Kinetic resolution of poly(ethylene glycol)-supported carbonates by enzymatic hydrolysis

Article abstract of DOI:10.1016/j.tet.2006.05.020

The enzyme-mediated enantioselective hydrolysis of poly(ethylene glycol) (PEG)-supported carbonates is disclosed. The water-soluble carbonates were prepared by immobilization of a racemic secondary alcohol (4-benzyloxy-2-butanol) onto low-molecular weight (av MW 550 and 750) monomethoxy PEG through a carbonate linker. For the screening of the hydrolytic enzymes, the substrate was enantioselectively hydrolyzed by commercially available lipase from porcine pancreas (PPL; Type II, Sigma) to afford the optically active compounds. In this system, the separation of the remaining (S)-substrate and the resulting (R)-alcohol was achieved by an extraction process without a laborious column chromatography. The (S)-carbonate was easily hydrolyzed with K₂CO₃ to afford the corresponding (S)-alcohol. Other MPEG-supported substrates were also hydrolyzed to afford the corresponding optically active alcohols.

Full text of DOI:10.1016/j.tet.2006.05.020

Tetrahedron 62 (2006) 7300–7306
Kinetic resolution of poly(ethylene glycol)-supported
carbonates by enzymatic hydrolysis
Masaki Nogawa,a Megumi Shimojo,a Kazutsugu Matsumoto,a,
*
Masayuki Okudomi,a Yuji Nemotoa and Hiromichi Ohtab
aDepartment of Chemistry, Meisei University, Hodokubo 2-1-1, Hino, Tokyo 191-8506, Japan
bDepartment of Biosciences and Informatics, Keio University, Hiyoshi 3-14-1, Yokohama 223-8522, Japan
Received 7 April 2006; revised 9 May 2006; accepted 11 May 2006
Available online 5 June 2006
Abstract—The enzyme-mediated enantioselective hydrolysis of poly(ethylene glycol) (PEG)-supported carbonates is disclosed. The water-
soluble carbonates were prepared by immobilization of a racemic secondary alcohol (4-benzyloxy-2-butanol) onto low-molecular weight
(av MW 550 and 750) monomethoxy PEG through a carbonate linker. For the screening of the hydrolytic enzymes, the substrate was enan-
tioselectively hydrolyzed by commercially available lipase from porcine pancreas (PPL; Type II, Sigma) to afford the optically active com-
pounds. In this system, the separation of the remaining (S)-substrate and the resulting (R)-alcohol was achieved by an extraction process
without a laborious column chromatography. The (S)-carbonate was easily hydrolyzed with K2CO3 to afford the corresponding (S)-alcohol.
Other MPEG-supported substrates were also hydrolyzed to afford the corresponding optically active alcohols.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
reaction causes a low reactivity and a difficult analysis of
the polymer-supported intermediate. Although enzymatic
transformation on a polymer support is also of contemporary
interest and can be potentially useful for the easy isolation of
the products, there have been relatively few reports on poly-
mer-supported reactions by enzymes so far.6–12 Recently,
poly(ethylene glycol) (PEG) has been recognized as an inex-
pensive and convenient soluble polymer.13,14 The synthetic
approach using a soluble polymer is termed as ‘liquid-phase’
chemistry and couples the advantages of homogeneous solu-
tion chemistry with those of solid-phase chemistry. We have
noted that a PEG-supported strategy could be suitable for
enzymatic transformation because the broad solubility of
PEG facilitates the analysis of the PEG-supported substrates
and could significantly enhance the reactivity under homo-
geneous conditions. Actually, PEG-supported esters and
amides have been studied as prodrugs, which are hydrolyzed
in vitro or in vivo to gradually release native drugs.15 In this
report, we disclose the first example of the kinetic resolution
of PEG-supported substrates with a carbonate linker by a
hydrolytic enzyme to afford the corresponding optically
active compounds, and the method enables us to achieve
the easy separation of the remaining substrates and the
resulting alcohols by an extraction process without laborious
column chromatography.16
Optically active secondary alcohols are versatile intermedi-
ates in organic syntheses. The use of enzymes in the prepa-
ration of such compounds is especially attractive due to its
benign effect on the environment. In particular, the kinetic
resolution of racemic alcohols and esters using hydrolytic
enzymes is one of the practical methods for the preparation
of the optically active compounds, and a significant number
of examples have been reported.1 In the reaction process, the
enantiomers, the remaining substrate and the resulting prod-
uct, could be separated mainly by column chromatography.
However, the tedious and wasteful separation step is the
bottleneck to an easy operation and a sustainable product.
Although, in order to resolve this irritating problem, several
studies of an easy separation have been published,2–9 facile
and efficient procedures are still desired.
