland
Enzyme reaction
Mechanism of transitions stat e st abilization
Catalysis by bond strain
Catal ysis by proximity and ori entation
enzyme-substrat e int eractions align reactive groups and hold
themclose together reduces t he overall loss of entropy
Catal ysis i nvolvi ng proton donors or acceptors
(acid/base catalysi s)
stabilization of developing charges in the t ransition state
activation of nuceophiles and electrophiles or
stabilization of leaving groups
Biocatalysis
Tanj a Gul der
2
Asp
O
O
H
N
N
His
H
O
Ser
R'
H
N
O
R
initial step of the serine protease catalytic mechanism:
As p
O
O
H
N
N
Hi s
H
O
Ser
R'
N
O
R
H
Electrostatic catalysis
stabilizat ion of charged transit ion st ates by f orming ionic bonds
with residues of the active site
Glu
O
O
H
O
H
Zn
2+
O
HN R'
R
CO
2
Glu
O
O
H
O
H
Zn
2+
O
HN R'
R
CO
2
initial step of the carboxypeptidase catalytic mechanism:
Covalent catalysis
substrate is forming a transient covalent bond with a residue in
the active site in order to reduce energy of later transitions
stat es of t he react ion
pyruvate decarboxylase mechanism
N
S
R
Me
R'
Me
O
OH
N
S
R
Me
R'
Me
thiami ne
py rophosphate
(TPP, vitami ne B1)
O
OH
N
S
R
Me
R'
Me
OH
Me H
O
Advantages of biocatalysts and enzymes
- very high enantioselectivity
- very high regioselectivity
- t ransf ormation under mild conditions
- ' green chemistry' e.g. solvent oft en water
Disadvantages of bi ocatal ysts and enzymes
- of ten low specif ic activity
- instability at extreme temperatures and pH values
- availability f or selected react ions only
- long development time f or new enzymes
advances in genomics, direct ed evolution, gene and genome
shuff ling and the explorat ion of Earths biodiversit y aided by
bioinf ormatics and high-t hroughput screening facilitate t he
discovery and optimizat ion of enzymes
It is estimated that biocatalysis and biotransformations
account for 30% of the chemical business by the year 2050
Baran Gr oup Meeti ng
07/11/2009
H
N
S
R
Me
R'
Me
OH
H
O
CO
2
OH
TPP
yl ide
N
S
R
Me
R'
H
H
Biocatalysis
Tanj a Gul der
Bar an Group Meeting
07/11/2009
3
Desymmetrizations
Biotransformations on an Industrial Scale
Pi g l iv er
esterase
98%, >99% ee
enantiot opos-diff erentiating hydrolysis
pH 8, 7d
CO
2
Me
CO
2
Me
H
2
O/
ac etone
89%, 95% ee
Literature
- K. Drauz, H. Waldmann, Enzyme Cat alysis in Or ganic
Synt hesis, Wiley-VCH, 2002
- V. Gotor, I. Alf onso, E. Garcia-Urdiales, Asymmet ric Organic
Synt hesis with Enzymes, Wiley-VCH, 2008
- E. Garcia-Junceda, Multi -Step Enzyme Catalysis, Wiley-
VCH,
2008
- D. Enders, K.-E. Jaeger, Asymmet ric Synthesi s wit h
Chemi cal and Bioblogical Met hods, Wiley-VCH, 2007
- A.S. Bommarius, B. R. Riebel, Biocat alysis, Wiley-VCH,
2004
- G. Carrea, S. Riva, Organic Synt hesis wit h Enzymes in Non-
Aqueous Media, Wiley-VCH, 2008
mes o
pr oc hi ral
MeO
2
C
MeO
2
C
HO
2
C
MeO
2
C
R
O
O
O
O
O
O
by ssoc hl amic
ac id
White et al., JACS.
