Preparation of (1R,4S)-4-hydroxycyclopent-2-en-1-yl acetate via Novozym-435 catalyzed desymmetrization of cis-3,5-Diacetoxy-1-cyclopentene

Article abstract of DOI:10.1016/j.tet.2018.09.052

Photooxidation of cyclopentadiene has been carried out in methanol using white light of LED lamp, rose bengal as photo initiator, and compressed air at 0 °C. Under conditions of [thiourea] ? [cyclopentadiene], the consumption of thiourea follows a pseudo-first-order reaction kinetics with half life of 75 ± 10 min; corr. coeff. r = 0.989. Slow addition of the monomer and maintaining excess thiourea concentration in reaction mass improves the yield. cis-3,5-Dihydroxy-1-cyclopentene is acetylated without isolation to obtain cis-3,5-Diacetoxy-1-cyclopentene of high purity (>99%) with overall isolated yield of 30%. Desymmetrization of the diacetate to (1R,4S)-4-hydroxycyclopent-2-en-1-yl acetate has been carried out via enzymatic transesterification with methanol in methyl tert-butyl ether (MTBE) at 5 °C using Novozym-435. The enantiomerically pure monoacetate (e.e. >99%) was obtained in 95% isolated yield. The recovered enzyme was reused for more than 10 times without loss in yield and selectivity. The entire protocol does not require purification of final product by chromatography.

Full text of DOI:10.1016/j.tet.2018.09.052

Accepted Manuscript
®
Preparation of (1R,4S)-4-hydroxycyclopent-2-en-1-yl acetate via Novozym-435
catalyzed desymmetrization of cis-3,5-Diacetoxy-1-cyclopentene
Shekhar Putta, Annem Mallikarjun Reddy, Gurrala Sheelu, B.V. Subba Reddy,
Thenkrishnan Kumaraguru
PII:
S0040-4020(18)31163-3
10.1016/j.tet.2018.09.052
TET 29824
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 3 July 2018
Revised Date: 26 September 2018
Accepted Date: 27 September 2018
Please cite this article as: Putta S, Reddy AM, Sheelu G, Reddy BVS, Kumaraguru T, Preparation
®
of (1R,4S)-4-hydroxycyclopent-2-en-1-yl acetate via Novozym-435 catalyzed desymmetrization of
cis-3,5-Diacetoxy-1-cyclopentene, Tetrahedron (2018), doi: https://doi.org/10.1016/j.tet.2018.09.052.
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ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
Preparation of (1R,4S)-4-Hydroxycyclopent-2-en-1-yl Acetate via
®
Novozym-435 Catalyzed Desymmetrization of cis-3,5-Diacetoxy-
1
-cyclopentene.
a,b
a
b
a
Putta Shekhar , Annem Mallikarjun Reddy , Gurrala Sheelu , B.V. Subba Reddy ,
b*
Thenkrishnan Kumaraguru
a
Department of Fluoro-Agrochemicals, CSIR-Indian Institute of Chemical Technology,
Uppal Road, Habsiguda, Hyderabad 500007. India.
b
Department of Organic Synthesis & Process Chemistry, CSIR-Indian Institute of Chemical
Technology, Uppal Road, Habsiguda, Hyderabad 500007. India.
Tel. 91-40-27191630, Fax: 91-40-27160512. E-mail: kumaraguru@iict.res.in
ABSTRACT
Photooxidation of cyclopentadiene has been carried out in methanol using white light of LED
lamp, rose bengal as photo initiator, and compressed air at 0 °C. Under conditions of
[
thiourea] >> [cyclopentadiene], the consumption of thiourea follows a pseudo-first-order
reaction kinetics with half life of 75 ± 10 min; corr. coeff. r = 0.989. Slow addition of the
monomer and maintaining excess thiourea concentration in reaction mass improves the yield.
cis-3,5-Dihydroxy-1-cyclopentene is acetylated without isolation to obtain cis-3,5-Diacetoxy-
1
-cyclopentene of high purity (>99%) with overall isolated yield of 30%. Desymmetrization
of the diacetate to (1R,4S)-4-hydroxycyclopent-2-en-1-yl acetate has been carried out via
enzymatic transesterification with methanol in methyl tert-butyl ether (MTBE) at 5 °C using
®
Novozym-435 . The enantiomerically pure monoacetate (e.e. >99%) was obtained in 95%
isolated yield. The recovered enzyme was reused for more than 10 times without loss in yield
and selectivity. The entire protocol does not require purification of final product by
chromatography.
1

