Microbial enantioselective reduction of acetylpyridine derivatives

Article abstract of DOI:10.1271/bbb.67.809

The microbial enantioselective reduction of acetylpyridine derivatives was studied. Many microorganisms were found to reduce 5-acetylfuro[2,3-c]pyridine (AFP) to (S)-5-(1-hydroxyethyl)furo[2,3-c]-pyridine (FPH). Candida maris IFO10003 reduced AFP to (R)-FPH with high enantioselectivity. The microbial reduction reaction was optimized. The aeration conditions and glucose concentration affected the yield and stereoselectivity. The cells accumulated 17.5 g/l (107 mM) of (R)-FPH with a 99% yield and 97% enantiomeric excess (e.e.). A cell-free extract of C. maris accumulated 91.5 g /l (559 mM) with over 99% e.e. with enzymatic NADH regeneration. (R)-FPH is an important intermediate for the synthesis of HIV reverse-transcriptase inhibitor, and other optically active 1-(pyridyl)ethanol derivatives are versatile chiral building blocks for asymmetric synthesis.

Full text of DOI:10.1271/bbb.67.809

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Microbial Enantioselective Reduction of
Acetylpyridine Derivatives
Shigeru KAWANOa, Miho HORIKAWAa, Yoshihiko YASOHARAa & Junzo HASEGAWAa
a Fine Chemicals Research Laboratories, Kaneka Corporation1-8 Miyamae, Takasago,
Hyogo 676-8688, Japan
Published online: 22 May 2014.
To cite this article: Shigeru KAWANO, Miho HORIKAWA, Yoshihiko YASOHARA & Junzo HASEGAWA (2003) Microbial
Enantioselective Reduction of Acetylpyridine Derivatives, Bioscience, Biotechnology, and Biochemistry, 67:4, 809-814, DOI:
10.1271/bbb.67.809
To link to this article: http://dx.doi.org/10.1271/bbb.67.809
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Biosci. Biotechnol. Biochem., 67 (4), 809–814, 2003
Microbial Enantioselective Reduction of Acetylpyridine Derivatives
Shigeru KAWANO, Miho HORIKAWA, Yoshihiko YASOHARA,† and Junzo HASEGAWA
Fine Chemicals Research Laboratories, Kaneka Corporation, 1-8 Miyamae, Takasago, Hyogo 676-8688, Japan
Received October 18, 2002; Accepted December 27, 2002
The microbial enantioselective reduction of acetyl-
pyridine derivatives was studied. Many microorganisms
were found to reduce 5-acetylfuro[2,3-
to )-5-(1-hydroxyethyl)furo[2,3- ]-pyridine (FPH).
Candida maris IFO10003 reduced AFP to ( )-FPH
c]pyridine (AFP)
(
S
c
R
with high enantioselectivity. The microbial reduction
reaction was optimized. The aeration conditions and
glucose concentration aŠected the yield and stereoselec-
Fig. 1. Enantioselective Reduction of 2-Acetylpyridine Deriva-
tive.
tivity. The cells accumulated 17.5 g l (107 m
M
) of (
enantiomeric excess
(e.e.). A cell-free extract of C. maris accumulated 91.5 g
l (559 m ) with over 99 e.e. with enzymatic NADH
regeneration. ( )-FPH is an important intermediate
R)-
W
FPH with a 99
% yield and 97%
M
%
W
both an eŠective and economical method.4) In this
paper, we report the results we obtained on screening
