Enzymatic kinetic resolution studies of racemic 4-hydroxycyclopent-2-en- 1-one using Lipozyme IM

Article abstract of DOI:10.1016/S0957-4166(99)00445-0

Enzymatic kinetic resolution studies of (±)-4-hydroxycyclopent-2-en-1- one 2 were taken up in organic solvents by transesterification with vinyl acetate and alcoholysis of its acetate 3 as an alternative to the desymmetrization of meso-cyclopentenediol to provide faster and economic access to enantiomerically pure 4-(R)-tert-butyldimethylsilyloxycyclopent-2- en-1-one 1. Parameters were screened using Lipozyme IM as catalyst. Although enantioselectivity observed was moderate (E=24, by alcoholysis of 3 with 2-butanol), trends in the effect of solvent, water content and alcohol structure showed useful directions for screening of other enzymes for optimization of the method to useful levels of efficiency.

Full text of DOI:10.1016/S0957-4166(99)00445-0

Pergamon
Tetrahedron: Asymmetry 10 (1999) 4115–4122
Enzymatic kinetic resolution studies of racemic
4-hydroxycyclopent-2-en-1-one using Lipozyme IM®
Sandeep R. Ghorpade,a Kulbhushan B. Bastawade,b Digambar V. Gokhale,b Popat D. Shinde,a
Vishal A. Mahajan,a Uttam R. Kalkote a,∗ and T. Ravindranathan a,∗
aDivision of Organic Chemistry: Technology, National Chemical Laboratory, Pune 411 008, India
bNCIM, Biochemical Sciences Division, National Chemical Laboratory, Pune 411 008, India
Received 30 August 1999; accepted 30 September 1999
Abstract
Enzymatic kinetic resolution studies of (ꢀ)-4-hydroxycyclopent-2-en-1-one 2 were taken up in organic sol-
vents by transesterification with vinyl acetate and alcoholysis of its acetate 3 as an alternative to the desym-
metrization of meso-cyclopentenediol to provide faster and economic access to enantiomerically pure 4-(R)-
tert-butyldimethylsilyloxycyclopent-2-en-1-one 1. Parameters were screened using Lipozyme IM® as catalyst.
Although enantioselectivity observed was moderate (E=24, by alcoholysis of 3 with 2-butanol), trends in the
effect of solvent, water content and alcohol structure showed useful directions for screening of other enzymes
for optimization of the method to useful levels of efficiency. © 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction
The importance of 4-(R)-tert-butyldimethylsilyloxycyclopent-2-en-1-one 1 in the synthesis of cyclo-
pentanoid natural products (prostaglandins, prostacyclins, thromboxane and nucleosides)1–4 has attracted
the attention of chemists and has resulted in development of various methods for its preparation, viz.: (i)
desymmetrization of meso-cyclopentenediol or meso-cyclopent-2-endiacetate using lipases and followed
by chemical conversion;5–7 (ii) classical resolution of racemic 4-hydroxycyclopent-2-en-1-one 2 by
forming diastereomers with enantiomerically pure caronaldehyde followed by separation;8 (iii) kinetic
resolution of 2 using lipases9 and chiral catalyst;10 and (iv) synthesis from D-tartaric acid.11 Even
though a substantial amount of literature is available on the subject, since there is increasing demand
for prostaglandins and their intermediates due to their varied biological activities, development of cost
effective methods for their large-scale preparation has been the major goal for our group.
∗
Corresponding authors. E-mail: kalkote@dalton.ncl.res.in
0957-4166/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(99)00445-0

