Enantioselective microbial reduction of 2-oxo-2-(1′,2′,3′,4′-tetrahydro-1′,1′, 4′,4′-tetramethyl-6′-naphthalenyl)acetic acid and its ethyl ester

Article abstract of DOI:10.1016/S0957-4166(02)00109-X

The chiral ester ethyl (2R)-hydroxy-2-(1′,2′,3′,4′-tetrahydro-1′, 1′,4′,4′-tetramethyl-6′-naphthalenyl) acetate 2 and the corresponding acid 4 were prepared as intermediates in the synthesis of the retinoic acid receptor gamma-specific agonist (R)-3-fluor

Full text of DOI:10.1016/S0957-4166(02)00109-X

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 13 (2002) 349–355
Enantioselective microbial reduction of 2-oxo-2-
(1%,2%,3%,4%-tetrahydro-1%,1%,4%,4%-tetramethyl-6%-naphthalenyl)acetic
acid and its ethyl ester
Ramesh N. Patel,* Linda Chu, Ramakrishna Chidambaram, Jason Zhu and Joydeep Kant
Process Research & Development, Bristol-Myers Squibb Pharmaceutical Research Institute, PO Box 191, New Brunswick,
NJ 08903, USA
Received 11 January 2002; accepted 25 February 2002
Abstract—The chiral ester ethyl (2R)-hydroxy-2-(1%,2%,3%,4%-tetrahydro-1%,1%,4%,4%-tetramethyl-6%-naphthalenyl) acetate 2 and the
corresponding acid 4 were prepared as intermediates in the synthesis of the retinoic acid receptor gamma-specific agonist
(R)-3-fluoro-4-[[hydroxy(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)acetyl]amino]benzoic acid 7. Enantioselective
reduction of ethyl 2-oxo-2-(1%,2%,3%,4%-tetrahydro-1%,1%,4%,4%-tetramethyl-6%naphthalenyl)acetate 1 to alcohol 2 was carried out using
Aureobasidium pullulans SC 13849 in 98% yield and with an enantiomeric excess (e.e.) of 96%. Among microorganisms screened
for the reduction of 2-oxo-2-(1%,2%,3%,4%-tetrahydro-1%,1%,4%,4%-tetramethyl-6%-naphthalenyl)acetic acid 3 to hydroxy acid 4, Candida
maltosa SC 16112 and two strains of Candida utilis (SC 13983, SC 13984) gave reaction yields of >53% with e.e.s of >96%. © 2002
Elsevier Science Ltd. All rights reserved.
1. Introduction
the desired hydroxy acid 417a was prepared by chemical
reduction of the corresponding keto acid 3 followed by
resolution with an enantiomerically pure amine.17b
A number of studies have demonstrated that retinoids
(vitamin A derivatives) are essential for normal growth,
vision, tissue homeostasis, and reproduction.1–5
Retinoic acid and its natural and synthetic analogs
(retinoids) exert a wide variety of biological effects by
binding to or activating a specific receptor or sets of
receptors.6,7 They have been shown to affect cellular
growth and differentiation and are promising drugs for
the treatment of cancers.8–10 A few retinoids are already
in clinical use for the treatment of dermatological dis-
eases such as acne and psoriasis.11,12 (R)-3-Fluoro-4-
[[hydroxy(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naph-
thalenyl)acetyl]amino]benzoic acid 7 is a retinoic acid
receptor gamma-specific agonist which is potentially
useful as a dermatological and anti-cancer drug.12–16
2. Results and discussion
Oxidoreductases from many organisms, including those
from yeast,18–23 horse liver,24 Thermoanerobic
brockii,25,26 Lactobacillus kefir,27 Pseudomonas sp.,28
Geotrichum candidum,29,30 Hansenula polymorpha,31,32
Mortierella rammaniana,33 and Sulfolobus solfataricus,34
have been used for the enantioselective reduction of
ketones to alcohols. A number of microorganisms were
screened for the enantioselective reduction of keto ester
1 to alcohol 2. The yields and product e.e.s obtained
with the six best cultures are shown in Table 1. A.
pullulans SC 13849 gave the highest yield (99%) and e.e.