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Keywords: cyclopentadiene, photooxidation, thiourea, rose bengal, enzyme,
desymmetrization, transesterification, cis-3,5-Dihydroxy-1-cyclopentene.
1
. Introduction
Enantiomerically pure (1R,4S)-4-hydroxycyclopent-2-en-1-yl acetate 6 and its related
isomers are common motifs in prostaglandins, antibiotics such as viridenomycin and
1
-5
pentenomycin, or antitumor compounds such as neocarzinostatin. The required isomers can
6
7
be prepared via chiral pool, asymmetric reduction of hydroxycyclopent-2-enone, Pd-
8
9
catalyzed asymmetric allylic nucleophilic substitution, or metathesis of chiral diol. These
methodologies are very interesting but not easy to scale-up.
For large scale preparation, the preferred routes are based on meso-diol 4 as starting
1
0-16
point which is prepared by either photooxidation of cyclopentadiene (2)
or reduction of 3-
1
7-18
hydroxy cyclopentenone 8
(Scheme1). Previously, we had prepared the compound 8
1
9
starting with 7 in a microreactor, but it would need several such reactors to make large
quantities. Besides, the compound 8 is also unstable and highly water soluble. In comparison,
the photochemical approach was easier. Commercially available dicyclopentadiene 1 is
heated to 200-300 °C to prepare cyclopentadiene 2. Photochemically generated singlet
oxygen reacts with 2 to form an endo-peroxide 3 via a [4+2] cycloaddition reaction which is
1
0
in situ reduced to diol 4 with thiourea. The meso-diol is converted to enantiomerically pure
20-24
monoacetate 6 using enzyme catalyzed desymmetrization
(Scheme 1).
2
4
Selective acylation of meso-diol 4 using vinyl acetate or related derivatives or
1
4, 25-28
hydrolysis of the meso-diacetate 5
provide the monoacetate in good enantiomeric
purity and high yields (Scheme 1). Enzymes employed are lipase from Mucor
2
5
26
27
sp., pancreatin, Mucor miehei lipase (Chirazyme), Pseudomonas fluorescens lipase
2
8
® 26b
(
PFL), Candida rugosa lipase and lipase B from Candida antarctica (Novozym-435 ).
2

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2
9
30
Acetyl choline esterase from electric eel and recombinant pig liver esterase have also been
used for the reaction.
Scheme 1. Preparation of enantiomerically pure (1R,4S)-4-hydroxycyclopent-2-en-1-yl
acetate 6.
In our laboratory, we required rather large quantities (>100 g) of (1R,4S)-6 for our
research purpose and considering the ease of preparation, we chose the route based on
photooxidation of cyclopentadiene followed by enzymatic desymmetrization of diol 4 for
preparation of 6. According to available literature, the singlet oxygen is usually generated by
3
1-32
employing rose bengal as photo initiator. The preferred reaction medium is methanol
but
the reaction can be carried out efficiently in chlorinated solvents such as dichloromethane or
1
4
33
carbon tetrachloride where the life time of singlet oxygen is much longer. The reported
reaction temperatures ranging from -78° to + 25 °C with varied yields (20-90%). The
reaction is usually performed in a stirred tank glass reactor irradiated with halogen or xenon
34
lamp. A falling film microreactor has also been reported for the reaction but it is not
suitable for multigram scale synthesis. Recently this reaction has been carried out very
3
5
efficiently in liquid and supercritical carbon dioxide at 0 °C with excellent yield of 94%.
3
6-38
Such high yields were attributed to longer life time of singlet oxygen
and stability of the
endo-peroxide 3 over several minutes in this medium. However, the reaction was carried out
3

