Chiral induction in cyclopentyl-derived 1,3-meso-diesters: enantioselective hydrolyses with electric eel acetylcholinesterase

Article abstract of DOI:10.1016/S0957-4166(99)00236-0

Eight 1,3-meso-diesters derived from a common cyclopentyl backbone were exposed to the hydrolase enzyme acetylcholinesterase from Electrophorus electricus. All eight compounds were hydrolyzed by the enzyme. The overall enantioselectivities were quite high, and the resulting e.e.s were generally >90%. The absolute configurations of the product monoesters were determined through stereochemical correlation. These data revealed that the preferred site for enzymatic hydrolysis in seven of the substrates was the pro-S ester function, with pro-R cleavage detected in the eighth.

Full text of DOI:10.1016/S0957-4166(99)00236-0

Pergamon
Tetrahedron: Asymmetry 10 (1999) 2139–2152
Chiral induction in cyclopentyl-derived 1,3-meso-diesters:
enantioselective hydrolyses with electric eel acetylcholinesterase
Donald R. Deardorff,∗,† Roberto B. Amador, James W. Morton, Henry Y. Kim,
Cullen M. Taniguchi, Arnel A. Balbuena, Sam A. Warren, Vadim Fanous and
S. W. Tina Choe ‡,§
Department of Chemistry, Occidental College, Los Angeles, CA 90041, USA
Received 20 April 1999; accepted 27 May 1999
Abstract
Eight 1,3-meso-diesters derived from a common cyclopentyl backbone were exposed to the hydrolase enzyme
acetylcholinesterase from Electrophorus electricus. All eight compounds were hydrolyzed by the enzyme. The
overall enantioselectivities were quite high, and the resulting e.e.s were generally >90%. The absolute configura-
tions of the product monoesters were determined through stereochemical correlation. These data revealed that the
preferred site for enzymatic hydrolysis in seven of the substrates was the pro-S ester function, with pro-R cleavage
detected in the eighth. © 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction
Enantiomerically pure cyclopentanoids are important building blocks for the preparation of biolo-
gically active molecules such as prostaglandins and carbocyclic nucleosides. Recently, we described1
the formal synthesis of the carbocyclic nucleoside (−)-aristeromycin from the optically active starting
material (1R,4S)-(+)-4-hydroxycyclopent-2-enyl acetate ((+)-2), a cyclopentanoid that shares the same
absolute configuration found in natural nucleosides. A search for an efficient synthetic route to (+)-2
led us to the discovery that the enzyme acetylcholinesterase from Electrophorus electricus (electric eel;
EeAChE) could differentiate enantiotopically between pro-R and pro-S acetoxy groups on meso-diester
1 to afford chiral (S)-hydroxy ester 2 with a selectivity of 96% e.e. (>99% e.e. after one recrystallization)
∗
†
‡
§
Corresponding author. E-mail: deardorf@oxy.edu
Camille and Henry Dreyfus Scholar
Camille and Henry Dreyfus Fellow
Present address: Loyola Marymount Unioversity, 7900 Loyola Blvd, Los Angeles, CA 90045-8225, USA.
0957-4166/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(99)00236-0

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and a chemical yield of 94% (Scheme 1).2 This enantioselective hydrolysis has been carried out on a 10
gram scale without difficulty.3
Scheme 1.
Since our inaugural work on this system, others have also reported successful chiral inductions with
EeAChE on different meso-diester substrates.4–8 In each case, the researchers found that EeAChE posted
impressive enantioselectivities that ranged from 92 to 100% e.e. These findings have prompted us to
explore further the scope and limitations of using EeAChE for the preparation of chiral, nonracemic
cyclopentanoids. Herein, we report on the remarkable substrate enantioselectivity that EeAChE exhibits
toward a series of structurally related meso-diesters derived from 1,3-cyclopentanediols. Enzyme enantio-
selectivity was also measured against structural variations in the acyl moieties. Details of the enzyme’s
substrate specificities and stereoselectivities gleaned from this study may eventually prove useful in the
development of an experimental active-site model of predictive merit.
2. Results and discussion
2.1. Substrate preparation
Table 1 illustrates the structurally related series of cyclopentanoid 1,3-meso-diester substrates used
in this study. With the exception of diacetates 3 and 11, all cis-diester substrates were constructed
from their monoprotected racemic counterparts using standard acylation technology. Accordingly, the
unsaturated hydroxyesters 2, 14, 16, and 18, precursors to meso-diesters 1, 13, 15, and 17, respectively,
were conveniently prepared in a single step via the palladium-catalyzed reaction between cyclopentadiene
monoepoxide and the appropriate carboxylic acid.9,10
Monoacetate 2 also served as the starting point for bicyclo-diesters 5, 7, and 9. Hydroxyl-directed
cyclopropanation of 2 via the Simmons–Smith reagent11 yielded the expected β-cyclopropane
monoacetate 6 which, upon acetylation with acetic anhydride in pyridine, produced the desired
meso-diacetate 5. β-Epoxy diacetate 7 was synthesized from 2 by a two-step procedure: stereoselective
MCPBA epoxidation of the double bond12 followed by acetylation. The preparation of the α-epoxy
diacetate 9 necessitated that both hydroxyl functions be protected with the bulky t-butyldimethylsilyl
group to discourage epoxidation from the β-face of the cyclopentanoid. Hence, the acetoxy group on
2 was hydrolyzed with K2CO3 in MeOH to give an intermediate meso-diol which was immediately
disilylated with TBDMSCl. Epoxidation with purified MCPBA gave satisfactory yields of the required
α-product.12 Subsequent removal of the protecting groups with tetrabutylammonium fluoride (TBAF)
and diacetylation with acetic anhydride and catalytic DMAP then afforded meso-diacetate 9.
Finally, diacetates 3 and 11 were fashioned directly from the unsaturated diacetate 1. Catalytic
hydrogenation of 1 with 10% palladium on carbon provided the corresponding saturated diester 3 in
useful yield. Alternatively, stereoselective osmylation of 1 followed by ketalization of the putative cis-
diol with cyclohexanone and catalytic PTSA led to the desired spiral acetal 11.