(97.9%) of the desired (R)-hydroxy ester.
Herein, we describe the enantioselective microbial
reduction of ethyl 2-oxo-2-(1%,2%,3%,4%-tetrahydro-
1%,1%,4%,4%-tetramethyl-6%-naphthalenyl)acetate 1 and acid
3 to the corresponding (R)-hydroxy ester 2 and
hydroxy acid 4, potential intermediates in the synthesis
of 7. We also demonstrate the reduction of ketoamide 5
to the corresponding (R)-hydroxy amide 6. Previously,
Aureobasidium pullulans SC 13849 was investigated fur-
ther. In a two-stage process: cells were grown in a 25 L
fermentor for 40 h and then harvested by centrifugation
and stored at −60°C until further use. Frozen cells were
suspended in 50 mM potassium phosphate buffer (pH
6.8) and the resulting cell-suspensions were used to
carry out bioreduction (as described in Section 4). After
* Corresponding author.
0957-4166/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(02)00109-X

350
R. N. Patel et al. / Tetrahedron: Asymmetry 13 (2002) 349–355
OH
O
Microbial
Reduction
OEt
OEt
O
O
(R)-2
1
OH
O
Microbial
Reduction
OH
OH
O
O
3
(R)-4
Microbial
Reduction
F
O
F
OH
H
N
H
N
OR
O
5
OR
O
O
O
(R)- 6
6
7
R=Met
R=H
16 h, a yield of 99% and e.e. of 97% were obtained for
(R)-hydroxy ester 2 (Table 2). At the end of the reac-
tion, hydroxy ester 2 was adsorbed onto XAD-16 resin
and, after filtration, it was recovered in 94% yield from
the resin by acetonitrile extraction.
Table 1. Stereoselective microbial reduction of keto ester 1
Microorganisms
Yield of
E.e. of
hydroxy
ester 2 (%)
hydroxy
ester 2 (%)
The recovered (R)-hydroxy ester 2 was treated with
Chirazyme L-2 or pig liver esterase (as described in
Section 4) to convert it to the corresponding (R)-
hydroxy acid 4 in quantitative yield. The enzymatic
hydrolysis was used to avoid the possibility of racem-
ization under chemical hydrolytic conditions.
Aureobasidium pullulans SC 13894
Bre6ibacterium linens SC 13959
Hansenula polymorpha SC 13865
Pichia methanolica SC 16116
Pseudomonas oleo6orans SC 13828
Trigonopsis 6ariabilis SC 16071
98
26
8
28
29
13
98
89
98
96
99
93
A simpler, single-stage fermentation–bioreduction pro-
cess (fermentation of microbial cultures to complete
growth and subsequent addition of substrate to the
fermentor to continue biotransformation) was devel-
oped for the conversion of keto ester 1 (5 g/L) to the
corresponding hydroxy ester 2 with cells of pullulans SC
The reaction yields and e.e.s were determined by HPLC using C-18
column and chiralcel OD column, respectively, as described in Sec-
tion 4.
Table 2. Stereoselective microbial reduction of keto ester 1 by A. pullulans: SC 13849: two-stage process
Reaction time (h)
Keto ester 1 (g/L)
Hydroxy ester 2 (g/L)
Yield (%)
E.e. of hydroxy ester 2 (%)
1
3
5
8
12
16
1.6
0.84
0.6
0.3
0.1
0
0.4
1.1
1.4
1.7
1.87
1.98
20
55
70
85
93
99
Not determined
Not determined
96
97
97
97
The reaction yields and e.e.s were determined by HPLC using C-18 column and chiralcel OD column, respectively, as described in Section 4.

