Kinetic resolution of vic-diols by Bacillus stearothermophilus diacetyl reductase

Article abstract of DOI:10.1016/S0957-4166(98)00032-9

The kinetic resolution of several racemic syn- and anti-1,2-diols by enzymatic oxidation with Bacillus stearothermophilus diacetyl reductase is described. The enantiomerically pure (R,R)- and (R,S)-diols are recovered in almost quantitative yield.

Full text of DOI:10.1016/S0957-4166(98)00032-9

Pergamon
Tetrahedron: Asymmetry 9 (1998) 647–651
Kinetic resolution of vic-diols by Bacillus stearothermophilus
diacetyl reductase
a
a
a,∗
a
a
Olga Bortolini, Elena Casanova, Giancarlo Fantin,
Alessandro Medici, Silvia Poli and
Stefania Hanau b
a
Dipartimento di Chimica, Via Borsari 46, 44100 Ferrara, Italy
Dipartimento di Chimica e Biologia Molecolare, Via Borsari 46, 44100 Ferrara, Italy
b
Received 12 December 1997; accepted 22 January 1998
Abstract
The kinetic resolution of several racemic syn- and anti-1,2-diolsby enzymatic oxidation with Bacillus stearother-
mophilus diacetyl reductase is described. The enantiomerically pure (R,R)- and (R,S)-diols are recovered in almost
quantitative yield. © 1998 Elsevier Science Ltd. All rights reserved.
1. Introduction
1
Chiral vicinal diols are highly versatile building blocks in asymmetric synthesis. The most important
chemical route to these molecules, in terms of stereospecificity and enantioselectivity, is represented by
2
the catalytic cis-dihydroxylation of olefins achieved by OsO in the presence of cinchona alkaloids. The
4
major drawbacks of the chemical method, however, are the moderate enantiomeric excesses obtained
3
in the synthesis of anti-1,2-diols from cis-olefins and the use of toxic metal catalysts, which may be
a source of pollution. A useful alternative for the synthesis of enantiopure vicinal diols is represented
by enzymes, as recently demonstrated by the enantioselective reduction of prochiral α-diketones med-
4
iated by Bacillus stearothermophilus diacetyl reductase. This enzyme is an efficient catalyst for the
stereoselective reduction of these substrates to (S,S) vicinal diols and (S)-α-hydroxy ketones, in the
presence of NADH as cofactor. The catalytic system was equally effective in single- or double-enzyme
fashion, the latter supported by the glucose 6-phosphate/glucose 6-phosphate dehydrogenase recycling
counterpart. As will be presented in this paper, however, the same enzyme can be successfully employed
to obtain vicinal diols in the (R,R) or (R,S) configuration by kinetic resolution of the racemic mixture
of diols via oxidation. There are few enzymes available that catalyze the oxidation of specific hydroxyl
5,6
groups in polyols. Glycerol dehydrogenase is specific for (R)-alcohols and oxidizes (R)-butan-1,2-diol
∗
Corresponding author.
0957-4166/98/$19.00 © 1998 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(98)00032-9

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O. Bortolini et al. / Tetrahedron: Asymmetry 9 (1998) 647–651
to 1-hydroxybutan-2-one. (S)-Butan-1,2-diol can be recovered unchanged, although in poor enantiomeric
excess (ee 36%). Similarly, cis-cyclohexan-1,2 diol or cis-cyclopentan-1,2 diol are oxidized to (S)-α-
hydroxy ketones in high enantiomeric excess but low chemical yield.5
2. Results and discussion
Bacillus stearothermophilus diacetyl reductase (BSDR) also proved to be a powerful catalyst in the
oxidative sense, affording the corresponding α-hydroxy ketone and the unreacted alcohol with excellent
enantiomeric excesses and good chemical yield. The overall reaction view is shown in Scheme 1.
Scheme 1.
Note that both the remaining alcohol and the product are chiral, thus avoiding the major drawback
of the kinetic resolution concerning the recovery of optically active products with a maximum yield of
7
5
0%. BSDR was confirmed as a very flexible enzyme, accepting a large variety of substrates bearing
aliphatic, cyclic or aromatic substituents. Furthermore, all substituents being equal, the study has been
carried out on the two distinct sets of substrates presented below: the syn and anti pairs.8

