Microbial synthesis of chiral intermediates for β-3-receptor agonists

Article abstract of DOI:10.1007/s11746-998-0081-0

Chiral intermediates were prepared by biocatalytic processes for the chemical synthesis of β-3-receptor agonists. These include: (i) the microbial reduction of 4-benzyloxy-3-methanesulfonylamino-2′-bromoacetophenone 1 to the corresponding (R)-alcohol 2 by

Full text of DOI:10.1007/s11746-998-0081-0

Microbial Synthesis of Chiral Intermediates
for β-3-Receptor Agonists
Ramesh N. Patel*, Amit Banerjee, Linda Chu, David Brozozowski,
Venkata Nanduri, and Laszlo J. Szarka
Department of Microbial Technology, Bristol-Myers Squibb Pharmaceutical Research Institute,
New Brunswick, New Jersey 08903
ABSTRACT: Chiral intermediates were prepared by biocat- ble, new drugs should be homochiral to avoid the possibility
alytic processes for the chemical synthesis of β-3-receptor ago- of side effects caused by an undesirable stereoisomer. There
nists. These include: (i) the microbial reduction of 4-benzyloxy-
-methanesulfonylamino-2′-bromoacetophenone 1 to the cor-
responding (R)-alcohol 2 by Spingomonas paucimobilis SC
is the opportunity to double the use of an industrial process
by obtaining a separate patent on the use of a single stereoiso-
mer as a more efficient drug. The physical advantages of
enantiomers over racemates may confer processing and for-
mulation advantages, as well as lower doses.
The advantages of microbial or enzyme-catalyzed re-
actions over chemical reactions are that they are stereoselec-
tive and can be carried out at ambient temperature and atmos-
3
1
>
6113. In the biotransformation process, a reaction yield of
85% and an optical purity of 99.5% were obtained for the
desired (R)-alcohol 2; (ii) the enzymatic resolution of racemic
α-methyl phenylalanine amide, 3, and α-methyl-4-hydroxy-
phenylalanine amide, 5, by amidase from Mycobacterium
neoaurum ATCC 25795 to prepare the corresponding (S)-amino
acids 4 and 6. Reaction yields of 49.9 and 49 M% (theoretical pheric pressure. These minimize problems of isomeriza-
maximum yield 50 M%) and optical purities of 99 and 94% tion, racemization, epimerization, and rearrangement, which
were obtained for the desired (S)-amino acids 4 and 6, respec- generally occur during chemical processes. Biocatalytic
tively; (iii) the asymmetric hydrolysis of methyl-(4-methoxy-
phenyl)-propanedioic acid, ethyl diester, 7, to the correspond-
ing (S)-monoester 8 by pig liver esterase. A reaction yield of 96
M% and an optical purity of 96% were obtained for (S)-mono-
ester 8 when reactions were carried out in a biphasic system
containing 10% ethanol at 10°C.
processes are usually carried out in aqueous solution. This
avoids the use of solvent and other environmentally harmful
chemicals used in the chemical processes. Furthermore, mi-
crobial cells or enzymes derived from biocatalysis can be im-
mobilized and reused for many cycles. Recently, a number of
review articles (1–9) have been published on the use of en-
zymes in organic synthesis.
JAOCS 75, 1473–1482 (1998).
β-Adrenoceptors have been classified as β1 and β2 (10).
KEY WORDS: Asymmetric hydrolysis, biocatalysis, chiral in-
termediates, pig liver esterase, β-3-receptor agonist, resolution Increased heart rate is the primary consequence of β1-recep-
using amidase, stereoselective reduction.
tor stimulation, while bronchodilation and smooth muscle re-
laxation are mediated by β2-receptor stimulation. Rat
adipocyte lipolysis was initially thought to be a β1-mediated
Much attention is currently focused on the interaction of process (10). However, recent results indicate that the recep-
small molecules with biological macromolecules. The search tor-mediated lipolysis is neither β1 nor β2, but “atypical” re-
for selective enzyme inhibitors and receptor agonists or an- ceptors, later called β3-adernergic receptors (12). β3-Adren-
tagonists is one of the keys for target-oriented research in the ergic receptors are found on the cell surfaces of both white
pharmaceutical industry. Increasing understanding of the and brown adipocytes, and are responsible for lipolysis, ther-
mechanism of drug interaction on a molecular level has led to mogenesis, and relaxation of intestinal smooth muscle
growing awareness of the importance of chirality as the key (13,14). Consequently several research groups are engaged in
to the efficacy of many drug products. It is now known that developing selective β3 agonists for the treatment of gastroin-
in many cases only one stereoisomer of a drug substance is testinal disorders, type II diabetes, and obesity (15–19). We
required for efficacy; the other stereoisomer is either inactive report here an efficient biocatalytic synthesis of chiral inter-
or exhibits considerably reduced acitivity. Pharmaceutical mediates required for the total chemical syntheses of β3
companies are aware that, where the switch from racemate receptor agonists (Scheme 1) such as BRL 37344, BMS-
drug substance to enantiomerically pure compound is feasi- 187257, and BMS 210620 (15,16). These include the stereo-
selective microbial reduction of 4-benzyloxy-3-methane-
*
To whom correspondence should be addressed at Department of Microbial
sulfonylamino-2′-bromoacetophenone, 1, to the correspond-
ing (R)-alcohol 2 (Scheme 1); the enzymatic resolution of
racemic α-methyl phenylalanine amide, 3, and α-methyl-4-
Technology, Bristol-Myers Squibb Pharmaceutical Research Institute, P.O.
Box 191, New Brunswick, NJ 08903.
E-mail: Ramesh_N._Patel@ccmail.bms.com
Copyright © 1998 by AOCS Press
1473
JAOCS, Vol. 75, no. 11 (1998)

1474
R.N. PATEL ET AL.
