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Chemoenzymatic Synthesis of Building Blocks for Statin Side Chains.

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Enzyme Catalysis
Chemoenzymatic Synthesis of Building Blocks for Statin
Side Chains
Michael Mller*
enzyme catalysis · inhibitors · natural products ·
statins · steroids
inhibit the enzyme HMGCoA reductase (HMG = 3-hydroxy-3methylglutaryl), which catalyzes the reductive conversion of HMG-CoA into
mevalonate, an early and rate-limiting
step in cholesterol biosynthesis. This
inhibition leads to decreased levels of
LDL cholesterol (LDL = low-density
lipoprotein). Large-scale clinical trials
with the statin class of cholesterol-lowering drugs have shown the relationship
between diminished levels of LDL cholesterol and decreased morbidity and
mortality from coronary heart disease.[2]
Moreover, statins also decrease levels of
triglycerides and increase levels of HDL
cholesterol (HDL = high-density lipoprotein).
The market for cholesterol-lowering
drugs, valued at more than US$ 20 billion and thus the largest in the pharmaceutical sector, is dominated by the
statins. In 2003, revenues of US$ 9.2 billion (2002: US$ 8.0 billion) and US$ 6.1
billion (2002: US$ 6.2 billion) were recorded for atorvastatin and simvastatin,
respectively, which means that these
pharmaceuticals are the two top-selling
drugs in the world. Relative to simvastatin, pravastatin (2003: US$ 2.8 billion), and lovastatin (the first statin
marketed in 1987), which are all derived
[*] Prof. Dr. M. Mller+
Institut fr Biotechnologie 2
Forschungszentrum Jlich GmbH
52425 Jlich (Germany)
Fax: (+ 49) 2461-613-870
Dedicated to Heinz G. Floss
on the occasion of his 70th birthday
from microorganisms through partial
synthesis, the synthetic HMG-CoA reductase inhibitors atorvastatin,[3] fluvastatin, and rosuvastatin (approved by the
US Food and Drug Administration in
August 2003)[4] are increasing in value.[5]
With pitavastatin (NK 104) the next
candidate is in phase III clinical trials.[2b]
All statins have in common a homochiral side chain pharmacophore in the
form of a 3,5-dihydroxy acid[6] and a
(hetero)aromatic or cyclic residue
(Scheme 1).
As a result of their extremely high
market value and the requirement for
high chemical and stereochemical purity
(> 99.5 % ee, > 99 % de), immense effort has been invested in the production
of synthetic statins by competing research groups. Pharmaceutical, chemical, and biotechnological companies
have recently developed various chemoenzymatic strategies in which different
biocatalysts are used for the stereoselective synthesis of the 3,5-dihydroxy
acid side chain (Scheme 2). Several
remarkable achievements have been
made in the optimization of the biocatalytic step for application on an industrial scale. These examples nicely demonstrate that for the synthesis of a
specific target molecule many different
enzymatic transformations are applicable.
The established strategy of enantioselective reduction of alkyl 4-chloroacetoacetates (Scheme 2 A), such as 1, has
been optimized by Shimizu and coworkers in collaboration with scientists
at Kaneka. Permeabilized cells of a
recombinant E. coli strain that overproduces both an alcohol dehydrogenase
(ADH, in the form of carbonyl reductase from Candida magnoliae) and glucose dehydrogenase from Bacillus megaterium were used in a biphasic organic
solvent/aqueous buffer system for the
production of ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-2) in enantiopure
form. Product concentrations of 63 g L1
were observed in the organic phase, and
the product was isolated in 95 % yield,[7]
but much higher product concentrations
might also be attainable.[7c] The main
disadvantages of this system are the high
concentration of the coproduct gluconate and the utilization of nonliving
cells. The use of living organisms that
express both the catalyst and the cofactor-regenerating enzyme might enable
continuous production with high total
turnover numbers for the catalyst and
the cofactor NAD(P)H.[8]
Chemists at Avecia Pharmaceuticals
(formerly Zeneca, formerly ICI) have
developed a biocatalytic alternative to
the problematic diastereoselective reduction of 3-hydroxyketones 3, which is
[+] Present address: Institut fr Pharmazeutische Wissenschaften
Lehrstuhl fr Pharmazeutische und
Medizinische Chemie
Albert-Ludwigs-Universitt Freiburg
Albertstrasse 25, 79104 Freiburg (Germany)
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/anie.200460852
Angew. Chem. Int. Ed. 2005, 44, 362 –365
Scheme 1. HMG-CoA reductase inhibitors.
