Recombinant baker's yeast as a whole-cell catalyst for asymmetric Baeyer - Villiger oxidations

Article abstract of DOI:10.1021/ja972942i

Cyclohexanone monooxygenase (E.C. 1.14.13.22) from Acinetobacter sp. NCIB 9871 has been expressed in baker's yeast (Saccharomyces cerevisiae) to create a general reagent for asymmetric Baeyer - Villiger oxidations. This 'designer yeast' approach combines

Full text of DOI:10.1021/ja972942i

VOLUME 120, NUMBER 15
APRIL 22, 1998
©
Copyright 1998 by the
American Chemical Society
Recombinant Baker’s Yeast as a Whole-Cell Catalyst for Asymmetric
Baeyer-Villiger Oxidations
,†
†
†
‡
‡
Jon D. Stewart,* Kieth W. Reed, Carlos A. Martinez, Jun Zhu, Gang Chen, and
Margaret M. Kayser*,‡
Contribution from the Department of Chemistry, UniVersity of Florida, GainesVille, Florida 32611, and
Department of Chemistry, UniVersity of New Brunswick, Saint John, New Brunswick E2L 4L5, Canada
ReceiVed August 21, 1997
Abstract: Cyclohexanone monooxygenase (E.C. 1.14.13.22) from Acinetobacter sp. NCIB 9871 has been
expressed in baker’s yeast (Saccharomyces cereVisiae) to create a general reagent for asymmetric Baeyer-
Villiger oxidations. This “designer yeast” approach combines the advantages of using purified enzymes (single
catalytic species, no overmetabolism, etc.) with the benefits of whole-cell reactions (experimentally simple,
no cofactor regeneration necessary, etc.). The yeast reagent was used to systematically examine a series of 2-,
3
-, and 4-substituted cyclohexanones (R ) Me, Et, n-Pr, i-Pr, allyl, n-Bu), almost all of which were oxidized
to the corresponding ꢀ-caprolactones in good yields and high enantioselectivities (typically g 95%). Mesomeric
-substituted cyclohexanones were oxidized to ꢀ-caprolactones in g 92% ee. The engineered yeast strain also
4
effected kinetic resolutions of 2-substituted cyclohexanones with enantioselectivity values g 200 for substituents
larger than methyl. The behavior of 3-substituted cyclohexanones depended upon the size of the substituent.
The engineered yeast strain cleanly converted the antipodes of 3-methyl- and 3-ethylcyclohexanone to divergent
regioisomers. On the other hand, for cyclohexanones with larger substituents (n-Pr, allyl, n-Bu), both antipodes
were oxidized by the enzyme to a single regioisomer. In these cases, the observed enantioselectivities were
due to a combination of a modest preference for one enantiomer by the enzyme and an unfavorable
conformational preequilibrium required prior to binding of the less-favored antipode, a phenomenon we refer
to as substrate-assisted enantioselectivity.
6
,7
Introduction
cofactors, which is the case for many hydrolytic enzymes and
aldolases.
8
-11
Because only a single catalyst is present,
Interest in integrating traditional organic chemistry and
biocatalysis has increased dramatically over the past decade.
The use of isolated enzymes can be very successful when the
catalyst is inexpensive, easily purified, and does not require
problems with competing reactions are avoided, and reaction
conditions can be easily varied. On the other hand, whole
microbial cells that produce the enzyme(s) of interest are more
appropriate for enzymes that require cofactors, in particular those
1-5
†
University of Florida. Phone (352) 846-0743, Fax (352) 846-2095.
University of New Brunswick. Phone (506) 648-5576, Fax (506) 648-
‡
(7) Schoffers, E.; Golebiowski, A.; Johnson, C. R. Tetrahedron 1996,
52, 3769-3826.
5
650.
(
(
(
(
1) Stewart, J. D. Biotechnol. Genetic Eng. ReV. 1996, 14, 67-143.
(8) Wong, C.-H.; Halcomb, R. L.; Ichikawa, Y.; Kajimoto, T. Angew.
Chem., Int. Ed. Engl. 1995, 34, 521-546.
2) Faber, K. Pure Appl. Chem. 1997, 69, 1613-1632.
3) Sheldon, R. A. J. Chem. Technol. Biotechnol. 1996, 67, 1-14.
4) Servi, S., ed. Microbial Reagents in Organic Synthesis; Kluwer
(9) Wong, C.-H.; Halcomb, R. L.; Ichikawa, Y.; Kajimoto, T. Angew.
Chem., Int. Ed. Engl. 1995, 34, 412-432.
Academic Publishers: Boston, 1992.
(10) Wong, C.-H. Pure Appl. Chem. 1995, 67, 1609-1616.
(11) Gijsen, H. J. M.; Qiao, L.; Fitz, W.; Wong, C.-H. Chem. ReV. 1996,
96, 443-473.
(
5) Hudlicky, T. Chem. ReV. 1996, 96, 3-30.
(6) Lortie, R. Biotechnol. AdV. 1997, 15, 1-15.
S0002-7863(97)02942-9 CCC: $15.00 © 1998 American Chemical Society
Published on Web 03/31/1998

3542 J. Am. Chem. Soc., Vol. 120, No. 15, 1998
Stewart et al.
that catalyze redox reactions.12-14 Because the growing cells
provide both the enzyme and the cofactors, this approach is
experimentally very simple. Unfortunately, competing enzymes
Results
Construction of the Recombinant Yeast Strain and
Optimization of the Biotransformation Conditions. The
Acinetobacter sp. NCIB 9871 cyclohexanone monooxygenase
gene was amplified from genomic DNA using primers designed
from the published sequence. Following amplification, the
structural gene was inserted into three different yeast expression
vectors that differed in their plasmid copy number and type of
and overmetabolism of the desired product can lessen the
attractiveness of this strategy.15
In preliminary communications we have described a new and
2
1
potentially general solution to these problems using common
baker’s yeast (Saccharomyces cereVisiae).16 Standard cloning
techniques are used to express a synthetically useful enzyme in
yeast, which allows whole recombinant yeast cells to be used
as the biocatalytic reagent. The first example of these “designer
yeasts” expresses cyclohexanone monooxygenase and performs
2
2
promoter (Figure 1). Each plasmid was used to transform S.
cereVisiae strain 15C. This strain was chosen because of its
2
3
2
4
deficiency in a major cellular protease, although later work
showed that this precaution was unnecessary, and all S.
cereVisiae strains tested to date have given satisfactory results.
