Preparation of a chiral synthon for an HBV inhibitor: Enzymatic asymmetric hydrolysis of (1α,2β,3α)-2-(benzyloxymethyl)cyclopent-4-ene-1, 3-diol diacetate and enzymatic asymmetric acetylation of (1α,2β, 3α)-2-(benzyloxymethyl)cyclopent-4-ene-1,3-diol

Article abstract of DOI:10.1016/j.tetasy.2005.08.061

Enantioselective asymmetric hydrolysis of (1α,2β,3α)-2- (benzyloxymethyl)-cyclopent-4-ene-1,3-diol diacetate 1 to the corresponding (+)-monoacetate 2 was carried out using lipase PS-30 from Pseudomonas cepacia or pancreatin. A reaction yield of 85 M % wit

Full text of DOI:10.1016/j.tetasy.2005.08.061

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 17 (2006) 175–178
Preparation of a chiral synthon for an HBV inhibitor:
enzymatic asymmetric hydrolysis of
(1a,2b,3a)-2-(benzyloxymethyl)cyclopent-4-ene-1,3-diol diacetate
and enzymatic asymmetric acetylation of (1a,2b,3a)-
2-(benzyloxymethyl)cyclopent-4-ene-1,3-diol
Ramesh N. Patel,* Amit Banerjee, Yadagiri R. Pendri, Jing Liang,
Chung-Pin Chen and Richard Mueller
Department of Enzyme Technology, Process Research and Development, Bristol-Myers Squibb Pharmaceutical Research Institute,
PO Box 191, New Brunswick, NJ 08903, USA
Received 14 July 2005; accepted 3 August 2005
Abstract—Enantioselective asymmetric hydrolysis of (1a,2b,3a)-2-(benzyloxymethyl)-cyclopent-4-ene-1,3-diol diacetate 1 to the cor-
responding (+)-monoacetate 2 was carried out using lipase PS-30 from Pseudomonas cepacia or pancreatin. A reaction yield of
85 M % with an enantiomeric excess (ee) of 98% was obtained. Using pancreatin, a reaction yield of 75 M % with an ee of
98.5% was obtained. Asymmetric acetylation of (1a,2b,3a)-2-(benzyloxymethyl)-cyclopent-4-ene-1,3-diol 3 to the corresponding
(ꢀ)-monoacetate 4 was carried out using lipase PS-30 with isopropenyl acetate as the acylating agent. A reaction yield of
80 M % with an ee of 98% was obtained for (ꢀ)-monoacetate 4.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
antagonist. Enzymatic hydrolysis of the (1a,2b,3a)-2-
(benzyloxymethyl)cyclopent-4-ene-1,3-diol diacetate
The current interest in the enzymatic production of chi-
ral compounds lies in the preparation of intermediates
for pharmaceutical synthesis.1–11 Lipases have been used
extensively in the preparation of non-racemic chiral
compounds by enantioselective enzymatic hydrolysis,
esterification, and transesterification reactions.12–24
has been demonstrated by Griffith and Danishefsky to
produce the corresponding monoester using the acetyl-
choline esterase from the electric eel25,26 and pocine pan-
creatic lipase by Legrand and Roberts.27 An efficient
synthesis of the carbocyclic nucleosides (ꢀ)-aristero-
mycin and (ꢀ)-neplanocin A has been developed in an
enantioselective manner starting from the Diels–Alder
adduct of cyclopentadiene and dimethyl acetylenedi-
carboxylate. The symmetric unsaturated dimethyl ester,
dimethyl (3aa,4b,7b,7aa)-3a,4,7,7a-tetrahydro-2,2-di-
methyl-4,7-methano-1,3-benzodioxole-5,6-dicarboxylate
was quantitatively hydrolyzed with pig liver esterase
to yield a half-ester with reasonably high ee (88%).28
Deardorff et al. have demonstrated the hydrolysis of
cis-3,5-diacetoxycyclopent-1-ene to the corresponding
(3R,5S)-acetoxy-5-hydroxycyclopent-1-ene using elec-
tric eel acetylcholinesterase in 94% yield with an ee of
96%.29 Pig liver esterase and porcine pancreatic lipase
(PPL) catalyzed the enantiotopic selective hydrolysis of
