Biocatalytic synthesis of chiral intermediates for antiviral and antihypertensive drugs

Article abstract of DOI:10.1007/s11746-999-0139-7

The chiral intermediate (1S,2R) [3-chloro-2-hydroxy-1-(phenylmethyl)propyl] carbamic acid, 1,1-dimethyl-ethyl ester 2a was prepared for the total synthesis of a human immunodeficiency virus protease inhibitor, BMS-186318. The stereoselective reduction of (1S) [3-chloro-2-oxo-1(phenylmethyl)propyl] carbamic acid, 1,1-dimethylethyl ester 1 was carried out using microbial cultures, among which Streptomyces nodosus SC 13149 efficiently reduced 1 to 2a. A reaction yield of 80%, enantiomeric excess (e.e.) of 99.8%, and diastereomeric purity of 99% were obtained for chiral alcohol 2a. Chiral L-6-hydroxy norleucine 3, an intermediate in the synthesis of antihypertensive drug, was prepared by reductive amination of 2-keto-6-hydroxyhexanoic acid 4 using beef liver glutamate dehydrogenase. The cofactor NADH required for this reaction was regenerated using glucose dehydrogenase from Bacillus sp. A reaction yield of 80% and e.e. of 99.5% were obtained for L-6-hydroxynorleucine 3. To avoid the lengthy chemical synthesis of the ketoacid, a second route was developed in which racemic 6-hydroxynorleucine [readily available from hydrolysis of 5-(4-hydroxybutyl) hydantoin 5] was treated with D-amino acid oxidase from Trigonopsis variabilis to selectively convert the D-isomer of racemic 6-hydroxynorleucine to 2-keto-6-hydroxyhexanoic acid 4 and L-6-hydroxynorleucine 3. Subsequently, the 2-keto-6-hydroxyhexanoic acid 4 was converted to L-6-hydroxynorleucine by reductive amination using glutamate dehydrogenase. A reaction yield of 98% and an e.e. of 99.5% were obtained.

Full text of DOI:10.1007/s11746-999-0139-7

Biocatalytic Synthesis of Chiral Intermediates
for Antiviral and Antihypertensive Drugs
Ramesh N. Patel
Department of Enzyme Technology, Process Research, Bristol-Myers Squibb Pharmaceutical Research Institute,
New Brunswick, New Jersey 08903
ABSTRACT: The chiral intermediate (1S,2R) [3-chloro-2-hy-
droxy-1-(phenylmethyl)propyl] carbamic acid, 1,1-dimethyl-
ethyl ester 2a was prepared for the total synthesis of a human
immunodeficiency virus protease inhibitor, BMS-186318. The
stereoselective reduction of (1S) [3-chloro-2-oxo-1(phenyl-
methyl)propyl] carbamic acid, 1,1-dimethylethyl ester 1 was
carried out using microbial cultures, among which Strepto-
myces nodosus SC 13149 efficiently reduced 1 to 2a. A reac-
tion yield of 80%, enantiomeric excess (e.e.) of 99.8%, and di-
astereomeric purity of 99% were obtained for chiral alcohol 2a.
Chiral drug intermediates can be prepared by asymmetric
synthesis by either chemical or biocatalytic processes using
microbial cells or enzymes derived therefrom. The advan-
tages of microbial- or enzyme-catalyzed reactions over chem-
ical reactions are: (i) they are stereoselective and can be car-
ried out at ambient temperature and atmospheric pressure.
This minimizes problems of isomerization, racemization,
epimerization, and rearrangement that generally occur during
chemical processes. (ii) Biocatalytic processes are generally
carried out in aqueous solution, thus avoiding environmen-
tally harmful chemicals, used in the chemical processes, and
Chiral
L-6-hydroxy norleucine 3, an intermediate in the synthe-
sis of antihypertensive drug, was prepared by reductive amina-
tion of 2-keto-6-hydroxyhexanoic acid 4 using beef liver gluta- solvent waste disposal. (iii) Microbial cells or their enzymes
mate dehydrogenase. The cofactor NADH required for this re-
action was regenerated using glucose dehydrogenase from
Bacillus sp. A reaction yield of 80% and e.e. of 99.5% were ob-
can be immobilized and reused many cycles.