Preliminary experiments in which the recombinant yeast cells
were used for the Baeyer-Villiger oxidation of cyclohexanone
revealed that the regulated GAL promoter and the 2 micron circle
origin of replication found in plasmid pKR001 provided the
best balance between high enzyme expression level and rapid
cell growth. These properties are critical because the cells must
be capable of healthy growth during the biotransformation
process.
1
6
a variety of enantioselective Baeyer-Villiger oxidations. In
this paper, we provide full details on the use of this recombinant
yeast strain for the oxidations of a complete series of 2-, 3-,
and 4-substituted cyclohexanones. The corresponding lactone
products are important chiral building blocks and our “designer
yeast” methodology makes them accessible to all chemists, not
just specialists. In addition, our results have highlighted the
importance of conformational equilibria in enantioselective
reactions of flexible substrates.
We chose cyclohexanone monooxygenase (E.C. 1.14.13.22)
to demonstrate the potential of our “designer yeast” strategy
because of its broad substrate specificity, high enantioselectivity,
and underexploited potential as a synthetic reagent. Since its
We next sought to optimize the biotransformation conditions
using our recombinant yeast strain and cyclohexanone as a
model ketone. Because of its well-known ability to reduce
carbonyl compounds,
Villiger oxidation of cyclohexanone rather than its reduction
to cyclohexanol by yeast enzymes, some of which have very
2
5,26
1
7
our first goal was to favor the Baeyer-
purification and initial characterization by Trudgill, this
enzyme has been shown to catalyze the asymmetric Baeyer-
Villiger oxidations of a wide variety of ketones.18 Despite these
2
7-29
broad substrate specificities.
Preliminary experiments
successful applications and the importance of the Baeyer-
Villiger reaction,19 cyclohexanone monooxygenase has not yet
demonstrated that the level of recombinant yeast cells initially
present in the biotransformation mixture had a dramatic impact
on the extent of cyclohexanone reduction. When the initial
concentration of recombinant yeast cells was varied between
0.5 and 20 mg/mL, the final ratio of ꢀ-caprolactone:cyclohexanol
ranged from 88:12 to 50:50. By following the time courses
for these reactions, it was clear that ketone reduction predomi-
nated at high cell densities. Thus, the key to success lay in
oxidizing the ketone to completion before the cell density
reached a high level. Using optimized conditions, cyclopen-
tanone and cyclohexanone were oxidized by our engineered
yeast to the corresponding lactones in 67 and 79% yields,
respectively, and ketone reduction was limited to ca. 2.5%
become a widely accepted part of the synthetic organic
repertoire. This is partly due to the requirement for NADPH
as a cofactor. While advances in NADPH regeneration technol-
ogy have been reported (for example, see ref 20), virtually all
of these regeneration systems require a second enzyme that
increases the complexity of preparative biotransformations.
Another factor is the relatively small amounts of enzyme
available, either from cyclohexanone-grown Acinetobacter cells
or from an engineered Escherichia coli strain.21 Finally, cell
growth and enzyme isolation require biochemical expertise and
specialized equipment not found in the typical organic labora-
tory.
(
Scheme 1).
(
12) Crout, D. H. G.; Christen, M. in Biotransformations in Organic
Synthesis; Scheffold, R., Ed.; Springer-Verlag: Berlin, 1989; pp 1-114.
13) Santaniello, E.; Ferraboschi, P.; Grisenti, P.; Manzocchi, A. Chem.
ReV. 1992, 92, 1071-1140.
14) Davies, H. G.; Green, R. H.; Kelly, D. R.; Roberts, S. M.
During our studies with 4-alkyl-substituted cyclohexanones,
(
we noticed a strong correlation between increasing substrate
hydrophobicity and difficulties in the yeast-mediated biotrans-
formations. Specifically, the yeast cultures failed to grow when
the calculated log P value (partitioning of substrate between
1-octanol and water) was greater than approximately 2.0.
Similar trends have been observed for biotransformations with
(
Biotransformations in PreparatiVe Organic Chemistry. The Use of Isolated
Enzymes and Whole Cell Systems in Synthesis; Katritzky, A. R., Meth-
Cohn, O., Rees, C. W., Eds.; Academic Press: New York, 1989.
(
15) Faber, K. Biotransformations in Organic Chemistry - A Textbook;
Springer-Verlag: Berlin, 1995.
16) (a) Stewart, J. D.; Reed, K. W.; Kayser, M. M. J. Chem. Soc., Perkin
3
0-32
other organisms.
To overcome these problems, stoichio-
(
Trans. 1 1996, 755-757. (b) Stewart, J. D.; Reed, K. W.; Zhu, J.; Chen,
G.; Kayser, M. M. J. Org. Chem. 1996, 61, 7652-7653.
(23) S. cereVisiae 15C: MATR, leu2, ura3-52, ∆trp1, his4-80, pep4-3.
(24) Jones, E. W. Methods Enzymol. 1991, 194, 429-453.
(
17) Donoghue, N. A.; Norris, D. B.; Trudgill, P. W. Eur. J. Biochem.
1
976, 63, 175-192.
18) For a review of Baeyer-Villiger reactions catalyzed by cyclohex-
anone monooxygenase, see Stewart, J. D. Curr. Org. Chem. 1998, in press.
(25) Ward, O. P.; Young, C. S. Enzyme Microb. Technol. 1990, 12, 482-
(
493.
(26) Servi, S. Synthesis 1990, 1-25.
(
19) Krow, G. R. Org. Reactions 1993, 43, 251-798.
(27) Shieh, W.-R.; Gopalin, A. S.; Sih, C. J. J. Am. Chem. Soc. 1985,
107, 7, 2993-2994.
