Enzyme directed diastereoselectivity in chemical reductions: Studies towards the preparation of all four isomers of 1-phenyl-1,3-butanediol

Article abstract of DOI:10.1016/j.tetasy.2004.04.022

Enzymes play an important role in guiding the diastereoselectivity of the final products during the chemical reduction of the intermediates (R)- and (S)-3-hydroxy-1-phenyl-1-butanone, prepared by bioreduction of 1-phenyl-1,3-butadione. For example, the pr

Full text of DOI:10.1016/j.tetasy.2004.04.022

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 15 (2004) 1685–1692
Enzyme directed diastereoselectivity in chemical reductions:
studies towards the preparation of all four isomers of
1-phenyl-1,3-butanediol
K. Ahmad, S. Koul, S. C. Taneja,* A. P. Singh, M. Kapoor, Riyaz-ul-Hassan, V. Verma
and G. N. Qazi
Biotechnology Division Regional Research Laboratory (CSIR), Jammu Tawi 180001, India
Received 9 February 2004; accepted 13 April 2004
Abstract—Enzymes play an important role in guiding the diastereoselectivity of the final products during the chemical reduction of
the intermediates (R)- and (S)-3-hydroxy-1-phenyl-1-butanone, prepared by bioreduction of 1-phenyl-1,3-butadione. For example,
the presence of an oxidoreductase such as Pichia capsulata during a one pot, two step, enzymo-chemical reduction of 1-phenyl-1,3-
butadione produced corresponding (1R,3S)-1-phenyl-1,3-butanediol (de ꢀ 98%), similarly Zygosaccharomyces rouxii furnished
(1S,3R)-1-phenyl-1,3-butanediol (de ꢀ 98%). On the other hand chemical reduction of (R)- and (S)-3-hydroxy-1-phenyl-1-butanone
after the exclusion of the enzymes resulted in complete loss of diastereoselectivity, leading to the formation of all four isomers of
1-phenyl-1,3-butanediol. Moreover the yields of the final products are higher in the one-pot transformations than in the two step
sequential reactions.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
stereoselectivity during the formation of diastereoiso-
mers of 1-phenyl-1,3-butanediol. Both acyclic and cyclic
1,3-diols are of much interest to synthetic chemists since
these molecules form the basic skeleton of natural
products such as polyene and polyol macrolide antibio-
tics, for example, Compactin, Rifamycin2a and Lono-
mycin A, etc.2b They are also the precursors in the
synthesis of some important natural products such as
pheromones. Thus protection of diols followed by con-
version to chiral hydroxyketones as intermediates, has
been found to be of wide application in the preparation
of natural products.3–7
Stereoselective transformations using biocatalysts1a are
now regularly exploited in the preparation of chiral
synthons in natural products synthesis. Use of inert
solid supports such as silica gel and alumina to improve
reactivity and yield in the reduction of keto functions to
the corresponding secondary alcohols by chemical as
well as enzymatic methods has been a much studied
reaction.1b The presence of a protein as a guide to regio-
selective reduction is a comparatively recent develop-
ment. Chemoreduction in the presence of BSA (Bovine
serum albumin) for improving regioselectivity particu-
larly of natural products such as sterols has been
reported.1c On the other hand the influence of crude
enzymes on the diastereoselectivity profile of chemical
reactions such as reductions has not been explored.
Herein we describe results obtained during one-pot two
step enzymo-chemical reduction reactions of the
important substrate 1-phenyl-1,3-butadione 1. The
results of these transformations unambiguously demon-
strate that crude enzymes play an important role in the
A versatile method of preparing 1,3-diols is by reduction
of b-hydroxyketones, synthesized from corresponding
silyl enol ethers and aldehydes through Mukaiyama
aldol reaction.8 There are a number of methods
described in the literature for the preparation of syn-1,3-
diols9 while the Tishchenko reduction10 produces anti-
diastereoisomers.
2. Results and discussion
In our attempts to obtain all the four isomers of enan-
tiomerically enriched 1-phenyl-1,3-butanediols we
* Corresponding author. Tel.: +91-191-2580329; fax: +91-191-25486-
07/2543829; e-mail: sc_taneja@yahoo.co.in
0957-4166/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.04.022

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K. Ahmad et al. / Tetrahedron: Asymmetry 15 (2004) 1685–1692
envisaged to initially select a suitable biocatalyst for the
preparation of intermediate 3-hydroxy-1-phenyl-1-
butanone in enantiomerically pure form. Consequently,
screening of several oxidoreductases from our culture
collection as well as commercial sources was under-
taken. In this study, 15 enzyme strains in the families of
yeasts, bacilli and fungi were shortlisted by preliminary
screening of reductase activity and among them nine
were found to be effective in reducing the substrate 1. In
the first step, as reported in literature,1b;11;12 bioreduction
of 1-phenyl-1,3-butadione with Saccharomyces cerevi-
siae furnished a monohydroxy ketone (+)-2 as the
product, comprising predominantly the (S)-enantiomer
of high enantiopurity, while Geotrichum candidum and
Aspergillus niger are reported to furnish the (R)-enan-
tiomer.13 In our experiments, which include yeast, bacilli
and fungi, bioreduction was carried out using resting
cells and a bubbler was used to maintain anaerobic
conditions at 30 °C. In all the bioreduction reactions of
the substrate 1, a single product (3R)- or (3S)-hydroxy-
1-phenyl-1-butanone (Scheme 1) with varying degree of
enantiomeric excess was obtained. In these transforma-
tions, the 3-oxo functional group adjacent to terminal
methyl was stereoselectively reduced by the biocatalysts
in moderate to excellent ee’s leaving the other keto
group at C-1 intact. Bacillus pseudomegatherium (RRL
Acc. No: RRLB 008), Candida crusei (RRLY 003), Pi-
chia capsulata (RRLY 002), Physarium policephalum
(RRLF 005) and S. cerevisiae (RRLY 007) furnished
(S)-2, with the best selectivity being displayed by P.
