Gelozymes in organic synthesis. Part IV: Resolution of glycidate esters with crude Mung bean (Phaseolus radiatus) epoxide hydrolase immobilized in gelatin matrix

Article abstract of DOI:10.1016/j.tetasy.2008.01.036

A crude extract of Mung bean meal (Phaseolus radiatus) possessing epoxide hydrolase activity immobilized in gelatin gel (gelozyme) is employed in the stereoselective epoxide ring opening of glycidate esters. Thus, ethyl trans-(±)-3-phenyl glycidate 1a and methyl trans-(±)-3-(4-methoxyphenyl) glycidate 1b gave (2S,3R)-glycidate esters (ee >99% and 45% yield) with gelatin immobilized enzyme in diisopropyl ether. The corresponding (2R,3S)-enantiomer of 1a was hydrolyzed by an epoxide hydrolase to predominantly give the anti-product, ethyl (2R,3R)-2,3-dihydroxy-3-phenylpropanoate, with a diastereomeric excess of 78% and ee 94% (40%). A small amount (5%) of racemic syn-product was also obtained as a result of the spontaneous hydrolysis. In the case of 1b, the hydrolysis product was racemic due to high reactivity of the glycidate toward water.

Full text of DOI:10.1016/j.tetasy.2008.01.036

Available online at www.sciencedirect.com
Tetrahedron: Asymmetry 19 (2008) 1139–1144
Gelozymes in organic synthesis. Part IV: Resolution of glycidate
esters with crude Mung bean (Phaseolus radiatus) epoxide
hydrolase immobilized in gelatin matrixI
Avala Vedamayee Devi, Challa Lahari, Lakshmi Swarnalatha and N. W. Fadnavis*
Biotransformations Laboratory, Indian Institute of Chemical Technology, Uppal Road, Habsiguda, Hyderabad 500 007, India
Received 15 January 2008; accepted 23 January 2008
Available online 13 May 2008
Abstract—A crude extract of Mung bean meal (Phaseolus radiatus) possessing epoxide hydrolase activity immobilized in gelatin gel (gelo-
zyme) is employed in the stereoselective epoxide ring opening of glycidate esters. Thus, ethyl trans-( )-3-phenyl glycidate 1a and methyl
trans-( )-3-(4-methoxyphenyl) glycidate 1b gave (2S,3R)-glycidate esters (ee >99% and 45% yield) with gelatin immobilized enzyme in
diisopropyl ether. The corresponding (2R,3S)-enantiomer of 1a was hydrolyzed by an epoxide hydrolase to predominantly give the anti-
product, ethyl (2R,3R)-2,3-dihydroxy-3-phenylpropanoate, with a diastereomeric excess of 78% and ee 94% (40%). A small amount (5%)
of racemic syn-product was also obtained as a result of the spontaneous hydrolysis. In the case of 1b, the hydrolysis product was racemic
due to high reactivity of the glycidate toward water.
Ó 2008 Published by Elsevier Ltd.
1. Introduction
(R)-diols from racemic styrene oxide and related com-
pounds in excellent yields and enantiomeric purities.3 Here-
in, we report the application of the crude enzyme
preparation from Mung beans for the resolution of racemic
trans-ethyl phenyl glycidate 1a and trans-methyl (4-
methoxyphenyl)glycidate 1b to obtain enantiomerically
pure (2S,3R)-glycidate esters in high yields (90% of theoret-
ical) and high enantiomeric purity (ee >99%). (Scheme 1).
Although enantiomerically pure glycidate esters are of
great interest to organic chemists,4 we have surprisingly
not come across any reference work where epoxide hydro-
lase has been used to resolve a glycidate ester. Further-
more, several procedures are available to prepare the
(2R,3S)-enantiomers of 1 but very few efficient enzymatic
procedures4e are available for the (2S,3R)-enantiomer.
