Effect of different strains of yeast on stereocontrolled reduction of 5-acetylisoxazolines

Article abstract of DOI:10.1016/S0045-2068(02)00500-X

The stereocontrolled reduction of 3-aryl-5-acetylisoxazolines (1) to the corresponding alcohols (2 and 3) in the presence of four different yeast strains, recognized as Baker's yeast (commercial), Candida krusei (ATCC 14243), Pichia farinosa (NRRL Y110) and Sacchromyces sp. (soil isolate) have been attempted. The C. krusei was found to be diastereoselective for the (R)-1 while the Sacchromyces sp. led to complete reduction to yield the RS- and SS-alcohol in 1:1 ratio at 10 g/L scale.

Full text of DOI:10.1016/S0045-2068(02)00500-X

BIOORGANIC
CHEMISTRY
Bioorganic Chemistry 30 (2002) 350–355
www.elsevier.com/locate/bioorg
Effect of different strains of yeast on
stereocontrolled reduction of
5-acetylisoxazolinesq
M.K. Tripathi,a U.K. Jinwal,a U. Roy,b A. Patra,c
P.K. Roy,a,1 S. Batra,c,* and A.P. Bhaduric
a
Fermentation Technology Division, Central Drug Research Institute, P.O. Box-173,
Lucknow 226 001, India
b
Biochemistry Division, Central Drug Research Institute, P.O. Box-173,
Lucknow 226 001, India
c
Medicinal Chemistry Division, Central Drug Research Institute, P.O. Box-173,
Lucknow 226 001, India
Received 3 April 2002
Abstract
The stereocontrolled reduction of 3-aryl-5-acetylisoxazolines (1) to the corre-
sponding alcohols (2 and 3) in the presence of four different yeast strains, recog-
nized as BakerÕs yeast (commercial), Candida krusei (ATCC 14243), Pichia farinosa
(NRRL Y110) and Sacchromyces sp. (soil isolate) have been attempted. The
C. krusei was found to be diastereoselective for the (R)-1 while the Sacchromyces
sp. led to complete reduction to yield the RS- and SS-alcohol in 1:1 ratio at
10 g/L scale.
Ó 2002 Elsevier Science (USA). All rights reserved.
q CDRI Communication No. 6192.
* Corresponding author. Fax: +91-522-223405, 229338.
E-mail addresses: prodyutr@yahoo.com (P.K. Roy), batra_san@yahoo.co.uk (S. Batra).
1
Also corresponding author.
0045-2068/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved.
PII: S0045-2068(02)00500-X

M.K. Tripathi et al. / Bioorganic Chemistry 30 (2002) 350–355
1. Introduction
351
The synthesis of optically pure 2-isoxazolines has been the focus of research for
various workers since these heterocycles serve as useful intermediate for the synthesis
of different classes of functionalized acyclic compounds and natural products [2–11].
Both enzymatic and chemical methods have been reported to obtain such isoxazoline
derivatives. The chemical methods in general utilize the chiral alkenes during the
intermolecular or intramolecular 1,3-dipolar cycloaddition of nitrile oxide [3,8,9,12–
15] while the enzymatic method [16,17] involves the stereocontrolled reduction of
isoxazolines derivatives in the presence of BakerÕs yeast. In our search on spermicidal
agents [18], it was desired to obtain these 2-isoxazolines in enantiomerically pure
form so as to carry out further transformations to obtain the chiral analogs. It has
been reported earlier [17] that the stereocontrolled reduction of 5-acetylisoxazolines
with bakerÕs yeast in 2-propanol/water mixture leads to alcohol (S,S)-2 and ketone
(R)-1 in 98% ee with no traces of anti isomer (R,S)-3 [16]. Unfortunately in our
hands, a similar reaction condition led to the recovery of all the substrate unreacted.
Therefore, it was decided to examine some of the strains of yeast available in our lab,
which could result in the desired transformation in stereocontrolled manner. The
details of our results are presented here.
2. Materials and methods
Culture. BakerÕs yeast (Commercial), Candida krusei (ATCC 14243), Pichia
farinosa (NRRL Y110) and Sacchromyces sp. (soil isolate) were maintained on a
medium containing potato infusion, 20%; dextrose, 2%; agar, 1.5%; and racemic
5-acetyl-3-substituted phenyl-2-isoxazoline, 0.1%. The cultures used in this study
are deposited in culture collection of the institute.
Growth of organisms and preparation of cells for biotransformation. The yeast(s)
were grown in medium containing yeast extract, 0.25%; glucose, 1.0%; peptone,
0.25%; KH2PO4, 1.0%; (NH4)2HPO4, 2.0%; MgSO4 ꢀ 7H2O, 0.025%; substrate, 0.1%,
for 36 h at 28 °C. The cell suspension obtained was used as an inoculum (10%) for
further cultivation under identical cultural conditions. The cells obtained after 5-
sequential transfers were used as inoculum (10%) for the final cultivation. After 24 h
of final growth, cells were harvested by centrifugation, washed with 10 mM phos-
phate buffer (pH 7.0). The washed cells (50 g) were suspended in 100 ml of reac-
tion buffer [50 mM phosphate buffer (pH 7.0) containing (NH4)2HPO4, 0.2%;
MgSO4.7H2O, 0.025%] in a 1 L Erlenmeyer flask and incubated at 37 °C for 8 h
under reciprocal agitation with an agitation speed of 50 strokes/min, prior to the
addition of substrate for reduction.
Reduction. The biotransformation was initiated by the addition of solution of
appropriate compound from 1a–b (1 g) in DMF (2 ml) to the preincubated yeast
cells. The mixture was incubated at 37 °C for 18 h under reciprocal shaking at
240 rpm. Thereafter the reaction broth was extracted with ethyl acetate (4 ꢁ 200 ml),
the organic layers were combined and washed with brine (400 ml). The organic layer
was dried over anhydrous sodium sulfate and concentrated under vacuo to obtain an

