A practical and scalable process for 4-(R)-hydroxycyclopent-2-en-1-(S)- acetate by desymmetrization of meso-cyclopent-2-en-1,4-diacetate catalyzed by Trichosporon beigelii (NCIM 3326), a cheap biocatalyst

Article abstract of DOI:10.1016/S0957-4166(00)00256-1

Various yeast and fungal cultures from NCIM, NCL, Pune, India were screened for the hydrolysis of meso-cyclopent-2-en-1,4-diacetate 2 to 4-(R)- hydroxycyclopen-2-en-1-(S)-acetate 1 to provide a cheaper and more effective alternative to PLE which is currently being used for the conversion. Yeast cultures of Trichosporon species were identified as having a pro-R preference in the hydrolysis of 2; but the enantioselectivity was poor. Hence detailed medium-engineering investigations were made for the hydrolysis of 2 to 1 using a culture of Trichosporon beigelii (NCIM 3326) as catalyst. Addition of 10% v/v ethanol was found to enhance the enantioselectivity of the enzyme, affording 1 of 85% optical purity (op) in 83% yield. Further exploration of inherent consecutive kinetic resolutions to the desymmetrization afforded 1 of > 98% op in 74% chemical yield. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0957-4166(00)00256-1

Tetrahedron: Asymmetry 11 (2000) 2965±2970
A practical and scalable process for 4-(R)-hydroxycyclopent-
2-en-1-(S)-acetate by desymmetrization of meso-cyclopent-2-
en-1,4-diacetate catalyzed by Trichosporon beigelii
(NCIM 3326), a cheap biocatalysty
Uttam R. Kalkote,a,* Sandeep R. Ghorpade,a Rohini R. Joshi,a
T. Ravindranathan,a,* Kulbhushan B. Bastawadeb and Digambar V. Gokhaleb
aDivision of Organic Chemistry: Technology, National Chemical Laboratory, Pune 411 008, India
bNCIM, Biochemical Sciences Division, National Chemical Laboratory, Pune 411 008, India
Received 26 May 2000; accepted 20 June 2000
Abstract
Various yeast and fungal cultures from NCIM, NCL, Pune, India were screened for the hydrolysis of
meso-cyclopent-2-en-1,4-diacetate 2 to 4-(R)-hydroxycyclopen-2-en-1-(S)-acetate 1 to provide a cheaper
and more e€ective alternative to PLE which is currently being used for the conversion. Yeast cultures of
Trichosporon species were identi®ed as having a pro-R preference in the hydrolysis of 2; but the enantio-
selectivity was poor. Hence detailed medium-engineering investigations were made for the hydrolysis of 2
to 1 using a culture of Trichosporon beigelii (NCIM 3326) as catalyst. Addition of 10% v/v ethanol was
found to enhance the enantioselectivity of the enzyme, a€ording 1 of 85% optical purity (op) in 83% yield.
Further exploration of inherent consecutive kinetic resolutions to the desymmetrization a€orded 1 of
>98% op in 74% chemical yield. # 2000 Elsevier Science Ltd. All rights reserved.
1. Introduction
Over the past decade, chemoenzymatic methods using microorganisms and isolated enzymes as
natural chiral catalysts have emerged as extremely ecient tools for the synthesis of
enantiomerically pure materials on the large scale.1 Various large-scale processes based on such
methods have become important sources of chiral products.2 We have been interested in the
development of economically viable large-scale processes for prostaglandins and their
intermediates due to their varied biological activities.
* Corresponding authors. E-mail: kalkote@dalton.ncl.res.in
y
NCIM is the National Collection of Industrial Microorganisms, NCL, Pune, India.
0957-4166/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(00)00256-1

