Asymmetric synthesis of β-hydroxy esters and α-alkyl-β-hydroxy esters by recombinant Escherichia coli expressing enzymes from baker's yeast

Full text of DOI:10.1021/jo9917036

2
586
J . Org. Chem. 2000, 65, 2586-2587
Asym m etr ic Syn th esis of â-Hyd r oxy Ester s
a n d r-Alk yl-â-h yd r oxy Ester s by
aimed at improving the stereoselectivities of baker’s yeast
mediated reductions,1
4-16
and we have recently reported
the rational design of genetically engineered yeast strains
Recom bin a n t Esch er ich ia coli Exp r essin g
En zym es fr om Ba k er ’s Yea st
1
7
with predictable stereoselectivities.
Biochemical18 and genome analysis studies of baker’s
yeast have shown that a number of enzymes may be
involved in the reduction of â-keto esters. In some cases,
19
†
†
Sonia Rodr ´ı guez, Kersten T. Schroeder,
‡
,†
Margaret M. Kayser, and J on D. Stewart*
the enzymes have been isolated and shown to possess
Departments of Chemistry, University of Florida,
Gainesville, Florida 32611, and University of New
Brunswick, Saint J ohn, NB E2L 4L5, Canada
very high stereoselectivities;1
2,20
however, the need for
enzyme isolation and cofactor regeneration has limited
the practical utility of these catalysts.
Received October 29, 1999
Among the reductase enzymes present in baker’s yeast,
YKER I (Ypr1p) has been characterized most completely
and has been reported as the major enzyme responsible
In tr od u ction
2
0
for production of syn (2R,3S) â-hydroxy esters. However,
using gene knock-out technology, we have shown that at
least one other yeast enzyme participates in producing
Optically pure â-hydroxy esters and R-alkyl-â-hydroxy
esters provide very versatile building blocks for chiral
synthesis,1,2 and several methods for producing these
1
7
these alcohols. There are two other yeast aldo keto
synthons have been explored. Asymmetric reduction of
â-keto esters, either by chemical or enzymatic methods,
has been the most extensively investigated route. Chemi-
cal approaches have included the use of chirally modified
reductases, Gcy1p21 and Gre3p, that share high amino
22
1
9
acid sequence identity with Ypr1p, but their potential
role in reducing â-keto esters has not been investigated.
To learn whether these two enzymes might be useful in
stereoselective reductions of â-keto esters, engineered E.
coli strains expressing Gcy1p and Gre3p were created.
These recombinant strains form the basis for simple
methods of producing chiral â-hydroxy esters with high
optical purities.
hydride reagents,3 transition metal catalysts,
4-7
and
8
Lewis acid mediated borohydride reductions. A number
of biocatalytic reagents have also been investigated,
encompassing both purified enzymes and whole microbial
cells.9
,10
Whole cells provide a continuous source of
enzymes and cofactors, which simplifies these reactions
significantly. In one example, Sugiyama screened several
strains of bacteria for the enantioselective reduction of
ethyl R-methyl acetoacetate and identified a Klebsiella
pneumoniae strain as a suitable reagent.11 Although some
other microorganisms have been shown to reduce â-keto
Resu lts a n d Discu ssion
Engineered E. coli strains expressing Gcy1p and Gre3p
were constructed by placing the appropriate yeast gene
under control of a tac promoter. In these strains, adding
IPTG (isopropyl-â-D-thiogalactoside) to the culture in-
duces enzyme expression. Whole cells of the two strains
were tested for the reductions of several â-keto esters
9
esters, the ease of use, broad substrate tolerance, and
good stereoselectivities have focused most attention on
baker’s yeast (Saccharomyces cerevisiae). Unfortunately,
yeast-catalyzed biotransformations sometimes result in
low optical purities as a result of the presence of enzymes
(Scheme 1); the results are summarized in Table 1. The
optical purities of the products were determined by
chiral-phase GC, and optical rotations were used to
establish the absolute configuration of each â-hydroxy
ester product. 13C NMR spectra confirmed that only a
single diastereomer was produced in all cases. Both
strains yielded the (3S) alcohols with high enantiomeric
excess values and acceptable yields for all the substrates
tested. When racemic R-substituted â-keto esters were
employed as substrates, facile racemization at the R-posi-
tion allowed for dynamic kinetic resolutions (Scheme
with overlapping substrate specificities but different
stereoselectivities.1
2,13
There have been extensive studies
*
Ph: (352) 846-0743. Fax: (352) 846-2095. E-mail jds2@
chem.ufl.edu.