O
OH
Br
Br
Spingomonas paucimobilis
O
O
SC 16113
NHSO CH3
2
NHSO CH3
2
Substrate 1
Ketone
Product 2
( RR )) -Alcohol
(
CH R
2
OH
OH
O
H
N
H
N
N
H
H C
3
CH3
OCH CO H
2
2
HO
Cl
NHSO CH3
2
BRL 37344
BMS-210620
OCH3
SCHEME 1
hydroxyphenylalanine amide, 5, to the corresponding (S)- search Department, Bristol-Myers Squibb Pharmaceutical
amino acids 4 and 6, respectively, by an amidase from Research Institute (New Brunswick, NJ). The physicochemi-
1
Mycobacterium neoaurum ATCC 25795 (Scheme 2); and the cal properties, including spectral characteristics [ H nuclear
1
3
enzymatic asymmetric hydrolysis of methyl-(4-methoxy- magnetic resonance (NMR), C NMR, mass spectra], were
phenyl)-propanedioic acid, ethyl diester, 7, to the correspond- in full accord for all these compounds.
ing (S)-monoester 8 by pig liver esterase (PLE) (Scheme 3).
Microorganisms. Microorganisms (Table 1) were obtained
from the American Type Culture Collection (Rockville, MD)
and from our culture collection in the Microbiology Depart-
ment of the Bristol-Myers Squibb Pharmaceutical Research
MATERIALS AND METHODS
Materials. Substrates 1, 3, 5, and 7 and reference standards 2, Institute. Microorganisms were stored at −90°C in vials.
4
, 6, and 8 were synthesized by the Chemical Process Re-
Growth of microorganisms. For screening purposes, one
O
S
O
R
O
H N
H N
2
2
H N
2
OH
NH2
NH2
H C
3
H C
3
H C
3
Mycobacterium neoaurum
+
ATCC 25795
Product 4
O
Substrate 3
O
H N
H N
2
O
R
2
H N
2
NH2
S
OH
H C
H C
3
NH2
3
H C
3
M. neoaurum ATCC 25795
+
OCH3
Substrate 5
OCH3
OCH3
Product 6
SCHEME 2
JAOCS, Vol. 75, no. 11 (1998)

CHIRAL INTERMEDIATES FOR β-3-RECEPTOR AGONISTS
CH3
1475
CH3
CO C H
2
2
5
COOH
CO C H
2 5
CO C H
2
2
5
2
Pig Liver Esterase
H CO
3
H CO
3
Diester 7
SS -(-)-Monoester 8
SCHEME 3
vial of each culture was used to inoculate 100 mL of medium (HPLC). The optical purity of compound 2 was determined
A containing 1% yeast extract, 1% malt extract, 2% glucose, by chiral HPLC.
and 0.3% peptone. The medium was adjusted to pH 6.8 be-
Fermentation of Spingomonas paucimobilis SC 16113.
fore sterilization. Cultures were grown at 28°C and 280 rpm Spingomonas paucimobilis SC 16113 was grown in a 350-L
for 48 h on a rotary shaker. Cultures were harvested by cen- fermenter. The fermentation process consisted of two inocu-
trifugation at 18,000 × g for 15 min, washed with 25 mM lum development stages and fermentation. Inoculum devel-
potassium phosphate buffer (pH 6.8), and then used for bio- opment consisted of F1 and F2 stages. In the F1 stage, frozen
transformation studies.
vials of S. paucimobilis SC 16113 culture were inoculated
Biotransformations of compound 1 by microbial cell sus- into 100 mL of medium A. The growth was carried out in
pensions. Cells of various microorganisms were suspended in 500-mL flasks at 28°C and 280 rpm for 48 h. In the F2 stage,
1
2
0 mL of 100 mM potassium phosphate buffer, pH 6.0, at 100 mL of F1 stage culture was inoculated into a 25-L germi-
0% (wt/vol, wet cells) cell concentration and supplemented nator that contained 16 L of medium B (2.2% cerelose hy-
with 10 mg of compound 1 in 0.2 mL of acetonitrile and 250 drate, 1% Hy-yest 412, 0.025% Dow Corning antifoam AF,
mg of glucose. The biotransformation of compound 1 was 0.025% SAG 5639 antifoam). The pH of the medium was ad-
conducted at 28°C, 280 rpm on a rotary shaker. Periodically, justed to 6.8 before sterilization. The germinator was run at
samples of 2 mL were taken and extracted with 8 mL of ethyl 500 rpm, 28°C, and 15 liters per minute (Lpm) aeration for
acetate. After centrifugation, the ethyl acetate phase was col- 24 h. Fermenters with 550 L of medium B were inoculated
lected and filtered through a 0.2 µm LID/X filter (Whatman with 16 L of germinator-grown inoculum. The fermentations
Inc., Fairfield, NJ). Clarified ethyl acetate filtrate was evapo- were conducted at 28°C and 185 rpm with 150 Lpm aeration.
rated under a gentle stream of nitrogen. The oily residue ob- Samples were taken during the course of the fermentation to
tained was dissolved in acetonitrile, filtered through a measure pH, partial volume of solids, relative viscosity, opti-
0
2
.2 µm LID/X filter and analyzed for substrate 1 and product cal density, and glucose concentration. The off-gas carbon
concentrations by high-pressure liquid chromatography dioxide from the fermenter was monitored continuously with
a mass spectral (MS) gas analyzer. The fermenter was har-
vested after the glucose concentration dropped below 3 g/L.
TABLE 1
Stereoselective Microbial Reduction of Ketone 1
To determine the specific enzyme activity of cells during fer-
mentation, the cells were periodically harvested by centrifu-
gation from 200 mL of culture broth. Cell suspensions (20%
wt/vol, wet cells) were prepared in 100 mM potassium phos-
phate buffer (pH 6.8) and supplemented with 1.0 mg /mL of
compound 1 and 25 mg/mL of glucose. The biotransforma-
tion of compound 1 was conducted in a 125-mL flask with a
reaction volume of 10 mL at 28°C and 200 rpm on a shaker.
Periodically, samples were analyzed for the reduction of com-
pound 1 to compound 2 by HPLC.
