Kayser and Clouthier
SCHEME 2
Ketones with relatively small, nonpolar groups in position 4
(1e, 1f, 1n-1p) were oxidized rapidly and with high (S)
selectivity by WT-CHMO and the mutants alike. The 4-n-
propyl-cyclohexanone 1g, which is effectively oxidized by WT-
CHMO (92% ee), was converted only by Phe432Ser, with a
slight enhancement in enantioselectivity (99% ee). These results
suggest the existence of a midsize hydrophobic “pocket”, which
has not been significantly affected by the mutations. The limited
size of the “pocket” is confirmed by the fact that a bulky 4-tert-
butylcyclohexanone is a poor substrate14 for either WT-CHMO
or any of the mutants (not shown in Table 1).
The 4-disubstituted substrates (1h-1j) with hydroxy and
nonpolar chains were expected to provide further information
on the characteristics of the active site. In this series, again, the
Phe432Ser mutant turned out to be a winner. It accepted and
transformed the four substrates with excellent enantioselectivity.
Neither WT-CHMO nor Phe432Ser accepted 4-hydroxy-4-
phenylcyclohexanone (not shown). It was particularly gratifying
to obtain an essentially enantiopure lactone 1k, which was
oxidized by WT-CHMO efficiently but with low selectivity
(Table 1).
Absolute Configuration of Lactones 2m and 2h. The
absolute configurations of the monosubstituted lactones shown
in Table 1 have been established earlier,7 with the exception of
carboethoxy-substituted compound 2l. The latter lactone was
assigned an (S) configuration when, upon reduction with LiAlH4,
it gave triol (S)-7, identical to that obtained for the reduction
of a previously reported (S)-2m15 (Scheme 3).
The specific rotations for 2h-2k produced in oxidations
catalyzed with WT-CHMO and Phe432Ser indicated that the
same configuration was obtained in both series of biotransfor-
mations (Table 2); since there were no data in the literature on
the absolute configuration of any of the disubstituted lactones
or their derivatives, the structures had to be determined.
To establish the absolute configuration of 2h, we carried out
an asymmetric synthesis of the acylated lactone (R)-2h as shown
in Scheme 4. Ichihara and co-workers reported the preparation
of enantiopure (R)-2h as an intermediate in the total synthesis
of (-)-betaenone C.16 Unfortunately, no rotation or experimental
details were provided. In our synthetic sequence, the com-
mercially available natural product nerol 8 was converted to
(S,R) epoxide 9 (79% ee) via Sharpless expoxidation.17 The
reduction with LiAlH4 in THF at -40 °C generated (R)-3,7-
dimethyl-oct-6-ene-1,3-diol. Acylation of the primary hydroxyl
group, followed by oxidative cleavage of the double bond,18
gave acid (R)-11, which spontaneously closed to the five-
member γ-lactone (R)-12. In the course of a biotransformation
of 1h, the resulting seven-member lactone also spontaneously
rearranged to the five-member lactone 2h, which was acetylated
to give compound 12. Direct comparison of the chiral phase
Preparation of Substrates 1a-1k. Ketones 1e-1g were
purchased from Sigma-Aldrich; 1l was obtained from TCI
America Co. The synthesis and characterization of compounds
1n-1p were reported earlier.13 Commercially available 1,4-
cyclohexanedione monoethylene ketal was the starting material
for the preparation of 1a-1d, as shown in Scheme 2. The
synthesis of 4,4-disubstituted cyclohexanones 1h-1k was
accomplished via the reaction of appropriate alkylmagnesium
halides with ketal 3, followed by acid hydrolysis (Scheme 2).
The construction of the E. coli strain overexpressing wild-
type cyclohexanone monooxygenase (WT-CHMO) and the
CHMO mutants has been described previously.7,10 All WT-
CHMO-catalyzed reactions reported here were carried out with
the E. coli BL21(DE3)(pMM4) expression system,7 and those
for CHMO mutants were carried out with the JM109(pET-22b)-
(CHMO mutants) expression system.10 The general procedure
for all biotransformations is described in the Experimental
Section.
Screening of CHMO Mutants. The results of the Baeyer-
Villiger oxidations of the 4-substituted ketones by WT-CHMO
and CHMO mutants are reported in Table 1. Control fermenta-
tions with E-coli host strains BL21 and JM109 showed no
competing reactions within 24 h. All transformations carried
out with growing cells were monitored by GC and were analyzed
by chiral phase GC; product lactones were not isolated in the
screening experiments unless indicated otherwise. Chemical
oxidations with m-chloroperbenzoic acid were performed on all
substrates prior to biotransformations to establish appropriate
conditions for the clean GC resolution of all lactones.
The screening of 4-alkoxycyclohexanones 1b-1d demon-
strated that the CHMO mutants, like WT-CHMO, accepted only
substrate 1b with a short, methoxy chain in position 4. Two of
the mutants showed increased enantioselectivity compared to
WT-CHMO: from 78% ee (S) to 99% ee (S) for both Phe432Ser
and Phe432Ile. In both mutants, there was only a single amino
acid exchange in position 432 where phenylalanine was replaced
with serine and isoleucine, respectively. A decrease in selectivity
was observed for the reaction catalyzed by mutant Leu143Phe,
while the double mutant Asp41Asn/Phe505Tyr provided a
product with the same selectivity as that of WT-CHMO. The
amino acid modifications did not improve the mutants’ capacity
to accept 4-alkoxycyclohexanones with longer chains, such as
1c and 1d; these compounds (Table 1) were clearly not suitable
substrates for either WT-CHMO or the mutants. We were
gratified to see that carboethoxy-substituted 1l, which was a
poor substrate for WT-CHMO, was rapidly and enantioselec-
tively oxidized by Phe432Ser. High selectivity was also achieved
with mutant Asp41Asn/Phe505Tyr, but the conversion was low.
(13) Wang, S.; Kayser, M. M.; Iwaki, H.; Lau, P. C. K. J. Mol. Catal.
B: Enzym. 2003, 22, 211-218.
(14) tert-Butylcyclohexanone was accepted by an isolated enzyme with
low conversion (17%) but very high enantiomeric excess (98%) (ref 15).
(15) Taschner, M. J.; Black, D. J.; Chen, Q. Tetrahedron: Asymmetry
1993, 4, 1387-1390.
(16) Ichihara, A.; Miki, S.; Kawagishi, H.; Sakamura, S. Tetrahedron
Lett. 1989, 30, 4551-4554. Unfortunately, this paper failed to provide
spectroscopic characteristics and the specific rotation of the putative (R)
lactone.
(17) Katsuki, T.; Sharpless, K. B. J. Am. Chem. Soc. 1980, 102, 5974-
5976.
(18) Whitehead, D. C.; Travis, B. R.; Borhan, B. Tetrahedron Lett. 2006,
47, 3797-3800. Travis, B. R.; Narayan, R. S.; Borhan, B. J. Am. Chem.
Soc. 2002, 124, 3824-3825.
8426 J. Org. Chem., Vol. 71, No. 22, 2006