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Table 1
Enantioselective hydrolysis of cyclopentanoid 1,3-meso-diesters with EeAChE

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2.2. Enzymatic hydrolysis
Exposure of the sparingly water-soluble substrates to EeAChE in an aqueous pH 6.9 phosphate buffer
initially afforded a two-phase system, but that gradually converged to a single phase as the diester was
converted into the more soluble monohydroxy product. The hydrolyses were monitored by TLC. In most
examples, hydrolysis of the second ester group was insignificant. The chiral hydroxyesters were isolated
from the aqueous media via multiple extractions with an emulsion-suppressing solvent system consisting
of 1:1 ether:ethyl acetate. The crude material was chromatographed over silica gel and fully characterized
spectroscopically.
2.3. Determination of enantiomeric excess
Enantiomeric purities were determined by 31P NMR measurements13 on the diastereomeric pairs (e.g.,
20-(S) and -(R)) generated from reaction of the chiral alcohols with optically active phosphorylating agent
19 (Scheme 2). The two phosphorus singlets that arise from the diastereomeric pairs have 31P ∆δ that
range from 0.5 to 2.9 ppm and are thus easily resolved and integrated on high-field NMR spectrometers.
The major advantage of this method is operational simplicity. The reaction and analysis are carried
out directly in the NMR tube making isolation or purification of the derivatized alcohols unnecessary.
All meso-diesters proved to be substrates for the enzyme and gave impressive enantioselectivities after
hydrolysis (Table 1). The accuracy of the 31P NMR technique was verified by comparison with the GC-
analyzed e.e. value2,3 for the Mosher-derived ester of (+)-2. A 1% margin of error was observed.
Scheme 2.
Interestingly, a correlation was found in this system between the relative 31P absorption frequency of
the derivatized chiral alcohols and the absolute configuration of the cyclopentanoid. In every example
analyzed, the derivatized (S)-hydroxycyclopentanoids were found to adsorb at slightly higher field than
their diastereomeric (R)-hydroxy counterparts (Table 2). This observation may prove valuable when
assigning absolute configurations across a similar series of analogs.
2.4. Stereochemical correlations
The absolute configurations of the chiral monohydroxy esters were assigned through stereochemical
correlation with the exception of α-epoxide 10, which was deduced from comparison with the literature
rotation.12 The thoroughly documented2 (1R,4S)-(+)-4-hydroxycyclopent-2-enyl acetate (+)-2 served as
the asymmetric benchmark upon which the absolute configurations for 4, 6, 8, and 12 were based.
Catalytic hydrogenation of (+)-2 with 10% Pd on activated carbon cleanly afforded authentic (1R,3S)-(+)-
3-hydroxycyclopentyl acetate (+)-4. (S)-Hydroxy acetates (+)-6 and (+)-8 were synthesized from (+)-2

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Table 2
Diastereomeric 31P NMR absorption frequencies for the phosphorylated cyclopentanols used to
determine enantiomeric puritya,b
as described earlier for their racemic analogs. Cyclohexylidene acetal (−)-12 was prepared from (+)-2
using the aforementioned osmylation–ketalization sequence.
The three unsaturated chiral esters 14, 16, and 18 were converted into the known14 (+)-(1S,4R)-4-
phthalimido-2-cyclopenten-1-ol 21 via a palladium-catalyzed allylic substitution reaction with potassium
phthalimide (Scheme 3). This stereospecific substitution conserves the chirality of the stereocenter over
the course of the reaction. In each case, the cyclopentylphthalimide 21 obtained rotated polarized light
in the positive direction, which implies the (S)-arrangement about the hydroxyl function in esters 14, 16,
and 18.
Scheme 3.
3. Conclusions
A few salient points have emerged from this study. Most startling is the singular reversal in enzyme
stereoselectivity within the series of related cyclopentanoid analogs. Breach occurred when the cyclo-
pentyl ring was symmetrically fused with a β-cyclopropyl functionality (entry 3). In this example, the
pro-(R)-acetoxy group on 5 was preferentially hydrolyzed to supply the (R)-hydroxy enantiomer 6 in
99% e.e. This result is in sharp contrast to the skeletally analogous, yet more polar β-epoxy cyclopentane
(entry 4) which yielded the (S)-hydroxy compound 8 predominantly, albeit with diminished selectivity
(67% e.e.). These data suggest that polarity can influence the selection of opposing enantioselective
pathways.
Still, this substrate-induced reversal in enzyme enantioselectivity on a cyclopentanoid is not an isolated
event with EeAChE. Griffith and Danishefsky6,15 experienced a similar outcome during their impressive
synthetic study on the potent chitinase inhibitor allosamadin. They reported that desymmetrization of
meso-diacetate 22 with EeAChE gave the unexpected (R)-hydroxy16 product 23 in >95% e.e. (Scheme 4).