2000, 8665
MeO
Pi g l iv er
esterase
CO
2
Me
CO
2
H
MeO
S
N
H
Cl HO
2
C
OMe
( -)-v irantmy cin
Wulff et al ., ACIE
2004, 6493
t/a product enzyme
> 1 000 000 high-f ructose corn syrup glucose isomerase
> 100 000 lactose-free milk lactase
> 10 000 acrylamide nitrilase
cocoa butter lipase
> 1 000 nicotinamide nitrilase
D-pantot henic acid aldonolactonase
(S)-chloropropionic acid lipase
6-aminopenillanic acid penicillin amidase
7-aminocephalosporanic glutaryl amidase
acid
aspartame t hermolysin
L-aspart ate aspartase
D-phenylglycine hydant oinase
D-p-OH-phenylglycine hydantoinase
> 100 ampicillin penicillin amidase
L-methionine, L-valine aminoacylase
L-carnitine dehydrase/
hydroxylase
L-DOPA !-tyrosinase
L-malic acid fumarase
(S)-met hoxyisopropyl- lipase
amine
(R)-mandelic acid nitrilase
L-alanine L-aspartate-!-de-
carboxylase
f urther applicat ions:
baby foods, brewing indust ry, fruit juice, dairy industry, starch,
paper, biofuels, detergents, rubber,.. ..
Biocatalysis
Tanj a Gul der
Bar an Group Meeting
07/11/2009
4
Dynamic kinetic resolut ions
Ph
O
Ph
Ph
Ph
O
Ru
OC
CO
Cl
enzyme-metal combination
R-sec-alcohol
lipase
Ac yl- OR
R-ester
met al
S-sec-alcohol
subtilisin
Acyl -OR
S-ester
Ph Me
OH
Ph Me
OAc
R
CALB,
OAc
[Ru], KOtBu, Na
2
CO
3,
toluene, rt
78-92%
>99% ee
[Ru] = 2, 31h
[Ru] = 3, 3h
Ph
H
N
Ph
Ph
Ph
Ru
OC
CO
Cl
2
Ph
Ph
Ph
Ph
Ph
Ru
OC
CO
Cl
3
Ph
O
Ph
Ph
Ph
H
Ru
OC
CO
H
Ph
O
Ph
Ph
Ph
Ru
CO
OC
Shov' s catal ys t
1
Ph Me
OH
Ph Me
OAc
R
CALB,
1
, toluene, 70C,
24- 72h
Cl
OAc
78- 92%
> 99% ee
used for the product ion of R-phenylethanol by DSM
R
O
R
R
R
H
Ru
OC
CO
H
R
O
R
R
R
Ru
CO
OC
4
R = p-MeO-C
6
H
4
Ph Me
NH
2
Ph Me
NH
R
CALB,
OAc
4, Na
2
CO
3,
toluene, 90C, 3d
90%
98% ee
Ac
Ph Me
OH
Ph Me
O
S
subtili si n,
2
, THF, r t
95%
92% ee
PrCO
2
CH
2
CF
3
Pr
O
Ru
Cl
Cl
2
5
6
Ru-catalysts:
Ruthenium-catalyzed reactions
Biocatalysis
Tanj a Gul der
Bar an Group Meeting
07/11/2009
5
Me Ph
OAc
Me Ph
OAc
Ph
Pd
AcO
L
+ Pd(0)
- Pd( 0)
CALB, i PrOH
THF, 25C,
1.5d
Me Ph
OH
i PrOAc
Me
OH
Me
OH
Me
OAc
CALB,
OAc EtO
VO(OSiPh
3
)
3
,
acetone, 25C, 4.5d
71%, 98% ee
91%, 99% ee
OH
EtO O
O CO
2
Et
CALB,
6, NEt
3
, MS, MeCN, 35C, 3d
O
O
CO
2
Et
81%, 97% ee
O
O CO
2
Et
Tandem-DRK-Diels-Alder reaction Meerwein-Ponndorf-Verley-Oppenauer reaction
Ph
OH
Me
CALB, Al Me
3
OAc
BINOL
tol uene, rt, 3h
Ph
OAc
Me
96%, 96% ee
Vanadium-catalyzed reactions
Palladium-catalyzed reactions
DRK wit h enzyme-base combinat ion
Hydantoinase-carbamylase system
( S)
HN
NH
R
O
O
HN
NH
R
O
O
S R
borate buffer,
pH 9, 40
o
C
L- hy dantoi nas e
D-hy dantoi nas e
CO
2
H
NH
R
H
2
N
O
CO
2
H
HN
R
O
NH
2
L-car bo-
myolase
CO
2
H
NH
2
R
D-carbo-
my ol as e
CO
2
H
NH
2
R
or
racemase
100 kg scale pil ot process
for tert -Leuci n at Degus sa
R
R
R
Biocatalysis
Tanj a Gul der
Bar an Group Meeting
07/11/2009
6
Cyanohydrine-mediated DRK
Cl
O
Mandel oni tril e
ly as e
KCN,
i Pr OH/H
2
O
Cl
CN
OH
R
HCl
Cl
CO
2
H
OH
R
quant., 83% ee
anti platel et
cl opi dogrel (Plavi x)
produced by DSM Chemie Linz,
Nippon Shokubai, Clariant
O
HCN
CN
OH
nitr il as e
pH 7.2,
40
o
C
CO
2
H
OH
R
>95%, >99% ee
applied by Lonza, BASF,
and Mitsubishi Rayon on
a multiton scale
Ps eudomonas
cepac ia li pas e
OAc
CN
OAc
S
>96%, >84% ee
nonselective nitrile hydratase: Rhodococcus r hodochr ous J1
- acrylamide production (Nitto process, > 20 000 t/a)
- nicotinamide synt hesis (Lonza, 3000 t/ a)
Cl CO
2
Me
N
S
Oxidations
substrate
(red.)