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at high pressure of 120 bar and requires specially designed dedicated equipment as well as
manpower skilled in handling such an equipment. Since our requirement did not warrant a
huge investment of money and manpower, we decided to perform the preparation of 5 via 4
photooxidation of cyclopentadiene 2 using rose bengal as initiator. Since solubility of
thiourea required for in situ conversion of the endo-peroxide to diol is very limited in
chloroform or dichloromethane, methanol was used as solvent. Although the current trend in
3
9
photochemical reactions is in favour of a continuous reactor our reaction work-up involves
removal of methanol from reaction mixture before acetylation of the diol 4 to get diacetate 5.
Since these operations are conveniently performed in a batch mode, we have studied
photooxidation of cyclopentadiene in a jacketed glass reactor.
Among all the enzymatic processes described above, desymmetrization of 5 catalyzed
®
by Novozym- 435 is very interesting since it is an immobilized form of Candida Antarctica
lipase B available on commercial scale. Moreover, preparation of (1R,4S)-6 in yields of over
1
4, 26b
9
6% with e.e. >98% have been reported.
This prompted us to explore this reaction in
more detail.
2
. Results and Discussion
After repeating the procedures reported in literature for photooxidation of
cyclopentadiene using a halogen lamp, we realized that conducting the reaction on a small
scale at -40 °C (1 g) was not difficult but on a larger scale, several problems were
encountered. After careful examination, we realized the following: a) the capacity of
temperature controller to maintain temperature diminished drastically as the reaction volumes
increased. Heat emitted by the halogen lamp compounded the problem; b) the monomer tends
to dimerize on long standing even at -20 °C and should be ideally used within few hours of
making it; c) the reaction requires a continuous stream of oxygen to be bubbled in the
reaction mixture. This causes the low boiling monomer (b.p. 41°C) getting carried over the
4

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vent resulting in low yields. Safety issues also needed consideration. The use of pure oxygen
and accumulation of endo-peroxide in the reactor is a dangerous combination. Present studies
were directed towards prevention of the possible hazards and obtain a fair yield of the
diacetate 5 on a preparative scale.
Cyclopentadiene monomer 2 was prepared by cracking dicyclopentadiene using
4
0
equipment similar to that described by Magnusson with modification. The dimer was added
4
1
slowly to hot silicone oil maintained at 200 °C and the monomer vapours were further
passed through a glass column filled with Raschig rings and heated to 300 °C. A downward
double walled condenser, maintained at 0 °C cooled the vapours and the monomer was
collected in a receiver maintained at -20 °C with acetone-dry ice. This arrangement avoided
wastage of the dimer and also allowed preparation of the monomer as and when required.
In the next stage, the photochemical reaction was performed in a 1 L glass reactor. We found
that compressed air served the purpose of supplying oxygen without any change in product
yield. This solved the problem of using pure oxygen in laboratory. Further experiments
showed that commercially available white LED lamps used as flood lights were as good as
halogen lamps for our purpose with advantage of not producing heat around the reactor. It
was now possible to maintain temperature around 0 °C inside the reactor. Finally, by slow
addition of the monomer to the reaction mixture (as a solution in 2-propanol) instead of one
time addition in the beginning, the loss of monomer through evaporation was minimized.
Interestingly, the reaction mass becomes turbid as the reaction proceeds indicating progress
of the reaction. The purity of cyclopentadiene monomer is also indicated by clarity of its
solution in 2-propanol. As the monomer starts dimerizing, the solution becomes cloudy.
Although these signs are only rough indications, we hope that they would be helpful to the
operator.
5

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2
.1. Effect of thiourea content on the reaction
Although there are several reports on the use of thiourea for hydrogenolysis of endo-
peroxides, very little is known about the mechanism of the reaction. Arifoglu and co-
4
2
13
workers have studied the reaction of hydrogen peroxide with thiourea in water by C-
NMR. They have suggested that thiourea dioxide 10 formed in the first step in turn can react
with a second molecule of hydrogen peroxide (endo-peroxide in our case) and further
decompose to sulfenic acid 11 and urea 12. This mechanism suggests consumption of half
mole of thiourea per mole of endo-peroxide. On the other hand, Spivey and co-workers have
suggested fragmentation of the sulfenate intermediate 9 to give the product diol 4 and
thiazirine 13. Ring opening of 13 followed by decomposition of nitrile sulphide 14 produces
43
cyanamide and elemental sulphur. This mechanism supports consumption of one mole of
thiourea per mole of endo-peroxide (Scheme 2).
Scheme 2. Effect of thiourea content on the reaction
We have studied the consumption of thiourea during the course of reaction. Reaction
was carried out with 0.2 moles of thiourea in methanolic solution and 0.18 moles of monomer
dissolved in 2-propanol were slowly added over 2 h. A slow stream of air was passed through
the reaction mass which was illuminated with 100 watts LED lamp. Samples were collected
at regular intervals and analysed for thiourea content by reverse phase HPLC. The curve for
6