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Since the substitution pattern on 22 is quite unlike those found on the substrates encountered in this study,
it would be unwise to draw any conclusions based upon structural considerations. However, this is an area
we plan to investigate in the future.
Scheme 4.
Also noteworthy is the finding that propionates, butyrates, and isobutyrates (entries 7, 8, and 9) also
serve as substrates for this enzyme. Curiously, these three homologs induce slightly better enantioselec-
tivities than the acetate group, the enzyme’s evolutionary target. However, the larger ester moieties,
especially the butyrates, suffered slower rates of enzymatic hydrolysis. For instance, the rate of mo-
nohydrolysis of dibutyrate 17 was in the order of days, while under identical conditions, diacetate 1
underwent the enzymatic hydrolysis in a matter of hours. This result was probably a manifestation of
both the dibutyrate’s decreased solubility in the aqueous buffer compared with the smaller, more polar
acetoxy group and the spatial constraints imposed by the enzyme’s acyl binding pocket. This latter point
has been previously argued17,18 to explain AChE’s diminished activity towards butyrylcholine relative to
the butyrylcholinesterase enzyme and is supported by site-specific mutagenesis and molecular modeling
studies.
Finally, the enantioselectivities posted for 6, 8, and 10 (entries 3, 4, and 5) correlate with those
obtained through Theil’s12 complementary approach of lipase-catalyzed transesterifications on meso-
cyclopentane-1,3-diols. The three chiral monoacetates produced in this manner possessed absolute confi-
gurations opposite to those observed in the present study. Moreover, it was found that transesterifications
across a range of substrate and lipase combinations always yielded (R)-hydroxy-enriched products except
in the case of the β-cyclopropyl derivative 6, which was formed preferentially in the (S)-hydroxy
configuration. It is indeed curious that in both studies the cyclopropyl substrate was the sole outlier
in an otherwise uniform stereochemical pattern of product formation.
4. Experimental
All non-aqueous reactions were carried in flame-dried glassware under a nitrogen atmosphere. The
acetic, propionic, isobutyric, and butyric acids and their corresponding anhydrides were purchased from
Aldrich Chemical Company. Tetrahydrofuran was distilled from a midnight blue sodium ketyl solution
under nitrogen. Methylene chloride and pyridine were distilled from CaH2 immediately prior to use.
Chromatography solvents were purchased from VWR or Fischer and were used as received. Acetylcholi-
nesterase (EeAChE, EC 3.1.1.7., 200 units/mg) was purchased from Sigma Chemical Company (C-3389)
as a lyophilized powder. The 0.58 M NaH2PO4 buffer was prepared from sodium dihydrogen phosphate
monohydrate obtained from Alpha Products (#307892) and adjusted to pH 6.9 with concentrated NaOH
solution (MCB Reagent, SX0593-1).
1H, 13C, and 31P NMR spectra were recorded on Bruker AC 200 or ARX 500 MHz spectrometers
with CDCl3 as solvent, except in the 31P experiments where d6-benzene was employed. Optical rotations
were measured at 589 nm on a JASCO DIP-369 polarimeter with spectral grade solvents using a 1 mL
capacity water-jacketed microcell connected to a constant temperature bath. FTIR spectra were obtained
on NaCl plates with a Mattson 3050 Galaxy spectrometer operating at 10 scans and a resolution of
4 cm−1. Preparative separations were performed by radial chromatography on a model 7924T Harrison

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Research Chromatotron. Analytical TLC analyses were conducted on Baker Si 250 precoated glass plates
(0.25 mm) and developed using either ethanolic p-anisaldehyde reagent or phosphomolybdic acid. New
compounds were distilled bulb-to-bulb and submitted to UC Riverside Mass Spectrometry Facility for
HRMS analysis. Sample homogeneity was verified by high signal-to-noise 13C NMR spectra.
4.1. Preparation of cis-cyclopent-1,3-diyl diacetate 3
A stirred suspension of 10% Pd on activated carbon (10 mg) and diacetate 1 (200 mg, 1.09 mmol)
in 3.6 mL of absolute ethanol was cooled to −78°C and degassed (four times) under vacuum. The flask
was fitted with a balloon containing hydrogen and allowed to return to room temperature. The reaction
was quenched after two hours by passing the suspension through a plug of 1 g MgSO4 and 2 g of SiO2
with 25 mL of 1:1 hexane:ethyl acetate followed by 25 mL of ether. The filtrate was concentrated under
vacuum and the residual oil was purified by SiO2 radial chromatography with 3:1 hexane:ethyl acetate
(Rf 0.25) to yield 38–61% of a colorless oil:19 IR (film) 2979, 1739 (C_O), 1400, 1250, 1000 cm−1; 1H
NMR (200 MHz) δ 5.07 (m, 2H), 2.31 (overlapping dt, J=15.4, 7.2 Hz, 1H), 2.01 (s, 6H), 1.70–1.95 (m,
5H); 13C NMR δ 170.6, 74.6, 38.8, 30.6, 21.1.
4.2. Preparation of (ꢀ)-cis-3-hydroxycyclopentyl acetate 4
The preparation of 4 was carried out as described for 3 substituting monoacetate 2 for the alkene
substrate. Colorless oil:20 IR (film) 3441 (br OH), 2971, 1725 (C_O), 1450, 1400, 1250, 1100 cm−1; 1H
NMR (200 MHz) δ 5.12 (m, 1H), 4.29 (m, 1H), 2.13 (overlapping dt, J=14.9, 8.7 Hz, 1H), 2.01 (s, 3H),
1.89 (m, 5H); 13C NMR δ 170.6, 75.7, 72.6, 41.9, 34.0, 30.6, 21.2; 31P data for the diastereomeric pair
derived from 19, 31P NMR δ 136.2 and 135.6.
4.3. Preparation of (1RS,2SR,4RS,5SR)-bicyclo[3.1.0]hex-2,4-diyl diacetate 5
The preparation of 5 was carried out as described for 13 using 6 as the monohydroxy substrate and
acetic anhydride as the acylating agent. The crude material was chromatographed over SiO2 with 2:1
hexane:ethyl acetate (Rf 0.40) to yield 50% of a colorless oil:12 IR (film) 2960, 1736 (C_O), 1400,
1362, 1250, 1238, 1021 cm−1; 1H NMR (200 MHz) δ 5.21 (dt, J=8.5, 3.4 Hz, 2H), 2.43 (overlapping dt,
J=13.6, 8.0 Hz, 1H), 2.01 (s, 6H), 1.76 (m, 2H), 1.27 (overlapping dt, J=13.6, 8.8 Hz, 1H), 0.93 (m, 1H),
0.59 (m, 1H); 13C NMR δ 170.9, 73.0, 30.2, 21.0, 20.1, 3.5.
4.4. Preparation of (1RS,2SR,4RS,5SR)-(ꢀ)-4-hydroxybicyclo[3.1.0]hex-2-yl acetate 6
A stirred suspension of zinc–copper couple21,22 (365 mg, 2.8 mmol), CH2I2 (114 µL, 1.41 mmol) in
ether (1 mL) was heated to reflux for 15 min. A solution of 2 (100 mg, 0.70 mmol) dissolved in ether (0.5
mL) was then added to the flask in one portion. After 3 h, the reaction was quenched by the addition of
sat. aq. NH4Cl and then passed through a plug of Celite with 50 mL of ethyl acetate. The organic layer
was washed successively with water (3×20 mL), sat. aq. NaHCO3 (3×20 mL), water (3×10 mL), and
brine (3×10 mL). The organic layer was dried over MgSO4, filtered, and concentrated under vacuum.
The crude material was purified over SiO2 by radial chromatography using 2:3 hexane:ethyl acetate as
the eluent (Rf 0.30) to yield 7–38% of a colorless oil:12 IR (film) 3409 (br OH), 2928, 1729 (C_O), 1378,
1254, 1025 cm−1; 1H NMR (200 MHz) δ 5.15 (dt, J=8.4, 4.5 Hz, 1H), 4.46 (dt, J=8.2, 4.5 Hz, 1H), 2.32
(overlapping dt, J=13.3, 7.8 Hz, 1H), 2.01 (s, 3H), 1.70 (m, 2H), 1.17 (overlapping dt, J=13.4, 8.8 Hz,