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Desymmetrization methods are an attractive and extensively studied (including two methods developed
by our group5b,6j) approach for the production of 1 since a 100% yield of one of the enantiomers
can be obtained theoretically whereas kinetic resolution suffers from the drawback of throwing away
half of the material, i.e. the unwanted isomer. But for the large-scale production, the desymmetrization
method becomes costly because preparation of the required meso-cyclopentene derivatives involves either
expensive and sensitive Pd(0) complexes as catalysts12 or photochemical reactions13 wherein yields are
low and scale-up is difficult. In addition, the method involves three to five additional chemical steps
wherein tert-butyldimethylsilyl chloride, which is a major cost component, is used at an early stage
of the synthesis.7 Comparatively, direct resolution of racemic 4-hydroxycyclopentenone 2 would be
an attractive alternative as the large-scale preparation of 2 is easier.14 We have already scaled up the
process using furfuryl alcohol to 3 kg scale. Also, there are literature reports wherein (S)-2 has been
inverted to (R)-2 by simple chemical steps.15 This resolved (R)-2 can then be directly converted to 1
using expensive tert-butyldimethylsilyl chloride as the silylating agent at the last step, saving on the
cost. The classical resolution approach involving caronaldehyde as the resolving agent is discouraging
on the large scale due to the exorbitant high cost of caronaldehyde. Another successful approach involves
kinetic resolution by enantioselective hydrogenation using chiral catalyst developed by Noyori et al.10
Although lipase-catalyzed resolution of (ꢀ)-2 would be an economical approach, very few reports are
available on the lipase-catalyzed kinetic resolution by hydrolysis of (ꢀ)-3 in aqueous media. The limiting
factors of the approach are mainly: (i) high solubility of 2 in aqueous medium makes its isolation tedious,
and requires continuous extraction with ethyl acetate for 3 days; and (ii) possibility of racemization in
aqueous media which would deteriorate enantiopurity at least to some extent. Winterfeldt et al. have
overcome the problem of racemization by derivatizing (ꢀ)-2 to a cyclic ketal prior to resolution.9b But
this protection step is very tricky and low yielding, hence not suitable to scale-up. These problems can be
circumvented by the judicious use of organic solvents for the conversion. But to our knowledge such an
attempt is not reported. Therefore, we decided to study the lipase-catalyzed kinetic resolution of 2 and 3
in organic solvents. Herein we report our preliminary results on: (i) Lipozyme IM®-catalyzed irreversible
transesterification of (ꢀ)-2 in organic solvents; and (ii) Lipozyme IM®-catalyzed alcoholysis of (ꢀ)-3 in
organic solvents.
2. Results and discussions
2.1. Transesterification of (ꢀ)-2 with vinyl acetate catalyzed by Lipozyme IM®
Transesterification of (ꢀ)-2 was attempted using vinyl acetate as acyl donor and lipozyme IM® as
catalyst (Table 1). Since the enantioselectivity of the enzyme is higher at lower temperatures, reactions
were carried out at 12°C. The initial experiment in dry diisopropyl ether (DIPE) exhibited high reaction
rates (80% conversion (c) in 4 h) but the enantioselectivity of the reaction was rather poor (E=4, entry
no. 1, Table 1).
Since an intrinsic water content (more precisely, the water activity, aw) as low as 1% is reported to

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Table 1
Transesterification of (ꢀ)-4-hydroxycyclopentenone 2 with vinyl acetate catalyzed by Lipozyme IM®
in various solvents at 12°Ca
be beneficial for enzyme selectivity in several cases,16 the conversion was attempted in diisopropyl ether
containing 1% water. Now the reaction rate was much slower (c=28% in 19 h, entry no. 2, Table 1), the
enantioselectivity improved significantly (E=11) by 2.8 times. Such kinds of beneficial effects of water
content were reported previously in a few cases; the reason, though not very clear, was attributed to an
increase in enzyme flexibility by added water in a dry system. The reaction was attempted in a few other
solvents containing 1% water (entry nos. 3–5, Table 1), but an E value of more than 11 could not be
achieved.
The effect of an acyl donor on the enantioselectivity of lipase-catalyzed transesterification reaction has
been well demonstrated by Ema et al.17 Therefore, we tested a few other acyl donors for the conversion.
With isopropenyl acetate the enantioselectivity as well as rate of reaction were poor (c=39.2% in 1 week,
E=3, entry 6, Table 1). Addition of 1% water did not prove to be beneficial, but rather inhibited reaction.
Reaction did not proceed with acyl donors such as isopropyl acetate, ethyl acetate, etc. (Table 1).
Thus, the enantiodiscrimination in kinetic resolution of (ꢀ)-2 with vinyl acetate using Lipozyme IM®
as catalyst was not satisfactory. The highest value of enantiomeric ratio obtained was 11 which is not

