Enantioselective enzymatic reductions of sterically bulky aryl alkyl ketones catalyzed by a NADPH-dependent carbonyl reductase

Article abstract of DOI:10.1021/jo061571y

(Chemical Equation Presented) The enantioselective reductions of aryl alkyl ketones, ArC-(O)R, with a diverse number of alkyl groups have been achieved with an isolated carbonyl reductase from Sporobolomyces salmonicolor. Of special interest is the observ

Full text of DOI:10.1021/jo061571y

Enantioselective Enzymatic Reductions of
Sterically Bulky Aryl Alkyl Ketones Catalyzed by
a NADPH-Dependent Carbonyl Reductase
Dunming Zhu and Ling Hua*
Department of Chemistry, Southern Methodist UniVersity,
Dallas, Texas 75275
lhua@smu.edu
ReceiVed July 28, 2006
FIGURE 1. Aryl alkyl ketones.
ketones other than acetophenone.8 However, enantioselectivity
studies have only been reported for cyclopropyl phenyl ketone
and 2-(1-pentanoyl)furan, with ee values being 92% and 45%,
and the corresponding yields being 41% and 5-10%, respec-
tively.8 The enantioselective reduction of aryl alkyl ketones with
sterically bulky alkyl groups, such as tert-butyl and isopropyl,
still represents a significant challenge in both the enzymatic
and chemical reductions of ketones.9
Recently, a carbonyl reductase gene from red yeast Sporobo-
lomyces salmonicolor AKU 4429 has been cloned and overex-
pressed in E. coli, and the encoded protein (SSCR) showed
activity toward the reduction of various ketones, including
aromatic and aliphatic ketones, as well as R- and â-ke-
toesters.10,11 It was found that bulky R-ketoesters, such as ethyl
3,3-dimethyl-2-oxobutyrate, could be reduced to the chiral
alcohols in very high ee values and yields.11 Thus it was
reasoned that SSCR might catalyze the enantioselective reduc-
tion of aryl alkyl ketones possessing sterically bulky alkyl
groups. Therefore, we have studied the enzymatic reduction of
a number of aryl alkyl ketones catalyzed by this NADPH-
dependent carbonyl reductase (SSCR) (see Figure 1). The
enzyme was found to catalyze the enantioselective reduction
of sterically bulky aryl alkyl ketones, such as 2,2-dimethylpro
piophenone, to give the corresponding chiral alcohols in high
optical purity. An interesting alkyl chain-induced enantiopref-
erence flip-flop was also observed in these studies and an
enzyme-substrate docking analysis was performed to under-
stand this observation at the molecular level.12
The enantioselective reductions of aryl alkyl ketones, ArC-
(O)R, with a diverse number of alkyl groups have been
achieved with an isolated carbonyl reductase from Sporobo-
lomyces salmonicolor. Of special interest is the observation
that ketones with sterically bulky alkyl groups could be
reduced to the corresponding alcohols in excellent optical
purity. An unusual alkyl chain-induced enantiopreference
reversal was observed but was shown to be consistent with
the enzyme-substrate docking calculations.
Chiral alcohols are important as bioactive compounds or as
precursors to such molecules. Therefore, the asymmetric reduc-
tion of ketones to their corresponding alcohols is becoming an
increasingly important transformation in organic synthesis.
Because of their environmentally benign reaction conditions and
unparalleled chemo-, regio-, and stereoselectivities, enzymatic
protocols have attracted more and more attention.1 Up to now,
with the exception of the R- and â-ketoesters, most ketone
reduction studies have been limited to those in which the ketones
possess at least one small group such as methyl or halomethyl.2-5
The reductions of aryl alkyl ketones with larger alkyl groups
using isolated carbonyl reductase enzymes have scarcely been
studied.6-8 An alcohol dehydrogenase from Pseudomonas sp.
has been evaluated toward the reduction of a few aryl alkyl
The carbonyl reductase gene (SSCR) from Sporobolomyces
salmonicolor AKU4429 was cloned and overexpressed in E coli.
(6) Inoue, K.; Makino, Y.; Itoh, N. Tetrahedron: Asymmetry 2005, 16,
2539-2549.
(1) Faber, K. Biotransformation in Organic Chemistry; Springer-Ver-
lag: Berlin, Germany, 2004.
(7) Itoh, N.; Mizuguchi, N.; Mabuchi, M. J. Mol. Catal. B: Enzymatic
1999, 6, 41-50.
(2) Nakamura, K.; Yamanaka, R.; Matsuda, T.; Harada, T. Tetrahedron:
Asymmetry 2003, 14, 2659-2681.