produc t
(ox.)
dehydrogenase
NAD(P) NAD( P)H
cosubstrate
e.g. pyruvate
(ox.)
by pr oduct
e.g. lactate
(r ed.)
lactate dehydrogenase
regeneration of enzyme
drawback: co-fact or dependence of oxidases/reductases
solutions: - closed-loop systems with an additional enzyme
for co-factor regeneration
- electrochemical co-f actor recycling
- application of metals f or regeneration
- living whole cells
Oxidations of alcohols and amines
HO
OH
OH
OH
Me
Me
R
3-HSDH
12-HSDH
7-HSDH
regioselective oxidations of
bile acid depending on
hydroxysteroid
dehydrogenase used
Biocatalysis
Tanj a Gul der
Bar an Group Meeting
07/11/2009
7
N
O
NHMe
N
N
Me
pH 7
Deracemization of tertiary amines
N
N
Me
H
S
N
N
Me
H
R
S- amine ox idase
vari ant
NaBH
4
Deracemization of secondary alcohols
Me CO
2
H
OH
Me CO
2
H
OH
L-Lac tate
D-Lactate
Me CO
2
H
O
py ruvate
L-l actateoxidase
NaBH
4
NaBH
4
Oxygenation of nonactivated carbon centers
R
2
Me
R
3
R
1
R
1
= O, OH
R
2
= H, Me
al mos t all C atoms at the steroid
nuc leus can be hy dr ox yl ated
stereos peci fic al ly
O
O
H
Me
Me
Me
HO
OH
NaO
2
C
O
O
H
Me
Me
Me
HO
OH
NaO
2
C
HO
pravastati n
(Pravachol)
St reptomy ces
c arbophi l us
ML 236B
from Peni ci l l i um ci tr i num
produced by BMS and Sankyo Pharma
3.6 billion US Dollar annual market value
70%
unnatural
enanti omer
95%, >95% ee
ni cotine
N HO
O
CO
2
H
Pseudomonas
s p. DSM 8653
N
N
Me
H
S
HO
Ar thr obac ter
ox ydans
N HO
H NH
epibati dine: anal gesi c
Epoxidation
R
O
R
O
O
R
Rhodoc oc cus
equi
R
O
OH
HN
metopr olol
Lopresor/Toprol-XL
hy pertens ion
R = CH
2
CH
2
OMe
70%
pr oduction of
corti coster one
Me
Me
O
HO
H
H H
O
OH
Biocatalysis
Tanj a Gul der
Bar an Group Meeting
07/11/2009
8
Baeyer-Villiger-Oxidations
O
O
c ycl opentanone
monoox ygenase
Comamonas
NCIMB 9872
O
O
O
S
S
70%, 95% ee
O
HO
HO
OH
NH
O
O
O
O
O
HO
HO
Ph
H
H
O
Br
H
OAc
( +)-showdomy ci n
(+)-tr ans-kumausy ne
goni ofufurone anal ogs
Rudroff et al . Chem.