O. Bortolini et al. / Tetrahedron: Asymmetry 9 (1998) 647–651
649
Table 1
Kinetic resolution of the racemic syn-alcohols 1a–f by oxidation with Bacillus stearothermophilus
diacetyl reductase
Table 2
Kinetic resolution of the racemic anti-alcohols 2a–f by oxidation with Bacillus stearothermophilus
diacetyl reductase
8
In the catalytic cycle of the syn pair, Scheme 1, the (S,S)-diol is stereospecifically oxidized on the
9
hydroxy group proximal to the smaller substituent, affording the unreacted (R,R)-diol in excellent
chemical yield and enantiomeric excess, and the corresponding (S)-α-hydroxy ketone, as shown in
+
Table 1. The NADH produced during the oxidation process is conveniently transformed to NAD in the
presence of the relatively inexpensive pyruvate and lactic dehydrogenase (LDH). Moreover, the recycling
system has the additional advantage of being highly soluble in water, also owing to the pH, thus making
the recovery of the products easy and practical.10
The specificity of BSDR for the (S) stereochemistry is further confirmed by the data obtained with the
anti pair, where the exclusive elaboration of the (S)-hydroxy function adjacent to the smaller substituent
9
is observed. As a consequence, the (R,S)-diol is kinetically resolved from the racemate by virtue of the
oxidation of the (S,R)-enantiomer to the (R)-α-hydroxy ketone, as shown in Table 2.
When this biocatalyzed oxidation is applied to the meso diol 2f, desymmetrization of the substrate
11
is achieved, with the advantage of transforming, in principle, all the starting material into the desired
homochiral product.
As expected, shorter reaction times increase the chemical yield of the α-hydroxy ketones to the
detriment of the enantiomeric excess of the diols. As an example, the kinetic resolution of 1d after
10 hours affords 3d in 34% yield (ee 99%), but the corresponding (R,R)-1d in 75% enantiomeric
excess. Despite the shorter reaction times, however, side reactions frustrate attempts to obtain most of
the α-hydroxy ketones on a multigram scale. It is well known that enzymatic oxidations work best at
elevated pH (8–9), where cofactors and products may be unstable.12 To avoid this instability to some