(20) Seelbach, K.; Riebel, B.; Hummel, W.; Kula, M.-R.; Tishkov, V.;
Egorov, A. M.; Wandrey, C.; Kragl, U. Tetrahedron Lett. 1996, 1377-
(28) Nakamura, K.; Kawai, Y.; Nakajima, N.; Ohno, A. J. Org. Chem.
1991, 56, 4778-4783.
1
(29) Nakamura, K.; Kondo, S.; Kawai, Y.; Nakajima, N.; Ohno, A.
Biosci. Biotech. Biochem. 1994, 58, 2236-2240.
(30) Bar, R. Trends Biotechnol. 1988, 6, 55-56.
(31) Bar, R. Trends Biotechnol. 1989, 7, 2-4.
(32) Anderson, B. A.; Hansen, M. M.; Harkness, A. R.; Henry, C. L.;
Vicenzi, J. T.; Zmijewski, M. J. J. Am. Chem. Soc. 1995, 117, 12358-
12359.
1
70, 781-789.
(
22) Plasmids used: M949 (David Stillman, University of Utah), ARS-
CEN origin of replication, GAL promoter; pYES2 (Invitrogen, Inc.), 2
micron circle origin of replication, GAL promoter; pG-3 (Kieth Yamamoto,
University of California, San Francisco), 2 micron circle origin of replication,
GPD promoter.

Recombinant Baker’s Yeast
J. Am. Chem. Soc., Vol. 120, No. 15, 1998 3543
Figure 1. Structures of plasmids used in this study. A. Plasmid pJS196 contains the structural gene encoding cyclohexanone monooxygenase
CHMO) under control of the yeast GAL1 promoter. The plasmid is maintained at a low copy number, and selection is accomplished by growing
(
the yeast cells in the absence of uracil. B. Plasmid pKR001 is similar to pJS196; however, it is maintained at a higher copy number within the yeast
cells by virtue of the 2 micron circle origin of replication. C. In plasmid pKR002, the CHMO gene encoding cyclohexanone monooxygenase is
constitutively expressed from the yeast glyceraldehyde 3-phosphate dehydrogenase promoter (PGPD). In addition, the plasmid is maintained at a
relatively high copy number by virtue of the 2 micron circle origin of replication.
included in the biotransformation mixtures.3
1,33-36
In this way,
Scheme 1
we were able to achieve substrate concentrations of 10 mM for
preparative biotransformations, and this concentration was
adopted as our standard throughout these studies.
Oxidations of Prochiral 4-Substituted Cyclohexanones.
To demonstrate that the results from our engineered yeast strain
were comparable to those obtained from purified cyclohexanone
monooxygenase, we investigated a series of 4-alkyl-substituted
cyclohexanones (5a-f; Scheme 1). With the exception of 5e,
which was synthesized in several steps from 1,4-cyclohex-
37
anediol, the ketones were commercially available. In all cases
except 5f, the ketones were oxidized to the corresponding
lactones in good yields (Table 1). Even extended reaction times
failed to afford any lactone from 5f; however, this was not
unexpected since Taschner has reported that 5f is a relatively
38
poor substrate for the isolated enzyme. The lactone structures
were confirmed by comparing spectral data to racemic standards
39
prepared by mCPBA oxidations. In the cases of 5c-f, 1 equiv
of â-cyclodextrin was required in the growth medium to allow
the yeast cells to grow in the presence of the hydrophobic
ketones. We saw no trace of olefin oxidation in the biotrans-
formation of 5e, which is consistent with the oxidizing strength
(
35) Goetschel, R.; Bar, R. Enzyme Microb. Technol. 1991, 13, 245-
51.
36) Singer, Y.; Shity, H.; Bar, R. Appl. Microbiol. Biotechnol. 1991,
2
(
metric amounts of â-cyclodextrin (relative to the ketone) were
35, 731-737.
(
37) Details of this synthesis will be published elsewhere.
(
33) Jadoun, J.; Bar, R. Appl. Microbiol. Biotechnol. 1993, 40, 477-
(38) Taschner, M. J.; Black, D. J.; Chen, Q.-Z. Tetrahedron: Asymmetry
1993, 4, 1387-1390.
(39) Meinwald, J.; Tufariello, J. J.; Hurst, J. J. J. Org. Chem. 1964, 29,
9, 2914-2919.
4
2
82.
(
40.
34) Jadoun, J.; Bar, R. Appl. Microbiol. Biotechnol. 1993, 40, 230-

3544 J. Am. Chem. Soc., Vol. 120, No. 15, 1998
Stewart et al.
Table 1. Lactones Produced by Yeast-Mediated Ketone Oxidations
a
Yields refer to chromatographically purified samples. b No oxidation was detectable, even after prolonged reaction times. c Values of enantiomeric
d
excess were determined by chiral-phase GC analysis on a Chirasil-Dex CB column. Optical rotations were measured in CHCl
temperature. The preference for the (S)-ketone isomer was confirmed by the sign of the optical rotation obtained from the lactone product isolated
from a reaction that had proceeded to 60% completion. This isomer was not detected in the product mixture. Yields of lactones isolated from
reactions that had proceeded to completion. Regioisomeric composition of reactions that had proceeded to completion. Enantioselectivity calculated
3
solutions at ambient
e
f
g
h
i
j
directly from plots of ee(substrate) versus fractional conversion. Enantioselectivity corrected for unfavorable conformational equilibria.
of the flavin 4a-hydroperoxide.40 The yields and optical purities
of lactones 6a-e produced by our engineered yeast strain were
references therein). Because racemic 2-alkyl-substituted cy-
clohexanones are readily available by ketone alkylation,42
a
similar to those reported by Taschner, who used partially
Baeyer-Villiger oxidation that afforded a kinetic resolution
would constitute an attractive entry into this series of compounds
(Scheme 1). Despite the large number of ketones that have
been tested as substrates for cyclohexanone monooxygenase,
the behavior of simple 2-alkyl-substituted cyclohexanones had
purified cyclohexanone monooxygenase.3
8,41
The absolute
configurations of lactones 6a-d were determined by comparing
the signs of their optical rotations to literature values. The
absolute configuration of lactone 6e was established by hydro-
genation over Pd on carbon to yield (S)-6c (as shown by chiral-
phase GC analysis).