On the other hand, organic synthesis based on polymer
supports has made rapid progress, especially in the field of
combinatorial chemistry. Because insoluble polymers (poly-
styrene, silica gel, and so on) are usually used, it is called
‘solid-phase’ chemistry. While the methodology provides
us an easy separation of the products, the heterogeneous
Keywords: Carbonates; Enantioselective hydrolysis; Enzymes; Hydrolase;
Poly(ethylene glycol)-supported substrate.
* Corresponding author. Tel./fax: +81 42 591 7360; e-mail: mkazu@chem.
meisei-u.ac.jp
In general, previously reported PEG use during enzymatic
synthesis has been restricted as the reagent for the modifica-
tion of enzymes17 and the additive for improving the enzyme
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.05.020

M. Nogawa et al. / Tetrahedron 62 (2006) 7300–7306
7301
activities.18 To the best of our knowledge, there has been
only one report on the PEG-supported substrate used for
enzymatic transformation.19 However, the substrate only
worked as a nucleophile to an acyl–enzyme intermediate,
and the example did not make the most of the advantage
of PEG. We now present a new aspect of PEG use.
pig liver esterase (PLE; Amano Enzyme, Inc.) smoothly
catalyzed the hydrolysis of 1a to afford (R)-2, the enantio-
selectivity was quite low (E value¼3).22 Interestingly, the
esterase SNSM-87 (Nagase & Co., Ltd) preferentially
hydrolyzed the opposite enantiomer with moderate enantio-
selectivity to give the alcohol (S)-2, but the conversion was
very low (conv.¼0.08, E value¼11). Finally, lipase from
porcine pancreas (PPL; Type II, Sigma) was found to be the
best enzyme. Under the given reaction conditions, the reac-
tion of (ꢀ)-1a with PPL proceeded with a higher enantio-
selectivity (conv.¼0.29, E value¼23) to afford the optically
active (S)-1a (50%, 36% ee) and (R)-2 (28%, 89% ee; [a]D26
ꢂ12.2 (c 0.24, MeOH)). The absolute configurations of
the products were determined by comparing the optical
rotation of 2 with that of an authentic sample ([a]2D7 +19.0
(c 0.95, MeOH)) derived from ethyl (S)-3-hydroxybutanoate
(Scheme 4). Changing the MPEG part to a lower molecular
weight MPEG550 did not negatively affect the reactivity.
The reaction of the MPEG550-supported 1b also proceeded
with a high enantioselectivity (conv.¼0.35, E value¼28).
In the reaction of (ꢀ)-1b at 10 ꢁC, the E value was up to 32
and (R)-2 with 93% ee was obtained, although the conversion
apparently decreased. On the other hand, the methyl carbon-
ate (ꢀ)-1c (R¼Me) was also hydrolyzed, but the enantio-
selectivity was very low (E value¼1.4). The substrate
(ꢀ)-1c is not supported on MPEG and basically insoluble
in water. These facts indicate that the hydrophilic MPEG
matrix could change the physical property of the alcohol 2
and that the substrate would favorably fit into the enzyme
active site.
2. Results and discussion
2.1. Screening test of enzymes
We used low-molecular weight monomethoxy PEG (MPEG,
av MW 750 and 550) as the matrix.20 It has the desired sol-
ubility profile and the higher loading capacity (MPEG750
,
1.3 mmol/g;MPEG550,1.8 mmol/g),whilethatofMPEG5000
(av MW 5000), which has been used in many previous
reports, is only 0.2 mmol/g. In addition, the terminal methyl
group becomes a reference for the determination of the load-
ing ratio in the reaction steps.
For the screening test of enzymes, we selected the carbonate
(ꢀ)-1a (MPEG750), which was afforded by the coupling of
racemic 4-benzyloxy-2-butanol ((ꢀ)-2) with MPEG750
–
OH through a carbonate linker. The carbonate is a typical
linker for organic synthesis on a polymer support and can
be easily constructed. In general, carbonate is recognized
as a poor substrate for hydrolase. However, we have already
succeeded in the development of the enzymatic hydrolysis of
cyclic carbonates,21 and realize that carbonate is merely
a kind of ester, which can be hydrolyzed by enzymes.
O
O
The substrate (ꢀ)-1a was readily synthesized as shown in
Scheme 1. The reaction of (ꢀ)-2 with N,N0-carbonyldiimida-
zole in CH2Cl2 proceeded to afford the corresponding (ꢀ)-3.
The compound (ꢀ)-3 was immobilized on MPEG–OH with
O
O
R
enzyme
O
*
O
R
OH
*
+
pH 6.5 buffer
24 h
Me
OBn
Me
OBn
Me
OBn
( )-1
Scheme 2.
1*
2*
DMAP in DMF at 120 ꢁC to give nearly the pure MPEG750
-
supported (ꢀ)-1a in 67% yield. In the same way, the
MPEG550-supported (ꢀ)-1b and other substrates were also
prepared. The yields of substrates were determined by the
weights with the assumption that the MW was 750 or 550
for MPEG–OH.
