Highly stereoselective reductions of α-alkyl-1,3-diketones and α-alkyl-β-keto esters catalyzed by isolated NADPH-dependent ketoreductases

Article abstract of DOI:10.1021/ol051166d

(Chemical Equation Presented) The biocatalytic reduction of α-alkyl-1,3-diketones and α-alkyl-β-keto esters employing 1 of 20 different isolated NADPH-dependent ketoreductases proved to be a highly efficient method for the preparation of optically pure keto alcohols or hydroxy esters.

Full text of DOI:10.1021/ol051166d

ORGANIC
LETTERS
2005
Vol. 7, No. 22
4799-4801
Highly Stereoselective Reductions of
-Alkyl-1,3-diketones and -Alkyl- -keto
Esters Catalyzed by Isolated
r
r
â
NADPH-Dependent Ketoreductases
Dimitris Kalaitzakis,† J. David Rozzell,‡ Spiros Kambourakis,*‡ and
Ioulia Smonou*,†
Department of Chemistry, UniVersity of Crete, Iraklio 71409, Crete, Greece, and
BioCatalytics, Inc., 129 North Hill AVe, Suite 103, Pasadena, California 91106
smonou@chemistry.uoc.gr; skambourakis@biocatalytics.com
Received May 18, 2005
ABSTRACT
The biocatalytic reduction of
r-alkyl-1,3-diketones and r-alkyl-â-keto esters employing 1 of 20 different isolated NADPH-dependent ketoreductases
proved to be a highly efficient method for the preparation of optically pure keto alcohols or hydroxy esters.
Optically active R-alkyl-â-hydroxy ketones and R-alkyl-â-
hydroxy esters are important compounds in asymmetric
organic synthesis, where they are used as building blocks
for synthesis of polyketides, statins, protease inhibitors, and
other important pharmaceuticals.1 They are of relatively small
molecular weight, bear chirality at two stereogenic centers,
and contain at least two reactive functionalities (an alcohol,
a ketone, and other reactive groups potentially present in
the side chain of the substituent).
Enzyme-based approaches are being increasingly explored
for the synthesis of these classes of compounds. Whole cells
of microorganisms2 with ketoreductase enzyme activities
(particularly Baker’s yeast) have been frequently utilized;
however, many problems are associated with their use.2
Significant efforts are required to grow and screen large
libraries of microorganisms in order to identify those with
useful reductases for every particular application.2 Whole
cells typically contain multiple ketoreductase enzymes,
leading to mixed stereoselectivity and side reactions by other
competing ketoreductases. Whole-cell reactions also suffer
from low reaction rates, limited concentrations of product
per liter of culture, and inhibition due to the toxicity of
† University of Crete.
‡ BioCatalytics, Inc.
(1) For general reviews, see: (a) Liou G. F.; Khosla C. Current Opinion
Chemical Biology 2003, 7, 279-84. (b) Hoffmann, Reinhard W. An-
gewandte Chemie 1987, 99, 503-17. (c) Paterson, I.; Doughty, V. A.;
Florence, G.; Gerlach, K.; McLeod, M. D.; Scott, J. P.; Trieselmann, T.
ACS Symp. Ser. 2001, 783 (Organoboranes for Syntheses), 195-206. (d)
Chartrain, M.; Salmon, P. M.; Robinson, D. K.; Buckland, B. C. Curr. Opin.
Biotechnol. 2000, 11, 209-214. (e) Staunton, J.; Weissman, K. J. Nat. Prod.
Rep. 2001, 18, 380-416. (f) Sawada, D.; Kanai, M.; Shibasaki, M. J. Am.
Chem. Soc. 2000, 122, 10521-10532. (g) Eustache, F.; Dalko, P. I.; Cossy,
J. Tetrahedron Lett. 2003, 44, 8823-8826. (h) Hoffmann, R. W.; Helbig,
W.; Ladner, W. Tetrahedron Lett. 1982, 23, 3479-3482. (i) Vicario, J. L.;
Job, A.; Wolberg, M.; Mu¨ller, M.; Enders, D. Org. Lett. 2002, 4, 1023-
1026. (j) White, J. D.; Hanselmann, R.; Jackson, R. W.; Porter, W. J.; Ohba,
Y.; Tiller, T.; Wang, S. J. Org. Chem. 2001, 66, 5217-5231. (k) Che, Y.;
Gloer, J. B.; Wicklow, D. T. Org. Lett. 2004, 6, 1249-1252. (l) Magnin-
Lachaux, M.; Tan, Z.; Liang, B.; Negishi, E.-i. Org. Lett. 2004, 6, 1425-
1427.
(2) (a) Faber, K. Biotransformations in Organic Chemistry; Springer-
Verlag: Berlin, 1997; pp 160-206. (b) Buisson, D.; Henrot, S.; Larchev-
eque, M.; Azerad, R. Tetrahedron Lett. 1987, 28, 5033-5036. (c) Fauve,
A.; Veschambre, H. J. Org. Chem. 1988, 53, 5215-5219. (d) For a recent
review, see: Nakamura, K.; Yamanaka, R.; Matsuda, T.; Harada, T.
Tetrahedron: Asymmetry 2003, 14, 2659-2681. (e) Homann, M. J.; Vail,
R. B.; Previte, E.; Tamarez, M. Morgan, B.; Dodds, D. R.; Zaks, A.
Tetrahedron 2004, 60, 789-797.
10.1021/ol051166d CCC: $30.25
© 2005 American Chemical Society
Published on Web 10/04/2005