ACCEPTED MANUSCRIPT
decrease in thiourea concentration (peak area) as a function of time is shown as curve A (Fig.
1
).
700000
600000
500000
400000
300000
200000
100000
B
A
0
50
100
150
200
250
300
350
Time (min)
Fig.1. (A) [Thiourea] = 0.2 moles (one-time addition), [rose bengal] = 0.5 g;
Cyclopentadiene] = 0.18 mole; Temp. 5 °C. Reaction volume 560 mL. (B) Initial [Thiourea]
0.1 mole followed by continuous addition of thiourea, total 0.2 moles. Total reaction
volume 760 mL.
[
=
Interestingly, the thiourea concentration decreases logarithmically and follows a
pseudo-first-order kinetics. The apparent pseudo-first-order rate constant was found to be
-1
0
.55 ± 0.07 h , half life 75 ± 10 min; Corr. coeff. r = 0.989. Surprisingly, only about 0.5
moles of thiourea were consumed per mole of cyclopentadiene used for the reaction as
evidenced from the concentration of thiourea in reaction mass at the beginning and end of
reaction. The isolated yield of diacetate 5 in this experiment was 6 g (18%) which was
somewhat better than our first experiment but still less than expected.
To understand the pathway towards formation of 4, the reaction mass was analyzed
for sulphur content since the mechanism suggested by Spivey and co-workers predicts
formation of 1 equivalent of elemental sulphur per mole of thiourea consumed. Only traces of
sulphur were found in the reaction mass suggesting that the reaction follows the mechanism
suggested by Arifoglu and co-workers.
7

ACCEPTED MANUSCRIPT
Although less than 0.5 equivalents of thiourea were consumed, the product yield was
low. Apart from possibilities of incomplete acetylation, dimerization of monomer, formation
of polymeric products etc., one major reason for the low yield of the reaction could be the
spontaneous decomposition of the endo-peroxide (3) to (Z)-3-(oxiran-2-yl)acrylaldehyde
3
5,43
1
6.
If a competition exists between formation of 16 and 4 from the endo-peroxide 3, the
rate of formation of 4 would decrease as thiourea in reaction mass is consumed. One way to
overcome the problem would be to maintain the thiourea concentration via a continuous
addition of thiourea to the reaction mass. Thus the reaction was performed by using two
dosing pumps. In the beginning, the reactor contained a solution of thiourea (0.1 mole) and
rose bengal in methanol. One dosing pump added the monomer solution at a rate of 0.5
mL/min while the second dosing pump added thiourea solution in methanol at a rate of 2
mL/min. At any given stage, the concentration of thiourea was always much higher than the
monomer. At the end of addition, a total of 0.2 moles of thiourea and 0.18 moles of
cyclopentadiene were added to the reaction mass. HPLC analysis showed that thiourea
concentration in reaction mass was maintained (curve B, Fig.1) but again, only half mole of
thiourea was consumed for one mole of monomer. However, above strategy lead to an
improved yield of 10.2 g (31%). Although we are pretty sure that it would increase with
further lowering of reaction temperature and reducing the formation of 16, the current
experimental setup is limited by temperature maintenance near 0-5 °C. Moreover, the low
temperatures caused severe frosting of the glass reactor through which light could not pass. A
configuration where the lamp is inside the reactor is being studied.
2
.2. Product Isolation
The reaction mass mostly contained methanol, 2-propanol, unreacted thiourea,
thiourea oxidation products, rose bengal, the diol 4, some dimerized cyclopentadiene and the
aldehyde 16 at the end of reaction. Isolation of pure 5 without chromatography was another
8