O
O
O
R
OH
K2CO3
MeOH
Me
OBn
Me
OBn
*
*
In the first screening test, 12 hydrolytic enzymes were used.
The selection of the enzyme was carried out on the basis of
hydrolytic activity without paying attention to the enantio-
selectivity. The assay was performed by checking the pro-
duction of 2 using TLC, and we selected three enzymes. In
the second screening, the enantiomeric excesses (ees) of the
products were determined after purification (Scheme 2). The
ee of 2 was determined by HPLC analysis (CHIRALCEL
OD-H, Daicel Chemical Industries, Ltd), and a similar anal-
ysis of 2 derived from 1a with K2CO3 was also performed
(Scheme 3). These results are shown in Table 1. Although
1*
2*
Scheme 3.
2.2. Separation of the products
During the reaction process, we succeeded in the establish-
ment of a more facile separation of the remaining substrate
(S)-1a,b and the resulting alcohol (R)-2 due to the suitable
water-solubility of the MPEG-supported substrates. Scheme
5 illustrates this extraction procedure. First, the extraction
O
O
O
N
N
N N N
MPEG OH
OH
O
N
O
O MPEG
OBn
CH2Cl2
r.t.
DMAP, DMF
Me
Me
OBn
Me
OBn
( )-3 (quant.)
120 °C
( )-2
( )-1a: MPEG750 (67%)
( )-1b: MPEG550 (67%)
MPEG = -(CH2CH2O)nCH3
Scheme 1.

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M. Nogawa et al. / Tetrahedron 62 (2006) 7300–7306
Table 1. Enantioselective hydrolysis of carbonates (ꢀ)-1a
Substrate
R
Temp (ꢁC)
Carbonate 1
Alcohol 2
Conv.e
Ef
Yield b(%)
ee (%)c
Yield (%)
ee (%)d
PLE
Esterase SNSM-87
PPL
PPL
PPL
PPL
1a
1a
1a
1b
1b
1c
MPEG750
MPEG750
MPEG750
MPEG550
MPEG550
Me
30
30
30
30
10
30
4
56
50
54
76
65
96(S)
7(R)
36(S)
47(S)
10(S)
5(S)
76
11
28
30
21
28
7(R)
82(S)
89(R)
89(R)
93(R)
12(R)
0.93
0.08
0.29
0.35
0.10
0.27
3
11
23
28
32
1.4
a
The reaction was performed using 5 mM of the substrate with an enzyme in 0.1 M phosphate buffer (pH 6.5) for 24 h.
Determined by its weight on the basis of the weight of the racemic substrate.
Determined by HPLC analysis after the hydrolysis of the carbonate.
Determined by HPLC analysis.
Calculated by ee(carbonate)/[ee(carbonate)+ee(alcohol)].
Calculated by ln[(1ꢂconv.)(1ꢂee(carbonate))]/ln[(1ꢂconv.)(1+ee(carbonate))].
b
c
d
e
f
CH3OCH2Cl
i-Pr2NEt
OH
O
MOMO
Me
O
OMOM
LiAlH4
Me
OEt
OEt
Me
OH
CH2Cl2
0 °C
THF
0 °C
(S)-4
(S)-5 (84%)
(S)-6 (86%)
OMOM
OH
2 M HCl
BnBr, NaH
THF
r.t.
THF
r.t.
Me
(S)-7 (65%)
OBn
Me
OBn
(S)-2 (52%)
Scheme 4.
(1) Extraction with hexane
(2) Extraction with ethyl acetate
Aqueous layer
O
Ethyl acetate layer
O
Hexane layer
OH
O
O MPEG
OBn
Me
Aqueous layer
MPEG-OH
(S)-1a,b
O
O MPEG
OBn
Me
OBn
+
(R)-2
Me
MPEG-OH
(S)-1a,b
Scheme 5.
process is performed with hexane after the enzymatic
reaction. In this step, only the alcohol (R)-2 is selectively ex-
tracted into the hexane layer. Second, the substrate (S)-1a,b
is successfully extracted from the aqueous layer with
AcOEt. MPEG–OH, which is removed from (R)-1a,b, still
remains in the aqueous layer. In order to purify these com-
pounds, only a pad of silica gel is needed.