The use of enzymes in organic synthesis has been re-
viewed (1–11). This report provides some specific examples
of the use of oxidoreductases in stereoselective catalysis and
preparation of chiral drug intermediates required for our an-
tiviral and antihypertensive agents (12,13).
An essential step in the life cycle of human immunodefi-
ciency virus (HIV-1) is the proteolytic processing of its pre-
cursor proteins. This processing is accomplished by HIV-1
protease, a virally encoded enzyme. Inhibition of HIV-1 pro-
tained for L-6-hydroxynorleucine 3. To avoid the lengthy chem-
ical synthesis of the ketoacid, a second route was developed in
which racemic 6-hydroxynorleucine [readily available from hy-
drolysis of 5-(4-hydroxybutyl) hydantoin 5] was treated with
D
-amino acid oxidase from Trigonopsis variabilis to selectively
convert the -isomer of racemic 6-hydroxynorleucine to 2-keto-
6-hydroxyhexanoic acid 4 and -6-hydroxynorleucine 3. Sub-
sequently, the 2-keto-6-hydroxyhexanoic acid 4 was converted
to
D
L
L
-6-hydroxynorleucine by reductive amination using gluta- tease arrests the replication of HIV in vitro. Thus, HIV-1 pro-
mate dehydrogenase. A reaction yield of 98% and an e.e. of
99.5% were obtained.
tease is an attractive target for chemotherapeutic intervention.
Recently, Barrish et al. (14) reported the discovery of a new
class of selective HIV protease inhibitors that incorporate a
C2 symmetric aminodiol core as their key structural feature.
Members of this class, and particularly BMS-186318
(Scheme 1), display potent anti-HIV activity in cell culture.
In this report, we describe the stereoselective microbial
reduction of (1S) [3-chloro-2-oxo-1-(phenylmethyl)propyl]
carbamic acid, 1,1-dimethylethyl ester 1 to 2a (Scheme 1).
Chiral alcohol 2a {(1S,2R)[3-chloro-2-hydroxy-1-(phenyl-
Paper no. J9300 in JAOCS 76, 1275–1281 (November 1999).
KEY WORDS: Antihypertensive drug, antiviral drug, chiral in-
termediates, formate dehydrogenase, glutamate dehydrogenase,
L
-6-hydroxynorleucine, reductive amination, stereoselective
reduction.
Increasing understanding of the mechanisms of drug interac- methy)propyl] carbamic acid, 1,1-dimethyl ethyl ester} is a
tions on a molecular level has led to the awareness of the im- key intermediate for the total chemical synthesis of BMS-
portance of chirality. In many cases, only one enantiomer is 186318.
efficacious, and the other either is inactive or exhibits consid-
L
-6-Hydroxynorleucine 3 is a chiral intermediate useful
erably reduced activity. Pharmaceutical companies know that, for the synthesis of a vasopeptidase inhibitor now in clinical
where appropriate, new drugs should be homochiral to avoid trial and for the synthesis of C-7-substituted azepinones as
possible side effects due to an undesirable stereoisomer.
potential intermediates for other antihypertensive metallopro-
tease inhibitors (15,16). It has also been used for the synthe-
sis of siderophores, indospicines, and peptide hormone
analogs (17–21). Reductive amination of ketoacids using
*Address correspondence at Department of Enzyme Technology, Process
Research, Bristol-Myers Squibb Pharmaceutical Research Institute, P.O. Box
191, New Brunswick, NJ 08903. E-mail: patelr@bms.com
Copyright © 1999 by AOCS Press
1275
JAOCS, Vol. 76, no. 11 (1999)

1276
R.N. PATEL
BOC = butoxycarbonyl
SCHEME 1
amino acid dehydrogenases has shown to be a useful method lase followed by the reductive amination procedure to con-
for synthesis of natural and unnatural amino acids (22,23). In vert the mixture to 3.