The specific enzyme activity was expressed as mg of com-
pound 2 produced per h per g of dry cells. The cells were har-
vested with the aid of a Sharples centrifuge (Alfa-Laval,
Springfield, PA) and the wet cell paste was collected and
stored at −60°C until further use. About 25 kg of wet cell
Reaction
yield
Optical
purity
of 2 (%)
a
Microorganisms
of 2 (%)
Agrobacterium tumefaciens ATCC 15955
Alcaligenes eutrophus ATCC 17697
Arthrobacter petroleophagus ATCC 21494
Debaryomyces hansenii ATCC 66354
Mycobacterium sp. ATCC 29676
Rhodococcus rhodochorous ATCC 14347
Hansenula anomala SC 13833
H. anomala SC 16142
H. saturnus SC 13829
Spingomonas paucimobilis SC 16113
7
15
5
99
86
99.1
99.2
99.5
99.9
96.3
97
7
10
21
16
18
20
58
99.1
99.5
aReaction mixture in 10 mL of cell-suspensions (20% wet cells) contained
0 mg of substrate 1 (dissolved in acetonitrile and supplied at 2% acetoni-
trile concentration) and 250 mg of glucose. Reactions were carried out at
1
2
8°C, 200 rpm on a rotary shaker for 18 h as described in the Materials and paste was collected from each fermentation.
Methods section. The reaction yield of 2 and optical purity of 2 were deter-
mined by high-performance liquid chromatography as described in the Ma-
terials and Methods section. 1, 4-benzyloxy-3-methanesulfonylamino-2′-
bromoacetophenone; 2, (R)-alcohol of 1.
Two-stage process: reduction of compound 1 in a fer-
menter. Frozen cells from the above batches were used to con-
duct the reduction of compound 1 in 5-L and 25-L fermenters
JAOCS, Vol. 75, no. 11 (1998)

1476
R.N. PATEL ET AL.
under aerobic or anaerobic (under argon) conditions. Cell sus- ume of 100 L, corresponding to about 20% (wt/vol, wet) cell
pensions (20% wt/vol, wet cells) in 80 mM potassium phos- suspensions. The microfiltered and diafiltered cells were used
phate buffer (pH 6.0) were used. Glucose (25 g/L) and com- in the biotransformation process. Glucose (25 g/L) and com-
pound 1 in N,N-dimethylformamide (2 g/L) were supple- pound 1 in N,N-dimethylformamide (2 g/L) were supple-
mented to the cell suspensions, and the reduction was mented to the cell suspensions and the reduction was con-
conducted at 37°C and 300 rpm with gentle sparging of argon ducted at 37°C and 300 rpm with gentle sparging of argon gas
gas or air at 1 Lpm. The pH was allowed to drop from an in- at 1 Lpm. The pH was allowed to drop from an intial pH of
tial value of 6.0 to 5.5; thereafter the pH was maintained at 6.0 to 5.5; thereafter the pH was maintained at 5.5 during the
5
.5 during the bioreduction process. Periodically, 2-mL sam- bioreduction process. Periodically, 2-mL samples were taken
ples were taken and analyzed for substrate 1 and product 2 and analyzed for substrate 1 and product 2 concentrations by
concentrations by HPLC. The optical purity of compound 2 HPLC. The optical purity of compound 2 was determined by
was determined by chiral HPLC.
chiral HPLC.
Preparative scale biotransformation of 1 was carried out in
Biotransformation of compound 1 in the presence of resin.
a 380 L fermenter. Wet cells (40 kg) of S. paucimobilis SC XAD-7 resin (50 g) (Amberlite nonionic polymeric adsor-
6113 were suspended in 200 L of 80 mM potassium phos- bent, 20–60 mesh, polyacrylate, Rohm and Hass, Philadel-
1
phate buffer (pH 6.0). Glucose (25 g/L) and compound 1 in phia, PA) was washed twice with 200 mL of methanol and
N,N-dimethylformamide (2 g/L) were supplemented to the then twice with 200 mL of water. The washed resin was sus-
cell suspensions, and the reduction was conducted at 37°C pended in 1 L of 100 mM potassium phosphate buffer (pH
and 300 rpm with gentle sparging of argon gas at 1 Lpm. The 6.0), and 5 g of substrate 1 in dimethylformamide was added
pH was allowed to drop from an intial value of 6.0 to 5.5; to the resin suspension. The substrate was allowed to adsorb
thereafter the pH was maintained at 5.5 during the bioreduc- onto resin for 18 h at 25°C with continuous mixing. The resin
tion process. Periodically, 2-mL samples were taken and ana- was collected by filtration. Substrate-enriched resin was used
lyzed for substrate 1 and product 2 concentrations by HPLC. in the biotransformation process using microfiltered and di-
The optical purity of compound 2 was determined by chiral afiltered cells in a 3-L bioreactor. Cell suspensions (1 L)
HPLC. At the end of bioreduction, the aqueous solution (20% wt/vol, wet cells) were supplemented with compound 1
(
209 L) was extracted with an equal volume of ethyl acetate, in N,N-dimethylformamide (2 g/L) and glucose (25 g/L), and
and the separated organic layer was concentrated under vac- the reduction was conducted at 37°C and 200 rpm with gen-
uum at 35°C to 12 L. The ethyl acetate concentrate was fur- tle sparging of argon gas at 0.5 Lpm. The pH was allowed to
ther concentrated under vacuum at 35°C to 750 mL, washed drop from an intial value of 6.0 to 5.5; thereafter the pH was
three times with 500 mL of aqueous saturated sodium bicar- maintained at 5.5 during bioreduction process. Periodically,
bonate, then washed three times with 500 mL of saturated 2-mL samples were taken and analyzed for substrate 1 and
sodium chloride, and finally washed three times with 500 mL product 2 concentrations by HPLC. The optical purity of
of 0.5 N HCl. The rich ethyl acetate was concentrated under compound 2 was determined by chiral HPLC. At the end of
vacuum at 35°C to dryness. The concentrate was suspended the biotransformation, the reaction mixture was filtered on a
in 200 mL of ethanol and heated to 50°C until all solids were 100 mesh (150 µ) stainless steel screen, and the resin retained
dissolved. To the hot solution, 82 mL of 40°C water was by the screen was washed with 1 L of water. The product was
added, followed by the addition of 480 mL of n-heptane. The then desorbed from the resin with 100 mL of acetonitrile. The
solution was stirred for an hour at room temperature and al- acetonitrile solution was then evaporated to dryness under
lowed to cool. The crystals formed were filtered, washed with vacuum; the crude product was further purified, crystallized,
1
L of water, and dried under vacuum at 45°C to yield 120 g and recrystallized in an overall 70 M% yield with 90% homo-
of crude compound 2. The crude product was further recrys- geneity and 99.8% optical purity.
tallized to yield 100 g of product 2 in overall 50% yield.