conversion was greater than those of 1 (conv.¼0.55, E
value¼29). In this case, the corresponding optically active
compounds, (S)-8a (36%, 95% ee) and (R)-9a (31%,
77% ee), were obtained. While the reaction of (ꢀ)-8c (R1¼
vinyl, R2¼CH2CH2OBn) showed a good enantioselectivity
(E value¼16), (ꢀ)-8b (R1¼Me, R2¼CH2CH2Ph) was
Table 2. Enantioselective hydrolysis of carbonates (ꢀ)-8 with PPLa
2.3. Application of the enzymatic reaction
Substrate R1
R2
Carbonate (S)-8 Alcohol (R)-9 Conv. E
Yield
(%)b
ee
(%)
Yield ee
(%)
We next examined the enzymatic reactions of several sub-
strates supported on MPEG550 under the same conditions
(Scheme 6). These results are summarized in Table 2. As
expected, thehydrolysisof1-phenylethanol derivative(ꢀ)-8a
(R1¼Me, R2¼Ph) enantioselectively proceeded, and the
(%)
8a
8b
8c
Me Ph
36
95c
23c
56e
31
47
41
77d 0.55 29
Me CH2CH2Ph 37
Vinyl CH2CH2OBn 47
27d 0.46
80f 0.41 16
2
a
The reaction was performed using 5 mM of (ꢀ)-8 with PPL in 0.1 M
phosphate buffer (pH 6.5) for 24 h.
Determined by its weight on the basis of the weight of the racemic sub-
strate.
O
O
b
PPL
O
O MPEG550
O
O MPEG550
OH
c
d
e
+
Determined by HPLC analysis after the hydrolysis of the carbonate.
Determined by HPLC analysis.
pH 6.5 buffer
30 °C, 24 h
R1 R2
R1 R2
(S)-8
R1 R2
(R)-9
Determined by 1H NMR analysis of the corresponding MTPA ester after
the hydrolysis of the carbonate.
( )-8
Determined by 1H NMR analysis of the corresponding MTPA ester.
f
Scheme 6.

M. Nogawa et al. / Tetrahedron 62 (2006) 7300–7306
7303
1
hydrolyzed with a very low enantioselectivity. Because the
bulkiness of the substituent of 8c is not much different from
those of 1 and 8b, the interaction between the enzyme and
the oxygen atom in the benzyloxy group should be important
for the enantioselectivity. In all cases, the substrates 8 and
the alcohols 9 were successfully separated by the two step-
extraction procedure as expected.
as the internal standard. H (500 MHz) NMR spectra were
measured on JEOL a-500. IR spectra were recorded with
a Shimadzu IR Prestige-21 spectrometer. Mass spectra
were obtained with a JEOL EI/FAB mate BU25 instrument
(EI method). The optical rotations were measured with
a Jasco DIP-1000 polarimeter. HPLC data were obtained on
Shimadzu LC-10ADVP, SPD-10AVP, and sic 480II data sta-
tion (System Instruments Inc.). Kieselgel 60 F254 Art.5715
(E. Merck) was used for analytical thin-layer chro-
matography (TLC). Preparative TLC was performed on
a Kieselgel 60 F254 Art.5744 (E. Merck). Flash column
chromatography was performed with Silica Gel 60N
(63–210 mm, Kanto Chemical Co. Inc.). MPEG550–OH
and MPEG750–OH were purchased from Aldrich and the
containing water was removed as the toluene azeotrope
prior to use. Racemic secondary alcohols were prepared
from the suitable starting material in the usual way. All other
chemicals and enzymes were also obtained from commer-
cial sources.
2.4. What is the real active enzyme?
Although commercially available PPL (Type II, Sigma)
works well in this enzymatic reaction, the crude enzyme con-
tains a number of hydrolases besides the true PPL. The exis-
tence of several active enzymes might affect the reactivity
and enantioselectivity. In addition, the active-site model pro-
posed by Jones for the PPL-catalyzed hydrolysis of a primary
ester does not predict the reaction mode in this case.23 In or-
der to research the accurate result of the reaction for MPEG-
supported substrates, we then investigated the reaction using
the commercially available purified PPL (lipase Type VI-S,
Sigma) and two kinds of major contaminant hydrolases,
a-chymotrypsin (Type II, Sigma) and cholesterol esterase
(Sigma). We selected (ꢀ)-8a as the substrate because it
was the most reactive amongst all the examined substrates
(Table 3). It is noteworthy that all enzymes show no or very
low enantioselectivities. These results suggest that the activ-
ity could be due, not to the true PPL, but to another unknown
enzyme. Further detailed investigations are now in progress.
4.2. Preparation of carbonates as the substrate
4.2.1. MPEG750-supported substrate coupled with 4-ben-
zyloxy-2-butanol (( )-1a). Under an argon atmosphere, to a
solution of N,N0-carbonyldiimidazole (1.98 g, 12.2 mmol) in
CH2Cl2 (10 mL) was added a solution of 4-benzyloxy-2-bu-
tanol ((ꢀ)-2, 2.00 g, 11.1 mmol) in CH2Cl2 (10 mL), and the
mixture was stirred overnight at room temperature. After the
mixture was diluted with CH2Cl2, the solution was washed
with brine and dried over Na2SO4. After evaporation under
reduced pressure, 4-(benzyloxy)butan-2-yl 1H-imidazole-
1-carboxylate ((ꢀ)-3) was obtained as a colorless oil
(3.17 g, quant). This was used in the following reaction with-
out further purification; IR (neat) 2862, 2359, 1759, 1472,
Table 3. Enantioselective hydrolysis of carbonates 8aa
Enzyme
Carbonate (S)-8a Alcohol (R)-9a Conv.