R. N. Patel et al. / Tetrahedron: Asymmetry 13 (2002) 349–355
351
enases from Lactobacillus kefir27 and two Pseudomonas
sp.28 exhibit anti-Prelog specificity, transferring the Pro-
R hydride to form (R)-alcohols. In this paper, we have
described the enantioselective reduction of keto ester 1
and keto amide 5 to the corresponding (R)-hydroxy
ester 2 and (R)-hydroxy amide 6 by A. pullulans SC
13848. NADPH is a better cofactor compared to
NADH giving higher reaction yields when cell extracts
were used for this bioreduction process. We have also
demonstrated the enantioselective reduction of keto
acid 3 to the corresponding (R)-hydroxy acid 4 by three
different Candida strains. A. pullulans was not effective
in catalyzing the reduction of keto acid 3. The enzyme-
catalyzed reduction is useful in the preparation of chiral
alcohols, hydroxy acids and their esters, and amino
acids which serve as key intermediates in the synthesis
of pharmaceutical compounds.35
13849 as described in the Section 4. A yield of 98% and
an e.e. of 98% were obtained (Table 3). However, due
to low cell concentration in this single-stage process, the
reaction was completed in 107 h compared to 16 h for
the two-stage process.
The enantioselective reduction of an alternative sub-
strate, keto acid 3, to hydroxy acid 4 was also investi-
gated using various microorganisms. The yields and
product e.e.s obtained with the three best cultures are
shown in Table 4. Candida utilis SC 13983 gave the best
reaction yield (64%) for the desired (R)-hydroxy acid 4.
We also evaluated the microbial reduction of the more
complex intermediate ketoamide 5 to the corresponding
hydroxy amide 6. Low reaction yields (<5%) were
obtained, although several of the cultures tested posi-
tive for the reduction (Table 5). For example, A. pullu-
lans SC 13894 and H. anamola SC 16158 gave 12 and
20% yields of the desired (R)-hydroxy amide 6 with
e.e.s of 98.6 and 85.2%, respectively.
Table 5. Stereoselective microbial reduction of keto amide
5
Microorganisms
Hydroxy
amide 6
yield (%)
E.e. of
(R)-hydroxy
amide 6 (%)
Conversion of ketoamide 5 to hydroxy amide 6 was
improved by using cell extracts of A. pullulans SC 13984
and glucose dehydrogenase in the presence of glucose
and NADP as shown in Table 6. The lower reaction
yield obtained with a cell suspension may be due to the
impermeability of ketoamide 5 through cells or may be
due to limited NADPH-regenerating enzyme within
cells of A. pullulans SC 13894. A higher yield of 6 (60%)
was obtained using cell extracts and for when NADPH
was used as a cofactor (Table 6). Data for reduction
using these systems are also provided for the conversion
of 1 to 2.
Aureobasidium pullulans
SC 13894
Aureobasidium pullulans
SC 13925
12
4
98
Not determined
Not determined
Not determined
85
Bre6ibacterium citreum
SC 14804
Cunninghemella enchinulata
SC 16024
Hansenula anamola
SC 16158
Mortierella ramanniana
SC 13843
Mycobacterium 6acca
SC 16204
Rhizopus oligosporous
SC 16197
4
5
20
3
Not determined
Not determined
50
3.5
9
3. Conclusion
The dehydrogenases from yeast,18–23 horse liver,24 and
T. brockii25 transfer the pro-R hydride to the re face of
the carbonyl group to give (S)-alcohols, a process
described by Prelog’s rule.36 In contrast, dehydrog-
The reaction yields and e.e.s were determined by HPLC using C-18
column and chiralcel OD column, respectively, as described in
Section 4.
Table 3. Stereoselective microbial reduction of keto ester 1 by A. pullulans SC 13849: single-stage process
Reaction time (h)
Keto ester 1 (g/L)
Hydroxy ester 2 (g/L)
Yield (%)
E.e. of hydroxy ester 2 (%)
20
44
71
95
107
2.8
1.15
0.4
0.14
Trace
2.1
3.8
4.3
4.6
4.9
42
76
86
92
98
Not determined
98
98
98
98
The reaction yields and e.e.s were determined by HPLC using C-18 column and chiralcel OD column, respectively, as described in Section 4.
Table 4. Stereoselective microbial reduction of keto acid 3
Microorganisms
Ketoacid 3 (g/L)
Hydroxy acid 4 (g/L)
Yield (%)
E.e. of hydroxy acid 4 (%)
Candida maltosa SC 16112
Candida utilis SC 13983
Candida utilis SC 13983
0.45
0.35
0.43
0.56
0.64
0.53
56
64
53
99
98
98
The reaction yields and e.e.s were determined by HPLC using C-18 column and chiralcel OD column, respectively, as described in Section 4.

