Diastereoselective microbial reduction of (S)-[3-chloro-2-oxo-1- (phenylmethyl)propyl]carbamic acid, 1,1-dimethylethyl ester

Article abstract of DOI:10.1016/j.tetasy.2003.07.016

The chiral intermediate (1S,2R)-[3-chloro-2-hydroxy-1-(phenylmethyl)propyl] carbamic acid, 1,1-dimethylethyl ester 2a was prepared for the total synthesis of the HIV protease inhibitor Atazanavir. The diastereoselective reduction of (1S)-[3-chloro-2-oxo-1-(phenylmethyl)propyl] carbamic acid, 1,1-dimethyl-ethyl ester 1 was carried out using microbial cultures among, which Rhodococcus, Brevibacterium, and Hansenula strains reduced 1 to 2a. Three strains of Rhodococcus gave >90% yield. A diastereomeric purity of >98% and enantiomeric excess of >99.3% were obtained for alcohol 2a.

Full text of DOI:10.1016/j.tetasy.2003.07.016

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 14 (2003) 3105–3109
Diastereoselective microbial reduction of
(S)-[3-chloro-2-oxo-1-(phenylmethyl)propyl]carbamic acid,
1,1-dimethylethyl ester
Ramesh N. Patel,* Linda Chu and Richard Mueller
Process Research and Development, Bristol-Myers Squibb Pharmaceutical, Research Institute, PO Box 191, New Brunswick,
NJ 08903, USA
Received 2 May 2003; accepted 25 July 2003
Abstract—The chiral intermediate (1S,2R)-[3-chloro-2-hydroxy-1-(phenylmethyl)propyl]carbamic acid, 1,1-dimethylethyl ester 2a
was prepared for the total synthesis of the HIV protease inhibitor Atazanavir. The diastereoselective reduction of (1S)-[3-chloro-2-
oxo-1-(phenylmethyl)propyl] carbamic acid, 1,1-dimethyl-ethyl ester 1 was carried out using microbial cultures among, which
Rhodococcus, Brevibacterium, and Hansenula strains reduced 1 to 2a. Three strains of Rhodococcus gave >90% yield. A
diastereomeric purity of >98% and enantiomeric excess of >99.3% were obtained for alcohol 2a.
© 2003 Elsevier Ltd. All rights reserved.
1. Introduction
As a result, the use of microbial reduction to produce
the desired isomer was investigated. A number of alco-
hol oxidoreductases from many organisms, including
those from yeast,18–23 horse liver,24Thermoanerobic
brockii,25,26 Lactobacillus kefir,27Pseudomonas sp.,28
Geotrichum candidum,29,30 Hansenula polymorpha,31,32
Mortierella rammaniana,33 and Sulfolobus solfataricus34
have proven useful for the enantioselective reduction of
ketones to alcohols.
An essential step in the life cycle of human immunodefi-
ciency virus (HIV-1) is the proteolytic processing of its
precursor proteins. This processing is accomplished by
HIV-1 protease, a virally encoded enzyme. Inhibition of
HIV-1 protease arrests the replication of HIV in vitro.
Thus, HIV-1 protease is an attractive target for
chemotherapeutic intervention.1–6 Atazanavir is an
acyclic aza-peptidomimetic that is a potent HIV
protease inhibitor.6–11 The original process was utilized
for the small scale preparation of Atazanavir to support
initial development12–14 and subsequently an improved
process for the large scale synthesis of Atazanavir was
developed.15
About 120 microorganisms were screened for the
diastereoselective reduction of 1 (typically with e.e.
>99%) to 2a. The reaction yield and diastereomeric
purity of 2a obtained with the eight best cultures are
shown in Table 1; lower diastereomeric purities (<90%)
and reaction yields (<12%) were obtained with other
cultures. Brevibacterium sp. SC 16101, Hansenula
anomala SC 13833, Hansenula saturnus SC 13829,
Rhodococcus erythropolis SC 13845, Rhodococcus ery-
thropolis SC 116236, Rhodococcus sp. SC 13810, and
Rhodococcus sp. SC 16002 gave >30% reaction yields
and >94% diastereomeric purities of product 2a; chloro-
hydrin 2b was formed as the minor diastereomer.
Herein we describe the diastereoselective microbial
reduction of (1S)-[3-chloro-2-oxo-1-(phenylmethyl)-
propyl]carbamic acid, 1,1-dimethylethyl ester 1 to
homochiral chlorohydrin 2a, a key intermediate in the
total synthesis of Atazanavir as illustrated in Scheme 1.
2. Results and discussion
Chemical reduction of chloroketone 1 using NaBH4
produces primarily the undesired chlorohydrin 2b.16,17
Rhodococcus erythropolis SC 13845 and Rhodococcus
sp. 16002 were investigated further. Cells of Rhodococ-
cus erythropolis SC 13845 and Rhodococcus sp. 16002
were grown in a 25 L fermentor for 48 h. The cells were
collected and suspended in 70 mM potassium phos-
* Corresponding author. E-mail: patelr@bms.com
0957-4166/$ - see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2003.07.016