Over the last decade, a number of epoxide hydrolases (EC
3.3.2.3) performing hydrolytic kinetic resolution of epox-
ides have been discovered.1 Examples of bacterial epoxide
hydrolases include those isolated from Agrobacterium
radiobacter, Rhodococcus sp., Corynebacteriuim sp., Myco-
bacterium paranicasm, Nocardia sp., Pseudomonas NRRL
B-2994, and some Streptomyces strains. Fungal epoxide
hydrolases have also been found in Aspergillus niger, Hel-
minthosporum sativum, Diploida gossypina, Beauveria sulf-
urescens, and some Fursarium strains. The best-known
yeast epoxide hydrolase is Rhodotorulla glutinis enzyme.2
Although the applications of epoxide hydrolases in the
preparation of chiral epoxides and diols have been well
demonstrated,1,2 these enzymes are essentially obtained
through fermentation and hence are not easily accessible
to organic chemists. Recently, epoxide hydrolases have
been discovered in Mung beans (Phaseolus radiatus L.), a
commonly available commodity from supermarkets. The
enzyme from these beans has been found to provide chiral
2. Results and discussion
2.1. Epoxide hydrolase in Mung beans
Enantiomerically pure trans-ethyl phenyl glycidate 1a and
trans-methyl (4-methoxyphenyl)glycidate 1b are important
drug intermediates. Both the (2S,3R)- and (2R,3S)-enantio-
mers of ethyl phenyl glycidate 1a are useful in the synthesis
of the Taxol side chain,5 while the (2R,3S)-enantiomer
q IICT Communication No. 040412.
*
Corresponding author. Tel.: +91 40 27160123x2631; fax: +91 40
27160512; e-mail addresses: fadnavis@iict.res.in; fadnavisnw@yahoo.
com
0957-4166/$ - see front matter Ó 2008 Published by Elsevier Ltd.
doi:10.1016/j.tetasy.2008.01.036

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A. V. Devi et al. / Tetrahedron: Asymmetry 19 (2008) 1139–1144
O
HO
OH
HO
COOR1
OH
O
COOR1
Mung bean EH
COOR1
+
COOR1
+
Diisopropyl ether
R2
R2
R2
syn-( )-3a (minor)
R2
1a: R1 = Et, R2 = H
1b: R1 = Me, R2 = OMe
(2S,3R)-1, 45%
Ee > 99%
anti-(2R,3R)-2a (Major)
Ee 94%; de 78%
Scheme 1.
of 1b is used in the synthesis of Diltiazem, a drug used to
treat hypertension.6 The resolution of racemic glycidate
esters by an esterase/lipase catalyzed enantioselective
hydrolysis is a well-known process.6 However, the glycidic
acid, which forms as the hydrolysis product in this process
is unstable and quickly decomposes to 4-(methoxy) phenyl
acetaldehyde, which acts as an inhibitor of the enzyme. It
is necessary to design a special bioreactor that allows the
continuous removal of the aldehyde as a bisulfite adduct.6e
Furthermore, this aldehyde cannot be used as raw material
for the same process. On the other hand, the product of
enantioselective opening of the epoxide ring would
produce the corresponding diol which can be converted
to the starting aldehyde via a periodate reaction and
reused. Thus a resolution process based on epoxide hydro-
lase would be economically and environmentally more
favorable than a lipase based process. The report of the
presence of epoxide hydrolase activity in Mung beans
which are readily available in supermarkets led us to
investigate the application of the enzyme in the resolution
of glycidate esters.
products. For example, if the enzyme is completely stereo-
selective toward only the (2R,3S)-isomer of 1, and the
attack of nucleophilic water molecule results in the
inversion of configuration, the epoxide hydrolase EHA
opening of the epoxide ring at C2 (k1) would produce
anti-(2S,3S)-diol 2, while attack at the benzylic position
(k2) would produce the anti-(2R,3R) diol 2. Similarly,
EHB selective toward (2S,3R)-isomer would produce either
the (2R,3R) or (2S,3S)-enantiomer of 2 depending upon the
site of attack (Scheme 2).
Thus, the products of the enzymatic ring opening were
expected to be anti-diols, either (2R,3R) or (2S,3S) or a
mixture of theirs depending upon the relative activities
and selectivities of the two enzymes. Experimentally, the
reaction of 1 with crude Mung bean paste following the
procedure described by Xu et al.3 indicated a selectivity
toward the (2R,3S)-enantiomer (ee 40% at 80% conver-
sion). The Mung bean paste was hence stirred with an
aqueous buffer and fractionated using ultrafiltration equip-
ment to obtain a concentrated enzyme solution.