4
3
not been investigated when we began our studies. The only
exception was 7a, which Schwab and co-workers reported was
Oxidations of Racemic 2-Substituted Cyclohexanones.
Chiral 6-substituted ꢀ-caprolactones have been widely used as
chiral building blocks in asymmetric synthesis, and several
synthetic approaches have been described (see ref 16b and
(
42) For a summary of leading references, see: Carey, F. A.; Sundberg,
R. J. AdVanced Organic Chemistry. Part B: Reactions and Synthesis;
Plenum: New York, 1983; pp 1-35.
(
43) During the course of our work, Alphand et al. published their
independent studies of the Baeyer-Villiger oxidations of 2-alkyl-substituted
cyclohexanones by whole Acinetobacter cells: Alphand, V.; Furstoss, R.;
Pedragosa-Moreau, S.; Roberts, S. M.; Willetts, A. J. J. Chem. Soc., Perkin
Trans 1 1996, 1867-1872. Our results are in good agreement.
(44) Schwab, J. M.; Li, W.-b.; Thomas, L. P. J. Am. Chem. Soc. 1983,
105, 5, 4800-4808.
(
40) Walsh, C. T.; Chen, Y.-C. J. Angew. Chem., Int. Ed. Engl. 1988,
7, 333-343.
41) Taschner, M. J.; Black, D. J. J. Am. Chem. Soc. 1988, 110, 0, 6892-
893.
2
(
6

Recombinant Baker’s Yeast
J. Am. Chem. Soc., Vol. 120, No. 15, 1998 3545
oxidized by cyclohexanone monooxygenase to 8a with modest
selectivity for the (S)-ketone.44
monooxygenase-catalyzed oxidations of this type of compound,
and, in these cases, other substituents were also present.
41,54
We suspected that larger alkyl substituents might lead to
greater selectivity and therefore utilized our engineered yeast
strain to oxidize ketones 7a-f (Scheme 1). Ketone 7a was
commercially available, 7b was prepared by oxidation of the
corresponding alcohol,45 and the remainder were synthesized
from the morpholine enamine of cyclohexanone.46 The yeast-
mediated oxidations were very specific: in all cases, the lactone
regioisomer obtained was identical to that produced by oxidizing
the ketones with mCPBA, and we were unable to detect the
alternative regioisomer in the crude product mixtures from our
biotransformations by 13C NMR (Table 1). As in the oxidation
of 5e, the olefin of 7e was stable to the reaction conditions,
and we observed only the desired Baeyer-Villiger oxidation.
Because all the starting ketones were racemic, the yeast-
mediated oxidations resulted in kinetic resolutions. To ac-
curately determine the enantioselectivity for these resolutions,
the optical purities of both the starting ketone and the lactone
product were monitored during the course of each reaction by
chiral-phase GC. These data was analyzed by an equation
derived from the work of Sih that relates the enantiomeric excess
However, given the success of cyclohexanone monooxygenase
in discriminating between enantiomeric 2-alkyl-substituted
cyclohexanones, we investigated an analogous series of 3-alkyl-
substituted cyclohexanones 9a-f (Scheme 1). Ketone 9a was
commercially available, while 9b-f were prepared by cuprate
55-57
addition to 2-cyclohexenone.
The outcome of the yeast-mediated oxidations depended upon
the size of the substituent. In the case of methyl or ethyl, the
two enantiomers of the starting ketone were cleanly oxidized
to alternate lactone regioisomers (Table 1). Such divergent
behavior of enantiomers has also been observed in the cyclo-
hexanone monooxygenase-mediated oxidations of racemic
5
8
54b
bicyclo[3.2.0]heptenones and dihydrocarvone.
The struc-
tures of the two regioisomeric lactones obtained from the yeast-
mediated oxidations (10a and 11a) could be confidently assigned
on the basis of their 2-D NMR spectra. While the enantiose-
lectivity of this reaction was only 1.8 when R ) CH3, optically
pure (R)- and (S)-9a are readily available by the retro-aldol
degradation of (R)- and (S)-pulegone, respectively.5
9,60
Thus,
one can easily prepare optically and regioisomerically pure (R)-
10a and (S)-11a by our “designer yeast” approach. Similar
results were obtained for the oxidation of ethyl-substituted 9b.
However, since the enantioselectivity value for this substrate
was 18, enriched samples of the two lactone regioisomers (10b
and 11b) were obtained by isolating the products after short
and long reaction times, respectively. As before, 2-D NMR
spectra conclusively established the appropriate structures.
By contrast, both enantiomers of cyclohexanones bearing
three- or four-carbon substituents in the 3-position were oxidized
to the same lactone regioisomer. By comparing the 13C NMR
spectra of the crude products from biotransformations with those
obtained by mCPBA-mediated oxidations, it was clear that the
yeast reagent had produced only a single regioisomer. Interest-
ingly, three-carbon substituents (9c and 9e) afforded better
enantioselectivities than 3-n-butylcyclohexanone 9f (Table 1).
The lactone structures were deduced from COSY NMR spectra.
In all the lactones, the two diastereotopic C-6 protons were well-
resolved and both showed clear coupling to only a single proton;
this is only consistent with oxygen atom insertion proximal to
the substituent.
4
7
of the residual starting material to the fractional conversion.
By analyzing the data in this way, there is no dependence on
catalyst concentration or rates of mass transport across cell
membranes. We performed nonlinear least-squares fits to the
theoretical curve in which the E value was the only adjustable
parameter. From the data in Table 1, it is clear that substituents
larger than methyl gave rise to virtually complete selectivity
for the (S)-ketone enantiomer.48 For each entry in Table 1, the
ketone and lactone were obtained from the same reaction
mixture; deviations from 100% ee were mainly due to the
difficulty in stopping the reactions at precisely 50% completion.
The absolute configurations of the ketones were determined by
comparing the signs of the optical rotation data with literature
values, which in turn allowed us to deduce the absolute
configurations of the lactones.4
9-53
To show that it was possible
to obtain both enantiomers of the 6-substituted ꢀ-caprolactones
from a racemic ketone in a preparatively useful fashion, 7b was
oxidized by our yeast to a mixture of (S)-8b (79% yield, 98%
ee) and (R)-7b (69% yield, 94% ee). After separation, the latter
was converted to (R)-8b by mCPBA in 80% yield with no loss
of optical purity.