E
ee (%)
ee (%)
Purified PPLb
2.1
1.2
30
21
0.4
0.07
0.06 1.5
0.77 w1
2
1393, 1319, 1290, 1242, 1180, 1096, 1003, 743 cmꢂ1; H
1
a-Chymotrypsinc
Cholesterol esterased 1.2
NMR (300 MHz, CDCl3) d 1.42 (d, J¼6.0 Hz, 3H), 1.94–
2.11 (m, 2H), 3.51–3.59 (m, 2H), 4.44 (d, J¼12.0 Hz,
1H), 4.50 (d, J¼12.0 Hz, 1H), 5.33 (dqd, J1¼5.0 Hz,
J2¼6.0 Hz, J3¼8.0 Hz, 1H), 7.04 (t, J¼0.8 Hz, 1H), 7.19–
7.34 (m, 5H), 7.36 (t, J¼1.3 Hz, 1H), 8.07 (s, 1H); 13C
NMR (75 MHz, CDCl3) d 20.1, 35.8, 65.8, 73.1, 73.9,
117.0, 127.6, 127.7, 128.3, 130.4, 137.0, 137.9, 148.2; MS
m/z (EI, rel intensities) 274 (M+, 6.1%), 168 (100), 162
(93), 107 (42), 91 (100); HRMS m/z (EI) 274.1315 (calcd
for C15H18O3N2: 274.1318, M+).
a
The reaction was performed using 5 mM of (ꢀ)-8a with the enzyme in
0.1 M phosphate buffer (pH 6.5, 4 mL) for 24 h at 30 ꢁC.
Using 0.3 mg (81,000 U/mg).
Using 5 mg (30,000 U/mg).
Using 1 mg (54 U/mg).
b
c
d
3. Conclusions
In summary, we have demonstrated the first example for the
hydrolase-mediated kinetic resolution of low-molecular
weight MPEG-supported carbonates. We succeeded in the
highly enantioselective hydrolysis of several substrates to
give the substituted methyl (2 and 9a) and vinyl (9c) carbi-
nols, which were optically active. In our method, the separa-
tion of the resulting alcohols from the remaining substrates
was achieved by an extraction process without time- and
solvent-consuming column chromatography. We anticipate
that the use of a soluble polymer as the matrix of the sub-
strates will provide an operationally simple and eco-friendly
protocol.
Under an argon atmosphere, to a solution of N,N-dimethyl-
aminopyridine (DMAP) (445 mg, 3.65 mmol) in DMF
(10 mL) were added a solution of MPEG750–OH (2.74 g,
3.65 mmol) in DMF (20 mL) and (ꢀ)-3 (1.00 g, 3.65 mmol)
in DMF (10 mL) at 0 ꢁC. After the mixture was stirred over-
night at 120 ꢁC, it was washed with 2 M HCl in order to
remove DMAP, the resulting imidazole, and the remaining
MPEG–OH. After evaporation under reduced pressure, the
residue was purified by column chromatography on silica
gel (AcOEt/AcOEt/MeOH¼3/1) to give the MPEG750
-
supported carbonate (ꢀ)-1a as a colorless oil (3.50 g,
67%). The yield was determined by the weight with the as-
sumption that the molecular weight was 750 for MPEG–
OH; IR (neat) 2870, 2359, 1742, 1454, 1350, 1263, 1105,
4. Experimental
4.1. General
949, 847 cmꢂ1; H NMR (300 MHz, CDCl3) d 1.31 (d,
1
1H (300 MHz) and 13C (75 MHz) NMR spectra were mea-
sured on JEOL JNM AL-300, with tetramethylsilane (TMS)
J¼6.5 Hz, 3H), 1.77–2.03 (m, 2H), 3.38 (s, 3H, CH3O–
PEG), 3.50–3.57 (m, 2H, PEG), 3.55–3.70 (m, ca. 62H,

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M. Nogawa et al. / Tetrahedron 62 (2006) 7300–7306
PEG), 3.70 (t, J¼4.5 Hz, 2H, PEG), 4.17–4.33 (m, 2H), 4.49
67.0, 71.1, 75.3, 127.8, 127.9, 128.5, 137.4, 170.5; MS m/z
(EI, rel intensities) 238 (M+, 5.5%), 162 (100), 147 (6.8),
131 (30), 105 (100), 91 (100), 77 (100); HRMS m/z (EI)
238.1205 (calcd for C13H18O4: 238.1205, M+).