R
for the synthesis of HIV reverse-transcriptase inhibitor,
and other optically active 1-(pyridyl)ethanol derivatives
are versatile chiral building blocks for asymmetric
synthesis.
for the enantioselective reduction of AFP to (
FPH, and the preparation of (
strain.
R
)-
R)-FPH with a potent
Key words: enantioselective reduction; acetylpyridine;
pyridylethanol; Candida maris
Materials and Methods
Chemicals. 5-Acetylfuro[2,3-
5-acetyl-7-chlorofuro[2,3- ]pyridine were prepared
according to previous reports.3,5) Racemic alcohols
were prepared by NaBH4 reduction. ( )-FPH was
c]pyridine (AFP) and
Recently, the importance of optically active com-
pounds has increasingly been recognized in the ˆeld
of pharmaceuticals.1) Optically active pyridylethanol
derivatives are useful as intermediates for phar-
maceuticals and ligands for catalysts of asymmetric
synthesis. In particular, 1-(2-pyridyl)ethanol deriva-
tives are notable compounds because there is a
c
S
prepared by the microbial reduction of AFP using
Candida viswanathii IFO10415 cells. Glucose de-
hydrogenase was purchased from Amano Enzyme,
Japan. All other chemicals used in this study were of
analytical grade and were commercially available.
heteroatom near the asymmetric center. (
R)-5-(1-
hydroxyethyl)-furo[2,3- ]pyridine (FPH) is an im-
c
portant intermediate for the synthesis of an inhibitor
of non-nucleoside HIV reverse-transcriptase inhibi-
tor (Fig. 1).2) Enzymatic kinetic resolution by a lipase
Microorganisms and cultivation. Microorganisms
were obtained from our laboratory collection, the
collection of the Institute for Fermentation, Osaka
(IFO), the Institute of Molecular and Cellular
Biosciences, the University of Tokyo (IAM), and the
Centraalbureau voor Schimmelcultures, the Nether-
lands (CBS). The Y medium used for the yeast was
has been reported for the synthesis of FPH, but the
3)
theoretical maximum yield is only 50
z
.
Enan-
tioselective reduction of the corresponding carbonyl
compound, 5-acetylfuro[2,3- ]pyridine (AFP), is an
c
economically better method. It is said, however, that
a nitrogen atom in the pyridine ring results in low
enantioselectivity on catalytic asymmetric reduction.
The use of a stoichiometrical reducing reagent such
as optically active DIP-chloride results in good selec-
tivity and a good yield, but is not economical,3) while
the enzymatic reduction of carbonyl compounds is
composed of 0.7
z
KH2PO4, 1.3
z (NH4)2HPO4,
･
･
ZnSO4 7H2O,
0.08
z
MgSO4 7H2O, 0.007
z
z
･
･
0.009
z
z
FeSO4 7H2O, 0.0005
CuSO4 5H2O,
NaCl, 0.3 yeast
glucose, pH 7.0. The B medium used
for the bacterium was composed of 1 meat extract,
peptone, 0.5 yeast extract, and 0.3 sodium
･
0.001
MnSO4 4H2O, 0.01
z
z
extract, and 4
z
z
1
z
z
z
†
To whom correspondence should be addressed. Fax:
81-794-45-2668; E-mail: Yoshihiko.Yasohara kaneka.co.jp
＋ ＠
Abbreviation: e.e., enantiomeric excess