In an alternative process, frozen cells of S. paucimobilis
Single-stage fermentation and biotransformation of 1. SC 16113 were used with resin-adsorbed substrate. Cell sus-
Spingomonas paucimobilis SC 16113 cultures were grown in pensions (2 L) (15% wt/vol, wet cells) in 80 mM potassium
5
50 L of medium B as described in the two-stage process. At phosphate buffer (pH 6.0) were supplemented with 200 g of
the end of the fermentation stage, cells were microfiltered, di- XAD-7 resin containing 20 g of compound 1 and 50 g of glu-
afiltered, and used immediately in the single-stage biotrans- cose. The reduction was conducted at 37°C and 200 rpm with
formation process. The fermentation broth was cooled to gentle sparging of argon gas at 0.5 Lpm. The pH was main-
about 8°C and transferred to a tank maintained at about 8°C. tained at 6.0 during the bioreduction process. Periodically, 2-
2
Cultures were concentrated to 100 L volume by a 40-ft ce- mL samples were taken and analyzed for substrate 1 and
ramic membrane crossflow microfilter (Millipore Corp., Bed- product 2 concentrations by HPLC. The optical purity of
ford, MA) at a transmembrane pressure of 45 psi and a recir- compound 2 was determined by chiral HPLC. At the end of
culation rate of 700 gal/min. The cells were then diafiltered the biotransformation, the reaction mixture was filtered on a
by adding 400 L of 80 mM potassium phosphate buffer (pH 100 mesh (150 µ) stainless steel screen, and the resin retained
2
6
.0) to the recirculation tank at 3.8 L/h-ft . The washed cells by the screen was washed with 2 L of water. The product was
were further concentrated, if necessary, to give retentate vol- then desorbed from the resin with 200 mL of acetonitrile. The
JAOCS, Vol. 75, no. 11 (1998)

CHIRAL INTERMEDIATES FOR β-3-RECEPTOR AGONISTS
1477
acetonitrile solution was then evaporated to dryness under F1 stage, a frozen vial of culture was inoculated into 100 mL
vacuum and the crude product was further purified, crystal- of medium A. The growth was carried out in 500-mL flasks
lized, and recrystallized in an overall 75 M% yield with 91% at 28°C and 280 rpm for 48 h. In the F2 stage, 100 mL of F1-
homogeneity and 99.8% optical purity.
stage culture was inoculated into 1.5 L of medium A in a 4-L
Biotransformation of 1 by cell extracts of S. paucimobilis flask and incubated at 28°C and 180 rpm for 24 h. Fermenters
SC 16113. Spingomonas paucimobilis SC 16113 cells were containing 125 L of medium A were inoculated with 3 L of
suspended in 1.3 L of buffer A (50 mM potassium phosphate F2 stage inoculum and grown at 30°C and 280 rpm with 125
buffer, pH 6.0, containing 20% glycerol and 1 mM phenyl- Lpm aeration. During fermentation, cells were periodically
methylsulfonyl fluoride) at 20% (wt/vol, wet cells) concen- harvested by centrifugation from 200 mL of culture broth.
tration and homogenized to prepare cell suspensions. The cell Cell suspensions (20% wt/vol, wet cells) were prepared in
suspension was disintegrated by two passages through a Mi- 100 mM potassium phosphate buffer (pH 7.0) and supple-
crofluidizer (Microfluidics, Newton, MA) at 10,000 psi at mented with 2.0 mg /mL of compound 3. The biotransforma-
8°C. Disintegrated cells were centrifuged at 20,000 × g for 40 tion was conducted in a 125-mL flask with a reaction volume
min at 4°C; the supernatant solution is referred to as cell ex- of 10 mL at 30°C and 200 rpm on a shaker. Periodically sam-
tracts. To 1 L of cell extracts, 1 g of substrate 1 in dimethyl- ples were removed, and after centrifugation, the separated su-
formamide, 1 g of glucose, 0.1 g of NADP, and 1 kilounit of pernatant solution was collected, filtered through a 0.2 µm
glucose dehydrogenase were added. The bioreduction was LID/X filter, and analyzed by HPLC for substrate 3 and prod-
carried out at 30°C, 200 rpm, for 90 min. Periodically 2-mL uct 4 concentrations. The activity was expressed as mmol of
samples were taken and analyzed for substrate 1 and product product 4 formed per min per g of dry cells. Cells were har-
2
concentrations by HPLC. The optical purity of compound 2 vested at an optimal activity period with the aid of a Cepa
was determined by chiral HPLC. centrifuge, and the wet cell pastes were collected and stored
Analytical methods for quantitation and optical purity de- at −60°C until further use. Portions of the cells were freeze-
termination. Analysis of compounds 1 and 2 was carried out dried and stored at 5°C.
using a Hewlett-Packard (HP) 1070 high-performance liquid
Biotransformations of compound 3 by microbial cell sus-
chromatograph (Palo Alto, CA). A Vydak 5 µm column (25 pensions. The cells of M. neoaurum ATCC 25795 were sus-
cm × 4.6 mm, 5 µ) was used at ambient temperature. The mo- pended in 100 mM potassium phosphate buffer, pH 7.0, at
bile phase consisted of solvent A (0.1% trifluoroacetic acid in 10% (wt/vol, wet cells) cell concentration and supplemented
distilled water) and solvent B (0.1% trifluoroacetic acid in with 1 mg/mL of compound 3. Freeze-dried cells of M.