this report we also describe the conversion of 2-keto-6-hy-
droxyhexanoic acid 4 to 3 by reductive amination using beef
liver glutamate dehydrogenase and glucose dehydrogenase
MATERIALS AND METHODS
from Bacillus sp. for regeneration of NADH. A second route, Materials. Starting substrate 1 and reference compounds
which avoids the lengthy chemical synthesis, was developed 2a–d were synthesized in the Chemical Process Research De-
to prepare the ketoacid by treatment of racemic 6-hydroxy partment, Bristol-Myers Squibb Pharmaceutical Research In-
norleucine [readily available from hydrolysis of 5-(4-hydrox- stitute (New Brunswick, NJ), as described previously (12).
ybutyl) hydantoin 5, Scheme 2 (24)] with D-amino acid oxi- The physicochemical properties, including spectral character-
dase from porcine kidney or Trigonopsis variabilis and cata- istics [1H nuclear magnetic resonance (NMR), 13C NMR,
SCHEME 2
JAOCS, Vol. 76, no. 11 (1999)

CHIRAL DRUG INTERMEDIATES BY BIOCATALYSIS
1277
mass spectra], were in full accord for all these compounds. development stages (F1 and F2) and fermentation. In the F1
Compound 5 was obtained from Hampshire Chemical Com- stage, a frozen vial of each culture was inoculated into 100
pany. Enzymes were purchased from the following sources: mL of medium A contained in a 500-mL flask. Growth was
D
-amino acid oxidase, catalase, glutamate, phenylalanine, carried out at 28°C and 280 rpm for 48 h on a rotary shaker.
and alanine dehydrogenases, Sigma (St. Louis, MO); formate In the F2 stage, 10 mL of F1 stage culture was inoculated into
dehydrogenase, Boehringer Mannheim (Indianapolis, IN); 1 L of medium A and incubated at 28°C and 280 rpm for 24
glucose dehydrogenase, Amano (Troy, VA). Compound 4 and h. Fermentors containing 15 L of medium A were inoculated
reference compound 3 were synthesized at the Bristol-Myers with 1 L of inoculum of each culture from a F2 stage. Fer-
Squibb Pharmaceutical Research Institute (13), and physico- mentation was conducted at 25°C and 500 rpm with 15 LPM
chemical properties including spectral characteristics (1H (liters per minute) aeration for 48 h. After 48 h fermentation,
NMR, 13C NMR, mass spectra) were in full accord for all cells were collected and stored at −90°C until further use.
these compounds. The 1H NMR and 13C NMR spectra were About 1 kg of wet cell pastes was collected from each fer-
recorded on a Bruker AM-300 spectrometer (Billerica, MA).
mentation.
Frozen cells from the above batches were used to conduct
Microorganisms. Microorganisms (Table 1) were obtained
from the culture collection of the Bristol-Myers Squibb Phar- the reduction of 1 in a 5-L reactor. Cell suspensions (10%
maceutical Research Institute and from the American Type wt/vol, wet cells) in 3 L of 0.1 M potassium phosphate buffer
Culture Collection (Rockville, MD). Microbial cultures were (pH 6.0) were used. Compound 1 (3 g) and glucose (30 g)
stored at −90°C in vials.
were added to the reactor, and the reduction was carried out
For screening purposes, one vial of each culture was used at 28°C and 160 rpm with 1 LPM aeration for 24 h. The pH
to inoculate 100 mL of medium A containing 1% malt extract, was maintained between 6.6 and 6.8. Periodically, samples
1% yeast extract, 2% glucose, and 0.3% peptone. The were prepared as described above and analyzed by HPLC to
medium was adjusted to pH 6.8 before sterilization. Cultures determine the percentage of conversion of 1 to 2a. The di-
were grown at 28°C and 280 rpm for 48 h. Cultures were har- astereomeric purity and the enantiomeric excess of 2a were
vested by centrifugation at 18,000 × g for 15 min, washed determined by HPLC.
with 0.1 M potassium phosphate buffer pH 7.0, and used for
reduction studies.