reactants and/or products to the cells. Various empirical
approaches have been tried to minimize these problems in
specific cases, but no generally applicable solutions exist.3,4
Use of isolated enzymes can minimize, and even eliminate,
all these problems.5 Screening sets containing a variety of
different ketoreductase enzymes covering broad ranges of
different ketones are now available,6 and screening of these
enzymes in parallel to identify the best enzyme for a given
target ketone can be carried out rapidly. In this paper, we
present the results of highly diastereo- and enantioselective
reductions of R-alkyl-1,3-diketones and R-alkyl-â-keto esters
utilizing twenty different commercially available ketoreduc-
tase enzymes6 (Figure 1).
Table 1. Enzyme-Catalyzed Stereoselective Reduction of
Diketones/Keto Esters to Keto Alcohols/Hydroxy Esters
Figure 1. Enzymatic reduction of R-alkyl-1,3-diketones 1-6 and
R-alkyl-â-keto esters 7 and 8 with NADPH-dependent ketoreduc-
tases
Using R-alkyl-1,3-diketones and R-alkyl-â-keto esters as
starting materials, ketoreductases offer an opportunity to carry
out reductions that are stereoselective and regioselective.
Consequently, as we show here, it is possible to prepare
single diastereomers of the product keto alcohols and
hydroxy esters by tailoring the choice of enzyme. In addition,
the product is frequently formed in quantitative yield. The
yields and stereochemical purities achieved in the reduction
of six R-alkyl-1,3- diketones and two R-alkyl-â-keto esters
are summarized in Table 1.
a Diastereomeric ratio was determined by chiral GC. In each case, the
racemic product was prepared for calibration. b,c Syn stereochemistry was
1
assigned by comparing H NMR results with literature data.9 d Determi-
nation of absolute configuration of the stereogenic center bearing the OH
group, was made by 1H NMR analysis of the corresponding MPA esters.10
1
The relative stereochemistry (syn/anti) was assigned by H NMR analysis
on the basis of literature method.11
In an achiral environment, the reduction of the diketones
chosen as substrates for this study will yield mixture of all
four possible diastereomers (Figure 1). Reductive enzymes,
on the other hand, are both stereo- and regioselective and
can distinguish between the faces of prochiral ketones
reducing them in a highly stereoselective fashion.
This is clearly demonstrated in the reduction of compounds
2 and 4 (Table 1), where two out of the four stereoisomers
are formed in optically pure form (>99% ee) using different
enzymes while the third diastereomer in both compounds
was also accessed in lower enantiomeric purities (Table 1).
This result is very impressive in showing that potentially all
diastereomers can be accessed using different enzymes. Two
out of the four diastereomers were also obtained in substrates
7 and 8 (Table 1), and in three of the substrates studied one
diastereomer was produced (entries 1, 3, and 6, Table 1).
The diastereomeric ratio, presented in Table 1, was derived
from chiral GC analysis.
(3) Rodriguez, S.; Kayser, M. M.; Stewart, J. D. J. Am. Chem. Soc. 2001,
123, 1547-1555.
(4) (a) Nakamura, K.; Takano, S.; Ohno, A. Tetrahedron Lett. 1993, 34,
6087-6090. (b) Ishihara, K.; Yamaguchi, H.; Nakajima, N. J. Mol. Catal.
B: Enzymol. 2003, 23, 171-189.
(5) (a) Nakamura, K.; Miyai, T.; Kawai, Y.; Nakajima, N.; Ohno, A.
Tetrahedron Lett. 1990, 31, 1159-1160. (b) Shieh, W.-R.; Sih, C. J.
Tetrahedron: Asymmetry 1993, 4, 1259-1269. (c) Kawai, Y.; Takanobe,
K.; Tsujimoto, M.; Ohno, A. Tetrahedron Lett. 1994, 35, 147-148. (d)
Habrych, M.; Rodriguez, S.; Stewart, J. D. Biotechnol. Prog. 2002, 18,
257-261.
In all but one of the substrates shown in Table 1, both
carbonyl carbons are enantiotopic, chemically equivalent, as
(6) KRED-101-120, BioCatalytics, Inc., Pasadena, CA.
4800
Org. Lett., Vol. 7, No. 22, 2005

