Enantioselective hydrolysis of (RS)-isopropylideneglycerol acetate with Kluyveromyces marxianus

Article abstract of DOI:10.1016/j.tetasy.2004.05.010

The hydrolysis of (RS)-isopropylideneglycerol acetate with whole cells of the yeast Kluyveromyces marxianus is reported. The biotransformation furnished (R)-isopropylideneglycerol as major enantiomer with good enantioselectivity (E=28) under optimised conditions. The reaction can be performed in an ultrafiltration-membrane reactor allowing for the obtainment of 19.2g/L of enantiomerically pure (R)-isopropylideneglycerol acetate starting from 60g/L of racemic mixture.

Full text of DOI:10.1016/j.tetasy.2004.05.010

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 15 (2004) 1945–1947
Enantioselective hydrolysis of (RS)-isopropylideneglycerol acetate
with Kluyveromyces marxianus
Francesco Molinari,a,* Kristin Solange Cavenago,a Andrea Romano,a Diego Romanoa and
Raffaella Gandolfib
aDipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Sezione Microbiologia Industriale,
Universitꢀa degli Studi di Milano, via Celoria 2, 20133 Milan, Italy
bIstituto di Chimica Organica ‘Alessandro Marchesini’, Facoltꢀa di Farmacia, via Venezian 21, 20133 Milan, Italy
Received 15 April 2004; accepted 11 May 2004
Abstract—The hydrolysis of (RS)-isopropylideneglycerol acetate with whole cells of the yeast Kluyveromyces marxianus is reported.
The biotransformation furnished (R)-isopropylideneglycerol as major enantiomer with good enantioselectivity (E ¼ 28) under
optimised conditions. The reaction can be performed in an ultrafiltration-membrane reactor allowing for the obtainment of 19.2 g/L
of enantiomerically pure (R)-isopropylideneglycerol acetate starting from 60 g/L of racemic mixture.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
myces marxianus, which had shown in preliminary
studies preference for the formation of (R)-IPG.9 The
kinetic resolution of (RS)-IPG acetate by enzymatic
hydrolysis has some advantages since the substrate has a
good solubility in water and its synthesis is cheap.
The resolution of racemic 2,2-dimethyl-[1,3]dioxolan-4-
methyl acetate (1,2-O-isopropylideneglycerol, IPG or
solketal) esters is an easy method for obtaining both the
enantiomers of IPG. Enantiomerically pure IPG is a
valuable chiral building blocks, for example, in the
synthesis of b-adrenoceptor antagonists, prostaglandins
and leukotrienes.1;2
2. Results
The hydrolysis of (RS)-isopropylideneglycerol acetate
was firstly studied using different strains of Kluyvero-
myces marxianus as biocatalysts. Hydrolysis was carried
out using 3.0 g/L of racemic substrate and 35 g/L of dry
yeast in 1/15 M phosphate buffer at pH 7.0, at the tem-
perature of 30 ꢁC. The ability to perform the rapid
hydrolysis of IPG acetate was widespread among this
yeast’ species and the enzymatic activity was found to be
mostly cell bound, with enantiomeric ratios in the range
of 12–16 (Table 1).
The enantioselective hydrolysis of (RS)-isopropylidene-
glycerol esters is difficult to achieve with commercially
available enzymes,3–6 while the best results have been
obtained by hydrolysis of IPG caprylate and benzoate
using esterases from Bacillus species: a carboxylesterase
from Bacillus coagulans is able to enantioselectively
hydrolyse IPG benzoate to produce (S)-IPG7 and the
same enantiomer is predominantly formed by a recently
isolated esterase from Bacillus subtilis in the hydrolysis
of IPG caprylate.8 Enzymatic methods with preference
for the hydrolysis of (S)-IPG esters have not yet been
developed.
K. marxianus CBS 1553 was used in further experiments
aimed at the optimisation of the biotransformation. The
yeast was lyophilised showing similar performances and
maintained for six months at ꢀ20 ꢁC with no significant
loss of activity/enantioselectivity; therefore, lyophilised
biomass from a single 10 L fermentation could be used
throughout this study. Optimisation was performed
following a Multisimplex experimental design:10;11 type
of growth medium, cell and substrate concentrations,
Herein we report the enantioselective hydrolysis of (RS)-
IPG acetate with the easily cultivable yeast Kluyvero-
* Corresponding author. Tel.: +39-0250316695; fax: +39-0250316694;
e-mail: francesco.molinari@unimi.it
0957-4166/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.05.010