810
S. KAWANO et al
.
chloride, pH7.0. In the screening experiments, each
strain was inoculated into 5 ml of medium in a test
extract, 2.8 g of glucose, 120 mg of NAD＋, 270 units
of glucose dehydrogenase, and 2.5 g of AFP (315 mg
×
tube (24 mm
q
200 mm), followed by incubation at
portions, 8 times) was stirred at 30
9
C. The pH of the
309C with reciprocal shaking, usually for 24 h.
mixture was kept at 6.0 with 5 sodium hydroxide.
N
The AFP-reducing enzyme assay involving a spectro-
metric method was done as follows: the assay
Screening method for AFP-reducing strains. Each
reaction mixture, composed of cells from 5 ml of
culture broth, 5 mg of AFP, and 80 mg glucose in
mixture, which was composed of 100 m
phosphate buŠer (pH 6.5), 0.2 m
NADPH, and 1 m AFP, and the reaction mixture
at 30 C in a total volume of 3 ml, was monitored for
M
potassium
M
NADH or
1 ml of 100 m
M
potassium phosphate buŠer (pH 7.0),
C. Five milliliters of ethyl
M
was shaken for 24 h at 30
9
9
acetate was added to the reaction mixture, followed
by centrifugation, and then the organic layer was
analyzed for AFP and FPH.
the decrease in absorbance at 340 nm. The FPH-
oxidating enzyme assay was done as described above
using FPH instead of AFP, and NAD＋ or NADP＋
instead of NADH or NADPH, respectively. One unit
of the enzyme was deˆned as the amount catalyzing
the oxidation or reduction of 1
minute.
Cultivation of Candida maris IFO10003. C. maris
IFO10003 was inoculated into a 500-ml shaking ‰ask
containing 100 ml of the Y medium for which the
mmol of coenzyme per
glucose concentration was 8
z
, and the mixture was
then incubated at for 24 h at 30
9
C with shaking. The
Preparation of (R)-FPH. The fermenter reaction
mixture was extracted twice with ethyl acetate. The
organic layer was dried with sodium sulfate and then
evaporated to dryness. Ethyl acetate and activated
charcoal were added to the residue, followed by
stirring at room temperature for 2 h. The activated
charcoal was removed by ˆltration, and the solvent
was evaporated oŠ. The residue was crystallized from
cultures were then transferred to a 5-l fermenter (B.
E. Marubishi, Japan) containing 2.5 l of the same
medium. The cultivations were thermostatted at
9
30 C, and the pH was set at 5.5 by automatic
titration with aqueous sodium hydroxide. Constant
stirring was maintained at 300 rpm, and the air-‰ow
rate was 0.75 l min during the cultivation. After
W
cultivation for 24 h, the cells were collected by
centrifugation and washed with physiological saline.
ethyl acetate and methylcyclohexane to give (
hydroxyethyl)furo[2,3- ]pyridine as a white cystal-
line substance (8.1 g, 81 yield, 98.7 e.e.); NMR
dH (CDCl3): 1.56 (3H, d, 6.35 Hz), 4.12 (1H, s),
R)-5-(1-
c
z
z
Whole-cell conversion by C. maris IFO10003.
Each reaction mixture, composed of the cells from
3 ml of culture broth, 7.5 mg of AFP or FPH, and
J
＝
＝
＝
5.00 (1H, q,
J
6.35 Hz), 6.80 (1H, d,
J
1.95 Hz),
＝
7.54 (1H, s), 7.77 (1H, d,
J
1.95 Hz), 8.80 (1H, s),
20
30 mg of glucose in 1.5 ml of 100 m
phosphate buŠer (pH 6.5), was stirred for 24 h at
30 C. Five milliliters of ethyl acetate was added to
M
potassium
[a
]
37.0
9
(c 0.56, CHCl3). The reported value for
D
20
D
the (
S
)-isomer is [
a
]
(c 0.51, CHCl3).3)
－
35.39
9
the reaction mixture, followed by centrifugation, and
then the organic layer was analyzed.
Analysis. The substrate and product concentra-
tions were measured with a GC equipped with a 10
PEG-20M on a 80 100-mesh Chromosorb WAW
z
W
×
1 m) column (GL Science, Japan)
Microbial reduction of AFP. Two liters of C.
maris culture broth, 10 g of AFP, and 60 g of glucose
were placed in a 5-l fermenter. The reaction mixture
DMCS (3.2 mm
and FID detection. The optical purities of FPH
and 7-chloro-5-(1-hydroxyethyl)-furo[2,3- ]pyridine
were determined with an HPLC equipped with a
Chiralpak AS (4.6 mm 250 mm) column (Daicel
Chemicals, Japan). The HPLC conditions included
q
c
was incubated at 309C with stirring and aeration.
The pH of the mixture was maintained at 6.0. Two
experiments in which the stirring and aeration condi-
q
×
tions were changed were done; 700 rpm, 0.5 l min
n-hexane ethanol diethylamine (92 8 0.1(v v v)) as
W
W
W
WW
WW
(0–4.5 h); 400 rpm, 0.25 l min (4.5–7 h); 400 rpm,
a mobile phase, a ‰ow rate of 1 ml min, ambient
W
W
0 l min (7–23 h); and 400 rpm, 0.25 l min (0–7.5 h);
column temperature, and detection at 254 nm. The
optical purity of 1-(pyridyl)ethanol derivatives were
measured with an HPLC equipped with a Chiralcel
W
W
400 rpm, 0.125 l min (7.5–22.5 h); 300 rpm, 0 l min
W
W
(22.5–45 h). AFP and glucose were added to the reac-
tion mixture as described in the ˆgure legend.
×
250 mm) column (Daicel Chemi-
OB-H (4.6 mm
q
cals, Japan). The HPLC conditions included
n
-
The enzymatic reduction of AFP. Cells of C. maris
hexane ethanol diethylamine (95 5 0.1(v v v)) as a
W
W
WW
WW
from 100 ml of culture suspended in 25 ml of 100 m
M
mobile phase, a ‰ow rate of 1 ml min, ambient
W
potassium phosphate buŠer (pH 6.0) were disrupted
with 0.25 mm-diameter glass beads (Dyno Mill KDL-
column temperature, and detection at 254 nm. The
absolute conˆgurations were identiˆed by compari-
son with reported results for baker's yeast reduc-
tion.6) Glucose concentrations were measured with a
commercial kit using glucose oxidase and peroxidase
9
A, Switzerland) at 4 C. After centrifugation, the
resulting supernatant was used as the cell-free
extract. A reaction mixture of 25 ml of the cell-free