Single-stage process for reduction of ketone 1. Strepto-
myces nodosus SC 13149 culture was grown in a 25-L fer-
Reduction of 1 by cell suspensions. Cells of various mi- mentor as described above. After 30 h of growth, 15 g of ke-
croorganisms were suspended separately in 100 mM potas- tone 1 was added to the fermentor, and the biotransformation
sium phosphate buffer (pH 7.0) at 20% (wt/vol, wet cells) cell process was continued for 48 h. The pH was maintained at 6.8
concentration and supplemented with 1 mg/mL of 1 and 30 during the biotransformation process. Periodically, samples
mg/mL of glucose. Reduction was conducted at 28°C and 150 were prepared as described above and analyzed by HPLC to
rpm. Periodically, 1-mL samples were taken and extracted determine the percentage of conversion of 1 to 2a. The di-
with 5 mL of tert-butylmethylether/toluene (60:40). After astereomeric purity and the enantiomeric excess of 2a were
centrifugation, the separated organic phase was collected and determined by HPLC.
dried with a nitrogen stream. The oily residue obtained was
Isolation of alcohol 2a. At the end of single-stage biore-
dissolved in 1 mL of ethanol, filtered through a 0.2 µm LID/X duction, 12 L of the reaction mixture was extracted with 24 L
filter (Whatman Inc., Tewksbury, MA), and analyzed by high- of tert-butylmethylether/toluene (60:40). The separated or-
performance liquid chromatography (HPLC).
ganic phase (20 L) was washed with 10 L of 0.1 M sodium
Two-stage process for reduction of 1. Streptomyces no- chloride, dried over anhydrous sodium sulfate, and evaporated
dosus SC 13149 and Mortierella ramanniana SC 13850 were under reduced pressure to obtain 7.5 g of crude product, which
grown in a 25-L fermentor containing 15 L of medium A con- was recrystallized from ethyl acetate to obtain 6.5 g (62%
taining 0.025% antifoam. Growth consisted of two inoculum overall yield) of white needle crystals of 2a. The diastere-
omeric purity and the enatiomeric excess of the isolated chiral
1
alcohol 2a were >99% and >99.8%, respectively. H NMR
TABLE 1
Stereoselective Microbial Reduction of Ketone 1a
(CDCl3) δ 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). Mass spec-
trum: m/z 302 (M + H)+ (calc. for C15 H22ClNO2, 301).
HPLC analysis of compounds 1, 2a–2d. Analysis of 1 and
2a–2d were carried out using a Hewlett-Packard (Palo Alto,
CA) high-performance liquid chromatograph. A YMC-
PACK-ODS-A column (Whatman Inc.; 100 × 4.5 mm, i.d.
5 µm) was used under the following conditions. Mobile phase
was 10% methanol (solvent A) and 90% methanol (solvent
B) used in a gradient. For the first 25 min the eluant was
100% A; from 25 to 25.2 min the eluant was 25% A and 7%
B; from 25.2 to 30 min the eluant was 100% A.
Reaction Diastereomeric e.e.
yield
purity
of 2a
(%)
Microorganisms
of 2a (%)
of 2a (%)
Streptomyces nodosus SC 13149
Pullularia pullulans SC 13849
Candida boidinii SC 13821
Nocardioides albus SC 13910
Mortierella ramanniana SC 13850
Caldariomyces fumigo SC 13901
a1, (1S)[3-chloro-2-oxo-1-(phenylmethyl)propyl]carbamic acid, 1,1-di-
methylethyl ester; 2a, (1S,2R)[3-chloro-2-hydroxy-1-(phenylmethyl)propyl]
carbamic acid, 1,1-dimethyl ethyl ester; e.e., enantiomeric excess.