Oxidations of Racemic 3-Substituted Cyclohexanones. In
general, the Baeyer-Villiger oxidations of racemic 3-substituted
cyclohexanones are not synthetically useful reactions since they
afford two enantiomeric pairs of regioisomeric lactones. Unlike
the 2-alkyl-substituted cases, there is no electronic preference
for migration of one of the two carbon-carbon bonds. To our
knowledge, there have been only a few reports of cyclohexanone
In cases where the absolute configurations of the ketones were
known, it was possible to show that the (R)-enantiomers were
oxidized more quickly. In the case of methyl-substituted 9a,
where both antipodes of the starting material were available,
we directly determined that the enantioselectivity was ap-
proximately 2. For 9b, the residual ketone was isolated from
a reaction that had proceeded nearly to completion, and the sign
of its optical rotation was compared to literature values to show
6
1
that it had been enriched in the (S)-ketone. Finally, in the
case of 9f, the ketone was isolated after the reaction had
(
45) Corey, E. J.; Suggs, J. W. Tetrahedron Lett. 1975, 2647-2650.
(46) Stork, G.; Brizzolara, A.; Landesman, H.; Szmuszkovicz, J.; Terrell,
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(
47) (a) Chen, C.-S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J. J. Am. Chem.
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E
1/(1 - E)
Soc. 1982, 104, 7294-7299. (b) c ) ([{1 + ee(S)} ]/[1 - ee(S)])
where c ) fractional conversion, ee(S) ) enantiomeric excess of the
remaining substrate, and E ) enantioselectivity value.
(48) Note that there is a change in numbering priority for ketones 7d
and 7f which inverts the (R/S) nomenclature.
(56) Bertz, S. H.; Dabbagh, G. J. Org. Chem. 1984, 49, 1119-1122.
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5406.
(
49) Fouque, E.; Rousseau, G. Synthesis 1989, 661-666.
(
50) Saigo, K.; Kasahara, A.; Ogawa, S.; Nohira, H. Tetrahedron Lett.
1
983, 511-512.
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53) Fellous, R.; Lizzani-Cuvelier, L.; Loiseau, M. A.; Sassy, E.
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(
1
(
(60) Both enantiomers of pulegone are commercially available from Fluka
Chemical Corp.
(
(61) Posner, G. H.; Frye, L. L. Israel J. Chem. 1984, 24, 88-92.

3546 J. Am. Chem. Soc., Vol. 120, No. 15, 1998
Stewart et al.
This simple model, along with calculated log P values, has
proven useful in our laboratories for predicting the behavior of
novel substrates for cyclohexanone monooxygenase.
The most striking finding in these investigations was that
certain 3-substituted cyclohexanones were oxidized by cyclo-
hexanone monooxygenase via high-energy conformations. The
following discussion uses 9c as an example, although similar
logic applies to 9e and 9f. In all of these cases, the microscopic
rate constants for the enzyme-catalyzed steps are not known;
however, the following analysis is based solely on free-energy
differences. In the case of (R)-9c, the enzyme can accommodate
the substrate with its side-chain in an equatorial position (Figure
Figure 2. Summary of cyclohexanone monooxygenase substrate
specificity. A. Summary of the allowed positions for alkyl substituents
on cyclohexanones. The migrating carbon-carbon bond is indicated
by a heavy line. B. Diamond lattice representation of the allowed
positions for alkyl substituents. Note that the actual conformations of
the alkyl chains within the active site of the enzyme are unknown.
71
3). The second-order rate constant, (V/K)R, describes the free
proceeded to approximately 50% completion. Comparing the
sign of its optical rotation to literature data showed that it had
also been enriched in the (S)-enantiomer.62 Unfortunately,
because the absolute configurations of (+)-9c and (+)-9e have
not yet been established, optical rotation data could not be used
to determine the configuration of the faster-reacting enantiomer.
On the other hand, because the chiral-phase GC elution patterns
of the lactones were consistent with those observed for 9a, 9b,
and 9f, it seems likely that the (R)-enantiomers of 9c and 9e
are also the preferred substrates for cyclohexanone monooxy-
genase.
energy difference between free enzyme and substrate up to and
including the first irreversible transition state, rearrangement
of the tetrahedral intermediate (boxed region in Figure 3, top).
On the other hand, (S)-9c must react via a tetrahedral intermedi-
ate whose propyl side-chain is axial. If the side-chain were
equatorial, the reaction would either be forced to proceed via a
chair conformation that is forbidden by the cubic space
66,67
model
or by a binding mode that would afford the
unobserved lactone regioisomer (Figure 3). The second-order
rate constant (V/K)S therefore represents the free energy differ-
ence between free enzyme and (S)-9cax and the transition state
for the irreversible rearrangement of the tetrahedral intermediate
Discussion
(boxed region in Figure 3, bottom). The observed E value (Eobs)
for the oxidation of 9c is thus composed of two terms: the
unfavorable conformational preequilibrium (∆GS,eq-ax) for the
Since the seminal report by Dumas that baker’s yeast
catalyzed the reduction of elemental sulfur to hydrogen sulfide,
63
(
S)-enantiomer as well as the actual enantioselectivity of the
this organism has been widely used for asymmetric reductions
of carbonyl compounds. Yeast is nearly ideal for organic
synthesis: it is nonpathogenic and simple to grow on inexpen-
sive media and the cells can be stored indefinitely in dried form.
The major disadvantage is that its chemical repertoire is
essentially limited to carbonyl reductions. However, by ex-
pressing foreign enzymes that catalyze desirable chemistry in
baker’s yeast, we can create novel whole-cell biocatalysts that
are as simple to use as ordinary baker’s yeast.
†
†
†
72
enzymatic conversion (∆∆G actual ) ∆G S - ∆G R). Both of
these free energy terms cooperate to favor the (R)-enantiomer.