a result of the symmetrical structure of the molecule (plane
of symmetry). In an asymmetric environment, a regio- and
stereoselective reduction of the keto group leads to the
formation of a single diastereomer. In 3-methyl-2,4 hexa-
dione (entry 6, Table 1), however, the two keto groups are
chemically different because the molecule is not symmetrical.
Upon reduction with KRED102, hydroxy ketone 6 was
obtained in quantitative chemical yield and with excellent
optical purity (ee >99%). This is only possible if an
equilibrium between the two enantiomeric diketones was
taking place during the enzymatic reaction conditions,7 while
the enzyme was selective for the reduction of only one
enantiomer. Furthermore, the enzyme was regiospecific with
respect to the two carbonyl groups, reducing the less
substituted one. Such an equilibrium has been previously
observed in the enzymatic reduction of 2-alkyl-3-ketoglut-
arate diester precursors in the synthesis of statines.8
Quantitative yields of product were obtained in 3-24 h
of reaction time, using 1-10% (g/g) of enzyme relative to
substrate. For example, diketone 1 and keto ester 7 (50-
100 mM) were reduced on a larger scale (100 mL) with
KRED102 affording optically pure keto alcohol 1 and
hydroxy ester 7, respectively, in high isolated yield and
optical purities (90% yield, >99% ee) (details in the
Supporting Information). In every reaction discussed in this
paper, the NADPH cofactor was used in catalytic amounts
(1% relative to the substrate) and was recycled in situ using
glucose dehydrogenase (GDH).12 Determination of the
absolute configuration of the produced keto alcohols and
hydroxy esters was accomplished based on literature meth-
ods.10,11 In all of its reactions KRED102 shows syn selectivity
(entries 1-8b), whereas KREDs116, 118, 119, and 120 show
anti selectivity (Table 1). All of the ketoreductases used in
this study follow Prelog’s rule, except KRED107, which
showed anti-Prelog selectivity in the reduction of the
diketones.
We have shown herein that the enzymatic transformation
of the above diketones and keto esters shows excellent
chemical and optical yields and can be tailored to afford most
of the four possible diastereomers from the same starting
substrate at will depending on the chosen enzyme.
These results clearly demonstrate the power of bioreduc-
tions compared to traditional chemical transformations.
Besides being regio- and stereoselective, these enzymes
exhibited high chemoselectivity by giving a keto alcohol and
not the diol. To the best of our knowledge, no such group
of related chemical catalysts exists that is both regio- and
stereoselective enough to reduce a diketone to an optically
pure single keto alcohol. Finally, the ability to synthesize
various single diastereomers of the same compound, such
as the keto alcohols and hydroxy esters presented in this
report, is desirable when libraries of pharmaceutical com-
pounds are synthesized and tested for biological activity.
In conclusion, the biocatalytic reduction of R-alkyl-1,3-
diketones and R-alkyl-â-keto esters employing 1 of 20
different isolated NADPH-dependent ketoreductases proved
to be a highly efficient method for the preparation of optically
pure keto alcohols or hydroxy esters. Using isolated enzymes
as catalysts for organic reactions is becoming a more
standardized and practical tool in the hands of synthetic
chemists. Further investigations are underway in our labo-
ratories.
Acknowledgment. Financial support for this work came
from the Greek Secretariat of Research and Technology
(Pythagoras 2004 and PENED 2003). D.K. thanks the Greek
National Scholarships Foundation (IKY) for providing a three
year fellowship.
(7) Pellissier, H. Tetrahedron 2003, 59, 8291-8327.
(8) (a) Kambourakis, S.; Rozzell, J. D. AdV. Synth. Catal. 2003, 345,
699-705. (b) Kambourakis, S.; Rozzell, J. D. Tetrahedron 2004, 60, 663-
669.
(9) (a) see ref 2b (b) Bestmann, H. J.; Liepold, B.; Kress, A.; Hofmann,
A. Chem. Eur. J. 1999, 5, 2984-2989.
(10) Seco, J. M.; Quinoa, E.; Riguera, R. Chem. ReV. 2004, 104, 17-
117.
(11) (a) Stiles, M.; Winkler, R. R.; Chang, Y.-L.; Traynor, L. J. Am.
Chem. Soc. 1964, 86, 3337-3342. (b) House, H. O.; Crumrine, D. S.;
Teranishi, A. Y.; Olmstead, H. D. J. Am. Chem. Soc. 1973, 95, 3310-
3324.
(12) GDH has been cloned and overexpressed by BioCatalytics and can
regenerate NADPH from NADP+ by the concomitant reduction of glucose
to gluconic acid.
Supporting Information Available: Detailed experi-
1
mental procedures, GC data and H NMR spectra. This
material is available free of charge via the Internet at
http://pubs.acs.org.
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