Reduction of Acetylpyridine
811
(Wako Pure Chemicals, Japan). 1H-NMR spectra
were recorded in CDCl3 with a FT-NMR JM-400
spectrometer (400 MHz; JEOL, Japan). Chemical
shifts are expressed in parts million (ppm), with
W
trimethylsilane as the internal standard. Optical
rotation was measured with a digital polarimeter
(SEPA-200, Horiba, Japan).
Results
Screening of AFP-reducing microorganisms
Approximately 600 strains were examined for their
ability to reduce AFP to FPH. The distributions of
the yields and stereoselectivities of AFP-reducing
yeasts are plotted in Fig. 2. Many AFP-reducing
Fig. 2. Screening Results of AFP-Reduction.
The yields and optical purities of FPH produced by 435 stains
that reduce AFP to FPH are plotted.
strains were found to have (
only three strains had the desired (
(Table 1). Regarding the ( )-speciˆc microorgan-
S
)-speciˆc activity, but
R
)-speciˆc activity
R
Table 1. AFP-Reducing Microorganismsa)
isms, Candida maris IFO10003 had a high yield and
high stereoselectivity at higher substrate concentra-
tions (Table 2). This strain was thus selected for
further investigation.
Yield
Strain
Stereoselectivity
z
e.e.
(
z
)
Candida maris IFO10003
Candida parapsilosis IFO585
Dipodascus ovetensis IFO1201 68
99
59
R
S
S
97.8
98.5
97.2
Table 2. EŠects of the Substrate Concentration on Microbial
Geotrichum candidum
CBS178.71
92
S
98.8
AFP-Reductiona)
Hyphopichia burtonii IFO844
Jensenia canicruria IFO13914
Ogataea wickerhamii IFO1706 77
Pichia farinosa IFO602
Rodosporidium toruloides
IFO559
Sporidiobolus johnsonii
IFO6903
63
49
S
S
R
R
96.7
98.6
77.6
39.2
5 g l
20 g l
W
W
Strain
Yield (
z) ze.e. Yield (z) ze.e.
72
Candida maris IFO10003
Candida pintolopesii IFO729
Cadida etchellsii IFO1229
Pichia farinosa IAM4312
99
53
43
42
98.4
96.5
97.1
95.6
86
10
98.8
96.0
96.7
92.4
65
S
99.9
8.0
6.0
56
S
99.2
a)
a)
The assay conditions are given in Materials and Methods.
The assay conditions are given in Materials and Methods.
Fig. 3. EŠects of the Glucose Concentration on the AFP-Reducing Reaction with Candida maris Cells.


