A "designer yeast" that catalyzes the kinetic resolutions of 2-alkyl-substituted cyclohexanones by enantioselective Baeyer-Villiger oxidations

Full text of DOI:10.1021/jo961028p

7652
J . Org. Chem. 1996, 61, 7652-7653
Sch em e 1
A “Design er Yea st” Th a t Ca ta lyzes th e
Kin etic Resolu tion s of 2-Alk yl-Su bstitu ted
Cycloh exa n on es by En a n tioselective
Ba eyer -Villiger Oxid a tion s
J on D. Stewart,*,† Kieth W. Reed,† J un Zhu,‡
Gang Chen,‡ and Margaret M. Kayser*,‡
Department of Chemistry, University of Florida,
Gainesville, Florida 32611, and Department of Chemistry,
University of New Brunswick, Saint J ohn,
New Brunswick E2L 4L5, Canada
Our expectation that the Acinetobacter monooxygenase
would provide a kinetic resolution of 2-alkyl-substituted
cyclohexanones was based on the earlier work of Furst-
oss15,16 and Schwab, who demonstrated that 2-methyl-
cyclohexanone was oxidized by the enzyme to the corre-
sponding lactone with modest enantioselectivity.17
Interestingly, despite the large number of ketones that
have been tested as substrates for this enzyme, simple
2-substituted cyclohexanones have not been systemati-
cally investigated, and only a single example (2-methyl-
cyclohexanone) has been reported.17 Reasoning that
larger substituents at the 2-position might lead to greater
enantioselectivities, we tested a number of racemic
cyclohexanones as substrates for this enzyme, and our
results are summarized in Table 1.18-20 All of the
reactions were performed by the engineered bakers’ yeast
using the previously-described procedure.14,21 Because
several of these substrates were insoluble in the aqueous
growth medium, stoichiometric amounts of â-cyclodextrin
were included in the reaction mixture.22 Chiral-phase
GC provided the enantiomeric compositions of both the
ketone and lactone during the course of the reaction. As
noted previously,14 ketone reduction was only a minor
side reaction. In addition, the olefin functionality was
inert in the yeast-mediated oxidation of 1f. For prepara-
tive purposes, the reactions were terminated shortly after
Received J une 3, 1996
Optically active lactones are valuable intermediates in
asymmetric synthesis. In particular, 6-substituted ꢀ-cap-
rolactones are especially useful, and several methods for
producing these compounds have been explored. These
include the chromatographic separation of diastereomeric
precursors,1 Baeyer-Villiger oxidations of optically-
enriched 2-substituted cyclohexanones,2,3 the use of es-
terases to enantioselectively hydrolyze the racemic
lactones,4-6 and the development of metal complexes that
perform enantioselective Baeyer-Villiger oxidations on
the corresponding substituted cyclohexanones.7,8 Unfor-
tunately, these methods are either experimentally cum-
bersome or afford lactones with only modest enantiose-
lectivities.
Since racemic 2-alkyl-substituted cyclohexanones are
readily available, a Baeyer-Villiger oxidation that ef-
fected a kinetic resolution of these ketones would be an
attractive strategy (Scheme 1). Enzymes that perform
enantioselective Baeyer-Villiger oxidations with broad
substrate specificities have been isolated from several
microbial species including Cylindrocarpon destructans,9
Pseudomonas putida,10-12 and Acinetobacter.13 Here, we
describe an experimentally simple method for producing
6-substituted ꢀ-caprolactones in high optical purities
using whole cells of “oxidizing yeast”sa strain of bakers’
yeast that has been engineered to express Acinetobacter
sp. NCIB 9871 cyclohexanone monooxygenase.14
(15) Alphand, V.; Archelas, A.; Furstoss, R. Biocatalysis 1990, 3,
73.
(16) Alphand, V.; Archelas, A.; Furstoss, R. J . Org. Chem. 1990, 55,
347-350. For a more exhaustive compilation of references describing
the application of cyclohexanone monooxygenase to organic synthesis,
see ref 14 and references therein.
* To whom correspondence should be addressed. (J .D.S.) Tel.: (352)
846-0743. Fax: (352) 846-2095. (M.M.K.) Tel.: (506) 648-5576. Fax:
(506) 648-5650.
(17) Schwab, J . M.; Li, W.-b.; Thomas, L. P. J . Am. Chem. Soc. 1983,
105, 4800-4808.