†
University of Florida.
University of New Brunswick.
1) Mori, K. Tetrahedron 1989, 45, 3233-3298.
2) Pereira, R. Crit. Rev. Biotechnol. 1998, 18, 25-83.
3) Soai, K.; Yamanoi, T.; Hikima, H.; Oyamada, H. J . Chem. Soc.,
‡
(
(
(
Chem. Commun. 1985, 138-139.
4) Noyori, R.; Ohkuma, T.; Kitamura, M. J . Am. Chem. Soc. 1987,
09, 5856-5858.
5) Noyori, R.; Ikeda, T.; Ohkuma, T.; Widhalm, M.; Kitamura, M.;
(
1
(
(14) Zhou, B.; Gopalan, A. S.; VanMiddlesworth, F.; Shieh, W.-R.;
Sih, C. J . J . Am. Chem. Soc. 1983, 105, 5925-5926.
(15) Kawai, Y.; Kondo, S.; Tsujimoto, M.; Nakamura, K.; Ohno, A.
Bull. Chem. Soc. J pn. 1994, 67, 2244-2247.
(16) D’Arrigo, P.; Pedrocchi-Fantoni, G.; Servi, S.; Strini, A. Tetra-
hedron: Asymmetry 1997, 8, 2375-2379.
(17) Rodr ´ı guez, S.; Kayser, M.; Stewart, J . D. Org. Lett. 1999, 1,
1153-1155.
(18) Sybesma, W. F. H.; Straathof, A. J . J .; J ongejan, J . A.; Pronk,
J . T.; Heijnen, J . J . Biocat. Biotrans. 1998, 16, 95-134.
(19) Stewart, J . D.; Rodr ´ı guez, S.; Kayser, M. M. In Enzyme
Technologies for Pharmaceutical and Biotechnological Applications;
Zmijewski, M. H, Kirst, H. A., Yeh, W.-K., Eds.; Marcel Dekker: New
York, in press.
(20) Kawai, Y.; Tsujimoto, M.; Kondo, S.; Takanobe, K.; Ohno, A.;
Nakamura, K. Bull. Chem. Soc. J pn. 1994, 67, 524-528.
(21) Oechsner, U.; Magdolen, V.; Bandlow, W. FEBS Lett. 1988, 238,
123-128.
(22) Kuhn, A.; van Zyl, C.; van Tonder, A.; Prior, B. A. Appl. Environ.
Biotechnol. 1995, 61, 1580-1585.
Takaya, H.; Akutagawa, S.; Sayo, N.; Saito, T.; Taketomi, T.; Kumoba-
yashi, H. J . Am. Chem. Soc. 1989, 111, 9134-9135.
(
6) Gen eˆ t, J . P.; Pinel, C.; Mallart, S.; J uge, S.; Thorimbert, S.;
Laffitte, J . A. Tetrahedron: Asymmetry 1991, 2, 555-567.
7) Burk, M. J .; Harper, T. G. P.; Kalberg, C. S. J . Am. Chem. Soc.
995, 117, 4423-4424.
8) Marcantoni, E.; Alessandrini, S.; Malavolta, M. J . Org. Chem.
999, 64, 1986-1992.
9) J ones, J . B. In Comprehensive Organic Synthesis: Selectivity,
(
1
1
(
(
Strategy & Efficiency in Modern Organic Chemistry; Trost, B. M.,
Flemming, I., Eds.; Pergamon Press: New York, 1991; Vol. 8, pp 183-
2
09.