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O. Bortolini et al. / Tetrahedron: Asymmetry 9 (1998) 647–651
extent, the pH of the medium has been shifted from the optimum value of 9.2 to pH 8.2, where the specific
13
activity of BSDR is only partially reduced. Also, under these experimental conditions, a progressive
disappearance of the α-hydroxy ketones is observed, as confirmed by control experiments carried out
on (R)-3-hydroxy-4-hexanone 3d. A possible explanation for this side reaction might be the formation
of an enediol, followed by further biotransformation.14 A notable exception is the (R)-1-hydroxy-2-
cyclohexanone 3f that is recovered from the reaction mixture in almost quantitative yield.
In conclusion, several enantiomerically pure (R,R)- and (R,S)-vic-diols may be obtained by kinetic
resolution via oxidation of the corresponding racemates, in the presence of Bacillus stearothermophilus
diacetyl reductase.
3. Experimental
3.1. Materials and methods
Bacillus stearothermophilus is available from American Type Culture Collection (ATCC 2027). For
synthetic reactions, the wet cells (20 g) obtained from four portions of 250 mL cultures are separated
by centrifugation and washed with 200 mL of 0.15 M NaCl. The cells are suspended in 100 mL of
TEA–HCl buffer, treated with 40 mg of lysozyme for 60 min at 22°C and recentrifuged. The supernatant
(
enzyme solution) was used without further treatment. Alternatively the supernatant was freeze-dried to a
15
powder by lyophilization, stored in a refrigerator, and used directly for chemical synthesis. Typically 1
mL of enzyme solution contains ca 0.2 units of enzyme (1 unit=1µmol of endo-bicyclo[3.2.0]hept-2-en-
4
6
-ol oxidized per minute). The TEA–HCl buffer is composed of 50 mM triethanolamine, containing
0
.1 mM EDTA, 1 mM β-mercaptoethanol and HCl to adjust the pH to 7.5. Further purification of
the crude extract via DEAE (diethylaminoethyl) sepharose CL 6B and Cibachron-blue 3GA agarose
column chromatography provided a pure protein.13 Note that almost identical results were obtained
+
using either the purified enzyme or the crude extract. Lysozyme, NAD and pyruvate were from Sigma,
lactic dehydrogenase (LDH) was from Boehringer Mannheim. Cyclohexanediol and cyclopentanediol
were purchased from Aldrich. All other diols, except 2,3-octanediol, have been obtained from the
commercially available corresponding diketones (Aldrich) by reduction with NaBH , followed by
4
separation of the syn and anti pairs by column chromatography. The same reduction and separation
4
methodology has been used to obtain the 2,3-octanediol from the 3-hydroxy-2-octanone. Enantiomeric
4
separations and excesses were determined by GC on a chiral column and ee≥99% means that the
corresponding enantiomer was not detected. The absolute configurations are determined by comparison
of the signs of the specific rotations with the literature values, when available. In the other cases, the
absolute configurations are assigned on the basis of the GC retention time in a homologous series, upon
analysis of the racemates. NMR spectra were obtained on a 300 MHz instrument.
3.2. General procedure of oxidation
A portion of the enzyme solution (2 mL) was added to a flask containing 1 mmol of diol, 20 mg NAD+,
1
mmol of pyruvate, 200 µg of lactic dehydrogenase in 100 mL of TEA–HCl buffer, pH 8.2. After the
proper time, the reaction mixture was saturated with NaCl and extracted with ethyl acetate. The products
were separated by chromatography on SiO with ethyl acetate:cyclohexane (1:1) as eluent. The yields,
2
absolute configurations and enantiomeric excesses are listed in the tables. Specfic rotations: (R,R)-1a
16
[
α] =+16 (c 1.2, CHCl ) confirmed by comparison with literature data on the (S,S)-enantiomer; (R,R)-
D
3

O. Bortolini et al. / Tetrahedron: Asymmetry 9 (1998) 647–651
651
1
7
1
1
b [α] =+18.5 (c 1.1, CHCl ) confirmed by comparison with literature data; (R,R)-1c [α] =−55.9 (c
D
3
D
18
.9, CHCl ) confirmed by comparison with literature data; (R,R)-1d [α] =+12 (c 2, CHCl ) confirmed
3
D
3
16
by comparison with literature data on the (S,S) enantiomer; (R,R)-1e [α] =−33.3 (neat) confirmed
D
19
by comparison with literature data; (R,R)-1f [α] =−38 (c 1.5, H O) confirmed by comparison with
D
2
20
literature data; (R,S)-2a [α] =−10.4 (c 1.5, CHCl ), the stereochemistry was assigned based on the
D
3
GC retention time; (R,S)-2b [α] =−22 (c 1.0, CHCl ) confirmed by comparison with literature data
D
3
2
1
on the (S,S)-enantiomer; (R)-3f [α] =+13 (neat) confirmed by comparison with literature data on the
D
S)-enantiomer.5 1H and C NMR are consistent with previously published data.
13
(
Acknowledgements
This research is supported by the Italian Ministry of University and Scientific Research (MURST).
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8
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1
0. The catalytic system is equally effective as a single enzyme, where BSDR catalyzes the desired reaction while
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1
1
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1
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