#
Reactions were done as described in Materials and Methods. , Glucose; , FPH; , pH of the reaction mixture. The initial concen-
trations of glucose were (A) 2; (B) 4; and (C) 8 , and the concentration of AFP was 5 g l.
z
W

812
S. KAWANO et al.
EŠects of glucose on microbial AFP-reduction
Microbial reduction of AFP to (R)-FPH
We attempted to prepare ( )-FPH with C. maris
The eŠects of the glucose concentration on the
AFP-reducing reaction of C. maris cells was exam-
ined. The pH of the reaction mixture, the glucose
concentration, the AFP concentration, and the FPH
yield are shown in Fig. 3. A high yield was obtained
in the presence of su‹cient glucose in the mixture
R
cells using a 5-l fermenter, the results being shown in
Fig. 4. Two experiments were done in which the stir-
ring and aeration conditions were 700 rpm, 0.5 l min
W
(A), and 400 rpm, 0.25 l min (B), initially. In the
W
initial stage of the reaction, the accumulation of (
FPH was smooth with lower aeration conditions
(400 rpm, 0.25 l min). ( )-FPH was then accumu-
lated with a decrease in AFP by reducing the aeration
and stirring rates in either reaction. ( )-FPH
accumulated slowly during the reactions. After 24 h
of reaction starting with 400 rpm, 0.25 l min (B),
R)-
(glucose concentration, 4
decrease in the yield was observed, however, when
the glucose concentration was 2 . ( )-FPH was
z z
and 8 ). A drastic
R
W
z
R
converted to AFP under the same reaction condi-
tions, and AFP and FPH were stable in the absence
S
of cells (Table 3). Thus, the produced (R)-FPH
might be converted to AFP with a low glucose
concentration.
W
approximately 17.5 g l (107 m
M
)
of
(R)-FPH
, and the opti-
W
accumulated. The yield was over 99
cal purity of FPH was 97 e.e.
z
z
Table 3. Whole Cell Conversion of AFP Derivatives by
Enzymatic reduction of AFP linked with NADH
regeneration
C. marisa)
The AFP-reducing enzyme assay involving a spec-
trometric method showed that the AFP-reducing
Product (g l)
W
Cytalyst
Cells
Substrateb)
AFP
AFP
N.D.c)
2.9
0.1
5.0
(
R
)-FPH
(S)-FPH
activity (3.2 units ml) and (R)-FPH-oxidating activi-
W
ty (0.19 unit ml) in a cell-free extract of C. maris
4.9
2.0
N.D.
N.D.
5.0
N.D.
0.1
4.9
N.D.
N.D.
5.0
W
(
(
R
S
AFP
)-FPH
)-FPH
required NADH as a cofactor. There was no oxido-
reducing activity with NADP＋ or NADPH. Also,
Noned)
(
S
)-FPH oxidating activity was not observed. The
(
(
R
S
)-FPH
)-FPH
N.D.
N.D.
cell-free extract accumulated approximately 91.5 g l
N.D.
W
(559 m ) of (R)-FPH with NADH regeneration due
M
a)
The assay conditions are given in Materials and Methods.
to glucose dehydrogenase. The molar amounts of
FPH and sodium hydroxide for neutralizing gluconic
acid derived through glucose oxidation were almost
b)
c)
d)
The initial concentration of substrate was 5 g l.
W
Not detected.
The reaction mixture did not contain cells.
Fig. 4. Reduction of AFP with C. maris Cells in a Jar Fermenter.
The reaction was done as described in Materials and Methods. The aeration conditions: (A) 700 rpm, 0.5 l min (0–4.5 h); 400 rpm,
W
0.25 l min (4.5–7 h); 400 rpm, no aeration (7–23 h); and (B) 400 rpm, 0.25 l min (0-7.5 h); 400 rpm, 0.125 l min (7.5–22.5 h); 300 rpm,
W
W
W
no aeration (22.5–45 h), AFP was added: (B) 5, 5, 10 and 5 g at 22.5, 26, 28.5 and 32 h, respectively, glucose was added: (A) 60 and 80 g
at 4.5 and 7.5 h, respectively; and (B) 60, 50 and 80 g at 4.5, 7.5 and 22.5 h, respectively, , concentration of (R)-FPH; , concentra-