352
R. N. Patel et al. / Tetrahedron: Asymmetry 13 (2002) 349–355
Table 6. Stereoselective reduction of keto ester 1 and ketoamide 5 by cell extracts of Aureobasidium pullulans SC 13848:
Regenerating system
Substrate
Product
Reaction yield (%)
E.e. (%)
Glucose dehydrogenase
and NADP
Glucose dehydrogenase
and NAD
1
5
1
5
2
6
2
6
98
60
12
10
98
96
98
97
The reaction yields and e.e.s were determined by HPLC using C-18 column and chiralcel OD column, respectively, as described in Section 4.
4. Experimental
aqueous sodium bicarbonate solution (100 mL) and
brine (100 mL). The organic extracts were dried over
sodium sulfate. The crude product was purified by
column chromatography using silica gel 60 (230–400
mesh, eluted with 20% ethyl acetate in hexanes) to give
Substrates 1, 3, and 5 and reference compounds 2, 4,
and 6 were synthesized by co-authors in the Process
Research and Development Department, Bristol–Myers
Squibb Pharmaceutical Research Institute. The
physico–chemical properties including spectral charac-
teristics (1H, 13C NMR, mass spectra), were in full
accord for all these compounds.17 The proton magnetic
resonance (1H NMR) and carbon magnetic resonance
(13C NMR) were recorded on a Brucker AM-300
spectrometer.
1
the desired product as an oil (18 g, 78%). H NMR
(CDCl3; 400 MHz): l 7.32 (d, J=2 Hz; 1H); d 7.28 (d,
J=8 Hz; 1H); 7.15 (dd, J=8, 2 Hz; 1H); 5.10 (d, J=6
Hz; 1H); 4.15–4.35 (m; 2H); 3.29 (d, J=2 Hz); 1.20–
1.28 (m; 15 H) ppm. MS (+ESI): 308 (M+NH4)+
4.3. Preparation of keto amide 5
4.1. Preparation of keto ester 1
To a 15 mL, two-necked flask equipped with a mag-
netic stirrer and nitrogen inlet adapter was charged
keto acid 3 (0.5 g; 1.92 mmol) followed by anhydrous
dichloromethane (5 mL) and DMF (two drops). The
reaction mixture was cooled to 0°C and oxalyl chloride
was added dropwise while maintaining the reaction
temperature at 0°C. The reaction mixture was stirred at
0°C for 1 h and then allowed to warm to room temper-
ature and stirred for a further 0.5 h. Methyl 3-fluoro-4-
aminobenzoate (0.33 g; 1.92 mmol) was dissolved in a
mixture of anhydrous dichloromethane (5 mL) and
anhydrous pyridine (0.51 mL; 6.3 mmol). This solution
was added dropwise to the reaction mixture while
maintaining the internal temperature at –5°C. After
completion of the reaction, the reaction mixture was
transferred to a separatory funnel and washed with 1N
aqueous HCl (2×25 mL) and brine (25 mL) and dried
over sodium sulfate. Removal of the solvent gave the
desired keto amide 5 as a yellow solid (0.66 g, 85%).
To a 250 mL, three-necked flask equipped with a
magnetic stirrer, addition funnel, argon inlet adapter
and temperature probe were charged in order 1,2,3,4-
tetrahydro-1,1,4,4-tetramethylnaphthalene (20 g; 106.2
mmol), ethyl oxalyl chloride (13 mL; 116 mmol) and
dichloromethane (110 mL). The reaction mixture was
cooled to 0°C and aluminum chloride (22.6 g; 169
mmol) was added portionwise while maintaining the
temperature at 0°C. The reaction mixture was allowed
to warm to room temperature and stirred for 2 h, at
which point the reaction was complete as indicated by
TLC (1:1 ethyl acetate/hexane). The reaction mixture
was poured into ice-water (500 mL) and the aqueous
layer was extracted with dichloromethane (110 mL).