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4-(R)-Hydroxycyclopent-2-en-1-(S)-acetate 1 is an important intermediate in the synthesis of
cyclopentanoid natural products3a e.g. prostaglandins, prostacyclins, thromboxanes and recently
in the synthesis of some anti-HIV drugs.3b This has resulted in various approaches for its
preparation.4 Enzyme viz. lipase/esterase-catalyzed desymmetrization of meso-cyclpent-2-en-1,4-diol
by transesteri®cation5 or enzymatic hydrolysis of its meso-diacetate 2,6 or kinetic resolution of
monoprotected cyclopentenediol7 appear to be the methods of choice in the preparation
of enantiomerically pure 1. The latter method, viz. kinetic resolution, su€ers from the drawback
of throwing half of the material away; whereas use of the desymmetrization method allows 100%
yield to be attained theoretically. In the case of the desymmerization of 2 through enzymatic
hydrolysis in aqueous medium, most of the ecient enzymes reported, with the exception of
porcine liver esterase (PLE),6c show a pro-S preference yielding ent-1 in high enantiomeric excess.
It is possible to get the desired (4R)-hydroxy con®guration through manipulation of this (4S)-
hydroxy enantiomer (meso-trick).3b,8 But this adds to the steps and involves use of diethylalumi-
nium chloride or DIBALH which are costly and hazardous, hence not recommended for large
scale. Therefore, direct desymmetrization of 2 to 1 through aqueous hydrolysis is very desirable.
PLE having a pro-R preference for the hydrolysis of 2 yields 1 on hydrolysis and is currently
used as the method of preparation. However, PLE is expensive and also gives 1 of only 81% ee
which has to be recrystallized to enhance the ee to >96%, resulting in overall low yields.6c Hence
replacement by other cheaper enzymes of microbial origin is required for the economization of
the process.
The large collection of over 300 yeast and fungal cultures at NCIM, India o€ered us a great
opportunity to explore the possibility of hunting a microbial culture of desired selectivity for
the conversion of 2 to 1. Herein we report our successful e€orts in ®nding a yeast culture having
the desired pro-R selectivity for the conversion of 2 to 1 through aqueous hydrolysis wherein the
desired level of eciency of the conversion in terms of enantiopurity and yield of the product was
achieved through medium-engineering.
2. Results and discussion
Various cultures grown on oil media were tested for hydrolytic activity on 2 in 0.1 M
phosphate bu€er. Reactions were monitored by TLC. Where monoacetate was the hydrolysis
product, its con®guration and optical purity (op) were determined from the sign and value of the
optical rotation, respectively. Most of the cultures having hydrolytic activity for 2 were pro-S
selective a€ording ent-1 in varying op. Only a few cultures belonging to the Trichosporon species
exhibited the pro-R selectivity; however, the optical purities obtained were poor (Table 1). Since
we could not ®nd any culture having high enantioselectivity for the pro-R acetate, we decided to

U. R. Kalkote et al. / Tetrahedron: Asymmetry 11 (2000) 2965±2970
2967
optimize the conversion using this poorly selective Trichosporon species of good hydrolytic
activity with a pro-R preference.
Table 1
Results of preliminary screeninga
Enhancement of enzyme eciency through medium engineering, i.e. optimization of the
solvent system, has been well documented in several cases.9 Addition of co-solvents, such as
alcohols, acetone, DMF, DMSO, etc. in small percentages are reported to have bene®cial e€ects
on hydrolytic reactions. Trichosporon beigelii (NCIM 3326) was considered as the best candidate
for selectivity enhancement studies as it showed the best results (88% yield, 29% op. Table 1) in
preliminary screening studies. Reactions were carried out by adding various water-miscible
co-solvents in 10% v/v in bu€er and the results are presented in Tables 2±4. The above study
indicates a strong e€ect of ethanol on the selectivity of the whole cell culture NCIM 3326. Thus,
by using 10% ethanol in solvent system, the op of product 1 shot up from 29.1 to 82.6%. But
Table 2
Medium-engineering e€ect on the hydrolysis of 2 catalyzed by NCIM 3326a
Table 3
E€ect of variation of ethanol concentration on the hydrolysis of 2 catalyzed by NCIM 3326