(18) Ketones that were not commercially available were prepared
by alkylation of the morpholine enamine of cyclohexanone: Stork, G.;
Brizzolara, A.; Landesman, H.; Szmuszkovicz, J .; Terrell J . Am. Chem.
Soc. 1963, 85, 207-221. While many ketones could be successfully
oxidized by our “designer yeast”, this reagent was unable to oxidize
either 2-benzyl- or 2-isobutylcyclohexanone since these hydrophobic
compounds prevented growth of the cells, even in the presence of
cyclodextrins.
† University of Florida.
‡ University of New Brunswick.
(1) Pirkle, W. H.; Adams, P. E. J . Org. Chem. 1979, 44, 2169-2175.
(2) Matsumoto, K.; Tsutsumi, S.; Ihori, T.; Ohta, H. J . Am. Chem.
Soc. 1990, 112, 9614-9619.
(3) Katoh, O.; Sugai, T.; Ohta, H. Tetrahedron: Asymmetry 1994,
5, 1935-1944.
(4) Blanco, L.; Guibe´-J ampel, E.; Rousseau, G. Tetrahedron Lett.
1988, 1915-1918.
(19) Saigo, K.; Kasahara, A.; Ogawa, S.; Nohira, H. Tetrahedron Lett.
1983, 511-512.
(20) Kim, H.-d.; Kawasaki, H.; Nakajima, M.; Koga, K. Tetrahedron
Lett. 1989, 6537-6540.
(5) Fouque, E.; Rousseau, G. Synthesis 1989, 661-666.
(6) Fellous, R.; Lizzani-Cuvelier, L.; Loiseau, M. A.; Sassy, E.
Tetrahedron: Asymmetry 1994, 5, 343-346.
(7) Bolm, C.; Schlingloff, G.; Weickhardt, K. Angew. Chem., Int. Ed.
Engl. 1994, 33, 1848-1849.
(21) Typical experimental procedure: A 0.20 g portion of frozen,
washed 15C(pKR001) cells was added to 100 mL of YEP-galactose (1%
Bacto-yeast extract, 2% Bacto-peptone, 2% galactose) along with 0.10
g (0.78 mmol) of 2-allylcyclohexanone and 0.70 g of â-cyclodextrin. The
culture was shaken at 200 rpm at 30 °C and sampled periodically for
GC analysis. After half of the substrate had been consumed (ap-
proximately 20 h after the start of fermentation), the cells were
removed by centrifuging at 4000g for 10 min at 4 °C. The supernatant
was extracted with CH2Cl2 (4 × 50 mL), and then the combined organic
extracts were dried (MgSO4), filtered, and evaporated. The residue was
chromatographed over silica gel using 1:1 ether:hexanes as the eluant
to afford 29 mg of the (S)-ketone (58% yield) and 44 mg of the (R)-
lactone (78% yield).
(8) Gusso, A.; Baccin, C.; Pinna, F.; Strukul, G. Organometallics
1994, 13, 3442-3451.
(9) Ko¨nigsberger, K.; Braunegg, G.; Faber, K.; Griengl, H. Biotech-
nol. Lett. 1990, 12, 509-514.
(10) Gagnon, R.; Grogan, G.; Levitt, M. S.; Roberts, S. M.; Wan, P.
W. H.; Willetts, A. J . J . Chem. Soc., Perkin Trans. 1 1994, 2537-2543.
(11) Grogan, G.; Roberts, S.; Wan, P.; Willetts, A. Biotech. Lett. 1993,
15, 913-918.
(12) Adger, B.; Bes, M. T.; Grogan, G.; McCague, R.; Pedragosa-
Moreau, S.; Roberts, S. M.; Villa, R.; Wan, P. W. H.; Willetts, A. J . J .
Chem. Soc., Chem. Commun. 1995, 1563-1564.
(13) Donoghue, N. A.; Norris, D. B.; Trudgill, P. W. Eur. J . Biochem.
1976, 63, 175-192.
(14) Stewart, J . D.; Reed, K. W.; Kayser, M. M. J . Chem. Soc., Perkin
Trans. 1 1996, 755-758.
(22) (a) Bar, R. Trends Biotechnol. 1989, 7, 2-4. (b) In general, 1
equiv of â-cyclodextrin was sufficient to solubilize the ketones in the
aqueous growth medium; however, the biotransformation of 2-isopro-
pylcyclohexanone required 2.0 equiv of hydroxypropyl-â-cyclodextrin
to afford a homogeneous mixture.