7
1
0% acetonitrile and 30% distilled water). The flow rate was neoaurum ATCC 25795 were suspended in 100 mM potas-
mL/min. The detector wavelength was 211 nm. A gradient sium phosphate buffer, pH 7.0, at 1% cell concentration and
elution was carried out for 30 min as follows: 0 min, 50% sol- supplemented with 1 mg/mL of compound 3. The biotrans-
vent B; 0–20 min, 100% solvent B; 20–25 min, 100% solvent formation was conducted at 30°C, 280 rpm on a rotary shaker.
B; 25–26 min, 50% solvent B. The retention times for the Periodically, samples of 1 mL were taken and after centrifu-
substrate 1 and product 2 were 10.2 min and 8.3 min, respec- gation, the supernatant solution was collected, filtered
tively. The separation of the two enantiomers of racemic al- through a 0.2 µm LID/X filter, and analyzed for substrate 3
cohol 2 were achieved by HPLC using a Chiracel OJ-R (46 and product 4 concentrations. The biotransformation of sub-
cm × 15 cm, 5 µ) column (J.T. Baker, Phillipsburgh, PA). The strate 5 was carried out in a similar manner.
mobile phase consisted of 30% acetonitrile in deionized
Samples (2 mL) from the reaction mixture were analyzed
water. The flow rate was 0.75 mL/min and the detector wave- for substrate and product concentration with a Hewlett-
length was 210 nm. The retention times for the two enan- Packard 1070 high-performance liquid chromatograph. A
tiomers of racemic 2 were 33.1 (S-isomer) and 18.9 min Phenomenex Cyanopropyl column (150 × 4.6 mm, 5 µ) was
(R-isomer), respectively.
used. The mobile phase consisted of 5% isopropanol in
Growth of M. neoaurum ATCC 25795. One vial of M. hexane. The flow rate was 230 nm. The optical purity of prod-
neoaurum ATCC 25795 culture was used to inoculate 100 mL ucts 4 and 6 was determined by chiral HPLC. The separation
of medium A containing 1% yeast extract, 1% malt extracts, of the two enantiomers of products 4 and 6 was achieved by a
2
% glucose, and 0.3% peptone. The medium was adjusted to Chiralcel WH column (250 × 4.6 mm, 5 µ). The mobile phase
pH 6.8 before sterilization. Cultures were grown at 28°C and was 10 mM CuSO . The flow rate was 1 mL/min, and the de-
4
2
80 rpm for 48 h on a rotary shaker. Cultures were harvested tector wavelength was 230 nm. The retention times for the
by centrifugation at 18,000 × g for 15 min, washed with 100 two enantiomers of 4 were 8.0 and 16.7 min, respectively.
mM potassium phosphate buffer (pH 7.0), and then used for The retention times for the two enantiomers of 6 were 12.9
biotransformation studies.
and 18.9 min, respectively.
Fermentation of M. neoaurum ATCC 25795. Mycobac-
Preparation of cell extracts of M. neoaurum ATCC 25795.
terium neoaurum ATCC 25795 was grown in 150-L fer- Cultures of M. neoaurum ATCC 25795 were grown in a
menters containing 125 L of medium A. Growth consisted of 150-L fermenter as described earliar. Cell suspensions (10%
two inoculum development stages and a fermentation stage. wt/vol, wet cells) in 100 mM potassium phosphate buffer (pH
Inoculum development consisted of F1 and F2 stages. In the 7.0) containing 1 mM dithiothreitol and 10% glycerol (buffer
JAOCS, Vol. 75, no. 11 (1998)

1478
R.N. PATEL ET AL.
B) were disintegrated by Microfluidizer M-110F (Microflu- membrane was washed with 30 mL of 25 mM potassium
idics, Inc.) at 10,000 psi at 4°C. The lysates were centrifuged phosphate buffer, pH 7.2. Combined permeate and wash (380
at 20,000 × g for 30 minutes at 4°C, and the clear supernatant mL) were acidified to pH 2.2 with 6 N HCl and 10.5 grams of
collected was referred to as cell extracts.
SP 207 resin was added to adsorb monoester product 8 onto
Purification of amidase. Cell extracts of M. neoaurum resin at ambient temperature. Product containing resin was
ATCC 25795 (200 mL) were loaded on a DEAE (diethyl- collected by filtration, and desorption of product 8 from the
aminoethyl) cellulose column (5 × 50 cm) previously equili- resin was carried out using 215 mL of methanol in the first
brated with buffer B. The column was washed with buffer B extraction and 105 mL of methanol in the second extraction.
and eluted with 2 L of buffer B containing sodium chloride in Combined methanol extracts (300 mL) were evaporated
a linear gradient of 0–0.5 M. Fractions containing amidase under reduced pressure at 40°C to obtain 2.6 g of pale yellow
activity were pooled and used for biotransformation of sub- oily residue. The monoester 8 was isolated in 86.3 M% over-
strates 4 and 6.
all yield. The optical purity of isolated S-(−)-monoester 8 was
1
Biotransformations of 3 and 5 by cell extracts. Cell ex- 96.9%. The H NMR and MS of the isolated product were
tracts from M. neoaurum ATCC 25795 were evaluated for the consistent with monoester 8, and the specific rotation of
biotransformations of compounds 3 and 5. The reaction mix- monoester [α] was −14.4 (c = 1.1 in methanol).
D
ture contained 40 mg of substrate in 20 mL of cell extracts.
The reactions were conducted at 30°C, 200 rpm on a shaker.