(
(
10) Csuk, R.; Glanzer, B. Chem. Rev. 1991, 91, 49-97.
11) Miya, H.; Kawada, M.; Sugiyama, Y. Biosci. Biotechnol. Bio-
chem. 1996, 60, 95-98.
(
12) Shieh, W.-R.; Sih, C. J . Tetrahedron: Asymmetry 1993, 4, 1259-
1
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(
13) Nakamura, K.; Kawai, Y.; Nakajima, N.; Ohno, A. J . Org. Chem.
991, 56, 4778-4783.
1
1
0.1021/jo9917036 CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/23/2000

Notes
J . Org. Chem., Vol. 65, No. 8, 2000 2587
Ta ble 1. Red u ction of â-k eto Ester s by Recom bin a n t E. coli Str a in s Exp r essin g Gr e3p a n d Gcy1p
E. coli expressing Gcy1p
E. coli expressing Gre3p
product characterization
substituents
R2
isolateda
yield
isolateda
yield
absolute
config
substrate
R1
R3
% ee, dea
% ee, de
[R]25589 (c, CHCl3)
1
1
1
1
1
1
1
1
a
b
c
d
e
f
Me
Et
Me
Et
Me
Me
Me
Me
H
Me
Me
Et
Et
Et
Et
Et
Et
28%
45%
51%
48%
68%
75%
66%
56%
>98, c
35%
40%
45%
40%
81%
41%
72%
87%
>98, c
S
+28.5, (c 1.5)
+20.5, (c 1.3)
+44.6, (c 1.0)
+23.3, (c 1.0)
+2.3, (c 5.4)
+6.8. (c 7.9)
+3.7, (c 4.6)
+7.4, (c 4.1)
H
91, c
90, c
S
H
H
Me
Et
allyl
propargyl
>98, c
96, c
>98, >98
>98, >98
>98, >98
>98, >98
>98, c
91, c
>98, >98
>98, >98
>98, >98
>98, >98
S
S
2R,3S
2R,3S
2R,3S
2R,3S
g
h
a
Yields refer to chromatographically purified samples. b Values of enantiomeric and diasteromeric excess were determined by chiral-
c
phase GC analysis on a Chirasil-Dex CB column. Diastereoselectivity is not applicable in this case.
Sch em e 1
sive biological reagents that can be used by nonspecial-
ists. This approach eliminates the need for enzyme
isolation and cofactor regeneration, and these new bio-
catalysts broaden the spectrum of reactions that can be
catalyzed with recombinant E. coli cells.
Exp er im en ta l Section
Ma ter ia ls. â-Keto esters 1a -f were purchased from Aldrich
and used without further purification. Substrates 1g and 1h
were synthesized by slight modifications of the method reported
by Dauben et al.25 Racemic â-hydroxy esters used as standards
in GC analysis were obtained by NaBH
4
reduction of the
corresponding â-keto ester.26
1
).23 Reduction of 1d -g with either recombinant strain
Gen er a l P r oced u r e for Biotr a n sfor m a tion w ith Recom -
bin a n t E. coli Str a in s. Fresh plates of engineered E. coli
strains were streaked from the appropriate frozen stock, and a
resulted in almost complete conversion of the substrate
to the syn (2R,3S) diasteromer.
2
7
single colony was used to inoculate 10 mL of LB-ampicillin in
a 125 mL Erlenmeyer flask. The culture was incubated in a
rotary shaker overnight (200 rpm, 37 °C), and 1 mL of this
culture was used to inoculate 100 mL of fresh LB-ampicillin in
a 500 mL Erlenmeyer flask. The fresh culture was grown under
the same conditions until it reached an OD600 of 0.5 (ap-
proximately 2.5 h), and then IPTG was added (final concentra-
tion of 1mM) along with the substrate (final concentration of 10
mM). The culture was shaken (200 rpm) at room temperature
and sampled periodically for GC analysis. After maximum
conversion was achieved (24-50 h), the cells were removed by
centrifuging at 4,000 × g for 15 min at 4 °C. The supernatant
E. coli provides an ideal microorganism for expression
of these yeast enzymes since the level of intrinsic reduc-
tase(s) is low. Unmodified E. coli has been shown to
reduce 1e,11 and our results indicated that it can also
reduce several other â-keto esters to a small degree.