2,5-bis(methoxy-carbonyl) and 2,5-bis(acetoxymethyl)
Previously we have demonstrated the asymmetric enzy-
matic hydrolysis of 2-cyclohexyl- and 2-phenyl-1,3-pro-
panediol diacetate.23 The chiral monoacetates obtained
are key intermediates for the synthesis of the angiotensin
converting enzyme (ACE) inhibitor Monoprilꢁ. The
asymmetric hydrolysis of (exo,exo)-7-oxabicyclo[2.2.1]-
heptane-2,3-dimethanol diacetate ester to the corre-
sponding monoacetate ester has been demonstrated with
lipase PS-30.24 The chiral monoacetate ester is a key
intermediate in the synthesis of a thromboxane A2
*
Corresponding author. Tel.: +1 732 227 6213; fax: +1 732 227
3994; e-mail: ramesh.patel@bms.com
0957-4166/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2005.08.061

176
R. N. Patel et al. / Tetrahedron: Asymmetry 17 (2006) 175–178
meso-diester derivatives of 3,4-(isopropylidenedioxy)-
tetrahydrofuran to the corresponding monoesters with
an ee of 72% being demonstrated by Hultin et al.30
Transesterification of the 2,5-bis(hydroxymethyl) deriv-
atives with trifluoroethyl laurate by PPL in ether also
showed stereoselectively but in the opposite stereochem-
ical sense from the hydrolysis of the corresponding
diacetate.31 The synthesis of 30-deoxycarbacyclic nucleo-
side analogues has recently been demonstrated by the
regioselective ring opening of the cyclopentene epoxide
with lithium di-n-propylamide in ether.32
2. Results and discussion
Various lipases and the acetylcholine esterase from the
electric eel were screened for the enzymatic hydrolysis
of (1a,2b,3a)-2-(benzyloxymethyl)cyclopente-4-ene-1,3-
diol diacetate 1 to the corresponding (+)-monoacetate
2. Among the enzymes evaluated, pancreatin, the immo-
bilized lipase PS-30 from Pseudomonas cepacia and
acetylcholine esterase all catalyzed the asymmetric
hydrolysis of diacetate 1 to the corresponding (+)-
monoacetate 2. Lipase MAP-10, penicillin V amidase,
and lipase from Rhizopus sp. did not hydrolyze diacetate
1. Further experimentation was carried out using lipase
PS-30 and acetylcholine esterase. Preparative scale
hydrolysis of diacetate 1 was carried out in a 2-L reactor
with a substrate input of 26 g/L (total input 52 g) and
90 g/L input of immobilized lipase PS-30. After 18 h
reaction time, 31.2 g of (+)-monoacetate 2 was obtained
with 78 M % yield with an ee of 98% (Table 1). A pre-
parative batch for the hydrolysis of diacetate 1 to the
corresponding monoacetate was carried out using ace-
tylcholine esterase. The reaction was carried out using
a 5 g/L substrate input and 2400 units of enzyme input
in a 0.6 L volume. After 50 h reaction time, 2 g
(82 M % yield with 98% ee) of (+)-monoacetate 2 was
obtained (Table 1).
Herein we report the enantioselective asymmetric hydrol-
ysis of (1a,2b,3a)-2-(benzyloxymethyl)cyclopent-4-ene-
1,3-diol diacetate 1 to the corresponding (+)-monoace-
tate 2 by lipase PS-30 and pancreatin. We also describe
the enzymatic asymmetric acetylation of (1a,2b,3a)-2-
(benzyloxymethyl)cyclopent-4-ene-1,3-diol 3 to the cor-
responding (ꢀ)-monoacetate 4 using lipase PS-30
(Fig. 1). Chiral monoacetate esters 2 and 4 are key inter-
mediates in the total synthesis of entecavir, recently
approved for treatment of hepatitis B viral infec-
tion.33–37 Entecavir is a carboxylic analogue of 20-deoxy-
guanosine in which the furanose oxygen is replaced
with an exocyclic double bond (Fig. 1).