Scheme 2. Biocatalytic transformations for the synthesis of (3R,5S)-dihydroxyhexanoates.
Bn = benzyl.
a price-limiting factor on an industrial
scale because of ecological and economic concerns. The bioreduction of (S)-3
with cells of Pichia angusta resulted in
the formation of the desired diastereomerically pure triol in 80 % yield (substrate concentration 25 g L1). Immobilized lipase from Candida antarctica
(Chirazyme L2) provided access in very
Angew. Chem. Int. Ed. 2005, 44, 362 –365
high yield to regioselectively acetylated
4, which was used in the synthesis of the
important building block 5.[9] Depending on the strain and on the substituent
at C6, either the syn or the anti 1,3-diol
(e. g. 6) is obtained.[10b] A similar approach has been developed by scientists
at Kaneka, for example with Candida
The research group of Patel (BristolMyers Squibb) used cell suspensions of
Acinetobacter calcoaceticus for the stereoselective direduction of ethyl 6-benzyloxy-3,5-dioxohexanoate. The corresponding diol was isolated in 85 % yield
with 97 % ee (Scheme 2 B).[11]
The known desymmetrization of
prochiral 3-substituted glutarates by
enzymatic hydrolysis (Scheme 2 C)[12]
has been optimized by chemists at Ciba
Speciality Chemicals for the synthesis of
8 on a kilogram scale.[13] a-Chymotrypsin, an extremely cheap and robust
biocatalyst, is used in the selective
hydrolysis of the triester 7. The process
is characterized by a high substrate
concentration (285 g L1)[14] and a 94 %
yield of the isolated product with
98.2 % ee. After chain elongation, the
methoxyacetyl ester is removed selectively by hydrolysis in the presence of
pig-liver esterase (PLE) (9!10); the
ethyl ester is left untouched.[14] Because
of the potential for flexible functionalization of the monoprotected glutarate
8,[13] this strategy, like the following, in
principle allows access to both enantiomers of 3-hydroxy-5-oxohexanoates
and 5-hydroxy-3-oxohexanoates.
Burk and co-workers (Diversa)
identified more than 200 new nitrilases
by using large genomic libraries created
by extracting DNA directly from environmental samples. Four of the enzymes
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
identified were employed successfully in
the desymmetrization of 3-hydroxyglutaronitrile (12), which is readily available
Scheme 2 D). Subsequently, a highly
enantioselective nitrilase was created
through site-saturation mutagenesis.
The optimized efficient enzymatic transformation, which was performed on a
gram scale with a substrate concentration of 3 m, is characterized by a high
volumetric productivity. After a reaction time of 15 h, the product
(98.5 % ee) was isolated in 96 % yield,
which corresponds to a space–time yield
of 619 g L1 d1.[15]
The dehalogenase-catalyzed racemic
resolution of rac-2 was described by
Kasai and co-workers (Daiso).[16] Prochiral 1,3-dichloro-2-propanol was used
in the synthesis of (R)-4-chloro-3-hydroxybutyrontrile ((R)-16) via (R)-epichlorohydrin ((R)-11). The dehalogenase enzyme catalyzes both the reversible removal of HCl from 1,3-dichloro2-propanol and the introduction of the
cyano group.[17] The enantiomer (S)-16
can be obtained in two steps starting
either from racemic epichlorohydrin or
from prochiral 1,3-dichloro-2-propanol.[18]
Recently, scientists at Codexis together with Roger Sheldon described
the combination of a ketoreductase- and
a dehalogenase-catalyzed one-pot transformation (Scheme 2 E).[19] Although
this is an interesting approach, the
published data are not yet adequate for
the application of this two-step transformation on a larger scale.
Another highly attractive approach
has been developed by scientists at
DSM[20] based on a transformation introduced by Wong and co-workers.[21]
The 2-deoxyribose-5-phosphate aldolase (DERA) catalyzed formation of
the pyran 17 proceeds through asymmetric CC bond formation starting
from the cheap bulk chemicals acetaldehyde and chloroacetaldehyde. Although the reaction characteristics published in the initial article were not very
promising (low substrate concentration,
high catalyst loading, 7-day reaction
time), this biotransformation is now in
operation on an industrial scale.[20c] The
optimized enzymatic transformation,
which is performed at low temperature
(2–4 8C), is characterized by a high final
product concentration (> 100 g L1).[20a]
Recently, scientists at Diversa applied the strategy of screening genomic
libraries prepared from environmental
DNA, as mentioned in the case of
nitrilases, for the identification of improved DERA enzymes.[22] More than
15 DERA enzymes were discovered,
one of which was used to optimize the
process on a 100-g scale. By using a fed-
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
batch strategy, which is necessary because of substrate inhibition, Burk and
co-workers obtained 17 (> 99.5 % ee,
96.6 % de) in a final concentration of
93 g L1 and were able to decrease the
amount of enzyme used to 2 wt %. In
addition, Wong and co-workers recently
reported the DERA S238D mutant
catalyzed transformation of 3-azidopropionaldehyde with two molecules of
acetaldehyde.[21d] Thus, the building
block 18 for the atorvastatin side chain
is accessible through a straightforward
chemoenzymatic five-step synthesis
starting from cheap bulk chemicals.