56
47
39
5
54
51
>99
81
>99
85
91
93
99.9
99.9
99.9
99.9
99.9
99.9
JAOCS, Vol. 76, no. 11 (1999)

1278
R.N. PATEL
The flow rate was 1 mL/min, and the detection wave-
(ii) D-Amino oxidase from T. variabilis. Trigonopsis vari-
lengths were 224, 250, and 280 nm. A diode array detector abilis ATCC10679 was grown in a 250-L fermentor at 28°C
from Hewlett-Packard was used. The retention times for sub- on medium containing (g/L): KH2PO4, 5; MgSO4·7H2O, 1;
strate 1, compounds 2a, 2c, and 2d were 24, 23.5, and 24.5 CaCl2, 0.5; H3BO3, 0.1; (NH4)2MoO4, 0.04; MnSO4·4H2O,
.
min, respectively. The enantiomeric excess of chiral alcohol 0.04; ZnSO4 7H2O; 0.04; CuSO4·5H2O, 0.045; FeSO4·7H2O,
2a was determined by chiral HPLC. A Bakerbond chiralpak 0.025; DL-methionine, 3; biotin, 0.02; thiamine, 0.1; cysteine,
AD column (100 × 4.5 mm,i.d. 5 m) was used at ambient tem- 0.72; cerelose hydrate, 22. Cell paste (129 g) was harvested
perature; injection volume was 10 µL; mobile phase was 50 h after inoculation and stored frozen. Racemic 6-hydrox-
97.5% hexane/1% cyclohexanol/1.5% ethyl acetate mixture; ynorleucine (0.514 g) in 70 mL of 50 mM potassium phos-
flow rate was 0.8 mL/min; and detection wavelength was 210 phate buffer, pH 7, was shaken with 14 g washed T. variabilis
nm. The retention times for the compounds 2a, 2b, and 2c–2d ATCC 10679 cells for 21 h at 28°C and 200 rpm to give 3
were 14, 15.5, and 21.5 min, respectively.
with e.e. > 99%. Cells were removed by centrifugation, and
Screening of strains for reductive amination of 4. Strains the supernatant was treated with glutamate dehydrogenase,
inoculated from frozen vials were shaken at 220 rpm at 28°C glucose dehydrogenase, NH3, glucose, and NAD as described
for 48 h in 50 mL of medium containing (g/L): -phenylala- above. Compound 3 (0.469 g, 91% yield) was obtained with
L
nine, 10.0; peptone, 10.0; yeast extract, 5.0; K2HPO4, 2.0; >99% e.e. After the completion of the reaction, 3 was isolated
NaCl, 1.0; and MgSO4·7H2O, 0.2. Cells were harvested by as described above. The isolated yield was 0.394 g (76.6%),
centrifugation, washed with 50 mM potassium phosphate and the e.e. was 99%. The 1H NMR spectrum agreed with the
buffer pH 7, and resuspended in 5 mL of the same buffer con- structure.
taining 1 mM dithiothreitol. Cells were sonicated for 2 min,
then centrifuged for 20 min at 101,000 × g. Extracts were as-
sayed spectrophotometrically for reductive amination. The
RESULTS
assay for amino acid dehydrogenase contained, in a volume Enzymatic synthesis of compound 2a. About 100 microorgan-
of 1.0 mL: 0.75 M NH4OH adjusted to pH 8.75 with HCl, 0.4 isms were screened for the stereoselective reduction of 1 to
mM NADH, 25 mg/mL compound 4, and extract. Ab- 2a. The reaction yields, diastereomeric purity, and e.e. of 2a
sorbance change per minute at 340 nm was used to calculate obtained with the best six cultures were as shown in the Table
the activity of the enzyme.
1. Lower diastereomeric purities (<70%) and reaction yields
HPLC analysis of 6-hydroxynorleucine. Analysis of enan- (<20%) were obtained with other cultures. The reaction yield
tiomeric purity and of the amount of 6-hydroxynorleucine and diastereomeric selectivity were dependent upon the mi-
was performed with a Chiralpak WH (Diacel Chemical In- croorganism used during the reduction of 1. Streptomyces no-
dustries, Ltd., Easton, PA) 25 × 0.46 cm column; the mobile dosus SC 13149, Candida boidinni SC 13821, M. ramanni-
phase was 0.3 mM CuSO4; flow rate was 1 mL/min; temper- ana SC 13850, and Caldariomyces fumago SC 13901 gave
ature was 40°C; and detection was at 230 nm.
