Desymmetrization of meso-cyclopenten-cis-1,4-diol to 4-(R)- hydroxycyclopent-2-en-1-(S)-acetate by irreversible transesterification using Chirazyme

Article abstract of DOI:10.1016/S0957-4166(99)00068-3

The parameter optimization study for the desymmetrization of meso- cyclopenten-1,4-diol 1 through irreversible transesterification using an immobilized lipase from Mucor meihei, i.e., Lipozyme/Chirazyme is presented. The enzyme was studied for the transesterification of 1 in various organic solvents by varying reaction parameters such as the nature of acyl donor, temperature, enzyme quantity etc., to afford optically active 4-(R)- hydroxycyclopent-2-en-1-(S)-acetate 2 of >98% enantiomeric excess in >60% yield.

Full text of DOI:10.1016/S0957-4166(99)00068-3

Pergamon
Tetrahedron: Asymmetry 10 (1999) 891–899
Desymmetrization of meso-cyclopenten-cis-1,4-diol to
4-(R)-hydroxycyclopent-2-en-1-(S)-acetate by irreversible
transesterification using Chirazyme®
Sandeep R. Ghorpade, Rajendra K. Kharul, Rohini R. Joshi, Uttam R. Kalkote ∗ and
T. Ravindranathan ∗
Division of Organic Chemistry, Technology, National Chemical Laboratory, Pune 411 008, India
Received 7 January 1999; accepted 16 February 1999
Abstract
The parameter optimization study for the desymmetrization of meso-cyclopenten-1,4-diol 1 through irreversible
transesterification using an immobilized lipase from Mucor meihei, i.e., Lipozyme®/Chirazyme® is presented. The
enzyme was studied for the transesterification of 1 in various organic solvents by varying reaction parameters such
as the nature of acyl donor, temperature, enzyme quantity etc., to afford optically active 4-(R)-hydroxycyclopent-2-
en-1-(S)-acetate 2 of >98% enantiomeric excess in >60% yield. © 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction
The importance of 4-(R)-hydroxycyclopent-2-en-1-(S)-acetate 2 in the synthesis of biologically active
cyclopentenoid natural products,1 e.g., prostaglandins, prostacyclins and thromboxanes, has attracted the
attention of synthetic chemists. This has resulted in a variety of approaches in its preparation.2 Enzyme
viz. lipase/esterase-catalyzed desymmetrization of meso-cyclopenten-1,4-diol 1 by transesterification,3
or by enzymatic hydrolysis of its diacetate,4 or kinetic resolution of monoprotected cyclopentenediol5
appear to be the preferred methods of choice in the preparation of enantiomerically pure 2. A kinetic
resolution suffers from the drawback of throwing half of the material away, whereas, by using the
desymmetrization method, 100% yield can be obtained theoretically. In the case of desymmetrization of 4
through enzymatic hydrolysis, most of the efficient enzymes reported, with the exception of PLE,4c have
pro-S preference. Although high enantiomeric excess and good yields are achievable for the enantiomer
of 2 by the hydrolysis method, manipulation of this (4S)-hydroxy enantiomer through a few chemical
steps is required to get the desired (4R)-hydroxy configuration.6 The same enzymes can catalyze the
∗
Corresponding author. E-mail: kalkote@dalton.ncl.res.in
0957-4166/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(99)00068-3