It has been reported3 that Mung beans contain at least 2
epoxide hydrolases, which prefer to attack different carbon
atoms and with opposing enantioselectivities. For example,
in the case of styrene oxide, one epoxide hydrolse (EHA)
attacks the primary carbon of the (R)-epoxide to produce
the (R)-diol. The other epoxide hydrolase (EHB) attacks
the secondary carbon at the benzylic position of the (S)-
epoxide, which again results in the formation of (R)-diol
with the inversion of configuration at the benzylic carbon.
Thus, for a compound such as styrene oxide with a single
stereogenic center, this situation actually leads to high
product yield (>85%) since both reactions are complimen-
tary and produce the same enantiomer.3 The reaction of
two different epoxide hydrolases with opposing regio-
and enantioselectivities on a glycidate ester possessing
two stereogenic centers can theoretically give a mixture of
2.2. Enzyme preparation
The Mung beans were soaked overnight in water, de-
husked, grounds, and stirred with a phosphate buffer
(0.05 M, pH 7.5) containing EDTA (1 mM). The crude ex-
tract was found to be active toward the (2R,3S)-isomers of
both 1a and 1b, although most of the ester (>85%) was
converted to a mixture of diols, due to the spontaneous
ring opening of the epoxide in water. The crude extract
was then subjected to fractionation with membranes of dif-
ferent molecular weight cutoffs (0.2 lm, 100–10 kD). After
incubation with racemic ester 1b, we observed that the
epoxide hydrolase activity was present in the retentates of
0.2 lm and 100 kD filters. The combined retentate was
freeze-dried to obtain a crude enzyme powder (145 mg pro-
tein/g).
HO
COOR1
OH
HO
COOR1
OH
O
EH-OH
k2
HO-EH
k1
COOR1
R2
R2
(anti-2R,3S)-1
R2
(anti-2S,3S)-2
(anti-2R,3R)-2
Scheme 2.

A. V. Devi et al. / Tetrahedron: Asymmetry 19 (2008) 1139–1144
1141
2.3. Enzyme immobilization
increasing water content, we concluded that the addition
of an extra 1 mL of phosphate buffer (0.05 M, pH 7.2) to
a suspension of 10 g of gelozyme in 100 mL of diisopropyl
ether was adequate enough for enzyme activity. The gel
strength of the matrix was not significantly reduced and
very little spontaneous hydrolysis took place.
The epoxide ring of the glycidate ester 1b was found to
hydrolyze readily in aqueous buffers (pH 6–7.5). This
necessitated the use of a water-immiscible solvent such as
diisopropyl ether or toluene for the reaction. However,
being a hydrolase, the enzyme does require a small amount
of water for enzyme activity. The addition of an aqueous
buffer to the freeze-dried powder suspended in diisopropyl
ether or using water saturated solvent caused the formation
of lumps of the biocatalyst that reacted very sluggishly. At-
tempts were then made to disperse the enzyme by a simple
adsorption on supports such as Celite, activated carbon,
starch, and carboxymethyl cellulose (CMC). However,
the enzyme activity was rapidly lost on these supports when
they came into contact with organic solvents. Over the past
few years, we have successfully demonstrated the use of
lipases immobilized in gelatin organogels (gelozymes) for use
in organic solvents.7 The procedure provides the technique
of enzyme immobilization at low temperature and the gel-
atin matrix provides a porous support. The water content
of the matrix can be controlled to a level just sufficient
enough for an enzyme to be active. We have thus carried
out the immobilization of the crude enzyme preparation
in gelatin organogel. The crude enzyme powder is then
mixed with a solution of gelatin in a microemulsion of
sodium bis(2-ethylhexyl dioctyl sulfosuccinate, AOT)–
isooctane–water, and crosslinked with glutaraldehyde.
The gelozyme thus obtained was allowed to set overnight,
crushed into small pieces, washed with diisopropyl ether
until the organic layer was free of impurities as evidenced
from HPLC analysis, and dried under vacuum at room
temperature.