ACCEPTED MANUSCRIPT
challenge. The solvents, methanol and 2-propanol, were easily recovered by distillation under
reduced pressure. Extraction of the residue with n-heptane removed cyclopentadiene dimer.
The residue was then suspended in ethyl acetate and acetylated using triethylamine-acetyl
chloride protocol to prepare diacetate 5. The solvent was removed at this stage on a rotavapor
and the residue was repeatedly extracted with n-heptane. The n-heptane extracts were washed
with sodium carbonate solution to remove traces of rose bengal and dried over anhydrous
magnesium sulphate. Removal of n-heptane gave the final diacetate 5 in 30% overall yield
and >99% purity (GC analysis).
The reaction was further scaled up in a 20 L glass reactor. Here, 4 LED lamps were
placed around the reactor. The general experimental conditions were similar to those in 1 L
reactor (see experimental section), only multiplied by factor of 10 except the aeration rate
which was maintained at 1 L/min to minimize solvent evaporation. We were able to obtain
1
00 (± 10) g of product from 120 g of cyclopentadiene in 3 repeated batches.
2
.3. Hydrolysis in aqueous buffer
®
Hydrolysis of the diacetate 5 in aqueous buffer catalyzed by Novozym-435 was
2
6b
performed as per literature procedure
using 100 g of substrate in 1 L of 0.1 M phosphate
buffer at pH 7.5 and room temperature. During enzymatic hydrolysis of the acetate group,
acetic acid is released in the reaction mass as the reaction proceeds. This lowers the pH below
pH-optimum (7.0-7.5) for the enzyme and reaction slows down considerably. The pH of
reaction mass was therefore maintained by intermittent addition of 2N NaOH. Although the
reaction did proceed smoothly, a few problems were encountered. In our hands, the yield was
only 70% and e.e. of crude (1R,4S)-6 was 95%. We also observed that the product (1R,4S)-6
was soluble in aqueous buffer and several extractions with ethyl acetate were needed to
obtain a good yield. The reaction was accompanied by formation of diol 4 as a side product
9

ACCEPTED MANUSCRIPT
(GC analysis of the reaction mass). This was either due to self hydrolysis of 5 or 6 or due to
inefficient mixing of NaOH added during the reaction which causes non-selective alkaline
hydrolysis of the substrate. Another drawback of the procedure was the loss of enzyme
4
4
activity after each recycle. Since the enzyme is immobilized via adsorption, it tends to leach
out in aqueous buffer and consequently the expensive enzyme cannot be reused many times.
It is also difficult to scale-up a three-phase: immobilized enzyme-water immiscible substrate-
aqueous buffer system.
2
.4. Enzymatic transesterification in organic solvents
The use of an organic solvent instead of aqueous medium for enzymatic reactions
offers advantages such as increased solubility of hydrophobic substrates and easy recovery of
reaction products. The biocatalyst is also recovered by simple filtration. However, a solvent
can markedly affect enzyme reactivity. A major factor is solvent hydrophobicity, generally
measured as logPoctanol which increases with increase in value of logPoctanol. Enzyme activity
is generally higher in hydrophobic solvents (log P >1.0) than hydrophilic solvents (log P <
1
.0). The hydrophilic solvents apparently “strip” tightly bound water essential for enzyme
catalytic activity causing decrease of the enzyme activity while the hydrophobic solvents
4
5
show no obvious effect on the enzyme structure. However, functional groups and molecular
structure of hydrophobic organic solvents do exert significant influences on enzymes activity.
Although simple alkanes such as hexane do not significantly change the enzyme 3-D
structure, solvents with functional groups such as -OH, -CO, -CN etc. have polar interactions
with the enzyme surface that may cause "stripping off" the essential water from enzymes and
4
6
reduce the enzyme activity. Interestingly, a solvent with its functional group in the terminal
carbon atom has higher denaturation capacity than that possessing functional group
4
7
elsewhere. It has been also reported that lipases recognize not only the structure of the
4
8
substrate, but also that of solvent primarily due to changes in the relative solvation
1
0