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transesterification of 1 to yield 2. Various enzymes have been attempted for the conversion using a
variety of irreversible acyl donors, generally in the THF–Et3N system.7 Monoacylated products with a
very high enantiomeric purity have been obtained in many cases in 50–60% yield along with remarkable
quantities of the diacylated product 4 indicating a low selectivity in the first acylation step which was then
compensated for in the second step. Unfortunately, the lipase from the Mucor species, which gave the best
results (yield 85%, ee >98%),7c,d is not available commercially,3 whereas the lipase from Mucor meihei,
i.e., Lipozyme IM®/Chirazyme®, which is available commercially in bulk, was found to be inefficient
for the conversion (yield <5%) under the conditions attempted by the authors (THF–Et3N).7d
Considering the good market demand for the high-cost intermediate 2, development of economically
viable enzymatic technology for its large-scale preparation has been the major goal of our group. We have
already reported on the enantioselective hydrolysis of meso-diacetate 4 to 3 using yeast (NCIM 3574).8
Enhancement of enzyme efficiency through medium engineering, i.e., optimization of the solvent
system and optimization of other parameters such as pH, temperature, etc., has been well reported in
several cases.9 The availability of Chirazyme® in bulk prompted us to consider the study of enhancement
in its selectivity towards desymmetrization of 1 by transesterification through medium engineering and
optimization of other parameters.
2. Results and discussion
2.1. Effect of solvent variation
The effect of solvent on activity and specificity of various enzymes has been well documented
in the literature.9,10 Attempts have been made to derive correlations between enantioselectivity and
physicochemical characteristics of the solvent such as hydrophobicity or dielectric constant etc.10,11
Initially, we carried out transesterification of meso-diol 1 with vinyl acetate in various organic solvents
using Chirazyme® as catalyst. The results are indicated in Table 1. Although we cannot conclude on
correlation of enzyme efficacy and physicochemical properties of the solvent with available data, the
nature of the solvent was found to have a profound effect on enzyme efficacy. In general, enzyme activity
was good in all the ether solvents tested. Enzyme efficacy in terms of yield and enantiopurity of product
2 was maximum in diethyl ether (entry 5, yield 45%, enantiomeric excess 88%) and tert-butyl methyl
ether (TBME) (entry 7, yield 35%, ee 93%). The reaction in ethyl acetate and butyl acetate afforded 2
in high enantiomeric excess, but in lower yields (entries 8 and 13, respectively). In the case of THF and
acetonitrile, although the second acylation step was inhibited, the enantioselectivity of the monoacylation
step was rather poor. TBME was found to have a potentiating effect on enzyme activity, thus exhibiting
fast reaction rates (2 h only) and good enantioselectivity (ee of 2=93%).

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893
Table 1
Various solvents attempted using vinyl acetate as acyl donora
2.2. Effect of an acyl donor
The effect of an acyl donor on enantioselectivity of the lipase-catalyzed transesterification reaction has
been well demonstrated by Ema et al.12 In order to study the effect of an acyl donor, we carried out the
reaction with different acyl donors (Table 2). The reaction proceeded slowly with low enantioselectivity
with the acyl donors, except for vinyl acetate. The inefficient reaction with isopropenyl acetate may be
attributed to steric reasons. Thus, vinyl acetate was the only suitable acyl donor for the conversion and
was used for further parameter optimization.
2.3. Effect of temperature
It is widely believed that enzymes, like other catalysts, generally exhibit their highest selectivity at
low temperatures. This assumption has been supported by several experimental observations, not only
with hydrolases9 but also with dehydrogenase. Sakai et al., in their lipase PS-catalyzed kinetic resolution
studies,13 examined temperature variation effects ranging from 30 to −60°C on the enantioselectivity of

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Table 2
Other acyl donorsa
the reaction and have found a linear increase in enantioselectivity up to −40°C. Therefore, we studied
the reaction at lower temperatures in several solvents (Table 3). Lower temperatures were found to have
a very much more beneficial effect on enzyme efficacy in terms of chemical yields and enantiopurity of
products. Here again, diethyl ether and TBME turned out to be the best choice, affording 2 of >95% ee
in 65% and 52% yield, respectively (entries 3 and 6) at 4°C. Again, reaction rates were much faster in
TBME than in any other solvent. Next, we turned our attention to optimization of the ratio of lipase to
substrate and ratio of acyl donor to substrate.
2.4. Ratio of enzyme to substrate
Table 4 indicates a surprising result showing that a reduction in enzyme to substrate ratio from 2:1
to 1:1 has a beneficial effect in both solvents, thus affording product 2 of >99% ee in >40% yield for
both solvents (entries 2 and 7). But at lower temperatures, the yields and ees dropped in ether with
a 1:1 enzyme:substrate ratio (entry 3); a reason may be that the reaction is much slower at the lower
temperature with lower amounts of the enzyme, as indicated by lower yields of monoacetate 2 as well as
diacetate 4; whereas reaction in TBME with a 1:1 enzyme:substrate ratio at 4°C afforded 2 of >98% ee
in >60% yield (entry 9).
2.5. Ratio of vinyl acetate to substrate
The quantity of vinyl acetate acyl donor was varied from 5 equiv. to 1.5 equiv., using ether and TBME
as the solvent with 0.1 g of enzyme. Table 5 indicates that a minimum of 5 equiv. of acyl donor is