#
$
tion of (S)-FPH; , concentration of AFP; , optical purity of (R)-FPH.

Reduction of Acetylpyridine
813
Discussion
The enzymatic reduction of carbonyl compounds
is a useful method for the synthesis of optically active
compounds. It is important to obtain a suitable
biocatalyst for the desired reaction. Regarding the
biological reduction of 2-acetylpyridine, baker's
yeast is (
lus keˆr is (
results showed that many microorganisms reduced
AFP to ( )-FPH, but Candida maris IFO10003
S
)-speciˆc with 85
z
e.e.,7) and Lactobacil-
z
e.e.8) The screening
R
)-speciˆc with 97
S
catalyzed the desired reaction with a high yield and
high stereoselectivity.
A high glucose concentration was important for
the reduction of AFP with whole cells of C. maris. A
low glucose concentration caused the oxidation of
(R)-FPH to AFP with a decrease in pH. In general,
the optimum pH for enzymatic oxidation is higher
than that for reduction. A lack of glucose may cause
further degradation of glucose metabolites by cells,
and may result in lowering of the pH.
Fig. 5. Enzymatic Reduction of AFP with the Coenzyme
Regeneration System.
The reduction of AFP to FPH, and the oxidation
of (R)-FPH were detected in a cell-free extract of C.
Reactions were done as described in Materials and Methods.
maris in the presence of NADH. NADH-dependent
dehydrogenase may catalyze the reduction of AFP.
In fact, a cell-free extract containing an NADH
regeneration system accumulated a higher concentra-
tion of FPH than in the whole-cell reaction. In other
words, the aeration and glucose conditions for the
whole-cell reaction may be important for the regener-
ation of a reduced form of coenzyme. The calculated
turnover of NAD＋ based on the amounts of NAD＋
and AFP was only about 107 in the cell-free reaction
with NADH regeneration. The coenzyme regenera-
tion e‹ciency may be improved by construction of a
microorganism producing AFP-deydrogenase and
glucose dehydrogenase.9)
AFP (78 m
FPH; , NaOH.
M) was added to the reaction mixture (arrow). ,


Table 4. Microbial Reduction of Acetylpyridine Derivatives by
C. marisa)
Yield
ze.e.
Substrate
Stereoselectivity
(
z
)
5-Acetylfuro[2,3-
(AFP)
5-Acetyl-7-chlorofuro[2,3-
pyridine
2-Acetylpyridine
3-Acetylpyridine
4-Acetylpyridine
c
]pyridine
99
R
97.8
90.5
c
]
51
98
99
99
R
R
R
À
99
58.0
99
À
The accumulation of (S)-FPH was observed in the
a)
The assay conditions are given in Materials and Methods.
whole-cell reaction of C. maris, but was not observed
in the cell-free extract reaction with an NADH
regeneration system. This (S)-speciˆc or low enan-
stoichiometrical (Fig. 5). The reduction then
proceeded together with the regeneration of NADH
tioselective enzyme may only be stable in cells or may
not require NADH. C. maris has at least a ( )-
speciˆc reducing enzyme with over 99 e.e., and
thus is a favorable enzyme for application to
high enantioselective production of )-1-
(pyridyl)ethanol derivatives. Our interest continues
in the properties of this ( )-speciˆc enzyme of C.
maris and its gene for a more eŠective production
system for ( )-FPH.
R
by glucose dehydrogenase. The optical purity of (
FPH was over 99 e.e.
R
)-
z
z
(
R
Reduction of acetylpyridine derivatives
Acetylpyridine derivatives were reduced to the
corresponding ( )-1-(pyridyl)ethanols by C. maris
R
R
cells with high yields under these screening conditions
(Table 4). The stereoselectivity was high except in the
case of 3-acetylpyridine. Baker's yeast reduces acetyl-
R
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S
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