capsulata (ee ꢀ 99%) followed by Candida crusei
(ee ꢀ 94%). Bioreductions using Arthrobacter crystallo-
poietis (RRLB 281), Pichia farinosa (RRLY 004), Pichia
pseudopastoris (RRLY 001) and Zygosaccharomyces
rouxii (RRLY 006), furnished predominantly (R)-2 and
Z. rouxii furnished practically an enantiopure product
(ee ꢀ 99%) followed by Arthrobacter crystallopoietis
(ee ꢀ 83%). Surprisingly in all the bioreductions only the
3-oxo functional group was reduced, displaying com-
plete regioselectivity and even after prolonged reaction
time (14 days) there was no formation of the dihydroxy
product that is, 1-phenyl-1,3-butanediol. The results of
these experiments are summarized in Table 1.
The data in Table 1 clearly demonstrates the efficacy of
Z. rouxii and P. capsulata, displaying excellent regio-
selectivity to produce enantiomers ())-2 and (+)-2 that is
(R)-(ꢁ)- and (S)-(þ)-3-hydroxy-1-phenyl-1-butanone,
respectively, in high enantiomeric excess and chemical
yields. After the completion of the first step of biocat-
alytic reductions, addition of a chemical reducing agent
that is, sodium borohydride or sodium cyanoborohy-
dride in the same reaction pot comprising the biocatalyst
in aqueous medium at 5 °C, resulted in smooth trans-
formation to optically active 1-phenyl-1,3-butanediols
())-4 and (+)-4, respectively, in high yields. Scheme 2
represents the best results obtained in one-pot enzymo-
chemical reduction reaction using yeast strains namely
P. capsulata and Z. rouxii.
The diastereoisomeric ratio (de) of syn- and anti-1-phe-
nyl-1,3-butanediols as determined by proton NMR and
total yields of the final products are shown in Table 2.14
The results demonstrate that the one-pot sodium boro-
hydride reduction in the presence of biocatalysts such as
P. capsulata and Z. rouxii, displayed improved diaste-
reoselectivity, resulting in the formation of anti products
(de ꢀ 98%) while sodium cyanoborohydride displayed
comparatively lower diastereoselectivity (de ꢀ 66–76%).
The possibility of any influence exerted by the presence
of glucose on the diastereomeric ratio was ruled out
when experiments were run with biocatalyst with or
without using 5% glucose in which the de remained
unaffected.
O
OH
O
OH
O
O
Biocatalyst
Biocatalyst
(S)-(+)-2
(R)-(-)-2
1
Scheme 1.
Table 1. Bioreduction of 1-phenyl-1,3-butanedione 1 with micro-organisms
25
½aꢂ CHCl3
Entry
Micro-organism
Yield (%)
Ee (%)a
Reaction time
(h)
Absolute
configurationb
D
1
2
3
4
5
6
7
8
9
Bacillus pseudomegatherium
Candida crusei
50
82
85
60
80
75
70
80
85
46
94
99
53
77
83
67
78
99
72
48
18
72
72
48
48
48
40
S
S
S
S
+31.5
+64
P. capsulata
Physarium policephalum
S. cerevisiae
+67.5
+36
S
R
R
R
R
+48.5
)56.4
)45.5
)49
Arthrobacter crystallopoietis
Pichia farinosa
Pichia-pseudopastoris
Zygosaccharomyces rouxii
)67.5
a The ees were determined by Chiral HPLC (Chiralcel OD-H, mobile phase; hexane–isopropanol ¼ 95:5) analyzed after isolation of the bioproducts
by column chromatography. Retention times of (R)- and (S)-enantiomers are 15 and 16.5 min, respectively.
b The absolute configurations were assigned by comparison of the sign of the specific rotation with that reported in the literature.1b;11

K. Ahmad et al. / Tetrahedron: Asymmetry 15 (2004) 1685–1692
1687
OH
OH
OH
OH
O
O
(i)
Pichia-
capsulata
(i) Zygosaccharo-
myces rouxii
(ii) NaBH4
(ii) NaBH4
(+)-4, ee=99%
1
(-)-4, ee=99%
Scheme 2.
Table 2. One-pot enzymo-chemical reduction of 1 to (ꢁ)-4 and (þ)-4
Micro-organism
Reaction time for the
Ee (%) (conf.) of
syn–antic
anti-Isomer ee%
(conf.)d
syn-Isomer ee%
(conf.)d
Total yield
(%)
formation of (ꢁ)-2/(þ)-2
(ꢁ)-2/(þ)-2
P. capsulata
P. capsulata
P. capsulata
P. capsulata
Z. rouxii
24
24
30
30
40
40
60
60
60
72
99 (S)a
99 (S)b
99 (S)a
99 (S)b
99 (R)a
99 (R)b
77 (S)a
77 (S)b
77 (S)b
47 (S)b
1:99
1:99
99 (1R,3S)
99 (1R,3S)
99 (1R,3S)
99 (1R,3S)
99 (1S,3R)
99 (1S,3R)
75 (1R,3S)
75 (1R,3S)
71 (1R,3S)
41 (1R,3S)
ND
ND
70
68
67
65
75
72
60
60
62
55
12:88
13:87
1:99
99 (1S,3S)
99 (1S,3S)
ND
Z. rouxii
1:99
ND
S. cerevisiae
S. cerevisiae
S. cerevisiae
B. gatherium
15:85
17:83
20:80
1:99
75 (1S,3S)
75 (1S,3S)
73 (1S,3S)
ND
ND––not determined.
aNaBH4/NaCNBH3 was used in presence of enzyme without nutrient.
bNaBH4/NaCNBH3 was used in presence of enzyme and 5% glucose solution.
c syn–anti Ratio determined by proton NMR.
d Ee% determined by Chiral HPLC. The ee% of (ꢁ)-2 and (þ)-2 was determined by analyzing the aliquots after the completion of the reaction as
monitored by TLC.