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1H), 0.92 (m, 1H), 0.54 (m, 1H); 13C NMR δ 170.9, 73.2, 70.1, 33.0, 22.7, 20.9, 19.9, 2.2; 31P data for
the diastereomeric pair derived from 19, 31P NMR δ 135.4 and 133.6.
4.5. Preparation of (1RS,2SR,4RS,5SR)-6-oxabicyclo[3.1.0]hex-2,4-diyl diacetate 7
The preparation of 7 was carried out as described for 13 using 8 as the monohydroxy substrate and
acetic anhydride as the acylating agent. Chromatographed over SiO2 with 2:1 hexane:ethyl acetate (Rf
0.56, 1:1 hexane:ethyl acetate) to give a 70% yield of a white solid:12 IR (KBr) 2925, 1732 (C_O),
1460, 1364, 1250, 1072, 1031 cm−1; 1H NMR (200 MHz) δ 5.00 (t, J=8.2 Hz, 2H), 3.66 (s, 2H), 2.39
(overlapping dt, J=12.7, 7.9 Hz, 1H), 2.09 (s, 6H), 1.51 (overlapping dt, J=12.7, 8.6 Hz, 1H); 13C NMR
δ 170.4, 71.3, 54.7, 27.8, 20.6.
4.6. Preparation of (1RS,2SR,4RS,5RS)-(ꢀ)-4-hydroxy-6-oxabicyclo[3.1.0]hex-2-yl acetate 8
To an ice-cold stirred solution of 2 (48 mg, 0.341 mmol) in 10 mL of CH2Cl2 was added MCPBA
(141 mg, 0.811 mmol) portionwise. The ice bath was removed and the reaction mixture was heated at
reflux overnight. The solid material was removed from the reaction mixture by gravity filtration and
rinsed with hexane (2×1 mL). The filtrate was washed sequentially with fresh sat. Na2S2O3 (3×5 mL)
and sat. NaHCO3. The aqueous layers were then combined, treated with brine, and extracted with 1:1
ether:ethyl acetate (10×3 mL), then with pure ethyl acetate (20×3 mL) until TLC analysis showed no
epoxide product remaining in the aqueous layer. The organic extracts were combined, dried over MgSO4,
and concentrated under reduced pressure. The crude material was chromatographed over SiO2 with 1:1
hexane:ethyl acetate (Rf 0.18) to give a 38% yield of a colorless oil:12 IR (film) 3450 (br OH), 3057, 1734
(C_O), 1374, 1247, 1064, 1032 cm−1; 1H NMR (200 MHz) δ 4.94 (dt, J=8.5, 1.4 Hz, 1H), 4.16 (br t,
J=7.8 Hz, 1H), 3.64 (dd, J=2.9, 1.3 Hz, 1H), 3.54 (dd, J=2.9, 1.4 Hz, 1H), 2.35 (overlapping dt, J=12.8,
7.8 Hz, 1H), 2.10 (s, 3H), 1.34 (overlapping dt, J=12.8, 8.4 Hz, 1H); 13C NMR δ 170.8, 71.8, 69.7, 57.3,
55.4, 31.0, 20.6; 31P data for the diastereomeric pair derived from 19, 31P NMR δ 137.7 and 137.3.
4.7. Preparation of (1RS,2RS,4SR,5SR)-6-oxabicyclo[3.1.0]hex-2,4-diyl diacetate 9
The preparation of 9 is a modification of the procedure used by Theil et al.12 To a stirred solution
of 2 in methanol (7.1 mL) and ether (1.8 mL) was added K2CO3 (195 mg, 20 mol%). The hydrolysis
reaction was monitored by TLC. After 4 h, the reaction mixture was passed through a plug of SiO2
and MgSO4 and then concentrated under vacuum to yield 644 mg of thick oil which was used without
further purification. The crude diol was dissolved in CH2Cl2 (32.3 mL) and triethylamine (3.2 mL) and
then treated with excess TBDMSCl (3.9 g, 25.8 mmol) and catalytic DMAP (78.8 mg, 10 mol%). The
reaction proceeded for 1 h and was then quenched by addition of H2O. The mixture was extracted with
ether, washed with sat. NaHCO3, and evaporated under aspirator pressure. The crude material was passed
through a plug of SiO2 and MgSO4 with a solution of 40:1 hexanes:ethyl acetate. After removal of
the solvent, 634 mg (30% yield) of reasonably pure cis-1,3-disilylated-4-cyclopentene remained behind
and was judged satisfactory for the next epoxidation step. Dissolution of the TBDMS-diprotected diol
(634 mg, 1.93 mmol) in CH2Cl2 (6.4 mL) was followed by the addition of freshly purified MCPBA
(567 mg, 6.8 mmol) and NaHCO3 (1.16 g, 6.8 mmol). The reaction proceeded for three days before
being terminated by passing the mixture through an SiO2–MgSO4 plug with hexanes. The filtrate was
concentrated under vacuum and the residual oil purified via radial chromatography over SiO2 to yield
167.8 mg (25%) of the desired α-epoxy product and 162.1 mg (24.5%) of the diastereomeric β-epoxide:

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Rf (35:1 hexanes:ethyl acetate) α-isomer=0.33; β-isomer=0.29. Deprotection of the hydroxyl groups
was accomplished as follows: the α-epoxide (165 mg, 0.48 mmol) was dissolved in THF (0.5 mL) and
subjected to the slow addition of 4 equiv. (2 mL) of 2 M tetrabutylammonium fluoride (TBAF) in THF.
The reaction was complete in 5 min as visualized by TLC. Removal of the solvent and subsequent
diacetylation of the crude diol with acetic anhydride provided, after chromatographic separation over
SiO2 with 1:1 hexane:ethyl acetate (Rf 0.59), a 57% yield of crystalline 9:12 IR (KBr) 2979, 2925, 2854,
1739 (C_O), 1427, 1376, 1239, 1077 cm−1; 1H NMR (200 MHz) δ 5.21 (br d, J=6.1 Hz, 2H), 3.62 (br s,
2H), 2.11 (dt, J=17.4, 6.1 Hz, 1H), 2.01 (s, 6H), 1.78 (d, J=17.4 Hz, 1H); 13C NMR δ 170.2, 72.5, 56.6,
35.4, 20.9.
4.8. Preparation of (1RS,2RS,4SR,5RS)-(ꢀ)-4-hydroxy-6-oxabicyclo[3.1.0]hex-2-yl acetate 10
To a 10 mL flask was added diacetate 9 (40 mg, 0.2 mmol) in 5 mL of dry methanol. The flask was
cooled in an ice bath and finely powdered K2CO3 was added in one portion. The stirred suspension
was held at 0°C and monitored closely by TLC until the appearance of diol became evident (∼13 min).
The reaction mixture was immediately passed through a plug of SiO2 with 10 mL of methanol. Excess
solvent was removed under vacuum to give a yellowish oil. Purification of the monohydroxy compound
was performed by radial chromatography over SiO2 with 1:1 hexane:ethyl acetate (Rf 0.22) to give a 29%
yield of a colorless oil:12 IR (film) 3456 (br OH), 2953, 1736 (C_O), 1374, 1244, 1082 cm−1; 1H NMR
(200 MHz) δ 5.19 (d, J=6.3 Hz, 1H), 4.30 (d, J=6.1 Hz, 1H), 3.59 (br d, J=7.3 Hz, 2H), 2.45 (br s, 1H),
2.03 (s, 3H), 2.00 (overlapping dt, J=15.5, 6.4 Hz, 1H), 1.68 (d, J=15.8 Hz, 1H); 13C NMR δ 170.1,
73.3, 70.8, 58.3, 56.0, 37.7, 21.1; 31P data for the diastereomeric pair derived from 19, 31P NMR δ 135.0
and 132.1.
4.9. Preparation of (1RS,2RS,3SR,4SR)-2,3-O-cyclohexylidenecylopent-1,4-diyl diacetate 11
To a 25 mL flask equipped with a glass-coated stir bar were added diacetate 1 (194 mg, 1.00 mmol), N-
methylmorpholine-N-oxide (NMO) (234 mg, 2.0 mmol), and 12.5 mL of an 8:1 acetone:water solution.
Three millilitres of a 2% OsO4 in water were added in one portion and the cis-hydroxylation reaction
was allowed to proceed for 24 h at rt. The reaction was quenched by the addition of Na2SO3. The
reaction mixture was passed through a 5×30 mm plug of SiO2 with 100 mL of ether. The filtrate was
concentrated under vacuum to give a semicrystalline oil which was used in the ketalization step without
further purification. The crude diol was transferred to a dry 15 mL flask, dissolved in 1.86 mL (2 mmol)
of freshly distilled cyclohexanone, and exposed to a small crystal of PTSA. The flask was heated at an
oil-bath temperature of 75°C until TLC analysis indicated the starting material no longer remained (∼20
h). The reaction mixture was passed through a plug of SiO2 with ether and the solvent was removed under
vacuum. The crude oil was purified over SiO2 with 9:1 hexanes:ethyl acetate using radial chromatography
to afford 82.4 mg (30% yield) of a colorless oil: Rf 0.6, 1:1 hexane:ethyl acetate; IR (film) 2938, 2861,
1740 (C_O), 1371, 1250, 1021 cm−1; 1H NMR (200 MHz) δ 5.06 (br dd, J=5.4, 1.2 Hz, 2H), 4.57 (d,
J=1.6 Hz, 2H), 2.38 (dt, J=15.7, 5.4 Hz, 1H), 2.01 (s, 6H), 1.95 (dt, J=15.7, 1.24 Hz, 1H), 1.3–1.65 (m,
10H); 13C NMR δ 169.7, 111.9, 83.8, 78.7, 36.2, 34.4, 33.5, 25.0, 23.8, 23.5, 20.9; HRMS (CI, NH3)
calcd for C15H23O6 (M+H+) 299.1495, found 299.1479.