ACCEPTED MANUSCRIPT
4
9
modes of the substrate in the transition state. A preliminary screening was thus performed in
®
various organic solvents routinely employed in Novozym-435 catalyzed reactions. Alcohols
such as methanol, ethanol, n-propanol and n-butanol were used for transesterification at 30
°
C. Surprisingly, methanol and ethanol were found to be effective while n-butanol, which is
the most preferred alcohol in lipase catalyzed transesterification reactions, did not cause
formation of product. The reactions with methanol and ethanol occurred in most solvents
except dichloromethane and dimethyl carbonate, but no enantioselectivity was observed.
Apart from formation of monoacetates (1R,4S)-6, (1S,4R)-6 and diol 4 were also formed
(Scheme 1).
It is well known that reaction temperature has a significant effect on enzyme
5
0-53
enantioselectivity.
Increase in enantioselectivity is generally observed with decrease in
‡
temperature. This is mostly due to difference in Gibbs free energy of activation, ꢀ ꢀG for
RS
stereoisomeric transition states formed by two enantiomers R and S in the rate-limiting step
with respect to the ground state. The transesterification reactions were thus carried out at
lower temperature of 5 °C using methanol. Although the reaction rate decreased, a dramatic
increase (e.e. 97-99%) in enantiomeric purity of the product was observed. The conversion
was low (15%) in toluene, possibly due to high solubility of the substrate in the reaction
medium and preferential partitioning of the substrate in bulk solvent phase rather than
enzyme active site. The lower conversion in hexane (56%) is mostly due to low solubility of
the substrate (from the peak area in GC analysis, it was apparent that some of the substrate
had deposited on the polymer and did not react) although the enantioselectivity was high. For
cyclic ethers such as THF and 2-MeTHF, the conversions remained low. A possible
competition of such solvents (5-membered ring) at the active site of the enzyme with the
substrate, which is also constituted by a 5-membered ring may cause this effect since
1
1

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noncyclic ethers such as MTBE and DIPE apparently do not compete for the active site with
the substrate and increase the conversion (Table 1).
Table 1
Effect of solvents and Temperatures on enzymatic desymmetrization
Entry Solvent
Conversion at
0 °C.
substrate:enzyme
e.e. of 6 at
Conversion at
°C
e.e. of 6
3
5
5 °C
[
at 30 °C
[substrate:enzyme
ratio (w/w) 5:1]
ratio (w/w) 5:1]
1
2
3
4
Hexane
56
15
70
10
5
98
96
97
Toluene
Tetrahydrofuran
2-Methyl
15
8
2
12
0
99
0
tetrahydrofuran
Dichloromethane
Methyl tert-butyl
ether (MTBE)
Diisopropyl ether
5
6
0
No
1
00
selectivity
100
100
100
>99
>99
>99
7
8
100
Methyl
isobutyl
8
5
ketone (MIBK)
Dimethyl
9
0
0
-
carbonate
Reaction conditions: 5 (1 g, 5.43 mmol), Solvent (10 mL), Methanol (0.44 mL, 0.01 mmol),
Enzyme (200 mg), 16h.
1
2

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For optimum reaction conditions, MTBE was chosen as solvent and methanol as
reagent for transesterification. The effect of methanol concentration on product distribution
was studied at 5 °C. Optimum ratio of molar concentration of methanol to substrate was
found to be 2.5. Above this concentration, diol formation was quite evident.
2
.5. Enzymatic resolution
®
Novozym-435 catalyzed desymmetrization of cis-3,5-Diacetoxy-1-cyclopentene 5
was finally carried out in MTBE at 5 °C in a jacketed stirred vessel. At 10% (w/v) loading of
substrate, mole ratio of methanol to substrate at 2.5 and substrate to enzyme ratio of 20%
(w/w), the reaction was complete in 16 h. GC analysis of the reaction mass did not show
presence of any other component other than the product 6, methanol, methyl acetate and the
solvent. Enzyme was separated by filtration and after concentrating the filtrate on rotavapor,
the residue was stored in refrigerator where it crystallized slowly to a white solid. The
recovered enzyme was reused 10 times without any appreciable change in conversion rate
and enantioselectivity. The combined reaction mass after 10 recycles provided 73 g of 6
(
yield 95%, e.e. >99%).
3
. Conclusion
In conclusion, we have developed an optimized methodology for preparation of cis-
,5-Diacetoxy-1-cyclopentene of high purity with overall yield of 30% without
3
chromatographic purification. The procedure described herein uses air instead of pure oxygen
and continuous feeding of the reactants with in situ reduction of endo-peroxide which reduces
the peroxide build-up in the reactor. The overall process is safer compared to earlier reports.
®
Novozym-435 catalyzed desymmetrization via transesterification in methyl tert-butyl ether
(MTBE) is an improvement over reported procedures in aqueous buffer. The methodology
does not involve solvent extraction. The recovered enzyme and the solvent can be recycled
1
3