1946
F. Molinari et al. / Tetrahedron: Asymmetry 15 (2004) 1945–1947
Table 1. Hydrolysis of racemic IPG acetate catalysed by different
strains of Kluyveromyces marxianus; results after 3 h
the resolution of (RS)-IPG acetate, furnishing (R)-IPG
with good yields. The stability of the biocatalyst is good
and multigram production of the (R)-enantiomer can be
obtained by using a simple membrane reactor.
Strain
Ee % (R)-IPG Ee % (R)-IPG Molar
acetate
E
conv. (%)
CBS 397
CBS 607
CBS 834
48
53
68
82
76
74
69
78
77
81
37
41
48
57
41
43
39
16
12
15
16
13
13
15
CBS 1553 90
CBS 2231 55
CBS 2762 58
CBS 4857 51
4. Experimental
4.1. Microorganisms, growth and biotransformation con-
ditions
Strains of K. marxianus were from CBS (Central Bureau
voor Schimmelcultures, Baarn, Holland) and routinely
temperature and pH of the biotransformation were
chosen as response variables. Biomass concentrations
ranging between 25–30 g/L (dry cells) used in tap water
at pH 7.5 at 28 ꢁC gave the highest enantioselective
activity. Under these reaction conditions, different
amounts of substrate were transformed (Table 2).
maintained on malt extract (8 g Lꢀ1, agar 15 g Lꢀ1
,
pH 5.5). They were cultured in 2.0 L Erlenmeyer flasks
containing 200 mL of malt broth pH 6.0 and incubated
for 48 h at 28 ꢁC on a reciprocal shaker (100 spm).
K. marxianus CBS 1553 was employed in optimisation
studies accomplished using cells from a 8 L fermenter
containing 4.0 L of liquid medium for 48 h, pH 6.0, at
27 ꢁC and agitation speed 100 rpm. 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.
The highest E value was observed at the lowest substrate
concentration, nevertheless the best compromise
between overall enantioselectivity, reaction rate, yields
and recovery of enantiomerically pure substrate was
obtained using 3 g/L.
Repeated-batch experiments aimed at improving the
productivity were firstly carried out with Kluyveromces
marxianus CBS 1553. The biocatalyst was recycled by
simply paper filtering or centrifuging the cells when the
biotransformation had reached the desired conversion,
but the process was complicated by the partial release of
the carboxylesterase activity into the medium. There-
fore, an ultrafiltration (cut-off 10,000 Da) membrane
reactor was employed for semi-continuous operation for
maintaining the cells and the released enzyme inside the
reaction vessel. Ultrafiltration was applied when the
enantiomeric excess of the substrate had reached 100%.
Repeated-batch operation of the membrane reactor was
maintained for 20 cycles, before decrease of the enzy-
matic activity could be observed; this operation allowed
the recovery of 19.2 g/L of enantiomerically pure (R)-
IPG acetate starting from 60 g/L of racemic mixture.
Biotransformations were carried out in 10 mL screw
capped test tubes with cells suspended in 5 mL of dif-
ferent aqueous media. After 45 min of incubation, neat
substrate was added and the incubation continued under
magnetical stirring.
A stirred ultrafiltration cell (Model 8050, capacity
50 mL) containing 20 mL of biotransformation medium
was employed as membrane reactor for repeated-batch
biotransformation. The membrane had a cut-off of
10,000 Da. The reaction was stopped when the enan-
tiomeric excess of the substrate was 100% by filtering the
suspension under nitrogen pressure. The recovered fil-
trate was extracted with ethyl acetate and (R)-isopropyl-
ideneglycerol acetate was purified as described for the
20
D
synthesis of the racemic substrate ½aꢁ ¼ ꢀ8:3 (c 1,
CHCl3).
4.2. 1,2-O-isopropylideneglycerol acetate
3. Conclusion
The synthesis of (RS)-isopropylideneglycerol acetate
was carried out by adding acetic anhydride (12 mL) and
pyridine (8 mL) to a solution of racemic 1,2-O-isopro-
In conclusion, it was found that the carboxylesterase
activity bound to the cells of K. marxianus is suited for
Table 2. Hydrolysis of different amounts of racemic IPG acetate catalysed by Kluyveromyces marxianus CBS 1553
(RS)-IPG acetate
Initial concentration (g/L)
(R)-IPG acetate
Time (h)
Ea
Conv. (%)a
Ee (%)
Yield (%)b
Recovery (g/L)c
1
62
62
70
75
62
>99
>99
>99
>99
92
76
76
60
50
––
0.30
1.05
1.32
1.65
––
3
4
28
28
15
11
11
3
5
15
24
24
8
10
a Conversion and enantioselectivity factor (E) calculated from the ee of the substrate and the product.
b Calculated taking into account the conversion.
c After purification by flash column chromatography.