The true E values were calculated by subtracting the contribution
of the conformational preequilibrium (Table 1).73 Because
experimental data for the conformational equilibria of 3-alkyl-
substituted cyclohexanones are sparse, the value for 3-methyl-
cyclohexanone (1.4 ( 0.2 kcal/mol) was used as an approxi-
mation for 9c, 9e, and 9f. When these corrections are made,
it is clear that the enzyme displays only relatively modest
enantioselectivity for these substrates, particularly 9f. However,
the combination of modest enantioselectivity and an unfavorable
conformational preequilibrium can still allow synthetically useful
selectivity.
7
4
The ability to rapidly examine a systematic series of cyclo-
hexanone homologues has allowed us to probe the active site
models that have been proposed for cyclohexanone mono-
oxygenase.54a,64-67
66,67
The cubic space model of Ottolina et al.
requires that cyclohexanones bind in a specific chair conforma-
tion so that attack by the flavin 4a-hydroperoxide produces a
tetrahedral Criegee-like intermediate in which the O(H) sub-
One assumption in the above analysis is that the energetic
penalty for achieving the axial conformation is completely paid
before formation of the tetrahedral intermediate. Since the
68
stituent is axial. Based on the stereoelectronic precedents of
2
the nonenzymatic Baeyer-Villiger reaction19,69 and the assump-
starting ketones contain an sp center that diminishes 1,3-diaxial
interactions, we calculated the enthalpic differences between
the diastereomeric tetrahedral intermediates. All of these values
were within experimental error of ∆GS,eq-ax (1.4 ( 0.2 kcal/
tion that the tightly bound flavin must occupy a fixed position
within the enzyme active site, we have overlaid our substrates
for cyclohexanone monooxygenase on their carbonyl carbons
and defined their orientations using the carbon-carbon bonds
that are known to migrate (Figure 2A). We have also sum-
marized the allowed positions for substituents on the cyclohex-
74
mol). The entropic contributions to the free energy differences
between the pairs of diastereomeric Criegee intermediates were
neglected since one would expect the side-chains of the bound
intermediates to occupy single, well-defined conformations
within the enzyme active site.
7
0
anone ring in a diamond lattice representation (Figure 2B).
(
62) Dieter, R. K.; Tokles, M. J. Am. Chem. Soc. 1987, 109, 2040-
The notion that conformational equilibria can play an
important role in enzyme-catalyzed reactions has been recog-
nized in other studies. For example, Knowles and co-workers
have proposed that chorismate mutase directly binds the less-
2
046.
(
(
(
(
63) Dumas, M. Ann. Chim. Phys. 1874, 3, 57-108.
64) Alphand, V.; Furstoss, R. J. Org. Chem. 1992, 57, 1306-1309.
65) Kelly, D. R. Tetrahedron: Asymmetry 1996, 7, 1149-1152.
66) Ottolina, G.; Pasta, P.; Carrea, G.; Colonna, S.; Dallavalle, S.;
Holland, H. L. Tetrahedron: Asymmetry 1995, 6, 1375-1386.
67) Ottolina, G.; Carrea, G.; Colonna, S.; R u¨ ckemann, A. Tetrahe-
dron: Asymmetry 1996, 7, 1123-1136.
68) Since it is not known whether the carbonyl oxygen is protonated
(71) The shape of the enzyme binding pocket in this diagram is only a
schematic and does not accurately reproduce the cubic space model of ref
67.
(
(
(72) Details of this derivation are given in the Supporting Information
for this article.
after formation of the tetrahedral intermediate, we use the notation O(H) to
-
(73) Eactual ) Eobs - e-[∆GS,eq,ax/(RT)].
represent either O or OH.
(
69) Krow, G. R. Tetrahedron 1981, 37, 2697-2724.
(74) Allinger, N. L.; Blatter, H. M.; Freiberg, L. A.; Karkowski, F. M.
J. Am. Chem. Soc. 1966, 88, 2999-3011.
(70) Prelog, V. Pure Appl. Chem. 1964, 9, 119-130.

Recombinant Baker’s Yeast
J. Am. Chem. Soc., Vol. 120, No. 15, 1998 3547
Figure 3. Oxidation of racemic 3-n-propylcylohexanone 9c. The (R)-enantiomer of 9c can productively bind to the cyclohexanone monooxygenase
active site with its side-chain in an equatorial location. Thus, the (V/K) term contains only rate constants for enzyme-catalyzed steps (boxed region,
R
S
top). By contrast, (S)-9c must bind to the enzyme with its side-chain in an axial orientation. The (V/K) term therefore contains rate constants for
both the unfavorable conformational equilibrium as well as the enzyme-catalyzed steps.
stable diaxial substrate conformer from solution.75 Also, in the
case of horse liver alcohol dehydrogenase, Dutler proposed a
similar conformational preequilibrium to explain the observed
stereoselectivity in the case of 2-alkyl-substituted cyclohex-
Experimental Section
General Methods. Proton NMR spectra were obtained from CDCl
solutions on General Electric QE-300, Varian Gemini, or VXR-300
instruments operating at 300 MHz and referenced to residual CHCl
3
3
13
anones.76-79
(7.26 ppm). C NMR spectra were taken on a Varian Gemini 300
operating at 75 MHz in CDCl and were referenced to solvent (77.0
ppm). IR spectra were recorded from thin films on a Perkin-Elmer
600 FT-IR spectrophotometer. Optical rotations were measured using
We anticipate that other cases of substrate-assisted
3
enantioselectivity will be discovered as more enzymes are
applied to conformationally mobile organic substrates. This
phenomenon has important implications in attempts to tailor
enzyme properties such as diastereo- and enantioselectivity
based on crystal structures. In such cases, directed evolution
strategies may be more appropriate.80
1
chloroform solutions and a Perkin-Elmer 241 polarimeter operating at
ambient temperature. High-resolution mass spectra were obtained using
either electron impact (70 eV) or chemical ionization with methane as
the reagent gas. Packed column gas chromatography was performed
on a Hewlett-Packard 5710 instrument equipped with a flame ionization
detector and a 0.3 × 300 cm column of 10% OV-17 on Chromosorb
WHP with helium as carrier gas. Capillary gas chromatography was
performed on Hewlett-Packard 5880A or 5890A instruments equipped
with flame ionization detectors and a 0.32 mm × 30 m DB-17 column
for nonchiral separations and a Chrompack 0.25 mm × 25 m CP
chirasil-Dex CB column for enantiomer separations. Chiral separations
used the following conditions: 100 °C (2 min) to 150 °C (5 min) at
(
(
75) Copley, S. D.; Knowles, J. R. J. Am. Chem. Soc. 1987, 109, 8-5013.