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Table 4
E€ect of 10% v/v ethanol in bu€er media on the hydrolysis of 2 catalyzed by cultures from Trichosporon species
high concentrations of ethanol (>50%) were inhibitory for the reaction. Similar e€ects were
observed with the cultures belonging to the same species (Table 4).
Even though the enantiopurity of 1 can be enhanced from 85 to >98% by low temperature
crystallization, which is a tedious process, the yields became low (65%). Sih et al. have
demonstrated the importance of an inherent consecutive kinetic resolution step6c in enhancing the
enantiomeric excess of chiral species obtained during enantioselective hydrolyses of meso-diesters.
In the case studied, desymmetrization produces a mixture of 1 and ent-1, enriched in 1. This
mixture would further undergo kinetic resolution catalyzed by the same enzyme system. Here an
enzyme having pro-R selectivity would preferentially catalyze hydrolysis of ent-1 to the
corresponding diol, thereby enhancing the optical purity of the remaining monoacetate 1. This
was demonstrated by carrying out the hydrolysis reaction for various time intervals and by
checking the yield and optical purity of product 1 (Table 5). Thus, at 26 h product 1 was obtained
in >98% optical purity with 74% isolated yield.
Table 5
E€ect of reaction time on the hydrolysis of 2 catalyzed by NCIM 3326 in 0.1 M (pH 7) bu€er with 10% v/v ethanol
Before scaling-up the process, it was necessary to optimize the substrate concentration
tolerated by the enzyme. We found that a substrate concentration of 3.33% w/v was well toler-
ated by enzyme whereas at concentrations above this enzyme inhibition was observed. Thus, the
process was successfully scaled-up to the 100 g scale using 3.33% substrate concentration and
product 1 of greater than 98% optical purity was isolated in 74% yield. It was further converted
to 4-(R)-t-butyldimethylsilyloxycyclopent-2-en-1-one 3 by known chemical transformations3b,8
and its ee was determined to be >99% by chiral HPLC.
Thus, our study has demonstrated Trichosporon beigelii (NCIM 3326),10 a new very e€ective
biocatalyst, as a signi®cantly cheaper alternative to PLE for hydrolysis of meso-diacetate 2 to the
expensive intermediate 1 in excellent optical purity. The study has also demonstrated the
importance of medium-engineering approach for optimization of enzymatic process for an
important chiral drug intermediate which can be scaled up.

U. R. Kalkote et al. / Tetrahedron: Asymmetry 11 (2000) 2965±2970
2969
3. Experimental
3.1. General
Optical rotations were recorded on a Jasco Dip-181 digital polarimeter using sodium vapor
lamp. Optical purities (op) were determined by comparing the speci®c rotation value [ꢀ]D
with the literature values. All the reagents were of LR quality and were used without further
puri®cation.
3.2. General procedure for cell biomass preparation (whole cell enzyme)
The inoculum (5±10 ml) was developed by growing the microorganisms in a medium containing
malt extract (0.3%), glucose (1%), yeast extract (0.3%) and peptone (0.5%) at pH 6.6±7.0 for 48
h with shaking at 150±180 rpm. This inoculum was transferred to medium containing K2H2PO4
.
(0.2%), yeast extract (0.1%), peptone (0.5%), KCl (0.05%), NaNO3 (0.05%), MgSO4 7H2O
(0.05%), olive oil (1%) at pH 5.5 and incubated at 28±30oC for 36±48 h on rotary shaker (180±
200 rpm). The grown cells were separated by centrifugation and the wet biomass was used for the
reaction.
3.3. General procedure for enantioselective hydrolysis of meso-cyclopent-2-en-1,4-diacetate 2 to 4-
(R)-hydroxycycloent-2-en-1-(S)-acetate 1
meso-Diacetate 2 (100 g, 0.543 mol) was dissolved in ethanol (300 ml) in a 5 l three-necked
round-bottomed ¯ask equipped with an overhead stirrer, pH electrode and a dropping funnel. To
the solution 0.1 M sodium phosphate bu€er (pH 7, 2.7 l) was added and the mixture stirred
vigorously using the overhead stirrer. To the stirred reaction mixture wet biomass (50 g) of
Trichosporon beigelii (NCIM 3326) was added with a small amount of bu€er. The whole mixture
was stirred vigorously to yield a uniform emulsion. The pH of the reaction was monitored
regularly and was maintained at 7 by adding 1 M aqueous sodium hydroxide solution through a
dropping funnel. Progress of the reaction was monitored by TLC (30% ethyl acetate in pet.
ether). Reaction was continued for 26 h. Then the reaction mixture was ®ltered through a Celite
bed and the ®ltrate was extracted with ethyl acetate (3Â2 l). The organic extracts were combined
and washed with brine. The aqueous layer was separated and the organic layer was dried on
anhydrous sodium sulfate. The solvent was removed on a rotary evaporator and dried at high
vacuum. Product 1 was obtained as white crystalline needles. It was melted on a water bath and
was stirred vigorously with pet. ether. The mixture was allowed to stand at room temperature for
a few hours. Product 1 separated as ®ne crystalline needles. Pet. ether was decanted o€ and
washing with pet. ether was repeated once again. These washings removed any unreacted
diacetate 2 and other non-polar impurities. Yield of 1 was 57.40 g (74%). [ꢀ]D=^68.9 (c 1,
CHCl3), lit.11 [ꢀ]D=^69.3 (c 1, chloroform) optical purity >98%.
Acknowledgements
S.R.G. is thankful to CSIR, New Delhi for ®nancial support.

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