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4.10. Preparation of (1RS,2RS,3RS,4SR)-(ꢀ)-2,3-O-cyclohexylidene-4-hydroxycyclopentyl acetate 12
The preparation of 12 was carried out as described for 11 substituting 2 as the alkene substrate.
Colorless oil: Rf 0.28, 1:1 hexane:ethyl acetate; IR (film) 3449 (br OH), 2937, 1721 (C_O), 1371, 1250,
1021 cm−1; 1H NMR (200 MHz) δ 5.15 (d, J=5.3 Hz, 1H), 4.59 (m, 2H), 4.22 (m, 1H), 2.31 (dt, J=15.2,
5.2 Hz, 1H), 2.01 (s, 3H), 1.85 (br d, J=15.2 Hz, 1H), 1.65–1.3 (m, 10H); 13C NMR δ 169.6, 111.5,
85.9, 83.8, 79.7, 36.4, 36.2, 33.4, 25.0, 23.8, 23.5, 21.0; 31P data for the diastereomeric pair derived
from 19, 31P NMR δ 134.5 and 133.5; HRMS (DCI, NH3) calcd for C13H21O5 (M+H+) 257.1389, found
257.1379.
4.11. Preparation of cis-cyclopent-2-en-1,4-diyl dipropionate 13
Into a dry 10 mL flask equipped with a magnetic stir bar was added (ꢀ)-cis-4-hydroxycyclopent-2-
enyl propionate ((14) 74.9 mg, 0.749 mmol) and 0.5 mL of pyridine. The solution was cooled to 0°C
with an ice-water bath. Following the dropwise addition of propionic anhydride (0.2 mL, 1.5 mmol) via
syringe, the ice bath was removed and the reaction was monitored by TLC (2:1 hexane:ethyl acetate).
After 24 h, the reaction mixture was recooled to 0°C, layered with ether, and quenched with ice water.
The organic layer was washed consecutively with sat. CuSO4 (three times), NH4Cl (three times), and
brine. The organic phase was then filtered through a plug comprised of MgSO4 (1 g) and SiO2 (2 g)
with 25 mL of 1:1 hexane:ethyl acetate followed by 25 mL of ether. The solution was concentrated under
vacuum and the crude oil chromatographed over SiO2 using radial chromatography with 2:1 hexane:ethyl
acetate (Rf 0.68) to give a 74% yield of a colorless oil:23 IR (film) 2983, 1739 (C_O), 1351, 1353, 1190,
1
1081 cm−1; H NMR (200 MHz) δ 6.05 (s, 2H), 5.53 (dd, J=7.5, 3.9 Hz, 2H), 2.86 (overlapping dt,
J=15.0, 7.5 Hz, 1H), 2.30 (q, J=7.5 Hz, 2H), 1.65 (dt, J=15.0, 3.8 Hz, 1H), 1.11 (t, J=7.5 Hz, 3H); 13
NMR δ 173.9, 134.5, 76.9, 37.2, 27.5, 8.9.
C
4.12. Preparation of (ꢀ)-cis-4-hydroxycyclopent-2-enyl propionate 14
Into a dry 25 mL flask equipped with a magnetic stir bar was added 6.25 mL of THF. The stirred
solution was cooled in an ice-water bath and 1 mol% Pd(PPh3)4 was added in one portion. After
dissolution of the catalyst, propionic acid (0.326 mL, 6.10 mmol) was added to the flask. This was
immediately followed by a 5 min dropwise addition of a solution of cyclopentadiene monoepoxide (0.5
mL, 6.109 mmol) dissolved in 2 mL of THF. The reaction was monitored by TLC (1:1 hexane:ethyl
acetate) and quenched after 15 min by passing the solution through a plug of SiO2 (2 g) and MgSO4
(1 g) with sufficient ether to ensure all of the product had been eluted. The solvent was removed
by rotary evaporation and the residue was dissolved in ether and washed (three times) with sat.
NaHCO3. The aqueous layer was back-extracted several times with 1:1 ether:ethyl acetate. The combined
organic extracts were dried over MgSO4, concentrated under reduced pressure, and the residual oil
chromatographed over SiO2 using radial chromatography with 2:1 hexane:ethyl acetate (Rf 0.21) to give
1
a 44% yield of a colorless oil:24 IR (film) 3423 (br OH), 2981, 1731 (C_O), 1191 (C–O) cm−1; H
NMR (200 MHz) δ 6.10 (m, 1H), 5.99 (ddd, J=5.5, 2.0, 1.1 Hz, 1H), 5.51 (m, 1H), 4.72 (m, 1H), 2.81
(overlapping dt, J=14.6, 7.3 Hz, 1H), 2.32 (q, J=7.5 Hz, 2H), 1.87 (br s, 1H), 1.64 (dt, J=14.6, 3.8 Hz,
1H), 1.14 (t, J=7.5 Hz, 3H); 13C NMR δ 174.1, 138.4, 132.6, 76.9, 74.8, 40.6, 27.7, 9.0; 31P data for the
diastereomeric pair derived from 19, 31P NMR δ 135.7 and 134.8.