To assess the role of the biocatalysts in influencing the
diastereoselectivity in the formation of 1,3-diols,
chemoreduction of bioreduced products (ꢁ)-2 and (þ)-
2, purified by column chromatography was carried out
in the absence of biocatalysts. The reducing agents used
are lithium aluminium hydride in dry THF/diethyl ether
at 0–5 °C (Table 3, entries 1–3), sodium borohydride
(Table 3, entries 4–7), sodium cyanoborohydride (Table
3, entries 8–11) (with or without the presence of glucose)
(Scheme 3).
be separated by column chromatography. However,
reduction with LAH in dry diethyl ether showed some
improvement in the diastereoselectivity, resulting in the
formation of syn- and anti-diols in 73:27 ratio (Table 3).
From above studies it appears that the presence of the
biocatalyst in the reaction medium does play an
important role in guiding the diastereoselectivity of the
reduced products during chemical reductions. Whether
the crude enzyme acts as a solid support or matrix
during the transformations or the chemical reducing
agent forms a weak complex with the biocatalyst is not
clear. It may also be possible that the cell surface and
not the enzyme (protein) helps in modifying the diastereo-
selectivity of the final product. To rule out the possibility
The reduction of (ꢁ)-2 and (þ)-2 with sodium borohy-
dride and sodium cyanoborohydride afforded almost
equimolar ratio of enantiomerically pure syn- and anti-
diols (42:58) and (50:50), respectively, which could easily
Table 3. Chemical reduction of 3-hydroxy-1-phenyl-1-butanone (ꢁ)-2 and (þ)-2 after eliminating the enzyme
Entry
Substrate
Reagent
Solvent
Product ratio
syn–antib
Conf.
syn diol–anti diol
Total yield (%)
1
(ꢁ)-2
(ꢁ)-2
(þ)-2
(ꢁ)-2
(ꢁ)-2
(ꢁ)-2
(þ)-2
(ꢁ)-2
(ꢁ)-2
(þ)-2
(þ)-2
LiAlH4
LiAlH4
Ethera
THFa
Ethera
MeOHa
H2Oc
73:27
74:26
72:28
42:58
50:50
45:55
50:50
50:50
30:70
28:72
50:50
(1R,3R):(1S,3R)
(1R,3R):(1S,3R)
(1S,3S):(1R,3S)
(1R,3R):(1S,3R)
(1R,3R):(1S,3R)
(1R,3R):(1S,3R)
(1S,3S):(1R,3S)
(1R,3R):(1S,3R)
(1R,3R):(1S,3R)
(1S,3S):(1R,3S)
(1S,3S):(1R,3S)
90
86
87
85
85
75
85
86
78
75
82
2
3
LiAlH4
NaBH4
4
5
NaBH4
NaBH4
NaBH4
6
H2Od
7
H2Oc
MeOHa
H2Oc
H2Oc
MeOHa
8
NaCNBH3
NaCNBH3
NaCNBH3
NaCNBH3
9
10
11
a Reactions performed at 0–5 °C.
b syn–anti Ratio was determined by 1H NMR.
c Reaction performed at room temp (20–25 °C).
d Reaction performed at room temp in 5% glucose solution.

1688
K. Ahmad et al. / Tetrahedron: Asymmetry 15 (2004) 1685–1692
OH OH
OH
OH
OH
O
chemical-
reduction
+
(-)-4
ee 99%
(-)-2
ee 99%
(+)-5
ee 99%
O
OH
OH
OH
OH
OH
chemical-
reduction
+
(+)4
ee 99%
(+)-2
ee 99%
(-)-5
ee 99%
Scheme 3.
of the cell surface playing any role we also carried out the
one-pot chemical reductions of (ꢁ)-2 and (þ)-2 with
sodium borohydride in the presence of a cell free extract
of biocatalysts. The final results in these experiments
matched with those obtained in presence of whole cells
(Table 2) as again formation of predominantly anti
products was observed. These experiments clearly dem-
onstrate that it is possible to carry out one-pot sequential
transformations advantageously without the need to iso-
late the intermediates or sacrifice overall yields of the final
products.
(+)-4
ee 99%
(i) Saccharomyces
cerevisiae(96 hrs)
Pichia capsulata(24 hrs)
(ii)
OH
O
(-)-5
ee 99%
(-)-4
(+)-6
ee 99%
Zygosaccharo-
myces rouxii (48 hrs )
(+)-5
ee 99%
The specific rotation values and the enantiomeric excess
of all the four diastereomers thus prepared along with
the reported ones in literature15;16 are given in the Table
4. It should be emphasized here that the present strategy
of one-pot enzymo-chemical reductions to prepare the
enantiomerically enriched syn- and anti-1-phenyl-1,3-
butanediols seems to be better than those reported ear-
lier so far as the yields and enantiopurity of (ꢁ)-4 and
(þ)-4 are concerned.
Scheme 4.
tiomeric excess (ee ꢀ 99%) though the isolated yields
were low (45–50%).
Bioreduction of (ꢃ)-6 using Z. rouxii was also carried
out in a diastereospecific manner though with reversal of
selectivity, thereby giving other pair of enantiomerically
pure diastereoisomers (ꢁ)-4 and (þ)-5 in good isolated
yields (ꢀ70%). The results of these experiments are
summarized in Table 5.