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R. N. Patel et al. / Tetrahedron: Asymmetry 14 (2003) 3105–3109
Scheme 1.
3. Experimental
phate buffer (pH 7.0), and the resulting cell-suspensions
were used to carry out the two-stage process for bio-
transformation of 1 as described in Section 3. After 49
h, reaction yields of >92%, diastereomeric purities of
>98% and enantiomeric excesses of 99.3–99.4% for
alcohol 2a were obtained (Table 2).
Starting substrate 1 and reference compounds 2a and
2b were synthesized by colleagues in the Chemical
Process Research Department, Bristol-Myers Squibb
Pharmaceutical Research Institute. The physico-chemi-
cal properties including spectral characteristics (1H
NMR, 13C NMR, mass spectra) were in full accord for
all these compounds.36 The proton magnetic resonance
(1H NMR) and carbon magnetic resonance (13C NMR)
were recorded on a Brucker AM-300 spectrometer.
A more efficient single-stage fermentation-biotransfor-
mation process was developed for the reduction of
ketone 1 with cells of Rhodococcus erythropolis SC
13845 as described in Section 3. A reaction yield of
95%, diastereomeric purity of 98.2% and enantiomeric
excess of 99.4% for alcohol 2a were obtained (Table 3).
From a 12 L reaction mixture, 9.1 g of alcohol 2a was
isolated in 76% overall yield.
3.1. Microorganisms
Microorganisms (Table 1) were obtained from the cul-
ture collection of the Bristol-Myers Squibb Pharmaceu-
tical Research Institute. Microbial cultures were stored
at −90°C in vials.
Cell extracts of Rhodococcus erythropolis SC 13945
were then evaluated for the reduction of ketone 1.
Glucose dehydrogenase in the presence of glucose and
NADP or formate dehydrogenase in the presence of
formate and NAD were used to regenerate the cofactor
(NADPH or NADH respectively) required for the
reduction reaction. After 6 hr of reaction, higher reac-
tion yields and diastereomeric purities were obtained
for chiral alcohol 2a (49% yield, 98.6% diastereomeric
purity) when NADH was used as a cofactor compared
to NADPH (Table 4).
Table 1. Diastereoselective microbial reduction of ketone 1
Microorganisms
Yield of 2a
Diastereomeric purity
(%)
of 2a (%)
Brevibacterium sp. SC
16101
Hansenula anomala SC
13833
Hansenula saturnus SC
13829
Rhodococcus erythropolis
SC 13845
Rhodococcus erythropolis
SC 16236
Rhodococcus sp. SC
13810
Rhodococcus sp. SC
16002
Trichoderma viridae SC
13826
99
33
35
98
74
91
97
12
94
98
The oxidoreductases from yeast,18–23 horse liver,24 and
T. brockii26 transfer the pro-R hydride to the re-face of
the carbonyl to give (S)-alcohols, a process described
by Prelog’s rule.35 In contrast, oxidoreductases from
Lactobacillus kefir27 and two Pseudomonas sp.28 exhibit
anti-Prelog specificity, transferring the pro-R hydride to
form (R)-alcohols. We have described above the reduc-
tion of ketone 1 to the corresponding (S)-alcohol by
Rhodococcus erythropolis. NADH was a better cofactor
compared to NADPH, affording higher reaction yields
and diastereomeric purities when cell extracts were used
for this reduction process.
98.5
98.9
98
98
98
98