Enzymatic preparation of 3 from racemic 6-hydroxynor- 99.9% e.e. of product 2a.
leucine. (i) -Amino acid oxidase from porcine kidney. Further research was conducted using S. nodosus SC
>39% reaction yields, >91% diastereomeric purities, and
D
Racemic 6-hydroxynorleucine (0.5 g) in 20 mL of 50 mM 13149 and M. ramanniana SC 13850 to convert ketone 1 to
potassium phosphate buffer (pH 7) was treated with porcine the corresponding chiral alcohol 2a. Cells were grown in a
kidney D-amino acid oxidase (49 U, 350 mg) and beef liver 25-L fermentor for 48 h, collected, and suspended in 100 mM
catalase (182 U, 8 mg) to give a mixture of ketoacid and potassium phosphate buffer pH 6.8; and the resulting cell sus-
0.236 g compound 3 with 97% e.e. The enzymes were added pensions were used to carry out the two-stage process for bio-
in increments, and the reaction took 10 d to complete. The re- transformation of 1 as described in the Materials and Meth-
ductive amination was then carried out as described above. ods section. After 24 h biotransformation, a reaction yield of
6-Hydroxynorleucine (0.484 g) was produced (97% yield) 67%, e.e. of 99.9%, and diastereomeric purity of >99% were
with >99% e.e.
obtained for chiral alcohol 2a using cells of S. nodosus SC
TABLE 2
Stereoselective Microbial Reduction of Ketone 1: Two-Stage Processa
Reaction
time
e.e.
Compound Yield Diastereomeric of 2a
Microorganism
Streptomyces nodosus SC13149
(h)
12
24
2a (g/L)
0.56
0.67
(%)
56
67
purity of 2a (%) (%)
>99
91
99.9
99.9
Mortierella ramanniana SC13850
12
24
0.22
0.54
22
54
aFor abbreviations see Table 1.
JAOCS, Vol. 76, no. 11 (1999)

CHIRAL DRUG INTERMEDIATES BY BIOCATALYSIS
1279
TABLE 3
TABLE 4
Stereoselective Microbial Reduction of Ketone 1: Single-Stage Process
2-Keto-6-hydroxyhexanoic Acid Dehydrogenase in Microbial Strains
Using Streptomyces nodosus SC 13149a
Microbe
Strain
U/mLa
Reaction time
(h)
Compound
2a (g/L)
Yield
2a (%)
Diastereomeric
purity of 2a (%) of 2a (%)
e.e.
Bacillus licheniformis
Bacillus licheniformis
Rhodococcus sp.
Sporosarcina ureae
Sporosarcina ureae
SC12772
SC12148
SC13810
ATCC 6473
ATCC 13888
ATCC 33205
0.0099
0.0368
0.0177
0.0103
0.0613
0.2161
24
48
0.56
0.80
56
80
>99
99.9
aFor abbreviations see Table 1.
Thermoactinomyces intermedius
aCells from a 50-mL culture were assayed as described in the Materials and
Methods section, except that Thermoactinomyces intermedius was grown at
53°C and assayed at 50°C. U, units.
13149. Mortierella ramanniana SC 13850 gave a reaction
yield of 54%, e.e. of 99.9% and diastereomeric purity of 90%
for chiral alcohol 2a (Table 2).
A single-stage fermentation–biotransformation process
was developed for conversion of ketone 1 to chiral alcohol 2a
with cells of S. nodosus SC 13149 as described in the Materi-
als and Methods section. A reaction yield of 80%, dias-
teromeric purity of >99%, and an e.e. of 99.8% were achieved
(Table 3) . From a 12-L reaction mixture, 6.5 g of chiral alco-
hol 2a was isolated as white needle crystals in overall 62%
yield. The diastereomeric purity and the e.e. of the isolated
chiral alcohol were >99% and >99.8%, respectively.
been shown to be a source of thermostable phenylalanine de-
hydrogenase (26) as well as leucine dehydrogenase (27).