As our attempts to reduce both the keto groups of the
substrate 1 by successive bioreductions using a single
biocatalyst to prepare all of the possible enantiopure
isomers of 1-phenyl-1,3-diols through the intermediates
(ꢁ)-2 and (þ)-2 did not succeed, we therefore envisaged
bioreduction of another intermediate that is, (ꢃ)-4-
hydroxy-4-phenyl-butan-2-one 6 (prepared by catalytic
hydrogenation of 1 in 84% yield) as an alternative route
to the formation of enantiopure 1,3-diols. Thus (ꢃ)-6
was subjected to bioreduction (Scheme 4) using P. cap-
sulata and S. cerevisiae, and, as expected, the biocata-
lytic reduction was facile with P. capsulata affording a
high enantiomeric excess (99%) and isolated yields (75%)
with an equal ratio of both the diastereoisomers (þ)-4
and (ꢁ)-5. S. cerevisiae on the other hand afforded
predominantly the anti-diastereoisomer of high enan-
3. Conclusion
All the four diastereoisomers of 1-phenyl-1,3-butanedi-
ols were successfully prepared from corresponding
1-phenyl-1,3-butadione, applying chemo-enzymatic and
enzymo-chemical methodologies using a number of
reductases, best among them are Z. rouxii, S. cerevisiae
and P. capsulata. It was also established conclusively
that the presence of enzymes in the reaction media
imparts an important role in influencing the diastereo-
selectivity of the products during one-pot enzymo-
chemical transformations. Yields of the final products
Table 4. Specific rotation values of isolated bioproducts (diols)
25
D
25ꢄ
D
Entry
Compound
Conf.
Ee (%)
½aꢂ (Concn)
Ee (%) lit.14;15
½aꢂ (Concn)
1
2
3
4
(ꢁ)-4
(þ)-4
(ꢁ)-5
(þ)-5
(1S,3R)
(1R,3S)
(1S,3S)
(1R,3R)
99
99
99
99
ꢁ61.6 (0.7)
þ61.0 (1.0)
ꢁ58.7 (1.2)
þ56.5 (2.2)
94
98
98
98
ꢁ65.0 (3.4)
þ61.1 (3.4)
ꢁ48.9 (0.8)
þ49.4 (0.8)
* Literature value.15;16

K. Ahmad et al. / Tetrahedron: Asymmetry 15 (2004) 1685–1692
Table 5. Preparation of (ꢁ)-4, (þ)-4, (þ)-5 and (ꢁ)-5 by the biotransformation of (ꢃ)-6
1689
25
D
Substrate
Micro-organism
Reaction time (h)
Product
Yield (%)
Conf.
Ee (%)
½aꢂ
(ꢃ)-6
P. capsulata
24
(þ)-4
(ꢁ)-5
38
38
(1R,3S)
(1S,3S)
99
99
þ61.0
ꢁ58.7
(ꢃ)-6
(ꢃ)-6
S. cerevisiae
Z. rouxii
96
48
(þ)-4
(ꢁ)-5
30
15
(1R,3S)
(1S,3S)
99
99
þ61.0
ꢁ55.0
(ꢁ)-4
(þ)-5
23
46
(1S,3R)
(1R,3R)
99
99
ꢁ61.6
þ56.5
are much higher in one-pot transformations than in two
step sequential reactions.
arately with diethyl ether (3 ꢅ 70 mL). The combined
organic layer was washed with water, dried over anhy-
drous sodium sulfate and concentrated under reduced
pressure to furnish the crude product, which on purifi-
cation by column chromatography on silica gel and
elution with hexane–ethyl acetate (85:15) afforded (R)-3-
4. Experimental
4.1. General
hydroxy-1-phenyl-butan-1-one (ꢁ)-2 as a colourless oil
25
(140 mg, 85%). ½aꢂ ¼ ꢁ67:5 (c 1.2, CHCl3) 99% ee (by
D
Chiral HPLC analysis.); IR (KBr): 3423, 2970, 1681,
1H NMR spectra were recorded as d values at 200 MHz
NMR and 13C NMR at 50 MHz using CDCl3 as a sol-
vent and TMS as internal standard. Infrared spectra
were recorded as KBr pellets in cmꢁ1 on a Hitachi 270-
30 model spectrophotometer. Mass spectra were
recorded on JEOL MSD-300 mass spectrophotometer.
Optical rotations were measured on Perkin–Elmer 241
polarimeter in CHCl3 solution. Chiral HPLC was car-
ried out on a Shimadzu LC-10 AT model. HPLC anal-
ysis of 1-phenyl-3-hydroxy-butan-1-one was performed
on Chiralcel OD-H (chiral column), using isopropanol–
hexane ¼ 5:95 as eluent, flow rate 0.8 mL/min where as
the HPLC analysis of 1,3-diols was carried out on (R,R)-
Whelk-O1, using hexane–isopropanol–acetic acid ¼
96.9:3.0:0.1 as eluent, flow rate 0.5 mL/min.
1449, 1374, 1003, 754, 690 cmꢁ1 1HNMR (200 MHz,
;
CDCl3): d 1.30 (3H, d, J ¼ 6:4 Hz, CH3), 3.10 (2H, d,
J ¼ 6:0 Hz, CH2), 3.24 (1H, OH), 4.42 (1H, m, CHOH),
7.50 (3H, m, ArH), 7.97 (2H, dd, J ¼ 2:0, 8.5 Hz, ArH);
13C NMR (50 MHz, CDCl3): d 22.47, 46.54, 64.08,
128.12, 128.75, 133.63, 136.75, 200.95; MS m=z (%): 164
(2.4), 146 (11.4), 120 (12.4), 106 (100), 91 (5), 77 (60), 52
(32). Anal. Calcd for C10H12O2: C, 73.17; H, 7.31.
Found: C, 73.55; H, 7.39.
4.4. Bioreduction with S. cerevisiae
To a stirring solution of glucose (8 g) and S. cerevisiae
(13 g) in distilled water (100 mL) was added ethanolic
solution of compound 1 (540 mg, 3.33 mmol, 1.0 mL)
and the contents stirred for 72 h at 30 °C. The mixture
was worked up as described in Section 4.4 to furnish
crude bioproduct (þ)-2, which on purification by col-
umn chromatography on silica gel with ethyl acetate–
4.2. Cultivation of microorganisms
For the general preparation of biomass the following
solution was used (g Lꢁ1); yeast 3 g, dextrose 20 g, pep-
tone 10 g Lꢁ1, pH adjusted to 6.5. The medium auto-
claved at 120 °C for 20 min. The dextrose solution
autoclaved separately and used prior to inoculation.