R. N. Patel et al. / Tetrahedron: Asymmetry 14 (2003) 3105–3109
Table 2. Diastereoselective microbial reduction of ketone 1: two-stage process
3107
Microorganisms
Reaction time
(h)
Yield of 2a
(%)
Diastereomeric purity of 2a (%)
Enantiomeric excess of 2a (%)
Rhodococcus erythropolis SC
20
62
13845
49
20
97
60
98.5
98.2
99.4
99.3
Rhodococcus erythropolis SC
16236
49
92
Table 3. Diastereoselective microbial reduction of ketone 1: single-stage process
Microorganisms
Reaction time
(h)
Yield of 2a
(%)
Diastereomeric purity of 2a (%)
Enantiomeric excess of 2a (%)
Rhodococcus erythropolis SC
21
38
13845
48
72
93
52
72
95
98.2
99.4
Table 4. Diastereoselective microbial reduction of ketone 1: cell-free system
Regeneration system
Reaction time (h)
Yield of 2a (%)
Diastereomeric purity of 2a (%)
NAD, formate, formate dehydrogenase
NADP, glucose, glucose dehydrogenase
6
6
49
22
98.6
60
3.2. Growth of microorganisms
erythropolis SC 16236 culture were grown in a 25 L
fermentor containing 15 L of medium A containing
0.025% UCON antifoam. Growth consisted of several
inoculum development stages and fermentation. Inocu-
lum development consisted of F1 and F2 stages. In the
F1 stage, a frozen vial of each 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.
For screening purposes, one vial of each culture was
used to inoculate 100 mL of medium A containing 1%
malt extract, 1% yeast extract, 2% glucose and 0.3%
peptone. 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 centrifugation at 18,000×g
for 15 min, washed with 70 mM potassium phosphate
buffer pH 7.0, and used for reduction studies.
Fermentors containing 15 L of medium A were inocu-
lated with 1 L of inoculum of each culture from an F2
stage. Fermentation was conducted at 25°C and 500
rpm with 15 lpm (liter per min) aeration for 36 h. After
48 h fermentation, cells were collected and stored at
−90°C until further use. About 1 kg of wet cell pastes
was collected from each fermentation.
3.3. Reduction of 1 by cell-suspensions
Cells of various microorganisms were suspended sepa-
rately in 70 mM potassium phosphate buffer (pH 7.0)
at 20% (w/v, wet cells) cell concentration and supple-
mented with 1 mg/mL of 1 and 30 mg/mL of glucose.
Reduction was conducted at 28°C and 150 rpm. Period-
ically, samples of 1 mL were taken and extracted with
5 mL of tert-butylmethylether: toluene (60:40). After
centrifugation, the separated organic phase was col-
lected and dried with a nitrogen stream. The oily
residue obtained was dissolved in 1 mL of ethanol,
filtered through a 0.2 mm LID/X filter and analyzed by
HPLC.
Frozen cells from the above batches were used to
conduct the reduction of 1 in a 5 L reactor. Cell
suspensions (10% w/v, wet cells) in 3 L of 70 mM
potassium phosphate buffer (pH 7.0) were used. Com-
pound 1 (30 g) and glucose (75 g) were added to the
reactor and the reduction was carried out at 28°C and
160 rpm with 1 lpm aeration for 49 h. The pH was
maintained between 6.8–7.0. Periodically, samples were
prepared as described above and analyzed by HPLC to
3.4. Two-stage process for reduction of 1
determine the
diastereomeric purity of 2a was determined by HPLC.
% conversion of 1 to 2a. The
Rhodococcus erythropolis SC 13845 and Rhodococcus