(8) Bradshaw, C. W.; Fu, H.; Shen, G. J.; Wong, C. H. J. Org. Chem.
1992, 57, 1526-32.
(3) Kaluzna, I. A.; Matsuda, T.; Sewell, A. K.; Stewart, J. D. J. Am.
Chem. Soc. 2004, 126, 12827-12832.
(9) Ohkuma, T.; Sandoval, C. A.; Srinivasan, R.; Lin, Q.; Wei, Y.; Muniz,
K.; Noyori, R. J. Am. Chem. Soc. 2005, 127, 8288-8289.
(10) Kita, K.; Fukura, T.; Nakase, K.-I.; Okamoto, K.; Yanase, H.;
Kataoka, M.; Shimizu, S. Appl. EnViron. Microbiol. 1999, 65, 5207-5211.
(11) Zhu, D.; Yang, Y.; Buynak, J. D.; Hua, L. Org. Biomol. Chem.
2006, 4, 2690-2695.
(4) Zhu, D.; Rios, B. E.; Rozzell, J. D.; Hua, L. Tetrahedron: Asymmetry
2005, 16, 1541-1546.
(5) Zhu, D.; Mukherjee, C.; Rozzell, J. D.; Kambourakis, S.; Hua, L.
Tetrahedron 2006, 62, 901-905.
10.1021/jo061571y CCC: $33.50 © 2006 American Chemical Society
Published on Web 11/11/2006
9484
J. Org. Chem. 2006, 71, 9484-9486

TABLE 1. Asymmetric Reduction of Aryl Alkyl Ketones
Catalyzed by Carbonyl Reductase (SSCR) from Sporobolomyces
salmonicolor
the enantiopreference did not reverse, but afforded the (R)-
enantiomers with dramatically higher enantioselectivity (96-
98% ee) than those found for the methyl (1a) or ethyl (1b)
groups. This is remarkable in that it is in contrast to the usual
observation that the enantioselectivity decreases when the R
group becomes branched.16 In general, high enantioselectivity
is difficult to achieve in the reduction of ketones with bulky
groups via metal-catalyzed hydrogen transfer or hydrogena-
tion.9,17,18 The reductions of butyrophenone (1c), 2,2-dimeth-
ylpropiophenone (1h), and phenyl cyclopropyl ketone (1i) were
carried out on a millimole scale and the corresponding chiral
alcohols were isolated in greater than 90% yields with high
optical purity, demonstrating the applicability of carbonyl
reductase SSCR in the preparation of these chiral alcohols.
A similar substrate-size-induced reversal of stereoselectivity
has previously been observed for the reduction of methyl alkyl
ketones (CH3COR, R is alkyl) catalyzed by an alcohol dehy-
drogenase from Thermoanaerobium brockii.19 Since the X-ray
structures of this carbonyl reductase SSCR and its complex with
a coenzyme, NADPH, have been determined,10,20 an enzyme-
substrate docking analysis was performed on the SSCR protein
structure (PDB file 1Y1P) wth the FlexX program. Such an
analysis would be useful in our understanding of the alkyl chain
induced enantiopreference reversal at the molecular level. Three
residues (Ser133, Try177, and Lys181) were proposed as the
catalytic triad in this short-chain dehydrogenase/reductase
enzyme. The carbonyl oxygen atom of the substrate formed
hydrogen bonds with the Ser133 and Tyr177 residues, and was
protonated from the Tyr177 residue, followed by the attack of
a hydride from the C4 atom of NADPH at the carbonyl carbon
atom of the substrate.20,21 Therefore, care was taken to orient
the hydrogens of hydroxyl groups of Ser133 and Tyr177 toward
the position of water molecule HOH175, which the carbonyl
oxygen of the substrate would occupy during docking. The
overlay of the lowest energy docked conformations of pro-
piophenone (1b) and butyrophenone (1c) into the enzyme active
site are shown in Figure 2 (for individual docking pictures, see
the Supporting Information). In both lowest energy conforma-
tional ensembles the carbonyl carbons were in a position
proximal to the C4 atom of the nicotinamide ring of the cofactor
NADPH (3.311 and 3.394 Å for propiophenone (1b) and
butyrophenone (1c), respectively). The ensembles differed from
one another by the orientations of the substrates at the enzyme
active site. The nicotinamide ring of the cofactor was located
at the Si-face of propiophenone, but at the Re-face of buty-
rophenone. Thus these ketones should be reduced to (R)-1-
phenylpropanol and (S)-1-phenylbutanol, respectively, which is
consistent with our experimental observations. The lowest
energy docked conformation of 2-methylpropiophenone (1g)
showed that the nicotinamide ring of NADPH was located at
specific
activitya
absolute
conf
ketones
ee (%)
1a
1b
1c
1d
1e
1f
1g
1h
1i
28
48
57
114
91
45
42
28
88
87
34
27
98
98
96
98
98
R
R
S
S
S
S
44
R
R
R
R
R
201
574
303
271
1j
1k
a The specific activity was defined in units of nmol·min-1·mg-1
.