2.6. Enzyme reaction
In a typical experiment, the glycidate ester 1a (960 mg,
5 mmol) dissolved in diisopropyl ether (20 mL) was stirred
with gelozyme powder (2.5 g) at room temperature on a
magnetic stirrer. The reaction was followed by an analysis
on a chiral stationary phase. The (2R,3S)-ester slowly dis-
appeared. The ee of the unreacted ester reached >99% in
72 h. Interestingly, the glycidate ester 1b was far more reac-
tive than 1a and the reaction was complete in 12 h. The
organic layer was then separated, the enzyme powder was
extracted with diisopropyl ether, and the combined organic
layer was evaporated on a rotavapor. In the case of 1a, the
oily residue was purified by column chromatography to
separate the unreacted glycidate ester 1a and the diastereo-
meric mixture of diol products 2a and 3a. In the case of 1b,
the oily residue was extracted with hot hexane to obtain
unreacted (2S,3R)-1b (45%), in the hexane fraction. The
diol formed during the reaction was quite insoluble in
hexane and hence easily separated.
2.7. Determination of the configuration of the products
The configuration of the unreacted glycidate esters was
established as (2S,3R) for both substrates 1a and 1b on
the basis of their specific rotations, retention times on a chi-
ral HPLC column, and comparison with literature values
(ee >99%). In the case of unsubstituted ethyl phenyl glyci-
date, 1a, the diastereomeric mixture of diols obtained after
chromatography was converted to the corresponding
acetonides to determine diastereomeric excesses. In the
1H NMR spectra of acetonide of syn-ethyl-2,3-dihydroxy-
3-phenylpropanoate 3, H-2 resonated as a doublet at d
5.15 (J = 16.7 Hz) and for the anti-ethyl-2,3-dihydroxy-3-
phenylpropanoate 2 acetonide, it resonated at d 4.75
(J = 15 Hz) and 5.4 (J = 15 Hz). The ratio of anti to syn
acetonide was found to be 8:1 (de 77.8%). From the nega-
tive specific rotation of the acetonide,8 the configuration of
the anti-diol was assigned as (2R,3R). The ee of anti-diol
was found to be 94% based on chiral HPLC analysis.
The syn-diol was obtained as a racemic mixture. In the case
of 1b, the diol obtained after the reaction was found to be
racemic.
2.4. Effect of gelatin concentration on enzyme stability
Protection offered to the enzyme by gelatin matrix strongly
depends upon the concentration of gelatin. Excess gelatin
however increases the bulk without any advantage. We
have thus studied the effect of gelatin concentration on
the stability and reusability of the enzyme. A fixed amount
(100 mg crude powder) was immobilized with varying gel-
atin concentration (varying volumes of gelatin solution in
AOT microemulsion with a fixed amount of enzyme pow-
der), and the reactivity of the enzyme preparation was stud-
ied using 1a as a standard substrate. Optimum enzyme
activity was observed when 2 g of gelatin was used for
the immobilization of 1 g of crude powder containing
145 mg of protein. The enzyme preparation could be recy-
cled three times without the loss of activity or change in
selectivity.
2.8. Product distribution
2.5. Effect of concentration of water on enzyme activity
The unreacted glycidate esters recovered in near quantita-
tive yields (45%, 90% of theoretical) have (2S,3R)-configu-
ration in both cases. This indicates that the epoxide
hydrolase present in our preparation is selective toward
only the (2R,3S)-enantiomer. In case of slow reacting 1a,
the major product is anti-(2R,3R)-diol. This suggests that
only one epoxide hydrolase is active in the enzyme prepa-
ration and that it opens the epoxide ring of the (2R,3S)-gly-
cidate ester by attacking at the benzylic carbon. The
Glycidate esters are susceptible to spontaneous epoxide
ring opening in the presence of excess water causing a de-
crease in the product yield and enantiomeric purity. It is
thus necessary to control the amount of water present in
the gel matrix. The vacuum-dried gelozyme did not show
any hydrolase activity although the determination of water
content by Karl-Fisher titration showed the presence of 7%
water in the gel. After studying the enzyme activity at

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A. V. Devi et al. / Tetrahedron: Asymmetry 19 (2008) 1139–1144
racemic syn-diastereomer is most probably the result of
non-specific ring opening of epoxide ring by water mole-
cules present in the enzyme preparation. This is further
supported by the observation that the diol obtained after
the reaction of 1b, which is highly susceptible to spontane-
ous hydrolysis, is racemic.