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Table 2
Preliminary screening of various alcohols in different solvents at 12°C for the alcoholysis of (ꢀ)-4-
oxocyclopenten-2-yl acetate 3
good enough for practical purposes. Therefore, the resolution of (ꢀ)-3 was planned by alcoholysis with
different alcohols in organic solvents using Lipozyme IM®.
2.2. Alcoholysis of (ꢀ)-4-oxocyclopenten-2-yl acetate 3
Alcoholysis of 3 was attempted initially in toluene using various alcohols (Table 2).
The rates of the alcoholysis reactions were good enough, but the enantioselectivity was not satisfactory
(E=3–5). Among all alcohols investigated, 2-propanol, which is a moderately hindered secondary
alcohol, reacted much more slowly (c=29.3% in 45 h, entry 5, Table 2) in toluene, but when DIPE and
methyl isobutyl ketone (MIBK) were used as solvents, reaction rates improved considerably (c=31.5% in
15 h and c=42.5% in 15 h, resp., entries 6 and 7, Table 2) with some improvement in enantioselectivity
(E=6 and 4, resp.).
We observed that when 2-propanol containing some water was used for alcoholysis, the rate improved
considerably with some improvement in the E ratio as compared to the anhydrous reaction. Subsequently,
the effect of water content was studied in DIPE using 2-propanol for alcoholysis (Table 3). A water
content of 1% was found to enhance the reaction rates as well as the enantioselectivity considerably.
Thus, 37.3% conversion was achieved with an E value of 17 (entry 3, Table 3), i.e. a 3-fold increase in
enantioselectivity at 1% water content. To study further the water effect, several reactions were conducted
using different alcohols in different solvents containing 1% water (Table 4). For 2-propanol reaction
(entries 5 and 7, Table 4), enantioselectivity was maximum in DIPE (E=17) and dibutyl ether (DBE)
(E=19). Beneficial effects of water were observed with most other alcohols.
Enantioselectivity is found to be associated with the alcohol structure. Thus, an E value of >10 was

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Table 3
Effect of water content on the alcoholysis of (ꢀ)-4-oxocyclopenten-2-yl acetate 3 by 2-propanol
catalyzed by Lipozyme IM® in diisopropyl ether at 12°C
Table 4
Alcoholysis of (ꢀ)-4-oxocyclopenten-2-yl acetate 3 with various alcohols in different organic solvents
containing 1% water at 12°C

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observed with all secondary alcohols, 2-butanol (entry 9, Table 4) showing the best results (E=24).
Such kinds of influence of alcohol structure on the enantioselectivity of enzymes during alcoholysis are
somewhat analogous to the effect of the structure of the acyl donor on enzyme enantioselectivity in trans-
esterification reactions studied by Ema et al.17 Thus, moderate steric hindrance provided by 2-butanol
is most suitable for the enzyme active site during the reaction, giving the highest enantiodiscrimination.
Further decrease or increase in the hindrance by addition or deletion of -CH3 groups deteriorates the
enantiodiscrimination in the case studied. With an E value of approx. 25 substrate of >95% ee can be
obtained at 60% conversion whereas product of 85% ee can be obtained near 40% conversion (vide infra
Eq. 1 and Adachi et al.18).
3. Conclusion
We could resolve (ꢀ)-2 in moderate enantioselectivity (E=24) by alcoholysis of its acetate (ꢀ)-3 in
organic solvents using commercially available enzyme Lipozyme IM®. Our study demonstrates strong
effects of solvent, alcohol structure and water content on the enzymatic alcoholysis reaction in organic
media. Even though the enantioselectivity obtained is not very high, this study has set the guidelines for
further optimization work. Currently we are investigating some other commercial lipases and in-house
cultures from the National Collection of Industrial Microorganisms, NCL, Pune for the conversion to
achieve high enantioselectivity and to get the (R)-enantiomer 1 in high enantiomeric excess. We hope
that this methodology would provide a more economic alternative to the desymmetrization method for
the large-scale production of 1.
4. Experimental
4.1. General
Optical rotations were recorded on a Jasco Dip-181 digital polarimeter using sodium vapor lamp.
Enantiomeric excesses (ee) were determined by comparing the specific rotation value [α]D with the
literature value. All reagents were purchased from Aldrich and were used without further purification.
Solvents used were of LR quality and were dried before use by standard methods. Lipozyme IM® was
obtained as a gift sample from Arun & Co., Mumbai. Enantiomeric ratio E was calculated using the
following formula (Eq. 1),18 where c, ees and eep are conversion, enantiomeric excess of substrate and
enantiomeric excess of product, respectively.
(1 − c)(1 − ees)
E =
(1)
(1 − c)(1 + ees)
ees
conversion : c =
ees + eep
4.2. Typical laboratory procedure for preparation of (ꢀ)-4-hydroxycyclopent-2-en-1-one 2
A 3 L three-necked round bottomed flask, equipped with long air condenser, thermometer pocket and a
bubbler, was charged with furfuryl alcohol (25 g, 0.255 mol), potassium dihydrogen orthophosphate (6.3
g, 0.022 mol) and distilled water (1.5 L). The reaction mixture was purged with a slow stream of nitrogen
along with magnetic stirring. It was heated to 95°C for 48 h while maintaining effective stirring and