S0022-3263(96)01028-6 CCC: $12.00 © 1996 American Chemical Society

Communications
J . Org. Chem., Vol. 61, No. 22, 1996 7653
Ta ble 1. Kin etic Resolu tion s of Ra cem ic 2-Alk ylcycloh exa n on es by Wh ole Cells of Ba k er s’ Yea st Th a t Exp r ess
Cycloh exa n on e Mon ooxygen a se
50% conversion and the lactone and the remaining ketone
were isolated by silica gel chromatography. In all cases,
the “normal” Baeyer-Villiger regioisomer was obtained
and the (S)-ketone was the more reactive enantiomer.23
The absolute configurations of the products were deter-
mined by comparing the optical rotations of the ketones
or the corresponding lactones to literature values.4-6,19,20
In general, the optical purities of both the lactones and
the remaining ketones were extremely high, making this
method attractive for preparative purposes. The enan-
tioselectivity can be quantitatively described by E, the
ratio of the second-order rate constants for the conversion
of the substrate enantiomers to the two-product enanti-
omers, a ratio that is independent of enzyme concentra-
tion or mass transport phenomena (Figure 1).24 For a
F igu r e 1. Yeast-mediated Baeyer-Villiger oxidation of 1b.
The reaction mixture contained 10 mM ketone and 0.20 g of
washed yeast cells in 100 mL of growth medium. The mixture
was shaken at 200 rpm (30 °C) and sampled periodically.
Chiral-phase GC analysis was used to determine the enan-
tiomeric excess values for the remaining ketone and the
lactone product. To determine the value of E , the enantiomeric
excess of the remaining starting ketone as a function of
fractional conversion was fit to an equation derived from the
model of Sih with E as the only adjustable parameter. The
calculated line (E ) 200) is superimposed on the data to show
the quality of the fit.
kinetic resolution to be preparatively useful, E should
be >30. In the case of cyclohexanone monooxygenase,
all but one of the E values were g200, which represents
essentially complete enantioselectivity. For this reason,
it was possible to isolate both the remaining ketone and
the lactone in very high optical purities from a single
reaction. The sole exception was the E value of 10 for
2-methylcyclohexanone. Presumably, the steric size of
the substituent is insufficient to completely enforce the
stereochemical imperative in this case. Because the
nonenzymatic Baeyer-Villiger oxidation proceeds with
retention of stereochemistry, the optically pure ketones
obtained from the biotransformations can be converted
to the corresponding lactones by peracid oxidation. Thus,
both lactone enantiomers can be produced from the
racemic ketone. For example, racemic 1b was converted
by our “designer yeast” into (R)-1b (94% ee, 69% yield)
and (S)-2b (98% ee, 79% yield). The recovered ketone
was then oxidized by m-CPBA to afford (R)-2b in 55%
overall yield and 94% ee.
eliminates the need to isolate the enzyme and sidesteps
problems associated with overmetabolism of the lactone
products. Yeast-mediated reactions are environmentally
compatible and do not product toxic byproducts, making
them ideal for large-scale applications.
Ack n ow led gm en t. Financial support by the Uni-
versity of Florida, the NSF (CHE-9513349, J .D.S.), the
University of New Brunswick (J .Z., G.C., and M.M.K.),
and the Natural Sciences and Engineering Research
Council (Canada) (M.M.K.) is gratefully acknowledged.
J .D.S. is a Camille and Henry Dreyfus New Faculty
Awardee. K.W.R. was supported by the U. S. Army
Advanced Civil Schooling Program. We also thank
Amaizo, Inc., for providing cyclodextrins.
This work provides a useful example of the very high
enantioselectivities that can be achieved by applying
enzyme catalysis to organic synthesis. Furthermore, by
expressing the enzyme in bakers’ yeast, we have created
a simple reagent that can be used by chemists with no
training in biochemistry or microbiology. This approach
Su p p or tin g In for m a tion Ava ila ble: Procedures for pre-
paring the yeast cells and chiral-phase GC analyses of the
reaction products (13 pages).
(23) Note that the (R/S) designation is reversed for 1d , 2d , 1f, and
2f as a result of a change in priority numbers for the substituents.
(24) Chen, C.-S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J . J . Am. Chem.
Soc. 1982, 104, 7294-7299.
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