(s, 2H), 4.89–5.01 (m, 1H), 7.26–7.37 (m, 5H).
Other substrates were synthesized by the same procedure.
4.2.2. MPEG550-supported substrate coupled with 4-ben-
zyloxy-2-butanol (( )-1b). Yield 61% from 4-benzyloxy-2-
butanol (dl-2); IR (neat) 2870, 2359, 1742, 1454, 1350,
4.3. First screening of enzymes
In the screening test, we used the following enzymes: Lipase
from porcine pancreas (PPL; Type II, Sigma), Lipase AK,
Lipase PS, Lipase D, Lipase AP, Lipase AY, Newlase F,
PLE (Amano Enzyme, Inc.), Lipase OF (Meito Sangyo
Co., Ltd), Esterase SNSM-87 (Nagase & Co., Ltd), Trypsin,
a-Chymotrypsin (E. Merck). The substrate (ꢀ)-1a (200 mg)
and 50 mg of enzyme were incubated in 40 mL of 0.1 M
phosphate buffer (pH 6.5) for 24 h at 30 ꢁC. The products
were extracted with AcOEt (ꢃ3) and detected by TLC (hex-
ane/AcOEt¼3/1).
1
1263, 1107, 949, 851 cmꢂ1; H NMR (300 MHz, CDCl3)
d 1.31 (d, J¼6.5 Hz, 3H), 1.77–2.04 (m, 2H), 3.38 (s, 3H,
CH3O–PEG), 3.51–3.57 (m, 2H, PEG), 3.57–3.67 (m, ca.
44H, PEG), 3.70 (t, J¼4.5 Hz, 2H, PEG), 4.17–4.33 (m,
2H), 4.49 (s, 2H), 4.89–5.01 (m, 1H), 7.26–7.37 (m, 5H).
4.2.3. MPEG550-supported substrate coupled with 1-phe-
nylethanol (( )-8a). Yield 53% from 1-phenylethanol ((ꢀ)-
9a); IR (neat) 2872, 2359, 1744, 1454, 1348, 1261, 1103,
1
949, 849 cmꢂ1; H NMR (300 MHz, CDCl3) d 1.59 (d,
J¼6.5 Hz, 3H), 1.77–2.03 (m, 2H), 3.38 (s, 3H, CH3O–
PEG), 3.53–3.57 (m, 2H, PEG), 3.60–3.72 (m, ca. 44H,
PEG), 3.70 (t, J¼5.0 Hz, 2H, PEG), 5.72 (q, J¼6.5 Hz,
1H), 7.25–7.40 (m, 5H).
4.4. Typical procedure of enantioselective hydrolysis of
MPEG-supported substrates
To a 200-mL Erlenmeyer flask containing 200 mg (ca.
0.208 mmol; sub. concn, 0.5 mM) of (ꢀ)-1a was added
40 mL of 0.1 M phosphate buffer (pH 6.5). To the mixture
was added 50 mg of Lipase from porcine pancreas (PPL;
Type II, Sigma) (994 U/mg, using olive oil at pH 7.7), and
the solution was incubated for 24 h at 30 ꢁC. First, only
the resulting alcohol 2 was extracted with hexane (ꢃ3),
and the hexane layer was dried over Na2SO4. After evapora-
tion, the residue was passed through a pad of silica gel with
hexane/AcOEt (3/1) to give the alcohol (R)-2 (10.1 mg,
28%, 89% ee). Second, the remaining carbonate 1a was
re-extracted with AcOEt from the water layer, and the
organic layer was dried over Na2SO4. After evaporation,
the residue was passed through a pad of silica gel (AcOEt/
MeOH¼3/1) to give the carbonate (S)-1a (99.7 mg, 50%,
36% ee). The yields of 1a and 2 were determined by their
weights on the basis of the weight of the substrate (ꢀ)-1a.
The carbonate (S)-1a was easily hydrolyzed with K2CO3
in MeOH to afford the corresponding alcohol (S)-2.
4.2.4. MPEG550-supported substrate coupled with
4-phenyl-2-butanol (( )-8b). Yield 52% from 4-phenyl-2-
butanol (dl-9b); IR (neat) 2870, 2359, 1740, 1454, 1350,
1
1267, 1107, 949, 847 cmꢂ1; H NMR (300 MHz, CDCl3)
d 1.31 (d, J¼6.0 Hz, 3H), 1.77–2.03 (m, 2H), 3.38 (s, 3H,
CH3O–PEG), 3.50–3.57 (m, 2H, PEG), 3.55–3.70 (m, ca.
46H, PEG), 3.70 (t, J¼4.5 Hz, 2H, PEG), 4.73–4.84 (m,
1H), 7.14–7.31 (m, 5H).