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4121
nitrogen purge. The solution developed brownish insoluble impurities during reaction. It was cooled to
room temperature and then washed twice with ethyl acetate. The aqueous layer was concentrated almost
to dryness under reduced pressure. The residue was then thoroughly extracted with ethyl acetate. The
combined organic extracts were then dried on anhydrous sodium sulfate and concentrated under vacuum.
The residue was distilled under high vacuum using a fractionating column. Product 2 was obtained as a
lemon yellow colored liquid distilling at 95–100°C at 0.5 mm vacuum. Yield=10 g (40%). Compound 2
was stored in a refrigerator (ca. −5°C).
4.3. Typical laboratory procedure for preparation of (ꢀ)-4-oxocyclopenten-2-yl acetate 3
Into a 100 mL two-necked round bottomed flask equipped with a two-way stopcock and a dropping
funnel (ꢀ)-2 (5 g, 51 mmol) was placed. The assembly was evacuated and flushed with argon. Dry
dichloromethane (50 mL) was added and the solution was cooled below 0°C using an ice-salt bath
along with magnetic stirring. To the cold solution dry pyridine (7.9 g, 0.1 mol) was added and stirred
at 0°C for 10 min. To the stirred solution, acetic anhydride (7.8 g, 76.5 mmol) was added dropwise while
maintaining the temperature below 0°C. The reaction mixture was stirred at room temperature for 15 h,
then quenched by adding cold, dilute hydrochloric acid. The organic layer was washed four times with
a 1:4 mixture of brine and dilute hydrochloric acid, followed by washing with cold water, 10% sodium
bicarbonate solution and finally with brine. The organic layer was dried over anhydrous sodium sulfate
and concentrated under vacuum. The residue was distilled under high vacuum. Product 3 was obtained
as a colorless liquid (b.p. 59–61°C at 0.2 mm vacuum) which solidified as colorless crystals on cooling.
Yield=5.71 g (80%).
4.4. General procedure for enzymatic transesterification of (ꢀ)-2
Compound (ꢀ)-2 (1.5 mmol, 0.150 g) was stirred with 5 equiv. acyl donor and Lipozyme IM® (0.3 g) in
10 mL organic solvent at 12°C. The reaction was monitored by TLC. At sufficient conversion the reaction
mixture was filtered, dried on anhydrous sodium sulfate and the solvent was removed under vacuum. The
residue was treated with tert-butyldimethylsilyl chloride (1.5 mmol, 0.225 g), p-dimethylaminopyridine
(0.15 mmol, 0.018 g) and triethylamine (2 mmol, 0.2 g, 0.28 mL) under an argon atmosphere in dry
dichloromethane at 0°C. The reaction mixture was stirred at room temperature for a further 3 h. It was
then washed with cold dil. hydrochloric acid followed by a water wash, bicarbonate wash and finally a
brine wash. Organic layer was dried on sodium sulfate and solvent was removed under vacuum. This
derivatized residue contained OTBDMS derivative 1 and acetate 3. These were separated on a silica gel
column using pet. ether and ethyl acetate (gradient elution).
4.5. General procedure for enzymatic alcoholysis of (ꢀ)-3
Compound (ꢀ)-3 (1.5 mmol, 0.2 g) was stirred with 5 equiv. alcohol and Lipozyme IM® (0.3 g) in 10
mL organic solvent at 12°C. The progress of reaction was monitored by TLC. At sufficient conversion
the reaction mixture was filtered, the solvent was removed under vacuum and the residue was treated as
described in Section 4.4.

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Acknowledgements
The authors are grateful to Arun & Co., Mumbai for a gift sample of Lipozyme IM®. The authors
are grateful to Dr. A. B. Sahasrabuddhe, OCT, NCL for his help in the preparation of (ꢀ)-2. S.R.G. is
grateful to CSIR, New Delhi for financial support.
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