The encoded protein was then purified using literature proce-
dures.10,11 The specific activity of carbonyl reductase SSCR
toward the reduction of a series of 1-phenylalkanones was
determined by spectrophotometrically measuring the oxidation
of NADPH at 340 nm at room temperature as previously
described.11 Activity assay with the control cell-free extract,
which was prepared by the expression of the pET15b vector
without the SSCR gene in E. coli BL21(DE3) strain, did not
show activity toward the substrates. The enantioselectivity of
the carbonyl reductase was studied by using a NADPH
regeneration system consisting of D-glucose dehydrogenase
(GDH) and D-glucose.11,13 The enantiomeric excess (ee) values
of the product alcohols were determined by chiral GC analysis.
The absolute configurations of the product alcohols were
assigned by comparing either their retention times with standard
samples14 or their specific rotation with reported data.9,15 The
results are summarized in Table 1.
As can be seen from Table 1, the carbonyl reductase (SSCR)
effectively catalyzed the reduction of all the aryl alkyl ketones
shown in Figure 1. The activity of this enzyme first increased
as the alkyl chain became longer (1a f 1d), reaching its highest
activity when R ) n-butyl (1d). However, the enzyme became
less active when the alkyl chain length was further increased
(longer than 4 carbons, 1e and 1f). Surprisingly, SSCR showed
a higher activity toward the reduction of ketones with bulky
alkyl groups, such as the tert-butyl group (1h). Indeed,
compound 1h showed a higher activity than those found for
the smaller alkyl groups (1a-c,g) and its linear counterpart (1d).
Among all the tested aryl alkyl ketones, the carbonyl reductase
was most active toward the reduction of the aryl cyclopropyl
ketones (1i-k).
An intriguing phenomenon was observed for the enantiose-
lectivity of SSCR-catalyzed reductions of the aryl alkyl ketones.
The reduction of acetophenone (1a) and propiophenone (1b)
produced the (R)-enantiomer as the major product, while the
(S)-enantiomers were obtained in 87-88% ee when the alkyl
group was n-propyl (1c) or n-butyl (1d). The enantioselectivity
decreased as the alkyl group became longer (C5 and C6 chain),
but (S)-configuration alcohols were still the major products.
Interestingly, when the alkyl group became branched, for
example, isopropyl (1g), cyclopropyl (1i), or tert-butyl (1h),
(15) Yamada, T.; Nagata, T.; Sugi, K. D.; Yorozu, K.; Ikeno, T.; Ohtsuka,
Y.; Miyazaki, D.; Mukaiyama, T. Chem.-Eur. J. 2003, 9, 4485-4509.
(16) Brandt, P.; Roth, P.; Andersson, P. G. J. Org. Chem. 2004, 69,
4885-4890.
(17) Hayes, A. M.; Morris, D. J.; Clarkson, G. J.; Wills, M. J. Am. Chem.
Soc. 2005, 127, 7318-7319.
(18) Wu, J.; Ji, J.-X.; Guo, R.; Yeung, C.-H.; Chan, A. S. C. Chem.-
Eur. J. 2003, 9, 2963-2968.
(19) Keinan, E.; Hafeli, E. K.; Seth, K. K.; Lamed, R. J. Am. Chem.
Soc. 1986, 108, 162-169.
(12) Kazlauskas, R. J. Curr. Opin. Chem. Biol. 2000, 4, 81-88.
(13) Eguchi, T.; Kuge, Y.; Inoue, K.; Yoshikawa, N.; Mochida, K.;
Uwajima, T. Biosci. Biotechnol. Biochem. 1992, 56, 701-3.
(14) Uray, G.; Stampfer, W.; Fabian, W. M. F. J. Chromatogr. A 2003,
992, 151-157.
(20) Kamitori, S.; Iguchi, A.; Ohtaki, A.; Yamada, M.; Kita, K. J. Mol.
Biol. 2005, 352, 551-558.
(21) Filling, C.; Berndt, K. D.; Benach, J.; Knapp, S.; Prozorovski, T.;
Nordling, E.; Ladenstein, R.; Jornvall, H.; Oppermann, U. J. Biol. Chem.
2002, 277, 25677-25684.