Concentrations of substrates 3 and 5 and products 4 and 6
RESULTS
were determined by HPLC. The optical purities of products 4 Microbial reduction of 1. Microorganisms were screened for
and 6 were determined by chiral HPLC as described above.
the transformation of ketone 1 to chiral alcohol 2. As shown
Hydrolysis of 7. The enzymatic asymmetric hydrolysis of in Table 1, among the cultures evaluated, Hansenula anomola
was carried out in 25 mM potassium phosphate buffer at pH SC 13833, H. anomola SC 16142, Rhodococcus rhodochrous
.2, at 10°C. The reaction mixture contained 18 mL of buffer ATCC 14347, and Spingomonas paucimobilis SC 16113 gave
7
7
and 2 mL of organic solvent (as indicated) containing 200 mg the desired alcohol 2 in >96% optical purity and >15% reac-
of diester 7 and 500 units of enzyme PLE (Sigma Chemicals, tion yield. Spingomonas paucimobilis SC 16113 catalyzed the
St. Louis, MO). The pH of the reaction was maintained at 7.2 efficient conversion of ketone 1 to the desired chiral alcohol
by addition of 5 N NaOH. Periodically, samples of 1 mL were 2 in 58% reaction yield and >99.5% optical purity.
taken and extracted with 4 mL of ethyl acetate. After centrifu-
Since substrate 1 is insoluble in water, the effect of sol-
gation, the separated organic layer was filtered through a 0.2 vents on dissolved substrate 1 and its supply in the biotrans-
µm LID/X filter (Whatman Inc.) . A portion of the clarified formation reaction mixture were evaluated. Dimethylfor-
ethyl acetate filtrate was dried under a stream of nitrogen. The mamide at 2–5% concentrations was found to be the best co-
resulting material was dissolved in mobile phase and ana- solvent to supply the substrate in the biotransformation
lyzed by HPLC for optical purity of monoester 8. The analy- process (Table 2).
sis of diester 7 and monoester 8 were carried out by gas chro-
matography (GC) assay. An HP-1 capillary column (12 m × carried out in a 750-L fermenter as described in the Materials
.2 mm × 0.33 mm) was used with the oven at 110°C and the and Methods section. From each fermentation batch, about
The fermentation of S. paucimobilis SC 16113 culture was
0
injector and detector at 250°C. The retention times for the di- 60 kg of wet cell paste was collected. Cells harvested from
ester 7 and monoester 8 were 19.05 and 9.5 min, respectively. the fermenter were used to conduct the biotransformation in
The separation of two enantiomers of monoester 8 was 1-, 10-, and 210-L preparative batches under aerobic or anaer-
achieved by chiral HPLC with a Chiralpak AD (Diacel
Chemical Co., Easton, PA) column (0.46 × 25 cm). Mobile
TABLE 2
phase was 10% isopropanol, 10% cyclohexanol, and 0.1%
formic acid; retention times of two enantiomers of racemic 8
were 9.04 and 11.2 min, respectively.
Effect of Cosolvent in Microbial Reduction of Ketone 1
Reaction yielda
Optical purity
Cosolvent (%)
of 2 (%)
of 2 (%)
Semipreparative-scale hydrolysis of diester 7. A semi-
preparative-scale asymmetric hydrolysis of diester 7 was car-
ried out in a biphasic system using 10% ethanol as a cosol- Dimethylformamide (2)
vent. The reaction mixture contained 270 mL of 25 mM Dimethylsulfoxide (10)
Dimethylformamide (10)
Dimethylformamide (5)
80
84
85
73
73
80
59
99
99.1
99.4
98
98.2
98.2
99.2
Dimethylsulfoxide (5)
potassium phosphate buffer (pH 7.2) and 30 mL of ethanol
Dimethylsulfoxide (2)
containing 3 g of diester 7 and 22,000 units of crude PLE
Acetonitrile (2)
(
Amano International Enzyme Co., Troy, VA). The reaction
aReaction mixture in 10 mL of cell suspensions (20% wet cells) contained
0 mg of substrate 1 (dissolved in solvent as indicated and supplied at sol-
was carried out at 10°C, 125 rpm agitation and pH 7.2 for 11
1
h. The pH was maintained at 7.2 with 5 N NaOH. At the end vent concentration as indicated) and 250 mg of glucose. Reactions were car-
ried out at 28°C, 200 rpm on a rotary shaker for 18 h as described in the Ma-
terials and Methods section. The reaction yield of 2 and optical purity of 2
were determined by high-performance liquid chromatography (HPLC) as de-
of reaction, the reaction mixture was centrifuged and filtrate
was passed through an ultrafiltration device using a 10,000
MW cut-off membrane to remove most of the protein. The scribed in the Materials and Methods section. See Table 1 for abbreviations.
JAOCS, Vol. 75, no. 11 (1998)

CHIRAL INTERMEDIATES FOR β-3-RECEPTOR AGONISTS
1479
TABLE 3
biotransformation, the reaction mixture was filtered on a 100
mesh (150 µ) stainless steel screen, and the resin retained by
the screen was washed with 2 L water. The product was then
desorbed from the resin and crystallized in an overall 75 M%
yield with 91% homogeneity and 99.8% optical purity.
The reduction of compound 1 to compound 2 was also car-
ried out using cell extracts of S. paucimobilis SC 16113. Glu-
cose dehydrogenase was used to regenerate the cofactor
NADPH required for the reduction. After 90 min reaction
time, 80% conversion of ketone 1 to chiral alcohol 2 was ob-
tained. About 0.6 g of chiral alcohol 2 was isolated from the
reaction mixture in 90% chemical purity. The isolated com-
pound 2 gave 99.4% optical purity.
a
Semipreparative Batches for Microbial Reduction of Ketone 1
Reaction Substrate
Reaction
yield of 2
(%)
Optical
purity of 2
(%)
Batch
size (L)
time
(h)
input
(g/L)
Condition
Aerobic
1
2
1
1
1
2
10
200
4
4
3
3
6
3
3
2
1
1
1
1
2
1
1
1
85.3
87
87.9
89.7
87
90
95
84
98.5
98.6
98.5
98.7
99.4
98.5
99.4
99.5
Aerobicb
Aerobic
Anaerobicb
Anaerobic
Anaerobic
Anaerobic
Anaerobic
aCells were suspended in 80 mM potassium phosphate buffer (pH 6.0) at
0% (wt/vol, wet cells) concentration. Cell suspensions were supplemented
with substrate 1 (in dimethylformamide) and glucose (25 g/L). Reactions were
2
Enzymatic resolution of 3 and 5 to the corresponding (S)-
carried out at 37°C, 300 rpm under anaerobic conditions. The reaction yield amino acids 4 and 6. The cells (10% wt/vol, wet cells) of M.
and optical purity of 2 were determined by HPLC as in the Materials and
neoaurum ATCC 25795 were evaluated for biotransformation
of compound 3 to compound 4. The reaction was completed
Methods section. See Tables 1 and 2 for abbreviations.
b
Microfiltered and diafiltered cells were used.
in 75 min with a reaction yield of 48 M% (theoretical max.