Beef liver glutamate dehydrogenase was used for prepara-
tive reactions at 10% total substrate concentration. As de-
picted in Scheme 3, compound 4, sodium salt, in equilibrium
with 2-hydroxytetrahydropyran-2-carboxylic acid, sodium
salt, is converted to 3, and the reaction requires ammonia and
NADH. NAD produced during the reaction was recycled to
NADH by the oxidation of glucose to gluconic acid using glu-
cose dehydrogenase from B. megaterium. The optimal pH for
glutamate dehydrogenase with this substrate was determined
to be about 8.76. We previously reported that glucose dehy-
drogenase had a broad pH optimum centered at about 8.5
(27). The kinetics of the reaction are shown in Figure 1. Re-
action was complete in about 3 h with reaction yields of
89–92%, and e.e. was >99%.
Enzymatic synthesis of 3. A variety of ketoacids can be
converted to
L-amino acids by treatment with a suitable
amino acid dehydrogenase. Initial screening using HPLC
analysis, with formate dehydrogenase for regeneration of
NADH, showed that phenylalanine dehydrogenase from
Sporosarcina sp. and beef liver glutamate dehydrogenase
converted 0.1 M compound 4, sodium salt mixture, com-
pletely to 3. Additional screening of amino acid dehydroge-
nases with spectrophotometric enzyme assays using 4,
sodium salt as substrate showed that glutamate dehydroge-
nases from Candida utilis and Proteus sp. were active using
NADPH but not NADH. Leucine dehydrogenase partially pu-
rified from B. sphaericus ATCC 4525 (25) and alanine dehy-
drogenase from B. subtilis were not active. Of 132 cultures
screened, an extract from Thermoactinomyces intermedius
ATCC 33205 was the most active (Table 4). This strain has
Chemical synthesis and isolation of 4 required several
steps. In a second, more convenient process (Scheme 4), the
ketoacid was prepared by treatment of racemic 6-hydroxy-
norleucine (produced by hydrolysis of 5) hydantoin] with
D
-amino acid oxidase and catalase. After the e.e. of the
remaining 3 increased to >99%, the reductive amination
procedure was used to convert the mixture containing 4 and
3 entirely to 3 with yields of 91 to 97% and e.e. of >99%.
3
SCHEME 3
JAOCS, Vol. 76, no. 11 (1999)

1280
R.N. PATEL
FIG. 1. Biotransformation of 2-keto-6-hydroxyhexanoic acid to L-6-hydroxynorleucine using
beef liver glutamate dehydrogenase. NADH was recycled using glucose dehydrogenase. At
3 h, the yield was 92% and the enantiomeric excess was >99%.
Sigma porcine kidney
D-amino acid oxidase and beef from Sporosarcina sp. or Th. intermedius extract was also
liver catalase or T. variabilis whole cells (source of oxi- effective. Either formate dehydrogenase or glucose dehydro-
dase and catalase) were used successfully for this transfor- genase was useful for NADH regeneration. Preparation of
mation.
Compound 3 was readily prepared from the correspond- found in which racemic 6-hydroxynorleucine was pre-pared
ing ketoacid by reductive amination using beef liver by hydrolysis of 5. Conversion of the -enantiomer to
-amino acid oxidase followed by
the ketoacid required several steps, but a shorter route was
D
glutamate dehydrogenase. Phenylalanine dehydrogenase the ketoacid using
D
SCHEME 4
JAOCS, Vol. 76, no. 11 (1999)

CHIRAL DRUG INTERMEDIATES BY BIOCATALYSIS
1281
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to 3.
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[Received July 1, 1999; accepted September 15, 1999]
JAOCS, Vol. 76, no. 11 (1999)