J. Org. Chem, Vol. 71, No. 25, 2006 9485

(10 mg) were mixed in a potassium phosphate buffer (50 mL, 100
mM, pH 6.5). A solution of the aryl alkyl ketone (500 mg in 2.0
mL of DMSO) was added to this mixture that was then stirred at
room temperature until conversion was complete (usually over-
night). During the reaction the pH was controlled at 6.5-6.6 with
the addition of a 0.5 M NaOH solution. After reaction, the mixture
was extracted with methyl tert-butyl ether and the organic extract
was dried over anhydrous sodium sulfate. Removal of the solvent
gave the particular alcohol product, which was identified by
comparison of 1H and 13C NMR with literature data.9,16,18,22 (S)-1-
Phenylbutanol (2c): 466 mg (93% yield); [R]22 -38.1 (c 1.0,
D
CHCl3) (lit.15 [R]28 -47.9 (c 0.34, benzene)). (R)-2,2-Dimethyl-
D
1-phenylpropanol (2h): 475 mg (95% yield); [R]22D +30.4 (c 1.0,
CHCl3) (lit.9 [R]25D +24.3 (c 2.2, benzene)). (R)-Cyclopropylphe-
nylmethanol (2i): 460 mg (92% yield); [R]22D -32.4 (c 1.0, CHCl3)
(lit.15 [R]28 +22.6 (c 0.234, CHCl3)) for S-enantiomer (90% ee).
D
FIGURE 2. The overlay of lowest energy docked conformations of
propiophenone (golden yellow) and butyrophenone (purple) into the
enzyme active site.
Enzyme-Substrate Docking Studies. The crystal structure of
the SSCR/NADPH (PDB code 1Y1P) was used in the calculations.
In the crystal structure, two nearly identical protein molecules
(Mol-1 and Mol-2) were found. The docking was performed on
Mol-1 where an NADPH was tightly bound. Since the carbonyl
oxygen atom of the substrate was assumed to form hydrogen bonds
with Ser133 and Tyr177, the torsion angles of Ser133 and Tyr177
were adjusted so that the hydrogens of their hydroxyl groups were
oriented toward the water molecule (HOH175) in the crystal
structure, which was coincident with the carbonyl oxygen of the
docked substrate. All docking calculations were performed with
the FlexX program in the SYBYL7.1 modeling package. A docking
algorithm that took into account ligand flexibility but kept the
protein rigid was employed. All docking runs were carried out with
the standard parameters of the program for interactive growing and
subsequent scoring.
the Si-face of substrate (see the Supporting Information), leading
to (R)-2-methyl-1-phenylpropanol. The enzyme-substrate dock-
ing of the other aryl alkyl ketones also showed the preferred
docking conformations to be consistent with the experimental
results.
In summary, a carbonyl reductase from Sporobolomyces
salmonicolor (SSCR) was found to effectively catalyze the
enantioselective reduction of aryl alkyl ketones having a number
of diverse alkyl groups. Significantly, ketones with sterically
bulky alkyl groups such as isopropyl, tert-butyl, and cyclopropyl
were enzymatically reduced for the first time to the correspond-
ing alcohols with greater than 96% ee. It was also found that,
while a linear alkyl chain induced an enantiopreference reversal
of the reduction, the enantiopreference remained unchanged and
enantioselectivity was dramatically enhanced when the alkyl
group became branched. The lowest energy conformations of
the aryl alkyl ketones in the enzyme active site, obtained from
enzyme-substrate docking studies, were found to be consistent
with these intriguing experimental results. Thus, enzyme-
substrate docking analyses may provide useful guidance in the
rational design of new carbonyl reductases with desired enan-
tioselectivity. Such studies are currently underway in our
laboratories.
Acknowledgment. We thank Southern Methodist University
for generous financial support, Professor John A. Maguire for
proof reading the manuscript, and Dr. Yan Yang for preparing
the SSCR enzyme.
Supporting Information Available: General methods, proce-
dures for activity assay and ketone reductions, details of chiral GC,
and 13C NMR spectra for the alcohols obtained in the preparative
scale ketone reductions. This material is available free of charge
via the Internet at http://pubs.acs.org.
Experimental Section
JO061571Y
Preparative Scale Ketone Reduction. The preparative syntheses
were carried out as follows: d-glucose (1.0 g), D-glucose dehy-
drogenase (10 mg), NADPH (10 mg), and carbonyl reductase SSCR
(22) Carter, M. B.; Schiott, B.; Gutierrez, A.; Buchwald, S. L. J. Am.
Chem. Soc. 1994, 116, 11667-70.
9486 J. Org. Chem., Vol. 71, No. 25, 2006