We also screened various lipases for the enantioselec-
tive acylation of meso-diol 3 to the corresponding
monoacetate using isopropenyl acetate as an acyl
donor. Pancreatin and lipase PS-30 from P. cepacia cat-
alyzed the enantioselective acylation of meso-diol 3 to
the corresponding (ꢀ)-monoacetate 4. Kinetics of the
acylation reaction using lipase PS-30 are shown in
Table 2.
OBn
OAc
OBn
OAc
AcO
HO
Lipase PS-30 from
Pseudomonas cepacia or
Pancreatin
Diacetate 1
(+)-Monoacetate 2
OBn
OBn
OH
Griffith and Danishefsky reported the asymmetric
hydrolysis of (1a,2b,3a)-2-(benzyloxymethyl)cyclopent-
4-ene-1,3-diol diacetate 1 by electric eel acetylcholines-
terase during the total synthesis of allosamidin, a chitin
synthase inhibitor potentially useful for preventing
fungal and insect growth.25 Acetylcholinesterase is an
expensive enzyme, which is not readily available in large
quantities. Lipase PS-30 is inexpensive and readily
available in large quantities. Immobilized lipase PS-30
used in the asymmetric hydrolysis of diacetate 1 can
be recovered and reused for many cycles. We have
prepared both (+)-monoacetate 2 and (ꢀ)-monoacetate
4 by lipase PS-30 catalyzed hydrolysis of diacetate 1
or acylation of diol 3, respectively, in very high yield
and ee.
Lipase PS-30 from
AcO
OH
HO
Pseudomonas cepacia
Diol 3
(-)-Monoacetate 4
N
O
N
NH
NH2
HO
N
HO
Entecavir
Figure 1.
Table 1. Enzymatic asymmetric hydrolysis of diacetate 1
Enzyme
Reaction
time (h)
Diacetate 1
Monoacetate 2
Diol
(g/L)
Monoacetate 2
M % yield
% ee of
(+)-monoacetate
(g/L)
(g/L)
Immobilized
Lipase PS-30
0.1
18
26
0.1
1.2
17.6
0.03
3.4
80
82
98
98
Acetylcholine
Esterase
0.1
50
2.85
0
0.56
2.0
Trace
0.09
Reactions were carried out as described in the Experimental section.

R. N. Patel et al. / Tetrahedron: Asymmetry 17 (2006) 175–178
Table 2. Enzymatic asymmetric acetylation of diol 3 by immobilized lipase PS-30
177
Reaction time (h)
Monoacetate 4 (g/L)
Monoacetate 4 M % yield
% ee of (ꢀ)-monoacetate 4
24
48
72
8.2
15.4
22.5
36
62
90
98.9
99
99
3. Experimental
1 concentration had dropped below 0.05 mg/mL. The
reaction mixture was extracted with two volumes of
ethyl acetate. The organic phase was separated and con-
centrated under reduced pressure to obtain (+)-mono-
acetate 2, which was purified by flash chromatography.
When the hydrolysis was performed using lipase PS-
30, (+)-monoacetate 2 was obtained in 85 M % yield
with 98% ee. When the hydrolysis was performed using
pancreatin, (+)-monoacetate 2 was obtained in 75 M %
yield with 98.5% ee.
The physico-chemical properties including spectral char-
acteristics (1H NMR, 13C NMR, mass spectra) were in
full accordance for all these compounds. The proton
magnetic resonance (1H NMR) and carbon magnetic
resonance (13C NMR) were recorded on a Brucker
AM-300 spectrometer. Racemic monoacetate 2 required
as an HPLC reference standard was prepared by base-
catalyzed deacetylation of 1 with potassium carbonate
in methanol.
The semi-preparative scale asymmetric hydrolysis of
diacetate 1 was carried out using lipase PS-30 from
P. cepacia immobilized on Accurel polypropylene. The
reaction mixture in 2 L contained 1.8 L of 25 mM
pH 7.0 potassium phosphate buffer, 200 mL toluene,
52 g diacetate 1, and 180 g immobilized lipase PS-30.
