Enantioselective oxidation of prochiral 2-methyl-1,3-propandiol by Acetobacter pasteurianus

Article abstract of DOI:10.1016/S0957-4166(03)00407-5

The microbial oxidation of prochiral 2-methyl-1,3-propandiol into (R)-3-hydroxy-2-methyl propionic acid with Acetobacter pasteurianus DSM 8937 is reported. The biotransformation was optimised furnishing (R)-3-hydroxy-2-methyl propionic acid with 97% enantiomeric excess and 100% molar conversion of 5 g/L within 2 h. A simple fed-batch procedure allowed for the obtainment of 25 g/L of the enantiomerically enriched acid. (R)-3-Hydroxy-2-methyl propionic acid is an important building block for the synthesis of Captopril, a widely used antihypertensive drug.

Full text of DOI:10.1016/S0957-4166(03)00407-5

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 14 (2003) 2041–2043
Enantioselective oxidation of prochiral 2-methyl-1,3-propandiol
by Acetobacter pasteurianus
Francesco Molinari,a,* Raffaella Gandolfi,a Raffaella Villa,a Eva Urbanb and Andreas Kienerb
aDipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Sezione Microbiologia Industriale,
Universita` degli Studi di Milano, via Celoria 2, 20133 Milano, Italy
bLonza A.G., CH-3930 Visp, Switzerland
Received 7 April 2003; accepted 21 May 2003
Abstract—The microbial oxidation of prochiral 2-methyl-1,3-propandiol into (R)-3-hydroxy-2-methyl propionic acid with
Acetobacter pasteurianus DSM 8937 is reported. The biotransformation was optimised furnishing (R)-3-hydroxy-2-methyl
propionic acid with 97% enantiomeric excess and 100% molar conversion of 5 g/L within 2 h. A simple fed-batch procedure
allowed for the obtainment of 25 g/L of the enantiomerically enriched acid. (R)-3-Hydroxy-2-methyl propionic acid is an
important building block for the synthesis of Captopril, a widely used antihypertensive drug. © 2003 Elsevier Ltd. All rights
reserved.
In this work we have studied the conditions for obtain-
1. Introduction
ing high yields and enantioselectivity in the microbial
oxidation of prochiral 2-methyl-1,3-propandiol into
(R)-3-hydroxy-2-methyl propionic with Acetobacter
and Gluconobacter strains.
(R)-3-Hydroxy-2-methyl propionic acid is a valuable
chiral intermediate1 and it has been obtained through
biotransformation by modification of isobutyric acid
with a mutant strain of Candida rugosa.2 3-Hydroxy-2-
methyl propionic acid can also be obtained by enan-
tioselective dehydrogenation of prochiral 2-methyl-
1,3-propandiol, a cheap and widely available substrate.
The transformation of prochiral molecules into enan-
tiomerically pure products is of great relevance in syn-
thetic
methods;3,4
enzymatic
reactions
might
discriminate between prostereogenic groups of prochi-
ral substrates thus giving high yields of enantiopure
products (meso-trick).5 The enantioselective oxidation
of 2-methyl-1,3-propandiol to give (R)-3-hydroxy-2-
methyl propionic acid has been reported with Glu-
conobacter and Acetobacter species, but complete
enantioselectivity has not been achieved.6,7
2. Results
Several acetic acid bacteria (45 strains both Acetobacter
and Gluconobacter) were tested for the oxidation of
prochiral 2-methyl-1,3-propandiol; the biotransforma-
tion was carried out by directly adding the substrate
(2.5 g/L) to liquid cultures of acetic acid bacteria, which
were grown for 24 h in different media containing
glucose or glycerol as the main carbon source. Most of
the strains furnished 3-hydroxy-2-methyl propionic acid
as the only product of biotransformation when grown
with glycerol and yeast extract, while very low activity
was observed with cells grown in the medium contain-
ing glucose.
Acetic acid bacteria are efficient enantioselective biocat-
alysts and can perform the oxidation of structurally
different primary and secondary alcohols or diols, pro-
vided that suite growth and biotransformation condi-
tions are employed.8–11
Acetobacter pasteurianus DSM 8937 resulted in being
the best candidate for further development: this strain
completely converted the substrate into (R)-3-hydroxy-
* Corresponding author. Tel.: +39-0250316695; fax: +39-0250316694;
e-mail: francesco.molinari@unimi.it
0957-4166/03/$ - see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0957-4166(03)00407-5