D. R. Deardorff et al. / Tetrahedron: Asymmetry 10 (1999) 2139–2152
2149
4.13. Preparation of cis-cyclopent-2-en-1,4-diyl diisobutyrate 15
The preparation was carried out as described for 13 using 16 as the monohydroxy substrate and
isobutyric anhydride as the acylating agent. Chromatographed over SiO2 with 2:1 hexane:ethyl acetate
(Rf 0.65) to give a 70% yield of a colorless oil: IR (film) 2975, 1735 (C_O), 1470, 1351, 1258, 1189,
1154, 1071 cm−1; 1H NMR (200 MHz) δ 6.08 (br d, J=0.7 Hz, 2H), 5.56 (ddd, J=0.7, 4.0, 7.5 Hz, 2H),
2.87 (overlapping dt, J=14.8, 7.5 Hz, 1H), 2.54 (septet, J=7.0 Hz, 1H), 1.67 (dt, J=14.8, 4.0 Hz, 1H),
1.15 (d, J=7.0 Hz, 6H); 13C NMR δ 176.6, 134.6, 76.3, 37.4, 33.9, 18.8, 18.7; HRMS (CI, NH3) calcd
+
for C13H24NO4 (M+NH4 ) 258.1705, found 258.1710.
4.14. Preparation of (ꢀ)-cis-4-hydroxycyclopent-2-enyl isobutyrate 16
Chromatographed over SiO2 with 2:1 hexane:ethyl acetate (Rf 0.27) to give a 47% yield of a colorless
oil: IR (film) 3411 (br OH), 2976, 1729 (C_O), 1159 (C–O) cm−1; 1H NMR (200 MHz) δ 6.07 (m, 1H),
5.92 (m, 1H), 5.50 (m, 1H), 4.72 (m, 1H), 3.44 (br s, 1H), 2.82 (overlapping dt, J=14.6, 7.3 Hz, 1H),
2.52 (septet, J=7.0 Hz, 1H), 1.60 (dt, J=14.6, 3.9 Hz, 1H), 1.15 (d, J=7.0 Hz, 6H); 13C NMR δ 176.9,
138.4, 132.3, 76.9, 74.6, 40.5, 33.9, 18.8; 31P data for the diastereomeric pair derived from 19, 31P NMR
+
δ 135.7 and 134.9; HRMS (CI, NH3) calcd for C9H18NO3 (M+NH4 ) 188.1287, found 188.1283.
4.15. Preparation of cis-cyclopent-2-en-1,4-diyl dibutyrate 17
The preparation was carried out as described for 13 using 18 as the monohydroxy substrate and butyric
anhydride as the acylating agent. Chromatographed over SiO2 with 1:1 hexane:ethyl acetate (Rf 0.96) to
give a 70% yield of a colorless oil: IR (film) 2966, 2877, 1737 (C_O), 1176 cm−1; 1H NMR (200 MHz)
δ 6.06 (d, J=0.8 Hz, 2H), 5.54 (ddd, J=7.5, 3.9, 0.8 Hz, 2H), 2.86 (overlapping dt, J=14.9, 7.5 Hz, 1H),
2.26 (t, J=7.4 Hz, 4H), 1.66 (m, 5H), 0.95 (t, J=7.3 Hz, 6H); 13C NMR δ 173.0, 134.5, 76.3, 37.3, 36.1,
+
18.3, 13.5; HRMS (CI, NH3) calcd for C13H24NO4 (M+NH4 ) 258.1705, found 258.1703.
4.16. Preparation of (ꢀ)-cis-4-hydroxycyclopent-2-enyl butyrate 18
Chromatographed over SiO2 with 1:1 hexane:ethyl acetate (Rf 0.48) to give a 36–49% yield of a
colorless oil: IR (film) 3422 (br OH), 2966, 2877, 1730 (C_O), 1183 cm−1; 1H NMR (200 MHz) δ 6.08
(m, 1H), 5.95 (m, 1H), 5.46 (m, 1H), 4.69 (m, 1H), 2.78 (overlapping dt, J=14.6, 7.3 Hz, 1H), 2.25 (t,
J=7.2 Hz, 2H), 1.71 (br s, 1H), 1.57 (m, 2H), 0.90 (t, J=7.4 Hz, 3H); 13C NMR δ 177.3, 138.4, 132.3,
76.8, 74.5, 40.5, 36.2, 18.3, 13.4; 31P data for the diastereomeric pair derived from 19, 31P NMR δ 135.4
+
and 134.6; HRMS (CI, NH3) calcd for C9H18NO3 (M+NH4 ) 188.1287, found 188.1286.
4.17. General procedure for the enzymatic hydrolysis of meso-diesters
Into a 10 mL round-bottom flask equipped with a stir bar free of any metal filings is added the meso-
diester (∼180–200 mg). The substrate is suspended in 6 mL of a 0.58 M NaH2PO4 buffer that had been
adjusted to pH 6.9. A trace amount of NaN3 was added to discourage bacterial growth. A small flake
of lyophilized EeAChE (1 mg enzyme/g substrate) was added to the gently stirred mixture by means
of a microspatula. The course of the reaction was monitored closely by TLC analysis (2:1 hexane:ethyl
acetate). Diacetate substrates react between 4 and 24 h, with butyrates taking substantially longer. As the
diester is consumed, the initially two-phase system converges into a single aqueous layer. The reaction is