At the end of the reaction (18 h), the immobilized
enzyme was filtered and the clear supernatant obtained
was extracted with two volumes of ethyl acetate. The
separated organic phase was concentrated under
reduced pressure to provide the desired product, which
was purified by flash chromatography, (+)-monoacetate
2 (32.5 g), in 80 M % yield with 98% ee.
3.1. (1a,2b,3a)-2-(Benzyloxymethyl)cyclopent-4-ene-1,3-
diol diacetate 1
A mixture of (1a,2b,3a)-2-(benzyloxymethyl)cyclopent-
4-ene-1,3-diol35 3 (2.2 g, 9.9 mmol), acetic anhydride
(4 mL), and pyridine (2.5 mL) was stirred at room tem-
perature for 12 h. The reaction mixture was concen-
trated under vacuum and the residue chromatographed
(silica gel, 30% ethyl acetate in hexanes) to provide
diacetate 1 (2.8 g, 92%) as a light yellow oil.
3.2. Source of enzymes
Lipase PS-30, lipase MAP10, and lipase from Rhizopus
sp. were obtained from Amano International Enzyme
Company. Pancreatin and acetylcholin esterase was
obtained from Sigma Chemicals. Lipase PS-30 was
immobilized on the hydrophobic resin Accurel poly-
propylene as described earlier.12
3.4. Enzymatic acetylation of diol 3
Enantioselective acetylation of diol 3 was carried out
using various enzymes. The reaction mixture in 10 mL
of heptane/tert-butyl methyl ether (10:1) contained
200 mg of diol 3, 50 mg of enzyme, and 1.1 M equiv of
isopropenyl acetate. The reaction was carried out
at 33 ꢂC in a 50-mL Teflon flask. The progress of the
reaction was monitored by high pressure liquid
chromatography.
3.3. Enzymatic hydrolysis of diacetate 1
Enantioselective asymmetric hydrolysis of (1a,2b,3a)-2-
(benzyloxymethyl)cyclopent-4-ene-1,3-diol diacetate 1
was carried out as described below.
Semi-preparative scale acetylation of diol 3 was carried
out using immobilized lipase PS-30 from P. cepacia.
The reaction mixture in 1 L of heptane/MTBE (10:1)
contained 25 g of diol 3, 25 g of immobilized lipase
PS-30, and 2 M equiv of isopropenyl acetate as acylating
agent. The reaction was carried out at 33 ꢂC. The pro-
gress of the reaction was monitored by HPLC. The reac-
tion was terminated when the remaining substrate 3
concentration was below 0.1 mg/mL. After 72 h of reac-
tion time, the product (ꢀ)-monoacetate 4 was isolated in
80 M % yield with 98% ee.
The reaction mixture contained 9 mL of 25 mM pH 7.0
potassium phosphate buffer pH 7.0, 1 mL toluene,
2.5 mg/mL diacetate 1, and 50 mg/mL of enzyme. The
reaction was carried out at 25 ꢂC and the pH of the reac-
tion mixture maintained at 7.0 by the addition of 1 M
NaOH as required. The progress of the reaction was
monitored by high pressure liquid chromatography.
Enantioselective asymmetric hydrolysis of diacetate 1
was carried out using pancreatin or lipase PS-30 from
P. cepacia. The reaction mixture in 40 mL contained
36 mL of 25 mM pH 7.0 potassium phosphate buffer,
4 mL toluene, 2.5 mg/mL diacetate 1, and 50 mg/mL
of lipase PS-30 or pancreatin. The reaction was carried
out at 25 ꢂC and the pH of the reaction mixture main-
tained at 7.0 by addition of 1 M NaOH as required.