ACCEPTED MANUSCRIPT
and the product with excellent enantioselectivity is easily isolated. Since the procedure
involves a simple two-phase system of immobilized enzyme and substrate solution in organic
solvent, it is easy to scale-up.
4
. EXPERIMENTAL SECTION
4
.1. General methods
HPLC analysis was carried out on Shimadzu LC-1080 VP system with SPD-M10A
VP diode array detector and SIL-10 AD VP auto injector. HPLC pump LC-10AT and LC-20
AP, Shimadzu, Japan were used for dosing. GC analysis was performed on Shimadzu
QP2010 Plus GC-Mass unit equipped with Mass detector.
Jacketed glass reactor Model RS-37, capacity 1 L, was obtained from Radleys, UK.
The 20 L jacketed reactor was manufactured locally. 100 Watts LED lamps were purchased
from local market. A Poly Science Digital Temperature Controller with 40 % ethylene
glycol-water (75 L capacity) was used for maintaining the temperature of the reactor.
®
Novozym-435 was purchased from M/s Brenntag Ingredients (India) Private Limited,
Mumbai and was used as received. All reagents and solvents used were of analytical grade
obtained from Alfa Aesar and Qualigens, India.
4
.2. Preparation of cyclopentadiene monomer 2
Dicyclopentadiene (300 g) was slowly added (2 mL/min) with a dosing pump to a
L, 2-necked round bottom flask, containing magnetically stirred silicone oil (250 mL)
1
heated at 200 °C. The monomer vapors formed due to cracking of the dimer were further
passed through a glass column filled with Raschig rings and heated to 300 °C. A downward
double walled condenser, maintained at 0 °C cooled the vapors and the monomer was
1
4

ACCEPTED MANUSCRIPT
collected in a receiver maintained at -20 °C with acetone-dry ice. Thus 255 mL (200 g) of
monomer was collected in 3 h.
4
.3. Photochemical reaction
The reaction was performed in a 1 L jacketed glass reactor maintained at 0-5 °C using
an ethylene glycol-water circulating water bath. A solution of thiourea (7.6 g, 100 mmol) and
rose bengal (0.5 g) in methanol (500 mL) was introduced and stirred at 150 rpm with aeration
(10 L/h) using an air compressor. A 100 watts LED lamp was used for illumination. The
monomer (25% (v/v) solution in 2-propanol maintained at -20 °C was continuously added to
the reactants at a rate of 0.5 mL/min with a dosing pump. Simultaneously, a solution of
thiourea (3 % w/v) in methanol was also pumped separately at the rate of 2 mL/min into the
reaction mass. Aliquots of reaction mass were collected at regular intervals of 30 min and
stored in refrigerator. The addition of monomer and thiourea was stopped after 2 h. The total
amount of monomer added to the reactor at this point was 12.0 g (180 mmol) and thiourea
(14.8 g, 195 mmol). The reaction was continued for 4 h more with the light switched on.
Afterwards the cooling and illumination were switched off and the reactants were stirred
overnight.
The reaction mass was then collected, the reactor was carefully rinsed with methanol
and combined methanolic solution was evaporated on rotavapor. The residue was extracted
with n-heptane (2 × 100 mL). After removal of the solvent, the residue was suspended in
dichloromethane (250 mL) in a 500 mL round bottom flask. The flask was placed in an ice
bath, the contents were stirred on a magnetic stirrer and triethylamine (40.4 g, 400 mmol) was
slowly added. After stirring for 15 min, acetyl chloride (31.4 g, 400 mmol) was added
dropwise with cooling. After stirring for 6 h, water (250 mL) was added to dissolve solids
and the layers were separated. The aqueous layer was extracted with dichloromethane (2 ×
1
00 mL), solvent was removed from combined organic extracts on rotavapor and the residue
1
5