352
M.K. Tripathi et al. / Bioorganic Chemistry 30 (2002) 350–355
oily residue, which was subjected to chromatography over silica gel (230–400 mesh).
Elution with hexanes:ethyl acetate (60:40, v/v) furnished the RS-alcohol (3) first
followed by SS-alcohol (2) as white solids.
Oxidation. To the stirred solution of appropriate compound from 2 (a–b) or 3
(a–b) (0.05 mmol) in dry DCM (10 ml) was added pyridinium chlorochromate
(0.075 mmol) at room temperature. The reaction was further continued for 18 h and
on completion was filtered through a small band of florisil using chloroform:ethyl
acetate (95:5, v/v). Evaporation of solvent under vacuo furnished the corresponding
ketones as white solids.
Analysis. Melting points were determined on hot stage apparatus and are un-
corrected. IR spectra were recorded on Perkin-Elmer 881 spectrophotometer.
1HNMR were measured on Bruker 300/200 FTNMR while EI mass spectra were
recorded on a JEOL-D-300 spectrometer. The ESMS spectra were recorded on
Merck 8000 LC/ 3DQMS system through flow injections. Elemental analysis were
carried out on Carlo–Erba EA1108 analyzer and ½aŠ were recorded on RudolphÕs
d
Autopol III. The ee were calculated by making the MTPA ester as reported earlier
[15].
3. Results and discussion
The 5-acetyl-4,5-dihydro-3-substituted phenyl-isoxazolines were obtained by 1,3-
dipolar cycloadditions of a nitrile oxide on methyl vinyl ketone as described earlier
[18]. The racemic 5-acetyl-isoxazolines (1a–b) were then subjected to enzymatic re-
duction in the presence of four different strains of yeast viz. BakerÕs yeast, C. krusei,
P. farinosa and Sacchromyces sp. It was decided to use the induced resting cells of
yeast available because we have observed that adaptation of the liquid-culture to the
substrate by sequential transfers increase the substrate-tolerance and provides high
yields of desired transformation. This is possibly due to the enhancement of the
cellular pool of the desired enzyme(s). The asymmetric reduction of wide spectrum of
ketones has been possible with the easy-to-use method of resting cells [19–23]. To
overcome problems in using free enzymes such as denaturation, replinshment of
cofactors, we also decided to use the whole cells since they offer advantage of co-
factors recycling in vivo and eliminate the expensive enzyme purification. Except for
the BakerÕs yeast, which was purchased from the market all other strains used for
reactions were maintained in our Fermentation division. The Sacchromyces sp. that
is the soil isolate is being regularly employed for the fermentation of molasses to
produce alcohol in our division. It was observed that all the yeast species reduced the
ketone in a stereospecific manner to the corresponding S-alcohol thereby affording
two diastereomers as SS-(2) and RS-(3) alcohols (Scheme 1). The percent conversion
and the relative ratio of the SS- and RS-alcohol are shown in Table 1.
Among all yeasts, Sacchromyces sp. showed maximum conversion as no ketone
was recovered unreacted. Both the diastereomers of alcohol could be easily separated
on silica gel using ethyl acetate:hexanes (40:60, v/v) mixture as eluent. The ratio of
both diastereomers was found to be 1:1 and the optical activity of both alcohols
matched the literature values suggesting that this enzyme though reduced the ketone