The combined organic layers were washed with satu-
rated aqueous bicarbonate solution (100 mL) and brine
(100 mL). The organic layer was dried over sodium
sulfate. Removal of the solvent gave the desired com-
1
Mp 131–133°C. H NMR (CDCl3; 400 MHz): l 9.39
1
pound as an oil (28 g, 92%). H NMR (CDCl3, 400
(br s; 1H); 8.59 (t, J=8 Hz; 1H); 8.41 (d, J=2 Hz; 1H);
8.15 (dd, J=8, 2 Hz; 1H); 7.90 (d; J=8 Hz; 1H); 7.82
(dd, J=11, 2 Hz; 1H); 7.43 (d, J=8 Hz; 1H); 3.92 (s;
3H); 1.70 (s; 4H); 1.34 (s; 6H); 1.31 (s; 6H) ppm. MS
(-ESI): 410 (M-H)−.
MHz): l 7.97 (d, J=2 Hz; 1H); 7.64 (dd, J=8, 2 Hz;
1H); 7.42 (d, J=8, Hz; 1H); 4.43 (q, J=7 Hz; 2H); 1.70
(s; 4H); 1.41 (t, J=7 Hz; 3H); 1.30 (s; 6H); 1.29 (s; 6H)
ppm. MS (+ESI): 306 (M+NH4)+, 289 (M+H)+.
4.2. Preparation of ( )-hydroxy ester 2
4.4. Preparation of keto acid 3
To a three-necked, round-bottomed flask equipped with
a magnetic stirrer, addition funnel, argon inlet adapter
and temperature probe was charged a solution of
NaBH4 (1.06 g; 0.35 mol equiv.) in ethanol (100 mL).
The reaction mixture was cooled to 0°C. A solution of
keto ester 1 (23.1 g) in ethanol (220 mL) was added to
the reaction mixture while maintaining the internal
temperature between 0 and 3°C and then the reaction
mixture was stirred at 0°C for 1 h. The reaction was
quenched with ice-water, acidified to pH 3 with 1N
aqueous HCl and extracted with ethyl acetate (200 mL).
The ethyl acetate extract was washed with saturated
To a 250 mL, three-necked flask equipped with a
magnetic stirrer, nitrogen inlet adapter and temperature
probe were charged keto ester 1 (36 g; 125 mmol), THF
(100 mL), water (50 mL) and LiOH·H2O (15.7 g; 373
mmol). The reaction mixture was stirred at room tem-
perature for 2.5 h. No starting material was detected at
this point by thin-layer chromatography using pre-
coated silica gel plates (GF-254; eluted with ethyl ace-
tate/hexanes 1:1). The pH of the reaction mixture was
adjusted to 2–3 with 1N aqueous HCl and extracted
with MTBE. The MTBE layer was extracted with satd.
NaHCO3, acidified with 1N aqueous HCl to pH 2–3,

R. N. Patel et al. / Tetrahedron: Asymmetry 13 (2002) 349–355
353
and extracted with ethyl acetate. Removal of the sol-
vent followed by drying gave the desired acid as a
4.7. Microorganisms
Microorganisms (Tables 1–5) were obtained from the
culture collection of the Bristol–Myers Squibb Pharma-
ceutical Research Institute. Microbial cultures were
stored at −90°C in vials.
yellow oil which solidified to a low melting solid upon
cooling (30 g, 94% yield). H NMR (CDCl3; 400 MHz):
l 8.37 (d; J=1 Hz; 1H); 8.14 (dd, J=8, 1 Hz; 1H); 7.44
(d, J=8 Hz; 1H); 1.71 (s; 4H); 1.32 (s; 6H); 1.30 (s; 6H)
ppm. HRMS calcd for C16H20O3 261.1491. Found
261.1501.
1
4.8. Growth of microorganisms
For screening purposes, one vial of each culture was
used to inoculate 100 mL of medium A (1% malt
extract, 1% yeast extract, 2% glucose and 0.3% pep-
tone). The medium was adjusted to pH 6.8 before
sterilization. Cultures were grown at 28°C and 280
RPM for 48 h. Cultures were harvested by centrifuga-
tion at 18,000×g for 15 min, washed with 0.1 M potas-
sium phosphate buffer pH 7.0, and used for reduction
studies.
