K. Uwai et al. / Bioorg. Med. Chem. 16 (2008) 1084–1089
1087
low q values (0.7–1.2) were observed in the reduction of
aryl ketones by crude carbonyl reductases of the human
liver.15 In the chemical reaction systems, low q values
were reported for the Meerwein–Ponndorf–Verly
(MPV) reaction by supercritical 2-propanol9 (q + 0.33)
and moderate q values were seen in the MPV reaction
by metal alkoxide (q + 1.3–1.7)10–12 and in the metal hy-
dride reaction13,14 (q + 1.9–3.1). Our present results
indicate that enzymatic reduction shows q values that
are similar to those with the MPV reaction by supercrit-
ical 2-propanol. In the present reaction, the enzyme dis-
plays an ordered bi–bi kinetic mechanism in which
binding of the nicotineamide cofactor is an obligatory
requirement before substrate can bind, and direct hy-
dride transfer occurs from the C4 position of the nicoti-
neamide ring to the acceptor carbonyl of the substrate
which is protonated by Y55 that acts as a general acid,
and this chemical event would be the rate limiting step.16
Thus, the strength of the acidity of the Y55 may also
contribute to determination of the rate of the carbonyl
reduction.
bonyl compounds to the corresponding alcohols. With
this in mind, new drugs can be designed that exploit this
reduction pathway by introducing an electron-with-
drawing group adjacent to the reduction site when a
reduction reaction is desired or by adding an electron-
donating group when minimization of the reduction
pathway is desired.
4. Materials and methods
4.1. Chemicals
Para-nitro- and methoxy-acetophenones and acetophe-
none were purchased from Wako Pure Chemical Indus-
tries, Ltd. (Osaka, Japan). Para-cyano- and N,N-
dimethylacetophenones were purchased from Tokyo
Kasei Kogyo Co., Ltd. (Tokyo, Japan). Para-bromo-
and methyl- acetophenones were purchased from Naka-
rai Tesque, Inc. (Kyoto, Japan). NADP+ and glucose
6-phosphate (G-6-P) were procured from Nacalai Tes-
que, Inc. Glucose-6-phosphate dehydrogenase (G-6-P-
D) was purchased from Sigma Chemical Co. (St. Louis,
MO, USA). All other chemicals and solvents were of the
highest commercially available grade.
In any case, our findings show that acetophenone deriv-
atives containing an electron-withdrawing group at the
para position were reduced faster than those with an
electron-donating group. This indicates that the reduc-
tive metabolism of drugs having aromatic ketones, such
as haloperidol, depends on the substituent at the para
position of the aromatic ring, and an electron-withdraw-
ing group promotes the metabolism.
4.2. Instruments
All chemical reactions were conducted in oven-dried
glassware. Silica gel 60 (70–230 mesh, ASTM, Merck)
was used for column chromatography. Precoated Kie-
selgel 60F-254 plates (0.25 mm, Merck) were used for
TLC analysis, and the spots were detected by the
absorption of UV light at 254 nm by spraying anisalde-
hyde–sulfuric acid reagent followed by heat treatment.
Optical rotations were measured on JASCO DIP-360
The abnormally large Vmax/Km values of the nitro and
cyano derivatives (1b and 1c) can be explained by the
relatively small Km value of the substrate. The Km value
increased according to the increase in the electron-
donating property of the substituent, except for the case
of the methoxy group. This suggests that the enzyme has
an amino acid residue(s) that can interact with the oxy-
gen moiety at the para position of the substrate. In that
case, nitro (1b) and methoxy substrates (1f) will show
smaller Km values compared to the other substrates.
1
polarimeters. H- and 13C-NMR spectra were recorded
on JEOL JNM EX270 (270 MHz), JEOL JNM 400
(400 MHz) and JEOL JNM 600 (600 MHz) spectrome-
ters. Mass spectra were recorded on JEOL JMS DX-
303/JMA-DA 5000 spectrometers. The quantitative
analyses, enantiomeric excess (ee) and absolute configu-
rations of the alcohols were determined by HPLC using
a Waters 510 HPLC Pump (Waters, Milford, MA,
USA) that was equipped with a chiral column. The elu-
ate was monitored at 254 nm using a Waters 486 Tun-
able Absorbance Detector (Waters, Milford, MA,
USA). Chromatographs were recorded using a Chro-
matocorder 21 (S.I.C., Tokyo, Japan).
The influence of the electronic effects on the enantiose-
lectivity can be surmised by comparison of the Vmax
/
Km value of the S-enantiomer with that of R. The
enantioselectivity of the reduction is subject to a remote
electronic effect on the substrate ketones. As seen in the
‘ratios’ presented in Table 1, R-enantiomers were not
recognized at all on HPLC in the reduction of elec-
tron-withdrawing moiety substituted acetophenones.
However, reduction of electron-donating methyl-(1e)
and N,N-dimethyl-(1g) substituted acetophenones gave
(R)-enantiomers at a relatively higher ‘ratio’. Thus, ke-
tones with an electron-withdrawing group in the
para-position of the aromatic ring might better afford
enantioselectively reduced alcohols compared to elec-
tron-donating groups.
4.3. Synthesis of authentic samples of reduced aryl
ketones
Alcohols were synthesized by reducing the aryl ketones
with NaBH4 (0.25 mol equivalent) followed by purifica-
tion by silica gel column chromatography. The pro-
duced alcohols were subjected to lipase catalyzed
transesterification to afford chiral alcohols, and their
optical rotatory powers were referenced to the reported
data.
3. Conclusions
This study demonstrated an electron-withdrawing effect
on the reduction rate in enzymatic conversion of car-