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895
Table 3
Variations in temperaturea
required for an efficient reaction. The reason may be explained as follows. The first acylation, which is
the desymmetrization step, would require some excess of acyl donor. The pro-S selectivity of the enzyme
is not very high; thus 2 and 3 are both formed in unequal quantities in the first step, 2 being the major one.
The second acylation is a kinetic resolution where the enzyme is still more selective for the pro-S-OH
group; therefore, it acylates 3 much faster than 2, thus enriching the enantiomeric excess of 2. Thus, to
have 2 with a desired enantiomeric excess of >98%, formation of diacetate 4 in a sufficient quantity is
required which in turn demands an excess quantity of acyl donor.
Previously, 2 has been prepared by the kinetic resolution of monoprotected cyclopentendiol using
pancreatin and Lipozyme IM® in 48% and 37% yield, respectively.5 Our results are superior to these as
we have obtained 2 of >98% ee in 64% yield (entry 9, Table 4); also extra protection–deprotection steps
are avoided, thus avoiding further losses in overall yield.
2.6. Effect of various additives
Addition of certain additives such as water, amines, DMF and DMSO in small percentages have been
reported to improve selectivity of hydrolytic enzymes in several cases.14 Especially the intrinsic water
content (more precisely, the water activity, aw) has been found to have an influence on the enzyme

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Table 4
Variations in enzyme:substrate ratioa
Table 5
Variations in ratio of vinyl acetatea

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897
Table 6
Effect of various additivesa
selectivity in several cases.10,14g,15 Our results with various additives in the reaction are presented
in Table 6. Unfortunately, none of the additives attempted were found to have any beneficial effect
on enzyme efficacy, on the contrary yields and ee had badly deteriorated in most of the cases. The
commercial enzyme preparation has a 2–3% moisture content which seems to be optimal in this case.
Added extra water was found to be detrimental to the reaction.
3. Conclusion
Desymmetrization of 1 has been demonstrated successfully through irreversible transesterification
using Chirazyme® by a parameter optimization approach. Thus meso-cyclopentene-1,4-diol was mo-
noacylated with vinyl acetate in the presence of Chirazyme® in TBME at 4°C to afford 2, an important
prostaglandin intermediate in >60% yield with >98% ee. Further studies regarding enzyme recycling
and scale-up of the process are in progress. The results indicate a strong possibility of exploitation of
Chirazyme® for the development of economically-viable technology for large-scale production of 2.
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

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literature value. All the solvents and reagents were of LR quality and used without further purification.
Chirazyme® was obtained as a gift sample from Boehringer Mannheim, Germany.
4.2. Typical example of desymmetrization experiment
In a typical experiment, meso-diol 1 (0.1 g, 1 mmol), vinyl acetate (0.430 g, 5 mmol, 0.46 mL) in 5 mL
TBME was stirred at 4°C for half an hour. Chirazyme® (0.1 g) was added to the reaction mixture. The
mixture was stirred at 4°C for 4.75 h. Filtered solvent was evaporated under reduced pressure. Residue
was chromatographed on silica gel (No. 60-120) column using ethyl acetate/petroleum ether as an eluent
to separate diacetate 4 (yield=0.051 g, 28%) and monoacetate 2 (yield=0.091 g, 64%; [α]D=−68.1 (c 1,
CHCl3), ee >98%, lit.7f −69.3 (c 1, CHCl3), ee >99%).
Acknowledgements
The authors are thankful to Boehringer Mannheim, Germany for the gift sample of Chirazyme®.
S.R.G. and R.K.K. are thankful to CSIR, New Delhi for financial support.
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