Commun. 2006, 3214
Aryl dihydroxylations
R
R
O
cytochrome-ty pe
monooxy genase
euk ar yots
bacterial
diox ygenase
prokaryots
R
OH
OH
or tho and meta hydroxyl ati on occurs us ing toluene (TDO, Ps eudomonas puti da
F39/D), naphthal ene (NDO, P. puti da 119), or bi phenyl dioxy genas es (BPDO,
Sphi ngomonas y anoi kuy ae B8/36)
R
TDO
R
OH
OH
NDO
OH
OH
BPDO
OH
OH
R
S
R
L
O
2
model f or predicting the regio- and
st ereochemical course for the cis
selective dihydroxylat ion reaction
R = H, Cl , Br, I, Me,
CN, CO
2
Et, etc
Br
TDO
Br
OH
OH
77%, 99% ee
CO
2
Et O
AcHN
NH
2
H
3
PO
4 Os el tamiv ir
(Tamif lu)
Me
TDO
Me
OH
OH
1. DMP
2. O
3
/DMS
O
O
O
Me
O
Al
2
O
3
O
O
O
O
HO
OH
Me
CO
2
H
PGE
2
!
Hudl ic ky et al . J ACS
1988
, 4735
Fang et al . ACIE
2008
, 5788
(OC)
3
Cr
Biocatalysis
Tanj a Gul der
Bar an Group Meeting
07/11/2009
9
ipso and ort ho dioxygenations possible with Ralstonia and
Pseudomonas mutant strains
CO
2
H
OH
OH
HO
2
C
O
O
HO
2
C
OH
OH
X
O
O
H
2
NO
2
SO
O
O
X = O: topiramate
anti- epi leps y, anti- mi gr ane
Par ker et al. Synl ett 2004, 2095
Reductions
Reduction of aldehydes
O
Me
(OC)
3
Cr
horse li ver alc ohol
dehy dr ogenas e
Me
HO
Me
O
(OC)
3
Cr
S R
33%, 91% ee 51%, 81% ee
NAD
+
, EtOH
N
H
S
O
O
H
OMe
N
H
S
O
O
H
OMe
R
S
baker' s
yeast
N
H
S
O
OH
H
OMe
S
S
80%,
>99% ee
dil tiazem
hyper tensi on, angina pectori s,
and s ome types of ar rhythmi a
Reduction of ket ones
Chada et al.
J . Mol . Catal. B
2004, 103
X
CO
2
R
O
c ar rot
X
CO
2
R
OH
X = H, o-Cl , p-Cl, p-Me; R = Me, Et
92-99% ee
R
Formation of the 3, 5-dihydroxy side chain in stat ins
3,5-dihydroxy side chain
common in all statins
N
O
N
H
F
OH
OH
HO
2
C
produced by Pf izer
12.4 billion US Dollar 2008
Cl
O
CO
2
Et
keto
r eductase
Cl
OH
CO
2
Et
S
NC
OH
CO
2
Et
R
halohydrin
dehydrogenase
H
2
N
O O
CO
2
tBu
ator vastati n
atorv astati n
(l ipitor)
Alc ali genes
eutr ophy us
str ai n B9.4.5
>95% ee
Biocatalysis
Tanj a Gul der
Bar an Group Meeting
07/11/2009
10
Carbon-Carbon coupling reactions
R
2
H
O
R
1
O
R
2
O
R
1
OH
Aldolase
(Lyase c lass )
HO
O
H
OH
R
2
R
1
O
HO
O
R
1
OH
O
R
2
Aldol ase/
Transaldolase
(Tr ansfer as e cl ass)
R
2
H
O
R
1
O
R
2
O
R
1
OH
Ketol ase
(Lyase cl as s)
Thiamine diphosphate dependent conversions
HO
O
R
1
O
OH
HO
O
R
1
OH
R
2
O
R
2
Ketol ase/
Trans ketol ase
(Transferase cl as s)
Aldolreactions
2-deoxyribose-5-phosphate aldolase (DERA)
st ereodivergent product generation possible using
stereocomplementary enzymes
Enzyme classification dependent on the nucleophile:
1. pyruvat e-dependent aldolase
2. dihydroxyacetone phosphate (DHAP)-dependend aldolase
3. acetaldehyde-dependent aldolase
4. glycine-dependent aldolase
dihydroxyacet one phosphate (DHAP)-dependend aldolase
HO
O
OPO
3
O
OMe
OMe OBn
1. 1,6- bi sphos phate
aldolase (FruA)
HO
OMe
OMe OBn
2. phosphatase
42%
O
OH
HO
HO
OMe
OH OH
Me
HO
OH OH
C
5
H
11
OH OH
OH
Me
O O
pentamyc in
Shi magak i et al. Chem.