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quenched by the addition of sat. NH4Cl and the hydrolyzed products are isolated by repeated extraction
with 1:1 ether:ethyl acetate until TLC analysis of the aqueous phase indicates the monohydroxyester is no
longer present. The combined extracts are dried over MgSO4, filtered, and concentrated under vacuum.
The crude residue is further purified by chromatography over SiO2.
4.17.1. (1S,3R)-(+)-3-Hydroxycyclopentyl acetate (+)-4
Colorless oil: 70% yield (86% yield based upon recovered starting material); [α]28=+35.8 (c 0.52,
D
CHCl3); 93% e.e. based upon 31P data for derivative with 19, 31P NMR δ 135.5.
4.17.2. (1R,2S,4R,5S)-(−)-4-Hydroxybicyclo[3.1.0]hex-2-yl acetate (−)-6
Colorless oil: 60% yield (74% yield based upon recovered starting material); rotation of 99% e.e. ent-6
(prepared from 99% e.e. (+)-2): [α]24=+46.9 (c 1.25, CHCl3); calculated rotation [α]24=−46.9 (c 1.25,
D
D
CHCl3) for 99% e.e., based upon 31P data for derivative with 19, 31P NMR δ 135.4.
4.17.3. (1S,2R,4S,5S)-(+)-4-Hydroxy-6-oxabicyclo[3.1.0]hex-2-yl acetate (+)-8
Colorless oil: 72% yield; [α]25=+32.0 (c 1.32, CHCl3); 67% e.e. based upon 31P data for derivative
D
with 19, 31P NMR δ 137.3.
4.17.4. (1R,2R,4S,5R)-(+)-4-Hydroxy-6-oxabicyclo[3.1.0]hex-2-yl acetate (+)-10
Colorless oil: 40% yield (95% yield based upon recovered starting material); [α]25=+15 (c 0.865,
D
CHCl3); 82% e.e. based upon lit. value12 for 82% e.e. [α]20=+15.0 (c 1.0, CHCl3); 31P data for derivative
D
with 19, 31P NMR δ 135.5.
4.17.5. (1R,2R,3R,4S)-(−)-2,3-O-Cyclohexylidene-4-hydroxycyclopentyl acetate (−)-12
Colorless oil: 61% yield (64% yield based upon recovered starting material); rotation of 99% e.e. (−)-
12 (prepared from 99% e.e. (+)-2): [α]25.6=−7.24 (c 2.4, CHCl3); [α]26=−5.9 (c 0.29, CHCl3); 81% e.e.
D
D
based upon 31P data for derivative with 19, 31P NMR δ 133.4.
4.17.6. (1R,4S)-(+)-4-Hydroxycyclopent-2-enyl propionate (+)-14
Colorless oil: 91% yield; [α]25=+63.1 (c 1.045, CHCl3); 98% e.e. based upon 31P data for derivative
D
with 19, 31P NMR δ 134.8.
4.17.7. (1R,4S)-(+)-4-Hydroxycyclopent-2-enyl isobutyrate (+)-16
Colorless oil: 48% yield (72% yield based upon recovered starting material); [α]25=+66.8 (c 1.315,
D
CHCl3); >99% e.e. based upon 31P data for derivative with 19, 31P NMR δ 134.9.
4.17.8. (1R,4S)-(+)-4-Hydroxycyclopent-2-enyl butyrate (+)-18
Colorless oil: 49% yield; [α]25=+57.0 (c 1.02, CHCl3); 97% e.e. based upon 31P data for derivative
D
with 19, 31P NMR δ 134.6.
4.18. The conversion of monohydroxy esters 14, 16, and 18 into (+)-(1S,4R)-4-phthalimidocyclopent-
2-en-1-ol 21
The preparation of 21 was carried out as previously described14 substituting 14, 16, and 18 for (+)-2.
Purification by chromatography over SiO2 with 1:1 hexane:ethyl acetate (Rf 0.47) gave a colorless solid:
mp 69–71°C; [α]26=+276 (c 1.03, CHCl3); IR (KBr) 3398 (br OH), 1697 (C_O), 1380, 720 cm−1; 1H
D

D. R. Deardorff et al. / Tetrahedron: Asymmetry 10 (1999) 2139–2152
2151
NMR (200 MHz) δ 7.90–7.65 (m, 4H), 6.22 (m, J=5.5 Hz, 1H), 5.72 (dd, J=5.5, 2.5 Hz, 1H), 5.23 (m,
J=9.6, 2.2 Hz, 1H), 4.74 (m, 1H), 4.07 (br s, 1H), 2.82 (ddd, J=15.4, 9.6, 7.8 Hz, 1H), 1.97 (br d, J=15.4
Hz, 1H); 13C NMR δ 168.4, 138.4, 134.2, 131.8, 130.1, 123.3, 75.8, 53.0, 38.2.
4.19. General method for the determination of enantiomeric excess with reagent 19
The chiral derivatizing agent 19 was prepared from trans-1,2-diaminocyclohexane as reported by
Alexakis.13 This reagent was stored at −20°C as a 0.20 M solution in benzene with 10% C6D6. To
determine the enantiomeric ratios, 0.11 mmol of 19 and 0.10 mmol of the chiral alcohol were added to a
5 mm NMR tube. The mixture was allowed to stand until no dimethylamine was detected with a litmus
paper held at the mouth of the tube. No isolation or purification of the derivatized chiral alcohol was
needed for this analysis. The sample was locked on deuterated benzene and the 31P signals recorded. The
signals were integrated to calculate the e.e. values reported in this paper.
Acknowledgements
The authors would like to thank the donors of the Petroleum Research Fund, administered by the
American Chemical Society, and the National Science Foundation (CHE-8908212) for support of this
research. D.R.D. and S.W.T.C. are especially grateful to the Camille and Henry Dreyfus Scholar/Fellow
Program for Undergraduate Institutions. Summer undergraduate research stipends were provided to
J.W.M. by the Ford Foundation, to V.F. (community college student participant) and H.Y.K. by the
NSF REU Program (CHE-9100606), and to R.B.A. by the NSF California Alliance for Minority
Participation Program. C.M.T. acknowledges academic-year support from the Howard Hughes Medical
Institute through the Undergraduate Biological Sciences Education Program and summer support from
GlaxoWellcome. We are indebted to Drs. C. L. Craney and S. Shambayati for their suggestions and
advice, and thank A. Nguyen for her initial work on the dipropionates.
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