Flask (1000 mL) filled with 250 mL of the medium
inoculated and cultivated under CO2 atmosphere on a
rotary shaker (200–220 rpm), temp 30 °C. The optical
density (OD) of the medium was observed at 610 nm and
the medium centrifuged at 8000 rpm with temp at 10–
15 °C for 8–10 min. The pellet washed with autoclaved
distilled water and centrifuged again. The cell pellet thus
obtained is used as such for biotransformation.
n-hexane (15:85) as eluant gave (þ)-2 (440 mg, 80%).
25
½aꢂ ¼ þ48:5 (c 2.8, CHCl3) 77% ee (HPLC analysis);
D
IR (KBr): 3427, 2971, 1681, 1449, 1373, 1002, 754,
690 cmꢁ1
.
4.5. Synthesis of racemic syn- and anti-1-phenyl-1,3-
butanediol; ( )-4 and ( )-5
Compound 1 (6.48 g, 40 mmol) was reacted with sodium
borohydride (2 g, 52.9 mmol) in methanol (110 mL) at 0–
5 °C to afford a mixture of syn- and anti-1-phenyl-1,3-
butanediol [(ꢃ)-5 and (ꢃ)-4, 6.0 g, 90% yield] after usual
workup. The diol mixture was divided into two parts.
The first part was processed for the isolation and sepa-
ration of racemic 4 and 5 and second part was used for
the preparation of (ꢃ)-2. Column chromatography of
part one on silica gel and elution with ethyl acetate–
n-hexane (20:80) affords (ꢃ)-5 (syn product as a semisolid
(900 mg, 27%). IR (KBr): 3260, 3027, 2967, 2866, 1601,
4.3. General method for the biocatalytic reduction of
benzoylacetone
In a typical experiment, to a suspension of Z. rouxii in
distilled water (7.5 g wet pellet in 75 mL) containing
glucose (5 g) was added ethanolic solution of compound
1 (162 mg, 1.0 mmol, 0.3 mL) and the contents were
shaken at 30 °C as such using a bubbler. After comple-
tion of the reaction (40 h), the contents were centrifuged
and the supernatant and cell pellet were extracted sep-
1491, 1452, 1373, 1321, 1133, 1073, 974, 760, 700 cmꢁ1
;
1H NMR (200 MHz, CDCl3): d 1.23 (3H, d, J ¼ 6:2 Hz,
CH3), 1.81 (2H, m, CH2), 4.15 (1H, m, CHCH3), 4.94

1690
K. Ahmad et al. / Tetrahedron: Asymmetry 15 (2004) 1685–1692
(1H, dd, J ¼ 3:54, 9.55 Hz, ArCH) and 7.31 (5H, m,
ArH); 13C NMR (50 MHz, CDCl3): d 23.90, 46.91,
68.86, 75.20, 125.67, 127.50, 128.43, 144.50; MS m=z (%)
166 (4.3), 148 (25.5), 133 (9.5), 107 (100), 105 (66.8), 104
(17.8), 79 (89.5), 77 (54.8). Anal. Calcd for C10H14O2: C,
72.28; H, 8.43. Found: C, 72.82; H, 8.50. Further elution
with the same eluent gave racemic (ꢃ)-4 as a crystalline
solid mp 80 °C (2.10 g, 63%). IR (KBr): 3395, 3308,
2972, 1586, 1494, 1455, 1423, 1378, 1350, 1333, 1319,
133 (12), 107 (96.4), 105 (68.1), 79 (100), 77 (72). Anal.
Calcd for C10H14O2: C, 72.28; H, 8.43. Found: C, 72.66;
H, 8.51.
4.8. One-pot synthesis of (+)-4
To a suspension of P. capsulata (wet pellet, 6.5 g) in
distilled water (65 mL) containing glucose (4 g) was
added ethanolic solution of compound 1 (100 mg,
0.61 mmol, 1 mL) and the contents shaken at 28 °C using
a bubbler. Reaction was monitored by TLC, the ee%
and the configuration of the intermediate (þ)-2 was
determined by taking out 5 mL test sample, extracting it
with diethylether, followed by desolventization and
analysis on Chiral HPLC using Chiracel OD-H. Bubbler
was removed and the contents were cooled to 15 °C and
to the stirring solution was added NaBH4 (25 mg,
0.64 mmol) in three small portions. After the completion
of the reaction, HPLC analysis showed de of 98% and ee
of 99%. Purification of the bioproduct by column
1129, 1049, 969, 762, 704, 548 cmꢁ1
;
1H NMR
(200 MHz, CDCl3): 1.20 (3H, d, J ¼ 6:2 Hz, CH3), 1.84
(2H, m, CH2), 4.03 (1H, m, CHCH3), 5.01 (1H, dd,
J ¼ 4:6, 6.5 Hz, ArCH) and 7.31 (5H, m, ArH). 13C
NMR (50 MHz CDCl3): d 23.50, 46.17, 65.36, 71.71,
125.63, 127.34, 128.46, 144.50; MS m=z (%): 166 (3.9),
148 (23), 133 (11), 108 (11.0), 107 (100), 105 (72), 104
(18.3), 91 (9.3), 79 (99), 77 (74). Anal. Calcd for
C10H14O2: C, 72.28; H, 8.43. Found: C, 72.61; H, 8.47.