ACCEPTED MANUSCRIPT
was repeatedly extracted with n-heptane (4 × 100 mL). The pale yellow n-heptane extract
was washed with 10% sodium carbonate solution till the aqueous layer was colourless (3 ×
2
5 mL) to remove last traces of rose bengal. The organic layer was dried over anhydrous
magnesium sulphate and evaporated on rotavapor. cis-3,5-Diacetoxy-1-cyclopentene 5 was
1
obtained as a pale-yellow oil. Yield 10 g, 30%. Purity >99%, GC. H NMR (CDCl δ) 6.09
3,
1
3
(
d, 2H), 5.54 (qd, 2H), 2.86 (quin, 1H), 2.05 (s, 6H), 1.73 (dt, 1H). C NMR (CDCl δ)
3,
+
1
1
70.61, 134.54, 76.54, 37.07, 21.06. MASS: [M+Na] 207; IR [KBR]: 3451, 2948, 1737,
-1
434 cm .
4
.4. Analysis of thiourea content by HPLC
Aliquots of samples collected at regular intervals of 30 min were diluted with mobile
phase (10 µL to 10 mL) and analysed for thiourea content by reverse phase HPLC. Column:
Phenomenex Luna C-18, 5 µ, 250 × 4.5 mm; injection volume 20 µL; mobile phase 10%
(v/v) methanol-water, flow rate 1 mL/min, detection at 245 nm. Retention time: 3.1 min.
4
.5. Analysis of sulphur content by HPLC
The reaction mass (1 g) was stirred with dichloromethane (100 mL) for 15 min,
centrifuged and the supernatant was analyzed by HPLC. Column: ZodiacSil 120-5-CN 5 µ,
50 × 4.5 mm, injection volume 20 µL; mobile phase: n-heptane-dichloromethane-2-
propanol-acetonitrile (80:6:10:4), flow rate 1 mL/min, detection at 240 nm. Retention time:
.1 min. A standard sample of sulphur (0.1 mg/mL in mobile phase) was used for
comparison.
2
3
4
.6. Enzymatic transesterification of cis-3,5-Diacetoxy-1-cyclopentene
cis-3,5-Diacetoxy-1-cyclopentene (10 g, 54.3 mmol) dissolved in MTBE (100 mL,
1
0% w/v) and MeOH (4.4 mL, 100 mmol) were stirred at 150 rpm with immobilized enzyme
1
6

ACCEPTED MANUSCRIPT
(2 g, w/w) at 5 °C in a double walled glass reactor using an overhead mechanical stirrer. The
reaction was followed by analysis on TLC and chiral GC. The reaction was complete in 16 h.
4
.7. Enzyme recycle
After 12 h of reaction (>99% conversion & >99% e.e), the enzyme was separated by
filtration and washed twice with MTBE (2 × 5 mL). The combined organic portion was
collected separately. The used enzyme was found to be recyclable for up to 10 times without
significant change in productivity and activity of the enzyme.
4
.8. Product separation
The combined reaction mass after 10 recycles was concentrated on rotavapor and the
residue was stored in refrigerator where it crystallized slowly to a white solid (yield 73 g, 95
2
5
26b
20
%
, e.e >99%). [α]D = + 65.80 (c = 1, CHCl ), (lit [α]D = + 66 (c = 1, CHCl , e.e 99%).
3
3
2
7
The Mass and NMR spectra were comparable with literature report.
4
.9. GC analysis
Analysis was performed on Shimadzu QP2010 Plus GC-Mass unit using mass
detector. Retention time 4: 47.97 min; 5: 55.85 min; (1R,4S)-6: 48.88 min; (1S,4R)-6: 50.69
min. Column SUPELCO β-Dex 120, length 30 mt, Thickness: 0.25 µm; Dia: 0.25 mm.
Linear velocity: 49.0 cm/sec; Column oven temp. 64°C, Injection temperature 230 °C,
Carrier gas: Helium. Initial column oven temperature was 64 °C hold 2 min and @ 1 °C/min
to 130 °C, hold 5 min and increased up to 200 °C at 10 °C/min hold 1 min.
ACKNOWLEDGEMENTS
We thank Council of Scientific and Industrial Research, New Delhi (Project No. FTT,
MLP-27); we also thank Dr. Nitin Fadnavis for helpful discussions.
1
7

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Notes
The authors declare no competing financial interests.
CSIR-IICT’s Communication number: IICT/Pubs./2018/190
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