M.K. Tripathi et al. / Bioorganic Chemistry 30 (2002) 350–355
353
Scheme 1.
Table 1
Resultsa of the various strains of yeast used for reduction
Yeast
strains
Substrate Ketone
level recovered
(g/100 ml) (g)b
Total yield of Conversion Relative
recovered (g) (%) ratio of
diastereomers
(syn:anti)
BakerÕs yeast
1
0:5615 Æ 0:0662 0:3486 Æ 0:0709 35
1:1
(mixture)
No ketone
detected
(mixture)
0:8564 Æ 0:0593 86
(mixture)
Sacchromyces sp 1
1:1
C. krusei
1
1
0:4175 Æ 0:0425 0:4326 Æ 0:0253 44
(R) (SS)
0:8129 Æ 0:0275 0:1238 Æ 0:0308 12
(mixture) (mixture)
1:0.005
3:2
P. farinosa
a Values are mean Æ SD of five different experiments.
b Mixture here refers to diastereomeric mixture of (R) and (S)-ketones.
in an enantiospecific manner to yield only S-alcohol, was not diastereoselective. It
was also observed that the rate of reaction varied from batch to batch of cells used,
even though the cells were grown under identical conditions. This may be attributed
to the fact that in growing cells, different enzymes are induced and often compete for
the substrate and many reactions occur at the same time often giving mixture of
products derived from different catalytic capabilities [24–27]. In contrast, the re-
duction with C. krusei was found to be diastereoselective for S isomer leading to the
formation of alcohol (S,S)-2 and ketone (R)-1; with only minor contamination of
alcohol (R,S)-3. In reactions with this enzyme, no significant batch-to-batch varia-
tion was observed. We examined the bakerÕs yeast from five different sources (five
different market brands) and found that the best yield achieved was around 35% but
the reaction was not stereoselective since alcohols (S,S)-2 and (R,S)-3 were obtained
in 1:1 ratio after column chromatography. The recovered starting substrate was
devoid of any optical activity confirming that the enzyme was not stereoselective. In
this case it was assumed that the ability of bakerÕs yeast to carry out reduction is
dependent on the fermentative activity of the yeast cells, which may decrease as a
result of gradual inactivation of enzymes and regulatory proteins during the storage

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M.K. Tripathi et al. / Bioorganic Chemistry 30 (2002) 350–355
[28]. The most unsatisfactory results were obtained during the reactions with P.
farinosa since the amount of reduction was less and also no selectivity was observed.
These alcohols were further oxidized in the presence of pyridinium chlorochromate
to furnish the pure (R)- and (S)-5-acetyl-3-substituted phenyl-isoxazolines.
In conclusion, we have described the effect of various yeasts strain towards the
stereocontrolled reduction of 5-acetyl-3-substituted phenyl-2-isoxazolines to obtain
the optically pure alcohols, which can be oxidized back to afford the enantiomeri-
cally pure isoxazolines. During our study we have found that C. krusei can also effect
stereocontrolled reduction at very high substrate ratio with no special reaction
conditions involved.
Acknowledgments
Two of the authors (MKT and UKJ) are grateful to CSIR, New Delhi while ADP
is grateful to Volksvagen, Germany for financial support in the form of fellowships.
References
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(1987) 529.
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Bioorg. Med. Chem. 7 (1999) 2607.
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Biotech. Bioengg. 35 (1990) 559.