4.2.5. MPEG550-supported substrate coupled with 5-
benzyloxy-1-hepten-3-ol (( )-8c). Yield 60% from
5-benzyloxy-3-ol (dl-9c); IR (neat) 3113, 2870, 2359,
1
1744, 1454, 1350, 1261, 1105, 947, 851 cmꢂ1; H NMR
(300 MHz, CDCl3) d 3.38 (s, 3H, CH3O–PEG), 3.50–3.60
(m, 2H, PEG), 3.60–3.72 (m, ca. 46H, PEG), 3.70 (t,
J¼5.0 Hz, 2H, PEG), 4.22–4.33 (m, 2H), 4.49 (s, 2H),
5.21 (td, J1¼1.5 Hz, J2¼10.5 Hz, 1H), 5.31 (td, J1¼1.5 Hz,
J2¼17.0 Hz, 1H), 5.81 (ddd, J1¼7.0 Hz, J2¼10.5 Hz,
J3¼17.0 Hz, 1H), 7.25–7.37 (m, 5H).
4.5. Chemical hydrolysis of (S)-1a
4.2.6. 4-(Benzyloxy)butan-2-yl methyl carbonate (( )-1c).
Under an argon atmosphere, to a solution of (ꢀ)-2 (400 mg,
2.23 mmol) in CH2Cl2 (20 mL) were added pyridine (1.08
mL, 13.3 mmol) and methyl chlorocarbonate (0.34 mL,
4.44 mmol), and the mixture was stirred overnight at room
temperature. After the addition of methyl chlorocarbonate
(0.68 mL, 8.88 mmol), the mixture was stirred overnight at
room temperature again. The reaction was stopped with
0.1 M phosphate buffer (pH 6.5) and the products were
extracted with CH2Cl2 (ꢃ3). The combined organic layer
was washed with 2 M HCl (ꢃ2), brine, satd NaHCO3
aqueous solution and brine, and dried over Na2SO4. After
evaporation under reduced pressure, the residue was purified
by flash column chromatography (hexane/AcOEt¼5/1) to
give (ꢀ)-1c as a colorless oil (454 mg, 86%); IR (neat)
To a solution of (S)-1a (40.3 mg, 0.053 mmol) in MeOH
(6 mL) was added K2CO3 (36.6 mg, 0.265 mmol), and the
mixture was stirred for 1 h at room temperature. After the
reaction was stopped with water, MeOH was evaporated in
vacuo. The products were extracted with AcOEt (ꢃ3), and
the combined organic layer was washed with brine and dried
over Na2SO4. After evaporation under reduced pressure,
the residue was purified by flash column chromatography
(hexane/AcOEt¼4/1) to give (S)-2 as a colorless oil
(8.1 mg, 85%).
4.6. Several data of alcohols
4.6.1. 4-Benzyloxy-2-butanol (2). Compound (R)-2, [a]D26
ꢂ12.2 (c 0.24, MeOH) (89% ee); IR (neat) 3416, 2965,
2955, 2859, 2359, 1746, 1443, 1271, 1096, 941 cmꢂ1
;
2864, 1494, 1454, 1368, 1206, 1099, 1028, 737, 698 cmꢂ1
;
1H NMR (300 MHz, CDCl3) d 1.45 (d, J¼7.0 Hz, 3H),
3.50–3.75 (m, 8H), 4.33 (q, J¼7.0 Hz, 1H), 4.47 (d,
J¼11.5 Hz, 1H), 4.59 (d, J¼11.5 Hz, 1H), 7.26–7.39 (m,
5H); 13C NMR (75 MHz, CDCl3) d 17.8, 42.5, 45.6, 66.7,
1H NMR (300 MHz, CDCl3) d 1.22 (d, J¼6.0 Hz, 3H),
1.66–1.85 (m, 2H), 2.82 (br s, 1H), 3.60–3.75 (m, 2H),
3.95–4.07 (m, 1H) 4.53 (s, 2H), 7.25–7.39 (m, 5H); 13C
NMR (75 MHz, CDCl3) d 23.3, 38.1, 67.4, 69.0, 73.2,

M. Nogawa et al. / Tetrahedron 62 (2006) 7300–7306
7305
127.6, 127.7, 128.4, 137.9; MS m/z (EI, rel intensities) 180
4.7. Preparation of authentic (S)-2
(M+, 14%), 161 (57), 107 (100), 91 (100), 89 (42); HRMS
m/z (EI) 180.1146 (calcd for C11H16O2: 180.1150, M+).
The spectral data were in full agreement with those re-
ported.24 HPLC conditions: column, CHIRALCEL OD-H
(Daicel Chemical Industries, Ltd); eluent, hexane/2-prop-
anol¼90/10; flow rate, 0.5 mL/min; 254 nm; temperature,
25 ꢁC; retention time, 13 (S) and 14 (R) min.