4.2. Determination of the enzyme activity in aqueous
fractions
The activity of the enzyme in different fractions collected
after membrane filtration was determined using methyl 4-
methoxyphenyl glycidate 1b as the substrate. In a typical
experiment, 100 lL of a stock solution (0.1 M in ethanol)
was added to an enzyme solution (1 mL, pH 6.5) and the
mixture was stirred at room temperature for 1 h. A sample
of the reaction mixture (100 lL) was diluted with an equal
volume of acetonitrile, centrifuged for 5 min at 5000 rpm,
filtered through a 0.2 lm syringe filter, and analyzed by
reverse phase HPLC to determine the conversion. Another
sample of the reaction mixture (100 lL) was mixed with
ethyl acetate (100 lL) and centrifuged. The ethyl acetate
layer was evaporated in a vacuum concentrator, and the
volume was made up with a mobile phase and analyzed
by chiral HPLC to determine the enantioselectivity of the
reaction.
3. Conclusion
Herein, we have reported the use of easily extractable epox-
ide hydrolase from Mung beans for the preparation of
(2S,3R)-enantiomers of glycidate esters such as 1. Enantio-
merically pure glycidate esters (ee >99%) are obtained in
high yield (90% of theoretical). The enzyme can be immo-
bilized in a gelatin matrix using water-in-oil microemulsion
system. The technique provides an enzyme preparation, in
which the amount of water present in the reaction medium
can be easily controlled and the enzyme can be recycled for
repeated use.
4.3. Immobilization of crude Mung bean epoxide hydrolase
Gelatin (5 g) was heated with distilled water (8.5 mL) at
60 °C for 15 min to complete gelation. AOT solution
(35 mL, 0.3 M in isooctane) was added with vigorous stir-
ring. The viscous and turbid gel thus obtained was cooled
in ice with shaking for 10 min to give a transparent free
flowing liquid. The crude enzyme powder (2.5 g) was slowly
added to the cold solution with vigorous stirring to achieve
a uniform distribution of the enzyme powder. The cooling
bath was removed and glutaraldehyde (1 mL, 25% solu-
tion) was added. The contents were stirred at room temper-
ature with a glass rod until the contents started to become
viscous, poured into petri dish, and left at room tempera-
ture overnight. The dry gel was cut into small pieces and
washed with isooctane and diisopropyl ether until the
supernatant was free of impurities (HPLC analysis). The
gel was then vacuum-dried at room temperature for 12 h
to obtain immobilized enzyme (9 g). Determination of
water content by Karl-Fisher titration showed that the
water content of the gel was 7.7% (w/w).
4. Experimental
Glycidate ester 1a was purchased from Aldrich. Glycidate
1b was a gift from M/s Godavari Drugs Ltd, Hyderabad.
Mung beans (P. radiatus) were purchased from supermar-
kets. HPLC analyses were carried out on a Hewlett Pack-
ard HP1090 unit with diode array detector and HP Chem
Station software. Ultrafiltration was carried out on Sarto-
rius Minisart.
4.1. Preparation of the crude epoxide hydrolase from Mung
beans
The beans (500 g) were soaked in distilled water (5 L) in a
refrigerator overnight, dehusked, and ground to a fine
paste. The paste was suspended in a Tris–HCl buffer
(5 L, 0.05 M containing 1 mM EDTA, pH 7.5) and stirred
in the cold for 6 h with a mechanical stirrer. The mixture
was then filtered through fine muslin cloth and the sedi-
ment was discarded. The supernatant containing a fine sus-
pension of the meal was filtered first through a 0.45 lm
filter on a Sartorius ultrafiltration unit (Minisart). The res-
idue was discarded. The filtrate was then filtered through a
0.22 lm filter. The concentrate (0.5 L) was collected and
stored in a refrigerator. The supernatent (4.5 L) was then
subjected to ultrafiltration with membranes of different
molecular weight cutoff (100 kD, 50 kD, and 30 kD). After
each filtration, the retentate (500 mL) was diluted with
phosphate buffer (5 L, 0.01 M, pH 6.5) and again concen-
trated to 500 mL. The retentates were collected after each
filtration step and stored in a refrigerator for the assay of
enzyme activity.