4.6. Synthesis of ( )-3-hydroxy-1-phenyl-butanone ( )-2
chromatography on silica gel gave (þ)-4 (70 mg, 70%)
25
½aꢂ ¼ þ61 (c 1.0, CHCl3); 99% ee; IR (KBr): 3355,
A mixture of (ꢃ)-4 and (ꢃ)-5 (1.39 g, 8.4 mmol) was
reacted with pyridinium dichromate (3.15 g, 8.4 mmol)
in dimethylformamide (8 mL), the contents stirred at
0 °C for 2.5 h and the reaction was monitored by TLC.
After completion of the reaction, the mixture was
diluted with diethyl ether (150 mL), filtered and the solid
washed with diethyl ether (2 ꢅ 50 mL) and the combined
organic layer was concentrated under reduced pressure.
The crude product was purified by column chromato-
graphy on silica gel to give (ꢃ)-2 as an oil (760 mg, 55%).
IR (KBr): 3423, 2971, 1680, 1449, 1374, 1002, 754,
690 cmꢁ1. IR of (ꢃ)-2 was super-imposable with that of
(ꢁ)-2.
D
3030, 2968, 2928, 1607, 1492, 1452, 1404, 1336, 1129,
1054, 974, 755, 700, 551 cmꢁ1 1H NMR (200 MHz,
;
CDCl3): d 1.18 (3H, d, J ¼ 6:2 Hz, CH3), 1.81(2H, m,
CH2), 4.01 (1H, m, CHCH3), 4.98 (1H, dd, J ¼ 6:6,
J ¼ 3:7 Hz Ar–CH) and 7.29 (5H, m, ArH); 13C NMR
(50 MHz, CDCl3): d 23.36, 46.26, 65.11, 71.39, 125.59,
127.23, 128.37, 144.46; MS m=z (%) 166 (4.2), 148 (28.4),
133 (10), 107 (98.9), 105 (71.8), 79 (83.2), 77 (59.6), 56
(10.4), 52 (30.4), 47 (30.4). Anal. Calcd for C10H14O2: C,
72.28; H, 8.43. Found: C, 72.62; H, 8.41.
4.9. LiAlH4 reduction of ())-2: preparation of ())-4 and
(+)-5
4.7. One-pot preparation of ())-4
To a solution of bioproduct (ꢁ)-2 (328 mg, 2.0 mmol) in
diethyl ether (70 mL), LAH (57 mg, 1.5 mmol) was
added in three installments at 0 °C and the reaction was
monitored by TLC. After the completion of the reac-
tion, the contents were worked up by quenching the
unreacted LAH by the addition of ethyl acetate (50 mL)
followed by addition of water (30 mL), separation of the
organic layer followed by drying over anhydrous
sodium sulfate and concentration in vacuo gave the crude
mixture of (ꢁ)-4 and (þ)-5. The products (ꢁ)-4 and (þ)-5
were separated by column chromatography over silica
gel using the same mixture of solvents as eluent as de-
scribed for the separation of racemic (ꢃ)-4 and (ꢃ)-5 to
give (ꢁ)-4 as a semisolid (72 mg, 22%) and (þ)-5 as a
solid mp 65 °C (225 mg, 68%); IR (KBr): 3401, 3254,
3026, 2966, 2926, 2865, 1637, 1492, 1453, 1378, 1322,
To a suspension of Z. rouxii (wet pellet, 6.5 g) in distilled
water (65 mL) containing glucose (4 g) was added eth-
anolic solution of compound 1 (100 mg, 0.61 mmol,
1 mL) and the contents were shaken at 28 °C using a
bubbler. The reaction was monitored by TLC, after the
completion of the reaction (40 h), the ee% of the inter-
mediate (ꢁ)-2 was determined by taking out 5 mL test
sample and analyzing it by Chiral HPLC using Chiralcel
OD-H. Bubbler was removed and the contents cooled to
15 °C and to the stirring solution was added NaBH4
(25 mg, 0.64 mmol) in three small portions. After the
completion of the reaction (2 h), workup and HPLC
analysis of the final product showed the diastereoiso-
meric excess of 98% and ee of >99%. Purification of the
crude product by column chromatography on silica gel
25
D
1
afforded sole product (ꢁ)-4 (75 mg, 75%). ½aꢂ ¼ ꢁ61:6
1258, 1198, 1134, 1070, 934, 759, 699 cmꢁ1. H NMR
(c 0.7, CHCl3) ee 99%; IR (KBr): 3405, 3030, 2970, 2928,
1603, 1494, 1454, 1376, 1334, 1289, 1237, 1208, 1135,
1048, 974, 908, 809, 700, 665 cmꢁ1; 1H NMR (200 MHz,
CDCl3): d 1.19 (3H, d, J ¼ 6:3 Hz, CH3), 1.83 (2H, m,
CH2), 4.02 (1H, m, CHCH3), 4.99 (1H, dd, J ¼ 4:5,
6.8 Hz, Ar–CH) and 7.28 (5H, m, ArH); 13C NMR
(50 MHz, CDCl3): d 23.36, 46.21, 65.14, 71.43, 125.59,
127.19, 128.35, 144.48; MS m=z (%) 166 (4.1), 148 (26.9),
(200 MHz., CDCl3): d 1.19 (3H, d, J ¼ 6:0 Hz, CH3),
1.82 (2H, m, CH2), 4.09 (1H, m, CHCH3), 4.89 (1H, dd,
J ¼ 3:0, 9.4 Hz, ArCH), 7.29 (5H, m, ArH); 13C NMR
(50 MHz, CDCl3): d 23.97, 46.91, 68.76, 75.15, 125.67,
127.53, 128.45, 144.47; MS m=z (%): 166 (3.9), 148 (23),
133 (11), 107 (100), 105 (72), 79 (99), 77 (74). Anal.
Calcd for C10H14O2:C, 72.28; H, 8.43. Found: C, 72.42;
H, 8.45.