76) Dutler, H. Structure-ActiVity Relationships Chemoreception 1975,
6
5-77.
(
(
(
(
77) Dutler, H. Biochem. Soc. Trans 1977, 5, 617-620.
78) Dutler, H. FEBS Symp. 1977, 49, 339-350.
79) Dutler, H.; Br a¨ nden, C.-I. Bioorg. Chem. 1981, 10, 1-13.
80) Zhao, H. M.; Arnold, F. H. Curr. Opinion Struct. Biol. 1997, 7,
4
80-485.

3
548 J. Am. Chem. Soc., Vol. 120, No. 15, 1998
Stewart et al.
were combined, washed with brine (100 mL), dried with MgSO , and
1
.0 °C/min, followed by a 10 °C/min gradient to 180 °C (5 min). The
injector and detector temperatures were maintained at 250° and 220
C, respectively. Thin-layer chromatography was performed on pre-
coated silica gel 60 plates. Reaction products were purified by flash
4
concentrated by rotary evaporation. The lactone was purified by flash
chromatography on a 15 cm silica column using 1:1 ether-hexanes.
Spectral Data for New Compounds. (S)-5-(2-Propenyl)-2-oxe-
°
8
1
1
chromatography using 60 Å silica gel. Tetrahydrofuran and diethyl
ether were distilled from sodium benzophenone ketyl. All other
reagents were obtained from commercial suppliers and used as received.
Recombinant DNA procedures were carried out essentially as
panone 6e. H NMR δ 1.26 (br s, 1 H), 1.30-1.46 (m, 1 H), 1.46-
1.58 (m 1 H), 1.62-1.77 (m, 1 H), 2.06 (br t, J ) 7 Hz, 3 H), 2.55-
2.74 (m, 2 H), 4.17 (dd, J ) 10, 13 Hz, 1 H) 4.31 (ddd, J ) 2, 6, 13
Hz, 1 H), 5.02 (d, J ) 7 Hz, 1 H), 5.07 (s, 1 H), 5.67-5.82 (m, 1 H);
8
2
13
described by Sambrook et al.
Restriction endonucleases were
C NMR δ 28.6, 33.2, 35.1, 40.1, 40.8, 68.1, 117.1, 135.8, 163.1 ppm;
-
1
purchased from New England Biolabs or Promega. T4 DNA ligase
was obtained from New England Biolabs. Plasmid DNA was purified
by density gradient ultracentrifugation with CsCl in the presence of
9 14 2
IR (neat) 3074, 1734, 1639, 1173 cm ; HRMS calcd for C H O
8
6
154.0990, M + H 155.1068, found 155.1072 (∆ ) 0.4 mmu).
(R)-6-Ethyl-2-oxepanone 10b. 1H NMR δ 0.88 (t, J ) 4 Hz, 3H),
1.30-1.50 (m, 4H), 1.52-1.70 (m, 3H), 2.29 (t, J ) 4 Hz, 2H), 3.98
82
ethidium bromide. Standard media and techniques for routine growth
82
83
13
and maintenance of E. coli and S. cereVisiae strains were used. YPD
contained 1% Bacto-Yeast Extract, 2% Bacto-Peptone, and 2% glucose.
YEP contained 1% Bacto-Yeast Extract and 2% Bacto-Peptone. Yeast
expression vector pYES2 was purchased from Invitrogen, pG-3 was a
(d, J ) 3 Hz, 2H); C NMR δ 11.5, 21.4, 24.6, 34.4, 34.5, 40.3, 72.5,
173.7 ppm; IR (neat) 2963, 1735, 1460, 1169, 1101, 1012, 917, 755
-
1
cm ; HRMS calcd for C
143.1069 (∆ ) 0.1 mmu).
8 14 2
H O 142.0990, M + H 143.1068, found
84
(S)-4-Ethyl-2-oxepanone 11b. 1H NMR δ 0.89 (t, J ) 4 Hz, 3H),
1.22-1.47 (m, 4H), 1.49-1.73 (m, 2H), 1.74-1.77 (m, 1H), 2.18-
2.38 (m, 2H), 3.64 (m, 1H), 4.05 (m, 1H); 13C NMR δ 11.3, 27.8,
28.9, 34.6, 35.7, 39.8, 69.3, 173.8 ppm; IR (neat) 2961, 1734, 1458,
generous gift of Keith Yamamoto, and M949 was provided by David
Stillman. Oligonucleotides were obtained from Integrated DNA
Technologies. S. cereVisiae were transformed by the lithium acetate
8
5
method.
-
1
Creation of Yeast Expression Plasmids for Acinetobacter sp.
Cyclohexanone Monooxygenase. The cloning of the Acinetobacter
sp. cyclohexanone monooxygenase gene (CHMO) and the construction
of plasmids pJS196 and pKR001 have been described in detail
elsewhere. Plasmid pKR002 was constructed in several steps. The
CHMO gene was excised from pKR001 by sequential digestion with
BamHI and SalI, and then it was purified by agarose gel electrophoresis.
The CHMO gene was ligated with BamHI, SalI-digested pG-3. After
transformation of E. coli XL1-Blue, the desired plasmid (designated
pKR002) was identified by restriction mapping from a collection derived
from 12 randomly picked transformants. The plasmid was purified by
CsCl ultracentrifugation, and then it was used to transform S. cereVisiae
strain 15C.
8 14 2
1383, 1250, 1166, 754 cm ; HRMS calcd for C H O 142.0990, M
+ H 143.1068, found 143.1071 (∆ ) 0.3 mmu).