4.5. Preparation of ( )-hydroxy acid 4
To a 250 mL, three-necked flask equipped with a
magnetic stirrer, nitrogen inlet adapter and temperature
probe were charged the racemic hydroxy ester 2 (45.6 g;
157 mmol), THF (200 mL), water (100 mL) and
LiOH·H2O (19.9 g; 473 mmol). The reaction mixture
was stirred at room temperature for 2 h. No starting
material was detected at this point by thin-layer chro-
matography using pre-coated silica gel plates (GF-254;
eluted with ethyl acetate/hexanes 1:1). The reaction
mixture was adjusted to pH 2–3 with 1N aqueous HCl
and extracted with ethyl acetate (200 mL). Removal of
the solvent followed by drying over magnesium sulfate
gave the desired acid as a white solid (45 g). Recrystal-
lization from 10% ethyl acetate in heptane gave the
desired compound as a white solid (30 g, 73% yield). 1H
NMR (CDCl3; 400 MHz): l 7.35 (d J=2 Hz; 1H); 7.29
(d J=8 Hz; 1H); 7.16 (dd; J=8, 2 Hz; 1H); 5.19 (s;
1H); 1.67 (s; 4H); d 1.26 1.30 (overlapping singlets; 12
H). Anal. calcd for C16H22O3: C, 73.25; H, 8.45. Found:
C, 73.05; H, 8.36%.
4.9. Reduction of keto ester 1 by cell-suspensions
Cells of various microorganisms were suspended sepa-
rately in 60 mM potassium phosphate buffer (pH 7.0)
at 20% (w/v, wet cells) cell concentration and supple-
mented with 1 mg/mL of keto ester 1 (in methanol) and
25 mg/mL of glucose. Reduction was conducted at
28°C and 150 RPM. Periodically, samples of 1 mL were
taken and extracted with 2 mL of acetonitrile. The
samples were filtered through a 0.2 mm LID/X filter and
analyzed using a Hewlett Packard 1070 high pressure
liquid chromatograph (HPLC). A Kromasil C-18 (150×
4.6 mm) column was used. The mobile phase consisted
of solvent A (0.1% trifluoroacetic acid in water) and
solvent B (0.1% trifluoroacetic acid in acetonitrile) used
in a linear gradient over a 50 min run time at a flow
rate of 1.0 mL/min as follows.
4.6. Preparation of ( )-hydroxy amide 6
A 500 mL, three-necked flask equipped with a mechan-
ical stirrer, gas inlet adapter and temperature probe was
flushed with argon. To the flask were charged the
hydroxy acid 3 (20 g; 76.3 mmol), 1,2,4-triazole (7.4 g;
106.8 mmol) and 150 mL of dichloromethane. The
reaction mixture was stirred until it turned clear and
then it was cooled to 0°C. Methyl 3-fluoro-4-(N-sul-
finyl)aminobenzoate [MeO2CC6H3(3-F)(4-NSO)] (23 g;
106.8 mmol) was charged in one portion and the reac-
Time
% B
0
40
70
70
100
100
40
25
30
35
40
45
50
tion mixture was stirred at 0°C for
3 h. The
dichloromethane was removed under reduced pressure
at 35°C and further displaced by distillation with hept-
ane (2×50 mL). The residual solids were dissolved in a
2:1 mixture of heptane/MTBE, washed with 3N HCl
(5×70 mL), saturated NaHCO3 (2×50 mL) and brine
(50 mL). The organic layer was dried over sodium
sulfate and the solvents were removed under reduced
pressure to give the crude product as a solid (31 g).
Recrystallization from cyclohexane gave the desired
compound as a white solid (24.2 g, 77% yield). Mp
40
The detection wavelength was 210 nm. The retention
times for keto ester 1 and hydroxy ester 2 were 29.3 and
19.6 min, respectively. Separation of the two enan-
tiomers of the racemic hydroxy ester 2 was achieved on
a Chiracel OD column. The mobile phase consisted of
2.5% isoproanol and 97.5% hexane. The flow rate was 1
mL/min and the detection wavelength was 230 nm. The
retention time for the desired (R)-enantiomer was 12.7
min and that for the undesired (S)-enantiomer was 9.47
min.