The progress of the reaction was monitored by high
pressure liquid chromatography. The reaction was ter-
minated after about 16 h when the remaining substrate
4. Analytical methods
Samples from enzymatic reactions were extracted with
three volumes of ethyl acetate. The solvent layer was
collected and evaporated under vacuum, and the residue
dissolved in mobile phase before analysis. Analysis of
diacetate 1, diol 3, and the monoacetate were carried

178
R. N. Patel et al. / Tetrahedron: Asymmetry 17 (2006) 175–178
13. Patel, R. N.; Mc Namee, C. G.; Szarka, L. J. Appl.
a Kromasil C-8 column
out using HPLC with
Microbiol. Biotechnol. 1992, 38, 56–60.
(100 · 4.5 mm, ID 5 m). Mobile phases A and B were
used in a gradient; solvent A: 25 mM KH2PO4
(pH 7.0), solvent B: acetonitrile.
14. Hanson, R. L.; Banerjee, A.; Comezoglu, F. T.; Mirfa-
khrae, D.; Patel, R. N.; Szarka, L. J. Tetrahedron:
Asymmetry 1994, 5, 1925–1934.
15. Patel, R. N.; Banerjee, A.; Mc Namee, C. G.; Szarka, L. J.
Tetrahedron: Asymmetry 1995, 6, 123–130.
Time (min)
Solvent A (%)
Solvent B (%)
16. Brieva, R.; Crich, J. Z.; Sih, C. J. J. Org. Chem. 1993, 58,
1068–1075.
17. Patel, R. N.; Banerjee, A.; Szarka, L. J. Tetrahedron:
Asymmetry 1997, 8, 1055–1059.
18. Patel, R. N.; Howell, J. M.; Banerjee, A.; Fortney, K. F.;
Szarka, L. J. Appl. Microbiol. Biotechnol. 1991, 36, 29–34.
19. Tombo, G. M. R.; Schar, H. P.; Busquets, X. F.;
Ghisalba, O. Tetrahedron Lett. 1986, 21, 5707–5710.
20. Sumune, H.; Takahashi, M.; Maeda, S.; Xie, Z.-F.; Sakai,
K. Tetrahedron: Asymmetry 1990, 1, 425–429.
21. Chenevert, R.; Disjardins, M. Can. J. Chem. 1994, 72,
2312–2317.
22. Mohr, P.; Waespe-Sarcevic, N.; Tamm, C. Helv. Chim.
Acta 1983, 66, 2501–2511.
23. Patel, R. N.; Robison, R. S.; Szarka, L. J. Appl. Microbiol.
Biotechnol. 1990, 34, 10–14.
24. Patel, R. N.; Liu, M.; Banerjee, A.; Szarka, L. J. Appl.
Microbiol. Biotechnol. 1992, 37, 180–183.
25. Griffith, D. A.; Danishefsky, S. J. J. Am. Chem. Soc. 1996,
118, 9526–9538.
0
30
35
40
70
25
25
70
30
75
75
30
The flow rate was 1 mL/min and the detection wave-
lengths were 210, 250, and 280 nm. The retention times
for diacetate 1, diol 3, and the monoacetate were 30.3,
12, and 20 min, respectively. The ee of the monoacetate
was determined by chiral HPLC. A Bakerbond Chir-
alpak AD column (100 · 4.5 mm, ID 5 m) was used at
40 ꢂC; injection volume was 10 lL; mobile phase was a
95% heptane/5% isopropanol mixture; flow rate was
1 mL/min; and detection wavelength was 210 nm. The
retention times for compounds (+)-monoacetate 2 and
(ꢀ)-monoacetate 4 were 15 and 22 min, respectively.
26. Danishefsky, S. J.; Cabal, M. P.; Chow, K. J. Am. Chem.
Soc. 1989, 111, 3456–3457.
27. Legrand, D. M.; Roberts, S. M. J. Chem. Soc., Perkin
Trans. 1 1992, 14, 1751–1752.
References
28. Arita, M.; Adachi, K.; Ito, Y.; Sawai, H.; Ohno, M. J.
Am. Chem. Soc. 1983, 105, 4049–4055.
29. Deardorff, D. R.; Matthews, A. J.; McMeekin, D. S.;
Craney, C. L. Tetrahedron Lett. 1986, 27, 1255–1256.