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24
[29] Data of compounds: (R)-1a m. p. 54–56 °C, ½aŠ ¼ À198 (c ¼ 1:5, CHCl3); (S)-1a m. p.
D
24
52–53 °C, ½aŠ ¼ þ197 (c ¼ 1:5, CHCl3); 2a (1S)-1-[(5S) 4,5-dihydro-3-phenyl-5-isoxaz-
D
24
D
olyl] ethanol m. p. 82–84 °C, ½aŠ ¼ þ165 (c ¼ 2:0, CHCl3), IR (KBr): 3365 cmÀ1
,
1H
NMR (300 MHz, CDCl3) d 1.29 (d, 3H, J ¼ 6:6 Hz), 2.18 (brs, lH), 3.18 (dd, lH, J ¼ 7:8,
16.6 Hz), 3.40 (dd, lH, J ¼ 10:8, 16.6 Hz), 3.79 (brs, lH), 4.59 (ddd, lH, J ¼ 4:8, 7.5,
10.8 Hz), 7.41 (m, 3H), 7.67 (m, 2H), 13C NMR (75 MHz, CDCl3): d 19.37, 37.44, 69.60,
85.14, 127.10, 129.11, 129.70, 130.60, 157.40 ppm, MS (EI): 191 (35.0), 146 (30.6), 104
(100); 3a (1S)-1-[(5R)4,5-dihydro-3-phenyl-5-isoxazolyl] ethanol m. p. 102–104 °C,
24
D
½aŠ ¼ À145 (c ¼ 2:0, CHCl3), IR (KBr): 3504 cmÀ1
,
1H NMR (300 MHz, CDCl3) d
1.22 (d, 3H, J ¼ 6:6 Hz), 2.03 (brs, lH), 3.23 (dd, lH, J ¼ 10:9, 16.6 Hz), 3.40 (dd, IH,
J ¼ 8:7, 16.6 Hz), 4.13 (brs, IH), 4.66 (ddd, IH, J ¼ 5:1, 7.8, 10.8 Hz), 7.41 (m, 3H), 7.67
(m, 2H), 13C NMR (75 MHz, CDCl3): d 18.51, 34.65, 67.23, 85.57, 127.11, 129.08, 130.53,
157.16 ppm, MS (EI): 191 (37.0), 146 (30.8), 104 (100). (R)-1b m. p. 102–104 °C,
24
24
D
½aŠ ¼ À188 (c ¼ 0:5, CHCl3); (S)-1b m. p. 94–95 °C, ½aŠ ¼ þ189 (c ¼ 0:5; CHCl3); 2b
D
(1S)-1-[(5S)4,5-dihydro-3-(4-methyl-phenyl)-5-isoxazolyl] ethanol m. p. 114–116 °C,
24
½aŠ ¼ þ158 (c ¼ 0:7, CHCl3), IR (KBr): 3384 cm-1, 1H NMR (200 MHz, CDCl3) d
D
1.28 (d, 3H, J ¼ 6:4 Hz), 2.17 (d, 1H, J ¼ 5:6 Hz), 2.37 (s, 3H), 3.14 (dd, 1H, J ¼ 7:5,
16.8 Hz), 3.40 (dd, 1H, J ¼ 10:6, 16.5 Hz), 3.77 (m, 1H), 4.56 (ddd, 1H, J ¼ 3:1, 7.8,
11.2 Hz), 7.21 (d, 2H, J ¼ 8:0 Hz), 7.55 (d, 2H, J ¼ 8:0 Hz), MS (ESMS): 206.20 (Mþ +1);
3b (1S)-1-[(5R)4,5-dihydro-3-(4-methyl-phenyl)-5-isoxazolyl] ethanol m. p. 102–104 °C,
24
½aŠ ¼ À138 (c ¼ 0:9, CHCl3), IR (KBr): 3420 cmÀ1, 1H NMR (200 MHz, CDCl3) d 1.22
D
(d, 3H, J ¼ 6:5 Hz), 1.94 (d, 1H, J ¼ 5:5 Hz), 2.37 (s, 3H), 3.21 (dd, 1H, J ¼ 10:7,
16.6 Hz), 3.39 (dd, 1H, J ¼ 8:7, 16.6 Hz), 4.13 (m, 1H), 4.64 (ddd, 1H, J ¼ 3:2, 7.8,
11.1 Hz), 7.21 (m, 3H), 7.56 (m, 2H), MS (ESMSI): 206.33 (Mþ þ 1).