1
120–121°C. H NMR (CDCl3, 500 MHz): l 1.25–1.30
(overlapping singlets; 12H); 1.68 (s; 4H); 3.51 (d, J=2
Hz; 1H); 3.89 (s; 3H); 5.17 (d, J=2 Hz; 1H); 7.22 (dd,
J=8, 2 Hz; 1H); 7.33 (d, J=8 Hz; 1H); 7.4 (d, J=2 Hz;
1H); 7.75 (dd, J=11, 2 Hz; 1H); 7.80 (d, J=11 Hz;
1H); 8.44 (apparent t, J=8 Hz; 1H); l 8.86 (d, J=2
Hz; 1H) ppm. HRMS calcd for C24H28FN4 (+1)
414.2081. Found 414.2066.
4.10. Procedure for two-stage reduction of keto ester 1
A. pullulans SC 13894 culture was grown in a 25 L
fermentor containing 15 L of medium A (1% malt

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R. N. Patel et al. / Tetrahedron: Asymmetry 13 (2002) 349–355
4.13. Hydrolysis of hydroxy ester 2 by pig liver esterase
extract, 1% yeast extract, 2% glucose and 0.3% pep-
tone) containing 0.025% UCON antifoam. Growth
consisted of several inoculum development stages and
fermentation. Inoculum development consisted of F1
and F2 stages. In the F1 stage, a frozen vial of culture
was inoculated into 100 mL of medium A contained in
a 500 mL flask. Growth was carried out at 28°C and
280 RPM for 48 h on a rotary shaker. In the F2 stage,
10 mL of F1 stage culture was inoculated into 1 L of
medium A and incubated at 28°C and 280 RPM for 24
h. The fermentor containing 15 L of medium A was
inoculated with 1 L of inoculum from an F2 stage.
Fermentation was conducted at 25°C and 500 RPM
with 15 LPM (liter per min) aeration for 48 h. After 48
h fermentation, cells were collected and stored at −90°C
until further use. About 1 kg of wet cell paste was
collected from the fermentation. A. pullulans SC 13894
culture was also grown in a very cheap medium in a 25
L fermentor containing 15 L of medium B (2%
ardamine PH, 2% glucose, and 2% nutrisoy) without
compromising activity for reduction of all substrates.
Hydroxy ester 2 was hydrolyzed to the corresponding
hydroxy acid 4 with pig liver esterase. At the end of the
reduction of keto ester 1 to hydroxy ester 2, 5 g/L of
pig liver esterase was added to the reaction mixture and
the mixture was incubated at 28°C for 16 h. Complete
conversion of hydroxy ester 2 to hydroxy acid 4 was
obtained.
4.14. Reduction of keto acid 3 and keto amide 5
For screening purposes, one vial of each culture was
used to inoculate 100 mL of medium A. Cultures were
grown at 28°C and 280 RPM for 48 h. Cultures were
harvested by centrifugation at 18,000×g for 15 min,
washed with 0.1 M potassium phosphate buffer pH 7.0,
and suspended separately in 60 mM potassium phos-
phate buffer (pH 7.0) at 20% (w/v, wet cells) cell
concentration. Cell suspensions were supplemented
with 1 mg/mL of keto acid 3 or keto amide 5 (in
methanol) and 25 mg/mL of glucose. The reduction was
conducted at 28°C and 150 RPM. Periodically, samples
of 1 mL were taken and extracted with 2 mL of
acetonitrile. Each sample was filtered through a 0.2 mm
LID/X filter and analyzed by HPLC as described ear-
lier. The retention times for keto acid 3 and hydroxy
acid 4 were 15.7 and 10.2 min, respectively.
Frozen cells (600 g wet cells) from the above batches
were suspended in 3 L of 70 mM potassium phosphate
buffer (pH 7.0) and used to conduct the reduction of
keto ester 1 in a 5 L reactor. Keto ester 1 (1 g/L or 5
g/L in methanol) and glucose (25 g/L) were added to
the reactor and the reduction was carried out at 28°C
and 200 RPM with 1 LPM aeration for 24–72 h. The
pH was maintained between 6.8 and 7.0. Periodically,
samples were prepared as described above and analyzed
by HPLC to determine the percent conversion of keto
ester 1 to hydroxy ester 2. The e.e. of hydroxy ester 2
was determined by chiral HPLC as described earlier.