4.4. Ethyl (2S,3R)-phenyl glycidate 1a
The racemic glycidate ester 1a (960 mg, 5 mmol) dissolved
in diisopropyl ether (20 mL) was stirred with crosslinked
enzyme powder (0.5 g) at room temperature. The reaction
was followed by an analysis on a chiral stationary phase.
The ee of the unreacted ester reached >99% in 72 h. The
organic layer was then separated, the enzyme powder was
washed with diisopropyl ether (10 mL), and the combined
organic layer was evaporated. The oily residue was chro-
matographed over silica gel (ethyl acetate/hexane, 5:95)
to recover the unreacted (2S,3R)-1a (432 mg, 45%) and a
diastereomeric mixture of ethyl 2,3-dihydroxy-3-phenyl-
propanoate (435 mg, 45%).
The combined retentate after filtration with 0.22 lm and
100 kD (1 L) where the maximum enzyme activity was ob-
served, was concentrated in a stirred cell with a 10 kD
membrane filter. The concentrated slurry was freeze-dried
to obtain a pale yellow powder of crude epoxide hydrolase
(70 g, protein content 145 mg/g).
1
1a: H NMR (CDCl3): d 1.2 (t, 3H, –OCH2CH3), 2.1 (d,
1H, J = 2.5 Hz, C(2)H); 2.6 (d, 1H, J = 2.5 Hz, C(3)H);
4.15 (q, 2H, OCH2CH3), 7.2–7.4 (br s, 5H, aromatic). IR
(neat): 3560–3360, 2960, 2840, 1723, 1690. MS (EI, M+)

A. V. Devi et al. / Tetrahedron: Asymmetry 19 (2008) 1139–1144
1143
25
m/z = 178, 121, 91 (base peak), 77, 51, 39. ½aꢀD ¼ 156, (c 1,
(EI, M+) m/z = 249, 149, 137, 121, 109, 94, 77, 66, 41,
51, 41.
CHCl3); (lit.4c = +155 (c 1, CHCl3)).
4.5. Ethyl 2,3-dihydroxy-3-phenylpropanoate
4.9. Reverse phase HPLC analysis
1H NMR (CDCl3): d 1.2 (t, 3H, –OCH2CH3), 2.9–3.2 (br,
OH), 4.15 (q, 2H, OCH2CH3), 4.4 (d, J = 4 Hz, 1H,
CH(OH)COOEt), 5.0 (d, J = 4 Hz, PhCH(OH)), 7.3 (m,
5H, aromatic). IR (neat): mmax 3450–3350, 2957, 1736,
The disappearance of glycidate ester 1b was followed by
reverse phase HPLC. Column C-18 (250 ꢂ 5 mm), Chrom-
pack, The Netherlands. Mobile phase 70% acetonitrile–
water. Flow rate 0.7 mL/min. Detection wavelength
230 nm. Retention times: 1b 6.24 min, diol (2 + 3)
4.39 min.
1452, 1198, 1092, 703 cmꢁ1
.
4.6. Ethyl anti-(2R,3R)-2,3-isopropylinyloxy-3-phenyl pro-
anoate, 2a, and ethyl syn-( ) isopropylidinyloxy-3-phenyl
propanoate 3a
4.10. HPLC with chiral stationary phase
Enantiomeric purity was determined by HPLC analysis on
Chiralcel AD-H column (250 ꢂ 5 mm), Daicel Chemical
Industries, Japan. Mobile phase 15% isopropyl alcohol in
hexane. Flow rate 0.7 mL/min; detection wavelength
230 nm. Retention times 1a: (2S,3R) 8.1 min, (2R,3S)
8.8 min; 1b: (2R,3S) 9.52, (2S,3R) 10.74 min. 2a: (2S,3S)
15.4, (2R,3R) 16.21, (2S,3R) 18.0, (2R,3S) 19.5 min. 2b:
Peaks at 20.2, 20.9, 22.5 and 24.6 min (configurations not
assigned since the product was racemic).