Phar m Bul l . 1993, 282
-> generation of 2 stereocenters
-> generation of 1 stereocenters
O
Cl
Me
O DERA
1, 6-bisphosphat e aldolase (FruA)
acetaldehyde-dependent aldolase
O
Cl
OH
OH
70%,
>99.5%ee, 96.6% de
CO
2
tBu NC
O O
4 s teps
atorv as tati n
Biocatalysis
Tanj a Gul der
Bar an Group Meeting
07/11/2009
11
HO
O
Me
Me
O
DERA
46%
O OH
Me
OH
O O
Me
OH
Me
PMPO
Me
O
Me
Me
CO
2
tBu
OTBS
Me
HO
Me
O
Me Me
OH
O
O
O
H
Me
N
S
Me
epothil one A
Wong et al. ACI E 2002, 1404
OH
Me
O
Me
O DERA
35%
O
HO
Me
OH
OAc
Me
N
S
Me
S S
Tot al Synthesis of Natural Products
Pyruvate decarboxylase (PDC, thiamin diphosphate depended)
O
Me CO
2
H
O
Me
O
PDC,
ThDP
-CO
2
OH
PDC,
ThDP
Me
O
R- pheny lacetyl c ar bi nol
> 98% ee
OH
Me
NHMe
H
2
NMe,
H
2
, Pt
(-) -ephedr ine
N
S
N
N
Me
H
2
N
Me
O
6
P
2
O
ThDP
i n vit ro reconstitution of complete biosynthetic pathways
N
H
CO
2
H
NH
2
N
H
CO
2
H
O
CO
2
H
O
CO
2
H
NH
2
L-tr yptophan:
pheny lpyr uv ate
ami notransferase
TdiD
bi si ndoly l-
qui none
s ynthetase
Tdi A
AT P
NH
HO
S
O
TdiA
O
O
OH
NH
TdiA
O
O
O
NH
O
HO
HN
O
O
HO
OH
HN
NH
Didemethy l-
asterri qui none D
Hoffmei ster
Cel l 2007,
635
21%
OH
OH
HO
OH
HN
NH
quinone
reductase
TdiC
NADPH
O
O
HO
HN
HN
Me
Me
pr enyl trans fer as es
Tdi B/TdiE
OPP
terr equinone
Wal sh et al.
Nat. Chem.
Bi ol . 2007,
584
Biocatalysis
Tanj a Gul der
Bar an Group Meeting
07/11/2009
12
acyl carri er
protein EncC
li gase EncN
ATP
S
O
EncC
ketosy nthase
Enc A-Enc B,
k etoreduc tase EncD,
transacy lase FabD
S EncC
O O O O
OH
O O
Ph
O
O
HO
OH
Ph
O
HO O
OH OH
Ph
O
HO O
wai lupemyci n F
HO
O
O
O
O
O
COSEncC
O
O Ph
" fav orsk iiase"
fl av oprotein
EncM
9
9
9
Fav or ski i
rear rangment
HO
O
O
O
O
Ph
OH
O
O
COSEncC
HO
O
O
OH
O
Ph
OH
H
O
HO
O
HO O
5
5-deoxy enteroci n
HO
O
O
OH
O
Ph
OH
H
HO
O
MeO O
5
methy l-
trans fer as e
EncK
NADPH
OH
O
HO
O
O
OH
O
Ph
OH
H
HO
O
MeO O
ferredoxin,
ferr edoxin-NADP
+
r eductase
EncR
SAM
HO
NADP
Moore et al. Nat. Chem. Biol .
2007
, 557
HO
O
S
O
EncC
HO
O
SCoA
O
7x 7x
wail upemyc in G
desmethyl -5-
deox yenteroci n
enteroci n
~ 25% overal l yi el d;
for mation of 10 C-C, 5 C-O,
and 7 stereo c enters i n one pot