Separation of the two enantiomers of the racemic
hydroxy acid 4 was achieved on a Chiracel OD column
as described earlier except the mobile phase consisted of
2.5% isopropanol, 98% hexane and 0.1% trifluoroacetic
acid. The retention time for the desired (R)-enantiomer
was 32.3 min and that for the undesired (S)-enantiomer
was 36.5 min. To measure the concentration of
ketoamide 5 and hydroxy amide 6, a sample was
extracted with 2 volumes of acetonitrile. The mixture
was then dried under a stream of nitrogen and resus-
pended in 10% isopropanol:90% hexane mixture, soni-
cated, filtered through 0.2 mm LID/X filter and
analyzed by HPLC as described earlier using 98% hex-
ane and 2% isopropanol as the mobile phase. The
retention times for keto amide, (R)-hydroxy amide, and
(S)-hydroxy amide were 6.62, 22.9, and 26.6 min,
respectively.
4.11. Single-stage process for reduction of 1
A. pullulans SC 13894 culture was grown in a 5 L
fermentor containing 3 L of medium B as described
above. After 16 h of growth, 15 g of keto ester 1 was
added to the fermentor and the biotransformation pro-
cess was continued for 107 h. The pH was maintained
at 6.8 during the biotransformation process. Periodi-
cally, samples were prepared as described above and
analyzed by HPLC to determine the % conversion of
keto ester 1 to hydroxy ester 2. The e.e. of hydroxy
ester 2 was determined by chiral HPLC.
4.15. Preparation of cell extracts of A. pullulans
SC 13894
4.12. Isolation of hydroxy ester 2
At the end of the single-stage bioreduction, 150 g of wet
XAD-16 (previously washed with methanol and then
with 70 mM phosphate buffer pH 7.0) was added to the
3 L of reaction mixture to adsorb the product onto the
resin under continuous mixing. After 4 h, the product
rich-resin was recovered from the reaction mixture by
filtration through a stainless steel screen (80 mesh). The
separated resin was washed with 1 L of 70 mM phos-
phate buffer (pH 7.0), and hydroxy ester 2 was de-
sorbed from the rich resin with 1 L of acetonitrile. The
acetonitrile extract was evaporated under reduced pres-
sure to obtain the crude product (12 g).
Preparation of cell extracts were carried out at 4–7°C.
Cells were washed with 50 mM potassium phosphate
buffer (pH 7.0) and washed cells (30 g) were suspended
in buffer (200 mL) containing 10% glycerol and 1 mM
ethylenediamine tetraacetic acid (EDTA). Cell suspen-
sions (20% w/v, wet cells) were disintegrated with a
Microfluidizer (Microfluidics, Inc) at 12,000 psi (two
passages), the disintegrated cells were centrifuged at
25,000×g for 30 min to obtain the cell extract. The
protein in cell extracts was estimated using Bio-Rad
protein reagent using bovine serum albumin as a stan-

R. N. Patel et al. / Tetrahedron: Asymmetry 13 (2002) 349–355
355
dard. The assay mixture contained 1–10 mL of enzyme
fraction, water (0.8 mL) and Bio-Rad reagent (0.2 mL).
After mixing, the absorbance of the solution was mea-
sured at 595 nm. Cell extracts were evaluated for
biotransformation of keto ester 1 and keto amide 5.
The reaction mixture contained 1 mg/mL substrate, 2
mM nicotinamide adenine dinucleotide (NAD) or
nicotinamide adenine dinucleotide phosphate (NADP),
8 units glucose dehydrogenase, and 2 mg/mL glucose to
regenerate reduced cofactor (NADH or NADPH)
required for the reduction reaction. Substrate and
product concentrations were estimated by HPLC. The
e.e.s of the products were determined by chiral HPLC
as described earlier.
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Acknowledgements
We would like to acknowledgement Dr. John Wasylyk
for development of the HPLC assay system for determi-
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