F. Molinari et al. / Tetrahedron: Asymmetry 15 (2004) 1945–1947
1947
pylideneglycerol (8 g) in dry benzene (50 mL). After 24 h
at room temperature, the reaction mixture was quen-
ched with 150 mL of 5% NaHCO3 solution and the
product extracted with ethyl acetate. The organic ex-
tracts were dried over Na2SO4 and the solvent removed.
The crude product was purified by flash chromatogra-
phy (ethyl acetate/hexane 1/1) yielding 9.35 g of pure
internal standard solution (1-hexanol 2 g/L) in water; the
resulting solution was extracted with ethyl acetate and
analysed. The enantiomeric composition of IPG acetate
and IPG was determined by gas-chromatographic
analysis using a chiral capillary column (diameter
0.25 mm, length 25 m, thickness 0.25 l, DMePeBeta-
CDX-PS086, MEGA, Legnano, Italy). The absolute
configurations of IPG were determined by comparison
with enantiomerically pure samples commercially
available (Sigma–Aldrich).
1
product. H NMR (CDCl3, 200 MHz): d 1.38 (s, 3H,
CH3), 1.44 (s, 3H, CH3), 2.12 (s, 3H, CH3), 3.36–3.41
(m, 2H, CH2), 4.01–4.11 (m, 2H, CH2), 4.13–4.19 (m,
1H, CH).
4.3. Optimisation by sequential simplex method
References and notes
The simplex optimisation method was based on
sequential experimental trials guided by the systematic
search strategies of the Multisimplexꢂ 2.0 program
(Multisimplex AB, Karlskrona, Sweden).9 The starting
experiments were selected with levels of each control
variable (substrate concentration, pH, temperature and
biomass concentration) within the following ranges:
pH 4–8, temperature 20–50 ꢁC, substrate concentration
1–5 g/L, biomass concentration 15–30 g/L. The control
responses to be optimised were the molar conversion
after 3 h and the corresponding enantioselectivity factor
(E). Each experiment was carried out in triplicate.
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Alcohol and ester concentrations were determined by
gas-chromatographic (GC) analysis on a Carlo Erba
Fractovap GC gas chromatograph equipped with a
hydrogen flame ionisation detector. The column
(3 · 2000 mm) was packed with Carbowax 1540 (10% on
Chromosorb 80–100 mesh). Samples (0.2 mL) were
taken at intervals and added to an equal volume of an