Fortunately, this low background activity did not com-
promise the stereoselectivity of the recombinant strains,
since control experiments using the unmodified E. coli
strain showed that it resulted in the same stereoisomer
in all cases examined.
These two new biocatalysts complement the existing
methodologies for reduction of â-keto esters by providing
simple access to syn (2R,3S) R-alkyl-â-hydroxy esters in
high optical purities. Catalytic hydrogenation of R-sub-
stituted â-keto esters has been limited to formation of
the (2R,3R) and (2S,3R) diasteromers.5,6 The (2S,3S) anti
2
8
was extracted with CH
organic extracts were washed with brine, dried with Na
filtered, and evaporated. The product was purified by flash
chromatography with CHCl /ether (9:1) as the solvent system.
2
Cl
2
(4 × 50 mL), and the combined
2
SO
4
,
3
The products were characterized by IR, NMR, GC-MS, and
optical rotations. Enantiomeric and diasteromeric excess values
were determined by chiral GC (Chrompack 0.25 mm × 25 m
CP Chirasil-Dex CB). Alcohols 2a and 2f required acetylation
for complete resolution on the chiral column. Analytical condi-
tions appropriate for each substrate, as well as copies of the
chromatograms, can be found in the Supporting Information.
diasteromer has been obtained through stereoselective
alkylation of ethyl 3(S)-hydroxybutanoate.24 Lewis acid
mediated reductions have produced the syn diasteromer;
however, the enantioselectivities of these reagents were
8
not reported. A further advantage of these new biocata-
Ack n ow led gm en t. Financial support by the Na-
tional Science Foundation (J .D.S., CHE-9816318), the
University of Florida, and the CONICYT-Uruguay is
gratefully acknowledged.
lytic reagents is their ability to provide good stereose-
lectivities in the absence of bulky substituents, which is
not always the case for chemical methods of â-keto ester
reduction. Using whole cells of the engineered E. coli
strains, two stereocenters can be set in one reaction from
simple, commercially available starting materials. In
addition, the reaction proceeds under mild conditions,
and the process is environmentally friendly.
In summary, two yeast enzymes (Gcy1p and Gre3p)
that catalyze stereoselective reductions of â-keto esters
have been identified and shown to be useful biocatalysts
for chiral synthesis. Furthermore, by expressing the
enzymes in E. coli, we have created simple and inexpen-
Su p p or tin g In for m a tion Ava ila ble: Detailed experi-
mental procedures and GC analysis of the reaction products.
This material is available free of charge via the Internet at
http://pubs.acs.org.
J O9917036
(
(
25) Dauben, W. G.; Hart, D. J . J . Org. Chem. 1977, 42, 3787-3793.
26) Harwood: L. M.; Moody, C. J . Experimental Organic Chemistry.
Principles and Practice; 2nd ed.; Blackwell Scientific Publications:
London, 1992.
(27) LB (Luria-Bertani medium)-ampicillin: per liter, Bacto-
Tryptone (10 g), Bacto-Yeast extract (5 g), NaCl (10 g), ampicillin (0.2
g; added after cooling), Bacto-Agar (15 g; included for solid media).
(28) Centrifugation of the extraction mixture (4000 × g, 10 min)
was useful to break the emulsions formed during some extractions.
(
23) Stecher, H.; Faber, K. Synthesis 1997, 1-16.
(24) Fr a´ ter, G.; M u¨ ller, U.; Gunther, W. Tetrahedron 1984, 40,
1
269-1277.