K. Ahmad et al. / Tetrahedron: Asymmetry 15 (2004) 1685–1692
1691
4.10. NaCNBH3 reduction of ())-2: preparation of ())-4
and (+)-5
three installments at 0 °C and the reaction was moni-
tored by TLC. After the completion of the reaction the
contents were worked up as described for the prepara-
tion of (ꢁ)-4 and (þ)-5 to give after purification by
Bioproduct (ꢁ)-2 (164 mg, 1.0 mmol) and sodium cyano-
borohydride (126 mg, 2.0 mmol) were dissolved in
methanol (8 mL) at 0 °C, a trace of bromocresol Green
was also added to monitor the reaction. The solution
was immediately turned to blue and 3 N HCl methanol
was added dropwise to restore the yellow colour. The
solution was stirred for 2 h and the contents were con-
centrated at reduced pressure and the residue was
extracted with diethyl ether (3 ꢅ 20 mL). The combined
extracts were dried over anhydrous sodium sulfate and
concentrated under reduced pressure. The mixture was
separated by column chromatography on silica gel and
column chromatography on silica gel (ꢁ)-5 as a solid,
25
mp 65 °C (104 mg, 63%) ½aꢂ ¼ ꢁ52 (c 0.8, CHCl3) 94%
D
25
D
ee and (þ)-4 as a semisolid (40 mg, 24%). ½aꢂ ¼ þ58 (c
0.8, CHCl3) 94% ee.
4.14. Synthesis of racemic 4-hydroxy-4-phenyl-butan-1-
one ( )-6
To a solution of 1 (11.340 g, 70.0 mmol) in ethanol
(80 mL) was added 10% Pd/C (650 mg) and the contents
hydrogenated at 45 psi. The reaction was monitored by
TLC. After usual workup, the crude product was puri-
fied by column chromatography on silica gel to give
racemic ketol (ꢃ)-6 (9.53 g, 86%). IR (KBr): 3419, 2919,
eluted with ethyl acetate–n-hexane; 20:80 to give (þ)-5 as
25
25
a solid (70 mg, 43%). ½aꢂ ¼ þ52:5 (c 1.2, CHCl3) 99%
D
ee; and (ꢁ)-4 as a semisolid (70 mg, 43%). ½aꢂ ¼ ꢁ61:6
D
(c 0.7, CHCl3) 99% ee.
1
1709, 1493, 1361, 1162, 1060, 755, 700 cmꢁ1; H NMR
(200 MHz, CDCl3): d 2.17 (3H, s, CH3), 2.83 (2H, d,
J ¼ 6:5 Hz, COCH2), 3.05 (1H, s, exchangeable with
D2O), 5.17 (1H, d, J ¼ 6:5 Hz, CHOH), 7.36 (5H, s,
ArH); 13C NMR (50 MHz, CDCl3): d 30.79, 52.04,
69.85, 125.65, 127.66, 128.53, 142.88, 209.04; MS m=z
(%): 164 (100) Mþ, 146 (94), 131 (37), 107 (99.2), 105
(99.1), 91 (5), 79 (99.1), 77 (60), 58 (97.7). Anal. Calcd
for C10H12O2: C, 73.17; H, 7.31. Found: C, 73.42; H,
7.41 and a mixture of (ꢃ)-4 and (ꢃ)-5 (440 mg, 2.5%).
4.11. NaBH4 reduction of ())-2: preparation of ())-4 and
(+)-5
To a solution of (ꢁ)-2 (113 mg, 0.70 mmol) in methanol
(50 mL), NaBH4 (19 mg, 0.5 mmol) was added at 0 °C
and the reaction was monitored by TLC. After the
completion of the reaction, the reaction was worked up as
usual and the products were separated by column chro-
matography on silica gel as described for LAH reduction
25
D
to give (þ)-5 as a solid (40 mg, 35%) ½aꢂ ¼ þ52:5 (c 1.2,
25
CHCl3) 99% ee and (ꢁ)-4 as a semisolid (55 mg, 48%).
4.15. Bioreduction of ( )-6 with Z. rouxii: preparation of
())-4 and (+)-5
½aꢂ ¼ ꢁ61:6 (c 0.7, CHCl3) 99% ee.
D
Wet pellet of Z. rouxii (12 g) was suspended in a flask
containing glucose (7 g) and distilled water (120 mL) and
solution of compound 6 (328 mg, 2.0 mmol) in ethanol
(1 mL) was added to it. The contents were stirred for 48 h
at 30 °C using a bubbler. Workup of the reaction mixture
furnished a crude product. Purification by column
4.12. NaCNBH3 reduction of (+)-2: preparation of (+)-4
and ())-5
Bioproduct (þ)-2 (492 mg, 3.0 mmol) in methanol
(8 mL) and sodium cyanoborohydride (440 mg,
7.0 mmol) was made to react in the presence of bromo-
cresol Green (1 mg to monitor the reaction) at 0 °C and
the reaction was worked up as described for the prepa-
ration of (ꢁ)-4 and (þ)-5, to give after column chro-
chromatography on silica gel gave (þ)-5 (150 mg, 45%);
25
25
½aꢂ ¼ þ52:5 (c 0.9, CHCl3) >99% ee (HPLC) and (ꢁ)-4
D
(72 mg, 22%), ½aꢂ ¼ ꢁ61:6 (c 0.7, CHCl3), >99% ee.
D
matography on silica gel (ꢁ)-5 (206 mg, 41%)
25
½aꢂ ¼ ꢁ49 (c 0.8, CHCl3) 94% ee; IR (KBr): 3254,
D
4.16. Bioreduction of ( )-6 with P. capsulata: preparation
of (+)-4 and ())-5
3026, 2967, 2929, 2865, 1603, 1491, 1453, 1377, 1321,
1134, 1071, 974, 759 and 700 cmꢁ1; 1H NMR (200 MHz,
CDCl3): d 1.19 (3H, d, J ¼ 6:1 Hz, CH3), 1.78 (2H, m,
CH2), 4.09 (1H, m, CHCH3), 4.87 (1H, dd, J ¼ 3:4,
3.3 Hz, ArCH) and 7.30 (5H, m, ArH); 13C NMR
(50 MHz, CDCl3): d 23.98, 46.91, 68.76, 75.15, 125.70,
127.55, 128.47, 144.50; MS m=z (%) 166 (4.3), 148 (25.3),
107 (99), 105 (66), 79 (88), 77 (54.8), 56 (8.6), 52 (30.4),
47 (30.4). Anal. Calcd for C10H14O2:C, 72.28; H, 8.43.