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R. N. Patel et al. / Tetrahedron: Asymmetry 14 (2003) 3105–3109
3.5. Isolation of 2a
3.8. Preparation of cell extracts of Rhodococcus ery-
thropolis SC 13845
At the end of two-stage bioreduction, 3 L of the
reaction mixture was extracted with 6 L of tert-butyl-
methylether: toluene (60:40). The separated organic
phase was washed with 2 L of 0.1 M sodium chloride,
dried over anhydrous sodium sulfate and evaporated
under reduced pressure to obtain 24 gram of crude
product which was recrystallized from ethyl acetate to
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 200 mL of buffer containing 10% glycerol and 1 mM
ethylenediamine tetraacetic acid (EDTA). Cell suspen-
sions (15% w/v, wet cells) were disintegrated with a
Microfluidizer (Microfluidics, Inc) at 12,000 psi (two
passages), and the disintegrated cells were centrifuged
at 25,000×g for 30 min to obtain the cell extract.
Protein in cell extracts was estimated using Bio-Rad
protein reagent with bovine serum albumin as a stan-
dard. The assay mixture contained 1–10 mL of enzyme
fraction, 0.8 mL water and 0.2 mL Bio-Rad reagent.
After mixing, the absorbance of the solution was mea-
sured at 595 nm. Cell extracts were evaluated for
biotransformation of ketone 1. The reaction mixture
contained 1 mg/mL substrate, 2 mM nicotinamide
adenine dinucleotide (NAD) or nicotinamide adenine
dinucleotide phosphate (NADP), 8 units glucose dehy-
drogenase, and 2 mg/mL glucose or 1 mM formate and
3 units formate dehydrogenase to regenerate reduced
cofactor (NADH or NADPH) required for the reduc-
tion. Samples were incubated at 28°C,105 rpm for 6 h.
Concentrations of 1, 2a, and 2b were estimated by
HPLC.
afford 22.5
g (75% overall yield) of 2a. The
diastereomeric purity and the enantiomeric purity of
the isolated chiral alcohol 2a were >98% and 99.5%,
1
respectively. H NMR (CDCl3) l 1.4 (S, 9H), 2.9 (d,
2H, J=13 Hz), 3.2 (S, 1H); 3.6 (m, 2H), 3.85 (m, 1H),
4.5 (S, 1H), 7.2–7.4 (m, 5H). MS m/z 302 (M+H)+
(calcd for C15H22ClNO2, 301).
3.6. Single-stage process for reduction of 1
Rhodococcus erythropolis SC 13845 was grown in a 25 L
fermentor containing 15 L of medium A as described
above. After 30 h, 15 g of ketone 1 was added to the
fermentor and the biotransformation process was con-
tinued for 93 h. The pH was maintained at 7.0. Period-
ically, samples were prepared as described above and
analyzed by HPLC to determine the % conversion of 1.
The diastereomeric purity of 2a was determined by
HPLC.
Acknowledgements
3.7. Analytical methods
Analysis of 1, 2a, and 2b were carried out using a
Hewlett Packard HPLC. A Vydak column (100×4.6
mm, ID 5 m) was used under the following conditions.
Mobile phase was 0.1% trifluoroacetic acid in water
(solvent A) and 0.1% trifluoroacetic acid in 70% aceto-
nitrile, 30% water (solvent B) used in a gradient:
We would like to acknowledge Dr. John Wasylyk for
development of the HPLC assay system and Dr.
Thomas LaPorte for fermentation of microbial cultures.
We would like to thank Dr. John Scott for reviewing
this manuscript and providing valuable suggestions.
Time (min)
0
0.1-20 (gradient) 50
20-25
25.1-26
26-30
Solvent A (%) Solvent B (%)
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