5
0%) and an optical purity of 95% for the desired product 4.
obic conditions. The cells were suspended in 80 mM potas- Freeze-dried cells of M. neoaurum ATCC 25795 were sus-
sium phosphate buffer (pH 6.0) to 20% (wt/vol, wet cells) pended in 100 mM potassium phosphate buffer (pH 7.0) at
concentration. Compound 1 (1–2 g/L) and glucose (25 g/L) 1% concentration, and cell suspensions were used for the bio-
were added to the fermenter, and the reduction reaction was transformation of compound 3. The reaction was completed
carried out at 37°C. Results are as shown in Table 3. In some in 60 min with a reaction yield of 49.5 M% (theoretical max.
batches, the microfiltered and diafiltered cells were used di- 50%) and an optical purity of 99% for the desired product 4.
rectly in the bioreduction process. In all batches of biotrans- The kinetics of reaction are shown in Figure 1. Biotransfor-
formation, reaction yields of >85% and optical purities of mation of compound 3 was also carried out using a purified
>
98% were obtained.
The isolation of compound 2 from the 200-L preparative 99.9% were obtained for the desired product 4 after 60 min
batch was carried out as described in the Materials and Meth- reaction time.
amidase. A reaction yield of 49 M% and an optical purity of
ods section to obtain 100 g of product 2. The isolated 2 gave
Freeze-dried cells of M. neoaurum ATCC 25795 and par-
a homogeneity index of 83% and an optical purity of 99.5% tially purified amidase were used for the biotransformation of
as analyzed by chiral HPLC. The MS and NMR data of iso- compound 5. A reaction yield of 49 M% and an optical purity
lated compound 2 and the standard compound 2 were virtu- of 78% were obtained for the desired product 6 using freeze-
ally identical.
In an alternative process, frozen cells of S. paucimobilis
SC 16113 were used with resin-adsorbed substrate at 5 and
1
0 g/L substrate concentrations as described in the Materials
and Methods section. Results are as shown in Table 4. In this
process, an average reaction yield of 85% and an optical pu-
rity of >99% were obtained for product 2. At the end of the
TABLE 4
Semipreparative Batches for Microbial Reduction of Ketone 1
a
in the Presence of Resin XAD-7
Reaction Substrate Product 2 Product 2 Reaction Optical
Batch
size (L)
time
(h)
input
(g/L)
on resin in aqueous yield
purity
(g/L)
(g/L)
of 2 (%) of 2 (%)
1
2
73
93
5
10
4
7.8
0.12
0.29
82
81
98.5
98.6
aCells were suspended in 80 mM potassium phosphate buffer (pH 6.0) at
0% (wt/vol, wet cells) concentration. Cell suspensions were supplemented
2
with substrate 1 (on XAD-7 resin; Rohm and Haas, Philadelphia, PA) and
glucose (25 g/L). Reactions were carried out at 37°C, 300 rpm under anaer-
obic conditions. The reaction yield and optical purity of 2 were determined
by HPLC as in the Materials and Methods section. See Tables 1 and 2 for
abbreviations.
FIG. 1. Resolution of racemic α-methyl phenylalanine amide 3 by My-
cobacterium neoaurum ATCC 25795. 3, racemic α-methyl phenylala-
nine amide; 4, (S)-amino acid of 3; OP, optical purity.
JAOCS, Vol. 75, no. 11 (1998)

1480
R.N. PATEL ET AL.
FIG. 2. Resolution of racemic α-methyl-4-hydroxyphenylalanine
amide, 5, by M. neoaurum ATCC 25795. 6, (S)-amino acid of 5; for
other abbreviations see Figure 1.
FIG. 3. Asymmetric hydrolysis of methyl-(4-methoxyphenyl)-propane-
dioic acid ethyl ester, 7, by pig liver esterase. 8, (S)-monoester of 7. For
other abbreviation see Figure 1.
dried cells. The reaction was completed in 50 h. By using par- est reaction yield (96.7%) and optical purity (96%) for
tially purified amidase, a reaction yield of 49 M%, and an monoester 8.
optical purity of 94% were obtained for desired product 6
after 70 h reaction time. The kinetics of reaction are shown in PLE-catalyzed hydrolysis of diester 7 in a biphasic system
Figure 2.
using ethanol as a cosolvent. It was observed that the optical
The effects of temperature and pH were evaluated for the
Asymmetric hydrolysis of 7. Various organic solvents purity of the desired monoester 8 increased with decreasing
were tested for the PLE-catalyzed asymmetric hydrolysis temperature from 25 to 10°C. The optimal pH for asymmet-
of diester 7 in a biphasic system. The results (Table 5) indicate ric hydrolysis of diester 7 in a biphasic system using ethanol
that the reaction yields and optical purities of monoester 8 as a cosolvent is 7.2 at 10°C (Table 6).
depended upon the solvent used in asymmetric hydrolysis.