Racemic 6 (100 mg, 0.61 mmol) in ethanol (1 mL) was
subjected to bioreduction with P. capsulata (6.5 g wet
pellet, 65 mL distilled water and 5 g glucose) at 25 °C.
The reaction was worked up after 24 h. Purification by
column chromatography on silica gel and elution with
ethyl acetate–n-hexane (20:80) gave (þ)-4 as a semisolid
25
25
(38 mg), ½aꢂ ¼ þ61 (c 0.9, CHCl3) 99% ee and (ꢁ)-5 as
D
Found: C, 72.12; H, 8.48. (þ)-4 (206 mg, 41%)
a solid (38 mg), ½aꢂ ¼ ꢁ58:7 (c 0.5, CHCl3) 99% ee.
25
½aꢂ ¼ þ58 (c 0.8, CHCl3) 94% ee.
D
D
4.13. LiAlH4 reduction of (+)-2: preparation of (+)-4 and
())-5
4.17. Bioreduction of ( )-6 with S. cerevisiae: preparation
of (+)-4 and ())-5
To a solution of (þ)-2 (164 mg, 1.0 mmol) in diethyl
A solution of racemic 6 (100 mg, 0.61 mmol) in ethanol
(1 mL) was subjected to bioreduction with S. cerevisiae
ether (50 mL), LAH (38 mg, 1.0 mmol) was added in

1692
K. Ahmad et al. / Tetrahedron: Asymmetry 15 (2004) 1685–1692
Vedejs, E.; Duncan, S. M.; Haight, A. R. J. Org. Chem.
1993, 58, 3046; Evans, D. A.; Hoveyda, A. H. J. Org.
Chem. 1990, 55, 5190;; Kiyooka, S.; Kuroda, H.; Shima-
saki, Y. Tetrahedron Lett. 1986, 27, 3009.
(8 g, distilled water 60 mL, glucose 4 g) at 25 °C and
worked up after 96 h and after purification by column
chromatography on silica gel, and elution with ethyl
acetate–n-hexane (20:80) to afford (þ)-4, (30 mg, 30%),
25
25
½aꢂ ¼ þ61 (c 1.0, CHCl3) 99% ee and (ꢁ)-5 as a
10. Umekawa, Y.; Sakaguchi, S.; Nishiyama, Y.; Ishii, Y.
J. Org. Chem. 1997, 62, 3409; Gillespie, K. M.; Munslow,
I. J.; Scott, P. Tetrahedron Lett. 1999, 40, 9371; Keck, G.
E.; Carrie, W. A.; Thorsten, S.; Wager, T. T. J. Org.
Chem. 1999, 64(7), 2172; Chatla, N.; Reddy, M. R.; Hair,
M.; George, W. Tetrahedron Lett. 1997, 38, 7705.
11. Jones, J. B. Tetrahedron 1986, 42, 3351.
D
solid (15 mg, 15%), ½aꢂ ¼ ꢁ55 (c 1.2, CHCl3) 99% ee.
D
References and notes
12. Chenevert, R.; Thiboutot, S. Can. J. Chem. 1986, 64,
1599.
13. Jian-Xin, G.; Zu-Yi, L.; Guo-Qiang, L. Tetrahedron 1993,
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53, 5215.
14. The racemic syn- and anti-diols (as standards for HPLC
and spectral analysis) were prepared by NaBH4 reduction
of compound 1 followed by column chromatography on
silica gel giving (ꢃ)-4 and (ꢃ)-5, respectively, in overall
yield of 90%. The diastereoisomers ())-4 and ())-5; (+)-4
and (+)-5 and the synthetic samples of (ꢃ)-4 and (ꢃ)-5
were analyzed by Chiral HPLC using (R,R)-Whelk-O1
column with mobile phase n-hexane–isopropanol–acetic
acid (96.9:3.0:0.1).
15. Takeshita, M.; Miura, M.; Unuma, Y. J. Chem. Soc.,
Perkin Trans. 1 1993, 2901.
16. Takeshita, M.; Miura, M.; Hongo, T.; Kosaka, K.;
Takeshita, Y. J. Mol. Catal. B: Enzymatic 1998, 5, 245;
Kawano, H.; Ishii, Y.; Saburi, M.; Uchida, Y. J. Chem.
Soc., Chem. Commun. 1988, 87.
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2. (a) Hoffmann, R. W. Angew. Chem., Int. Ed. Engl. 1987,
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3. Fuganti, C.; Servi, S. Bioflavour 1988, 87, 555.
4. Gruyter, D.; Fuganti, C.; Grasselli, P.; Spreafico, F.;
Zirotti, C. J. Org. Chem. 1984, 49, 543.
5. Bonini, C.; Righi, G.; Rossi, L. Tetrahedron: Asymmetry
1994, 5, 173.
6. Evans, D. A.; Hoveyda, A. H. J. Am. Chem. Soc. 1990,
112, 6447.
7. Oishi, T.; Nakata, T. Synthesis 1990, 635.
8. Mukaiyama, T.; Banno, K.; Narasaka, K. J. Am. Chem.
Soc. 1974, 96, 7503.
9. Narasaka, K.; Pai, F. C. Tetrahedron 1984, 40, 2233;
Yamashita, H.; Narasaka, K. Chem. Lett. 1996, 539;