To a stirred solution of 2a and 3a (420 mg, 2 mmol) and
camphor sulfonic acid (1 mg) in dichloromethane (10 mL)
was added 2,2-dimethoxypropane (1 mL, 6 mmol). After
stirring for 12 h at room temperature, aqueous sodium
bicarbonate solution (5 mL) was added. The organic layer
was separated, and the aqueous layer was extracted with
dichloromethane (2 ꢂ 20 mL). The combined organic lay-
ers were washed with brine (10 mL), dried over anhydrous
sodium sulfate, and evaporated under reduced pressure.
The residue was purified over column chromatography
[ethyl acetate/hexane, 1:100] to afford syn (24 mg, 5%)
Acknowledgments
1
and anti (390 mg, 78%). syn 3a. H NMR (CDCl3) d 1.2
(t, 3H, –OCH2CH3), 1.6 (2s, 6H, –C(CH3)2), 4.3 (m, 3H,
–OCH2CH3 + CH(O)COOEt), 5.15 (d, J = 16.7 Hz, 1H,
PhCH(O)), 7.3 (m, 5H, aromatic). anti-(2R,3R)-2a: 1H
NMR (CDCl3) d 0.75 (t, 3H, –OCH2CH3), 1.5 (s, 3H,
–C(CH3)2), 1.8 (s, 3H, –C(CH3)2); 3.45–3.8 (m, 2H,
–OCH2CH3), 4.75 (d, J = 15.0 Hz), 5.4 (d, J = 15 Hz,
1H, PhCH(O)), 7.3 (m, 5H, aromatic). IR (neat) mmax
2990, 1760, 1200 cmꢁ1. [a]D = ꢁ52 (c 1, CH2Cl2) [lit. =
ꢁ67.3 (c 1.0, CH2Cl2)]. MS (EI, M+) m/z = 210, 141,
135, 122, 105, 91, 71, 57, 43, 41.
We thank the Indo-French Center for the Promotion of
Advanced Research and CSIR, New Delhi, for financial
support.
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4.7. (2S,3R)-Methyl (4-methoxyphenyl) glycidate
Reaction was performed (with 1.11 g, 5 mmol) as described
in Section 4.6. The reaction was complete within 12 h and
enantiomerically pure glycidate ester (466 mg, 42%) was
obtained. 1H NMR (CDCl3, 200 MHz): d 3.5 (d, 1H,
J = 2.5 Hz, C(2)H); 3.9 (s, 3H, COOCH3), 4.1 (d, 1H,
J = 2.5 Hz, C(3)H); 6.8–7.2 (dd, 4H, aromatic). IR
(KBr): 3560–3360, 2960, 2840, 1720, 1600, 1520, 1440,
1280, 1240, 1100, 1000, 840. MS (EI, M+) m/z = 208,
´
40, 221–227; (c) Kotik, M.; Brichac, J.; Kyslık, P. J. Biotech-
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151, 148, 121 (base peak), 120, 105, 91, 77, 51, 39.
25
½aꢀD ¼ þ196 (c 1, ethanol); {lit.4b = ꢁ196.2 (c 1, ethanol)
for the (2R,3S)-enantiomer}.
4.8. Methyl 2,3-dihydroxy-3-(4-methoxyphenyl) propanoate
1H NMR (CDCl3, 200 MHz): 2.57 (d, J = 6.70 Hz, 1H,
CH(OH)COOMe), 3.04 (d, J = 6.04 Hz, 1H, PhCH(OH)),
3.79 (s, 6H, COOMe + PhOMe), 4.24–4.29 (dd,
J1 = 3.02 Hz, J2 = 6.04 Hz, 1H, CH(OH)COOMe), 4.84–
4.91 (dd, J1 = 3.02 Hz, J2 = 6.04 Hz, 1H, PhCH(OH)),
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6.84 (d, J = 8.30 Hz, 2H, aromatic),
d
7.25 (d,
J = 8.30 Hz, 2H, aromatic). IR (neat) mmax 3497, 3383,
3012, 2958, 2842, 1713, 1612, 1516, 1447, 1105 cmꢁ1. MS

1144
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