A semipreparative scale asymmetric hydrolysis of diester
Tetrahydrofuran, methylisobutyl ketone, and hexane inhibited 7 was carried out in a biphasic system using 10% ethanol
PLE. Lower reaction yields (28–56 M%) and lower optical as a cosolvent. Substrate (3 g) was used in a 300-mL reac-
purities (59–72%) were obtained using t-butylmethyl ether, tion mixture. The reaction was carried out at 10°C, 125 rpm
dimethylformamide, and dimethylsulfoxide as cosolvents. agitation, and at pH 7.2 for 11 h. Kinetics of the reaction are
Higher optical purities (>91%) were obtained using methanol, as shown in Figure 3. A reaction yield of 96 M% and an op-
ethanol, and toluene as cosolvents. Ethanol gave the high- tical purity of 96.9% were obtained. From the reac-
TABLE 5
Effect of Solvent on Asymmetric Hydrolysis of Methyl-(4-methoxyphenyl)-propanedioic
a
Acid, Ethyl Diester, 7
Reaction
time
Yield of
Optical purity
Diester 7 Monoester 8 monoester 8 of monoester 8
Solvent
(h)
(mg/mL)
(mg/mL)
(M%)
(%)
Methanol
Ethanol
Acetonitrile
22
22
22
22
22
22
48
64
22
48
22
64
0
0
0
0
0.61
0
0
2.01
0.76
2
0.18
2.05
0.65
1.7
0.5
0.85
1
1.44
1.36
0
0.8
0
0.59
0
37
92
96
96.7
28.2
48.3
56.9
81.9
77.3
0
46
0
33.6
0
59.3
68.5
72
65.1
82.1
Dimethylformamide
Dimethylsulfoxide
Acetone
Methylethylketone
Methylisobutylketone
t-Butylmethylether
Tetrahydrofuran
Toluene
64.4
91
Hexane
a8, (S)-monoester of 7.
JAOCS, Vol. 75, no. 11 (1998)

CHIRAL INTERMEDIATES FOR β-3-RECEPTOR AGONISTS
1481
TABLE 6
Effect of Temperature and pH on Asymmetric Hydrolysis of 7
a
Yield
of monoester 8
Optical purity
of monoester 8
(%)
Temperature
°C)
Diester 7
(mg/mL)
Monoester 8
(mg/mL)
(
pH
(M%)
2
5
5
5
5
0
0
0
7
7.2
8
7.2
6.8
7.2
8
0
0
0
0
0
0
0
1.3
80
91
93
80.3
80.5
95
85
85
82
90.5
80.5
96.3
94
2
2
1
1
1
1
1.41
1.45
1.35
1.38
1.54
1.58
96
aFor abbreviations see Table 5.
tion mixture, 2.6 g of monoester 8 was isolated in 86.3 M% have developed a process for the stereoselective reduction of
overall yield. The optical purity of isolated S-(−)-mono- 1 to the corresponding (R)-alcohol 2 . In a batch process in
1
ester 8 was 96.9%. The H NMR and MS of the iso- the absence of resin, a reaction yield of 85–95% was obtained
lated product were consistent with monoester 8 and the spe- under either aerobic or anaerobic conditions. Substrate was
cific rotation of monoester [α]D was −14.4 (c = 1.1 in used at 1–2 g/L. To increase the substrate concentration up to
methanol).
10 g/L and to develop an efficient recovery process, the bio-
transformation process was carried out in the presence of
resin. In this process, substrate was adsorbed onto resin
XAD-7 and supplied to the reaction. At the end of the bio-
DISCUSSION
The asymmetric reduction of carbonyl compounds by baker’s transformation process, product-rich resin was easily sepa-
yeast has been reviewed (3,20–22). The synthetically useful rated from cell suspensions of organisms, and rich resin was
alcohol dehydrogenases from yeast (23), horse liver (2), and extracted with solvent to recover product.
secondary alcohol dehydrogenase (24,25) usually reduce car-
Optically pure α-methyl-substituted amino acids are chiral
bonyl compounds to give an (S)-alcohol. Recently, Bradshaw synthons for drugs (33–35). α-Methyl-substituted amino
et al. have demonstrated the use of alcohol dehydrogenase acids can be prepared by enzymatic resolution using esterases
from Lactobacillus kefir (26) and Pseudomonas sp. (27) as (36), acylases (37), hydantoinases (38), or amidases (39–41).
catalysts for synthesis of chiral aromatic, cyclic, and aliphatic In this report, we have demonstrated the preparation of α-
alcohols from their corresponding ketones.
methyl-substituted amino acids by a resolution process using
In our continuing effort to prepare chiral synthons for drug amidase from M. neoaurum ATCC 25795. Cell suspensions
development, we have demonstrated the microbial reduction of organisms and partially purified amidase were used in bio-
of N-(4-1-oxo-2-chloroacetyl ethyl) phenyl methane sulfon- transformation to obtain (S)-amino acids in >49 M% yield
amide to the corresponding R-(+)-alcohol (28), an intermedi- (theoretical maximum yield is 50 M%). The enzymatic reso-
ate for the synthesis of -(+)-sotalol (β-blocker). We have lution of 5 by cell suspension of organisms gave lower opti-
D
prepared R-(+)-BMY-14802, an effective antipsychotic drug, cal purity (74%) of the corresponding (S)-amino acid com-
by the stereoselective microbial reduction of 1-(4-fluo- pared to purified enzyme, which gave 94% optical purity of
rophenyl)-4[4-(5-fluoro-2-pyrimidinyl)-1-piperazinyl]butan- the desired product. In a resolution process, the maximum
1
-one by Mortierella ramanniana (29). We have developed theoretical yield obtained is 50%. Hence, we have developed
an enzymatic process for the preparation of chiral alcohol (re- an alternative process in which α-methyl-substituted amino
quired for the synthesis of calcium channel antagonist) by the acid was also prepared by the asymmetric synthesis by the hy-
stereoselective reduction of 4,5-dihydro-4-(methoxy phenyl)- drolysis of the prochiral molecule 7 to the corresponding (S)-
6
-(trifluoromethyl-1H-1)-benzazepin-2-one (30). A chiral in- monoester by PLE. A reaction yield of >95% was obtained
termediate of (3S,5R)-dihydroxy-6-(benzyloxy)hexanoic for the desired (S)-monoester.
acid, ethyl ester was enzymatically prepared by the stereose-
lective reduction process (31). Chiral dihydroxy compound is
an intermediate for our new anticholesterol drug. Recently,
we have prepared C-13 side-chain of an anticancer drug, pac-
litaxel, by the stereoselective microbial reduction of 2-keto-
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[Received July 6, 1998; accepted August 25, 1998]
JAOCS, Vol. 75, no. 11 (1998)