30. Hultin, P. G.; Mueseler, F.-J.; Jones, J. B. J. Org. Chem.
1991, 56, 5375–5380.
1. Jones, J. B. Tetrahedron 1986, 42, 3351–3403.
2. Crout, D. H. G.; Davies, S.; Heath, R. J.; Miles, C. O.;
Rathbone, D. R.; Swoboda, B. E. P. Biocatalysis 1994, 9,
1–30.
3. Davies, H. G.; Green, R. H.; Kelly, D. R.; Roberts, S. M.
Biotechnology 1990, 10, 129–152.
4. Crosby, J. Tetrahedron 1991, 47, 4789–4846.
5. Kamphuis, J.; Boesten, W. H. J.; Broxterman, Q. B.;
Hermes, H. F. M.; van Balken, J. A. M.; Meijer, E. M.;
Schoemaker, H. E. Adv. Biochem. Eng. Biotechnol. 1990,
42, 133–186.
6. Sih, C. J.; Gu, Q.-M.; Holdgrun, X.; Harris, K. Chirality
1992, 4, 91–97.
7. Santaneillo, E.; Ferraboschi, P.; Grisenti, P.; Manzocchi,
A. Chem. Rev. 1992, 92, 1071–1140.
8. Margolin, A. L. Enzyme Microb. Technol. 1993, 15, 266–
280.
9. Wong, C.-H.; Whitesides, G. M. In Enzymes in Synthetic
Organic Chemistry. In Tetrahedron Organic Chemistry
Series; Pergamon, Elsevier Science Ltd: USA, 1994; Vol.
12.
31. Matsuo, K.; Tanaka, M.; Sakai, K.; Suemune, H. Tetra-
hedron: Asymmetry 1997, 8, 3089–3094.
32. Ruediger, E.; Martel, A.; Meanwell, N.; Solomon, C.;
Turmel, B. Tetrahedron Lett. 2004, 45, 739–742.
33. Innaimo, S. F.; Seifer, M.; Bisacchi, G. S.; Standring, D.
N.; Zahler, R.; Collono, R. J. Antimicrob. Agents Chemo-
ther. 1997, 41, 1444–1448.
34. Yamanaka, G.; Wilson, T.; Innaimo, S. F.; Bisacchi, G.
S.; Egli, P.; Rinhart, K. J.; Zahler, R.; Colonno, R. J.
Antimicrob. Agents Chemother. 1999, 43, 190–193.
35. Seifer, M.; Hamatake, R. K.; Colonno, R. J.; Standring,
D. N. Antimicrob. Agents Chemother. 1998, 42, 3200–3208.
36. Innaimo, S.; Jacobs, G. A.; Kocy, O.; Lapointe, P.;
Martel, A.; Merchant, Z.; Slusarchyk, W. A.; Sundeen, J.
E.; Yound, M. G.; Colonno, R.; Zahler, R. Bioorg. Med.
Chem. Lett. 1997, 7, 127–132.
10. Patel, R. N. Adv. Appl. Microbiol. 1997, 43, 91–140.
11. Patel, R. N. In Stereoselective Biocatalysis; Patel, R. N.,
Ed.; Marcel Dekker: New York, 2000.
12. Patel, R. N.; Banerjee, A.; Ko, R. Y.; Howell, J. M.; Li,
W.-S.; Comezoglu, F. T.; Partyka, R. A.; Szarka, L. J.
Biotechnol. Appl. Biochem. 1994, 20, 23–33.
37. Pendri, Y. R.; Chen, C-H.; Patel, S. S.; Evans, J. M.;
Liang, J.; Kronenthal, D. R.; Powers, G. L.; Prasad, S. J.;
Bien, J. T.; Shi, Z.; Patel, R. N.; Chan, Y. Y.; Rijhwani, S.
K.; Singh, A. K.; Wang, S.; Stojanovic, M.; Polniaszek,
R.; Lewis, C.; Thottathil, J.; Krishnamurty, D.; Zhou, M.
X.; Vemishetti, P. 2004, US20040192912 A1.