These examples demonstrate that
state-of-the-art enzymatic transformations have reached an extraordinary
level, making them valuable and competitive methods for use in the chemical
and pharmaceutical industry. Clearly,
Angew. Chem. Int. Ed. 2005, 44, 362 –365
biocatalysis is “growing up” in terms of
its acceptance by chemists. Biocatalytic
strategies offer innovative solutions to
problems such as crossed-aldol reactions, the regio- and stereoselective
reduction of 1,3-diketones, the stereoselective formation of 1,3-diols, and the
desymmetrization of prochiral compounds. These approaches will also have
an impact on the development and
elucidation of non-enzymatic transformations, as well as on the training of
chemistry, pharmacy, and biology students.
Published Online: December 9, 2004
[1] According to the Anatomical Therapeutic Chemical (ATC) classification of the
World Health Organization, the International Nonproprietary Names (INN)
of the so-called “statins” are assigned
the root “-vastatin”.
[2] Coronary heart disease is a leading
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[3] B. D. Roth, Prog. Med. Chem. 2002, 42,
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[4] a) J. Quirk, M. Thornton, P. Kirkpatrick,
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Angew. Chem. Int. Ed. 2005, 44, 362 –365
[5] Cerivastatin, marketed by Bayer, was
withdrawn from the market in 2001
because of reported cases of rhabdomolysis (severe muscular toxicity).
[6] Fluvastatin is marketed as a racemate.
[7] a) Y. Yasohara, N. Kizaki, J. Hasegawa,
M. Wada, M. Kataoka, S. Shimizu,
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[10] N. Kizaki, Y. Yamada, Y. Yasohara, A.
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[11] a) R. N. Patel, A. Banerjee, C. G. McNamee, D. Brzozowski, R. L. Hanson, L. J.
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[12] F.-C. Huang, L. F. H. Lee, R. S. D. Mittal, P. R. Ravikumar, J. A. Amigo, C. J.
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[13] R. hrlein, G. Baisch, Adv. Synth. Catal.
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[14] See also: S. J. C. Taylor, R. C. Brown,
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[15] a) G. DeSantis, K. Wong, B. Farwell, K.
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L. W. Nicholson, C. L. Rand, D. P. Weiner, D. E. Robertson, J. Burk, J. Am.
Chem. Soc. 2002, 124, 9024 – 9025; c) see
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[17] T. Nakamura, T. Nagasawa, F. Yu, I.
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[18] E. J. de Vries, D. B. Janssen, Curr. Opin.
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[19] S. C. Davis, J. H. Grate, D. R. Gray, J. M.
Gruber, G. W. Huisman, S. K. Ma, L. M.
Newman, R. Sheldon, L. A. Wang (Codexis Inc.), WO 2004/015 132.
[20] a) J. G. T. Kierkels, D. Mink, S. Panke,
F. A. M. Lommen, D. Heemskerk
(DSM), WO 03/006 656; b) J. M. H. H.
Kooistra, H. J. M. Zeegers, D. Mink,
J. M. C. A. Mulders (DSM), WO 02/
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biocatalyst development for custom manufacturing of fine chemicals, Biocat,
Barcelona, Spain, 2003.
[21] a) H. J. M. Gijsen, C.-H. Wong, J. Am.
Chem. Soc. 1994, 116, 8422 – 8423; b) C.H. Wong, E. Garcia-Junceda, L. Chen,
O. Blanco, H. J. M. Gijsen, D. H. Steensma, J. Am. Chem. Soc. 1995, 117, 3333 –
3339; c) G. DeSantis, J. Liu, D. P. Clark,
A. Heine, I. A. Wilson, C.-H. Wong,
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[22] W. A. Greenberg, A. Varvak, S. R. Hanson, K. Wong, H. Huang, P. Chen, M. J.
Burk, Proc. Natl. Acad. Sci. USA 2004,
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2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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block, synthesis, side, chemoenzymatic, chains, building, statia
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