2042
F. Molinari et al. / Tetrahedron: Asymmetry 14 (2003) 2041–2043
2-methyl propionic acid within 3 h and no further
modification of the product was observed at prolonged
times (Scheme 1).
Table 1 reports the results obtained with cells of Aceto-
bacter pasteurianus DSM 8937 grown on different
media. The reaction was enantioselective (e.e. in the
range of 90–97%) when grown with cultural media
containing 1,3-dihydroxyacetone, glycerol, mannitol,
1,3-propandiol or sorbitol. The use of glycerol gave a
high concentration of biomass and this was employed
as main carbon source in further experiments.
Experiments with resting cells of Acetobacter pasteuri-
anus DSM 8937 in different aqueous systems showed
that the removal of the cells from the growth medium
did not improve the performances; the whole cultural
medium was, therefore, employed in further experi-
ments aimed at the optimization of the bioconversion;
the growth and the biotransformation were performed
in the same reactor.
Figure 1. Biotransformation of 2-methyl-1,3-propandiol at
different concentrations with Acetobacter pasteurianus DSM
8937.
Bioconversion carried out without pH adjustment
showed a general decrease of microbial activity after
4–5 h when the acid concentration was above 12 g/L,
while the control of the pH allowed for the accumula-
tion of 25 g/L of the product (e.e. 96–97%) within 10 h.
The initial optimization was carried out following the
Multisimplex experimental design, which has previously
been employed for enantioselective oxidation with ace-
tic acid bacteria.8 The response variables were the
growth time of the microbial cells and the more crucial
parameters of the biotransformation (pH, temperature
and aeration rate). The best results were obtained with
cells grown for 36 h and employed at pH 7.2, 32°C with
an aeration rate of 1.5 vvm. The influence of the
substrate concentration was evaluated under these con-
ditions (Fig. 1). Similar reaction rates were observed in
the range of 5–10 g/L and showed that the stereobias
was not affected by the different reaction conditions.
Experiments with a semi-continuous addition of the
substrate (5 g/L after 2 h) were also performed. Parallel
experiments were carried out with or without adjust-
ment of the pH to a constant value of 7.0 (Fig. 2).
3. Conclusions
The key intermediate of the synthesis of Captopril is
(R)-3-hydroxy-2-methyl propionic acid that has been
obtained by microbial oxidation of prochiral 2-methyl-
1,3-propandiol. The bioconversion occurred with high
molar conversion and enantioselectivity using Aceto-
bacter pasteurianus DSM 8937, a wild type and gener-
ally regarded as a safe microorganism. A simple
fed-batch procedure allowed for the obtainment of 25
g/L of the enantiomerically enriched (97% e.e.) acid.
Scheme 1.
Table 1. Growth and biotransformations with Acetobacter pasteurianus DSM 8937 grown for 24 h with different carbon
sources and substrates (5 g/L) added directly to submerged cultures. Dry weights of the cells and pH of the culture after 24
h; molar conversion and enantiomeric excess of (R)-3-hydroxy-2-methyl propionic after 3 h
Carbon source
Dry weight (g/L)
pH
Molar conversion (%)
E.e. (%)
1,3-Dihydroxyacetone
Ethanol
Glucose
Glycerol
3-Glycerophosphate
Mannitol
3.7
2.4
4.0
4.4
1.2
2.0
0.9
1.9
6.0
3.5
3.7
6.1
7.0
6.5
4.0
6.5
75
<5
5
>95
10
80
90
65
90
–
–
97
80
94
92
92
1,3-Propandiol
Sorbitol

F. Molinari et al. / Tetrahedron: Asymmetry 14 (2003) 2041–2043
2043
35°C, substrate concentration 25–35 mM, speed of
agitation 100–200 rpm.
The control responses to be optimized were the molar
conversion into acid after 2 h and the enantiomeric
excess of the product obtained. Each experiment was
carried out in triplicate.
Analytical methods. The molar conversion was routinely
determined by HPLC analysis using a Polyspher OA
HY column (Merck, Darmstadt, Germany) and an
aqueous acidic solution (H2SO4 0.005N) as eluent
which allowed for the determination of the concentra-
tion of the substrates and products. Samples (0.5 mL)
were taken at intervals, brought to pH 1 by the addi-
tion of 1 M HCl and extracted with an equal volume of
ethyl acetate. The enantiomeric composition was rou-
tinely determined by gas chromatographic analysis of
the corresponding methyl ester using a chiral capillary
column (diameter 0.25 mm, length 25 m, thickness 0.25
m, DMePeBeta-CDX-PS086, MEGA, Legnano, Italia).
The absolute configuration of the obtained acid was
determined after methylation by comparison with the
specific rotation of the commercially available (Sigma-
Aldrich) enantiomerically pure (R)-3-hydroxy-2-methyl
propionic methyl ester.
Figure 2. Batch-fed biotransformation of 2-methyl-1,3-
propandiol to (R)-HIBA with Acetobacter pasteurianus DSM
8937. The reactions were carried out with the pH maintained
at 7.0 (", diol; ꢀ, acid) or without the adjustment of the pH
(2, diol; ꢁ, acid).
4. Experimental
Microorganisms, growth and biotransformation condi-
tions. Acetic acid bacteria were routinely maintained on
GYC slants (glucose 50 g L−1, yeast extract 10 g L−1,
CaCO3 30 g L−1, agar 15 g L−1, pH 6.3) at 28°C. The
strains, grown on GYC slants for 24 h at 28°C, were
inoculated into 500 ml Erlenmeyer flasks containing 50
ml of the liquid medium containing yeast extract (10 g
L−1) and different carbon sources (25 g L−1) at pH 5 in
distilled water and incubated on a reciprocal shaker
(100 spm). Acetobacter pasteurianus DSM 8937
(Deutsche Sammlung von Mikroorganismen) was
employed in optimization studies accomplished using
cultures grown in a 1 L air-lift reactor.10 Biotransfor-
mations were accomplished using bacteria grown
directly inside the reaction vessel. Neat substrate was
directly added onto suspensions. The control of the pH
was performed by continuous addition of aqueous
NaOH via a multichannel Watson-Marlow 503 U/R
peristaltic pump connected to a pH controller (pH/
ORP Controller 3675, Jenco Electronics). The dry
weights were determined after centrifugation of 100 mL
of cultures, cells were washed with distilled water and
dried at 110°C for 24 h.
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