1
6-Propyl-2-oxepanone 10c. H NMR δ 0.92 (t, J ) 4 Hz, 3H),
1.20-1.48 (m, 5H), 1.61-1.70 (m, 1H), 1.79 (m, 1H), 1.88 (m, 2H),
2.63 (t, J ) 3 Hz, 2H), 4.04 (dd, J ) 8, 5 Hz, 1H), 4.15 (d, J ) 8 Hz,
1H); 13C NMR δ 14.0, 19.9, 21.3, 33.7, 34.3, 34.7, 38.3, 72.7, 176.1
1
6a
-1
ppm; IR (neat) 2930, 1736, 1459, 1282, 1169, 1046 cm ; HRMS calcd
for C 156.1146, M + H 157.1224, found 157.1254 (∆ ) 3.0
mmu).
9 16 2
H O
6-(2-Propenyl)-2-oxepanone 10e. 1H NMR δ 1.60-1.74 (m, 2H),
1.87-1.95 (m, 3H), 2.02-2.10 (m, 2H), 2.56-2.70 (m, 2H), 4.02 (dd,
J ) 8, 13 Hz, 1H), 4.19 (d, J ) 13 Hz, 1H), 5.05 (m, 1H), 5.10 (br s,
1H), 5.69-5.82 (m, 1H); 13C NMR δ 21.4, 34.3, 34.6, 36.3, 38.4, 72.2,
117.4, 135.3, 180.5 ppm; IR (neat) 3075, 2924, 1736, 1640, 1458, 1167,
General Procedure for Biotransformations. S. cereVisiae 15C-
-1
(pKR001) was maintained on SD plates containing 20 mg/L L-
9 14 2
913 cm ; HRMS calcd for C H O 154.0990, M + H 155.1068, found
tryptophan, 20 mg/L L-histidine and 30 mg/L L-leucine. Fresh plates
were streaked weekly from a frozen stock. A single colony was used
to inoculate 25 mL of YPD in a sterile 250 mL Erlenmeyer flask, and
the culture was shaken at 200 rpm at 30 °C until the OD600 value was
between 4 and 6. Cells were then harvested by centrifuging at 3000
155.1081 (∆ ) 1.3 mmu).
6-Butyl-2-oxepanone 10f. 1H NMR δ 0.89 (m, 3H), 1.16-1.36
(m, 4H), 1.36-1.46 (m, 1H), 1.59-1.70 (m, 1H), 1.70-1.78 (m, 1H),
1.82-1.91 (m, 2H), 2.60 (m, 2H), 4.01 (dd, J ) 8, 5 Hz, 1H), 4.13 (d,
J ) 8 Hz, 1H); 13C NMR δ 14.0, 21.4, 22.7, 29.0, 31.3, 34.4, 34.9,
38.6, 72.8, 176.1 ppm; IR (neat) 2928, 1736, 1458, 1351, 1270, 1168,
×
g for 10 min. The cell pellet was resuspended in 10 mL of 10 mM
-
1
Tris-Cl, 1 mM EDTA (pH 7.5) by vortexing. This washing procedure
was repeated an additional two times. The final cell pellet was
resuspended in 10 mM Tris-Cl, 1 mM EDTA, and 15% glycerol at a
concentration of 0.1 g/mL (wet weight). At this stage, cells were either
used directly for a reaction or frozen in aliquots at -80 °C for later
use. Standard reaction mixtures for preparative biotransformations
contained 90 mL YEP, 10 mL 20% galactose, and 10 mM substrate.
One equiv of â-cyclodextrin (1.14 g) was added if substrate solubility
or toxicity were a problem. Freshly prepared or frozen 15C(pKR001)
cells were added to a final concentration of 2 mg/mL (200 µL of the
above cell suspension per 10 mL of medium). Reaction flasks were
shaken at 200 rpm at 30 °C, and the conversion was monitored by
GC. Analytical samples were prepared by mixing 100 µL of the
reaction mixture with an equal volume of ethyl acetate. After vortexing
for 30 s, the mixture was centrifuged in a microcentrifuge for 2 min,
the organic layer was removed, and a 1-2 µL sample was analyzed
by GC. After the reaction was complete, the reaction mixture was
centrifuged at 3000 × g for 10 min at 4 °C to remove yeast cells. The
supernatant was extracted with ethyl acetate (4 × 100 mL). The cell
pellet was vortexed with 20 mL of ethyl acetate. The organic extracts
18 2
1049 cm ; HRMS calcd for C10H O 170.1302, M + H 171.1380,
found 171.1406 (∆ ) 2.6 mmu).
Acknowledgment. This work was supported by the National
Science Foundation (CHE-9513349), the Helen Hay Whitney
Foundation, the University of Florida (J.D.S.), the Natural
Sciences Engineering and Research Council, and the University
of New Brunswick (M.M.K.). J.D.S. is a New Faculty Awardee
of the Camille and Henry Dreyfus Foundation. K.W.R. was
supported by the U.S. Army Advanced Civil Schooling Program.
The authors thank Drs. Christopher Walsh for providing
Acinetobacter sp. NCIB 9871, Eric de Witts for chiral G.C.
analysis, and Nigel Richards for helpful discussions. We also
thank Cerestar, Inc. for generously supplying the cyclodextrins
used in this study and Oxford Molecular, Ltd. for a software
license for Tsar.
Supporting Information Available: Table of log P values
for substrates and products described in this study and a
derivation for the quantitative analysis of substrate-assisted
enantioselectivity and computational methods for calculating
∆∆H° values for diastereomeric Criegee intermedaties (6 pages,
print/PDF). See any current masthead page for ordering
information and Web access instructions.
(
81) Still, W. C.; Kahn, M.; Mitra., A. J. Org. Chem. 1978, 14, 2923-
925.
82) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning. A
Laboratory Manual; Cold Spring Harbor: Cold Spring Harbor, 1989.
2
(
(83) Sherman, F. Methods Enzymol. 1991, 194, 3-21.
(
84) Schena, M.; Picard, D.; Yamamoto, K. R. Methods Enzymol. 1991,
JA972942I
1
94, 389-399.
85) Becker, D. M.; Guarente, L. Methods Enzymol. 1991, 194, 182-
87.
(
(86) Although the high-resolution mass spectra were obtained under
electron-impact conditions, only the M + H ions were observed.
1