Under an argon atmosphere, to a solution of ethyl (S)-(+)-
3-hydroxybutanoate (4, 1.00 g, 7.57 mmol) in CH2Cl2
(20 mL) were added diisopropylethylamine (5.27 mL,
30.3 mmol) and a solution of chloromethylmethylether
(1.82 g, 22.8 mmol) in CH2Cl2 (5 mL) at 0 ꢁC. The reaction
was stopped with 0.1 M phosphate buffer (pH 6.5) and the
products were extracted with AcOEt (ꢃ3). The organic layer
was washed with brine (ꢃ2) and dried over Na2SO4. After
evaporation under reduced pressure, the residue was purified
by column chromatography (hexane/AcOEt¼5/1) to give
ethyl (S)-3-(methoxymethoxy)butanoate as a colorless oil
(5, 1.12 g, 84%); IR (neat) 2978, 1738, 1449, 1377, 1300,
Enantioselective hydrolysis of the other cases was carried
out by the same procedure. In the case of (ꢀ)-1c, the re-
maining 1c and the resulting 2 were separated by flash
column chromatography (hexane/AcOEt¼5/1/hexane/
AcOEt¼3/1).
1
1186, 1150, 1103, 1036, 918 cmꢂ1; H NMR (300 MHz,
4.6.2. 1-Phenylethanol (9a). Compound (R)-9a, [a]2D1 +24.8
(c 0.85, MeOH) (77% ee), lit.25 [a]D20 +45 (c 5.15, MeOH)
for the (R)-enantiomer. The spectral data were in full agree-
ment with that of commercial source. The ee of (R)-9a was
determined by GLC analysis. GLC conditions: column, CP-
Cyclodextrin-B-236-M19 (Chrompack), 0.25 mmꢃ50 m;
injection, 160 ꢁC; detection, 160 ꢁC; oven, 140 ꢁC; carrier
gas, He; head pressure, 2.4 kg/cm2; retention time, 8.9 (R)
and 9.2 (S) min.
CDCl3) d 1.25 (d, J¼6.0 Hz, 3H), 1.26 (t, J¼7.0 Hz, 3H),
2.41 (dd, J1¼5.5 Hz, J2¼15.0 Hz, 1H), 2.60 (dd, J1¼
7.5 Hz, J2¼15.0 Hz, 1H), 3.36 (s, 3H), 4.08–4.22 (m, 1H),
4.15 (q, J¼7.0 Hz, 2H), 4.66 (d, J¼7.0 Hz, 1H), 4.67 (d,
J¼7.0 Hz, 1H); 13C NMR (75 MHz, CDCl3) d 14.1, 20.5,
42.4, 55.3, 60.3, 70.3, 95.3, 171.2.
Under an argon atmosphere, to a suspension of LiAlH4
(200 mg, 5.26 mmol) in THF (5 mL) was added a solution
of (S)-5 (901 mg, 5.12 mmol) in THF (10 mL) at 0 ꢁC. After
the mixture was stirred for 1 h at room temperature, the re-
action was quenched with water (200 mL), 15% NaOH aque-
ous solution (200 mL), and water (400 mL). After filtration
thorough a Celite pad and evaporation, the residue was puri-
fied by column chromatography (hexane/AcOEt¼1/1/
AcOEt) to give (S)-3-(methoxymethoxy)-1-butanol as a
colorless oil (6, 588 mg, 86%); IR (neat) 3428, 2963,
4.6.3. 4-Phenyl-2-butanol (9b). Compound (R)-9b, [a]D27
ꢂ3.4 (c 0.69, CHCl3) (27% ee), lit.26 [a]D27 +17.45 (c 2.04,
CHCl3) for the (S)-enantiomer; IR (neat) 3358, 2965,
2926, 2361, 1713, 1603, 1495, 1454, 1373, 1128, 1055,
746 cmꢂ1
;
1H NMR (300 MHz, CDCl3) d 1.23 (d, J¼
4.0 Hz, 3H, CH3), 1.72–1.82 (m, 2H), 2.60–2.82 (m, 2H),
3.83 (tq, J1¼J2¼6.0 Hz, 1H), 7.14–7.32 (m, 5H); 13C
NMR (75 MHz, CDCl3) d 23.5, 32.1, 40.8, 67.5, 125.8,
128.4, 142.0; MS m/z (EI, rel intensities) 150 (M+, 25%),
132 (100), 117 (100), 105 (47), 91 (100); HRMS m/z (EI)
150.1042 (calcd for C10H14O: 150.1045, M+). HPLC condi-
tions: column, CHIRALCEL OD-H (Daicel Chemical
Industries, Ltd); eluent, hexane/2-propanol¼90/10; flow
rate, 0.5 mL/min; 254 nm; temperature, 25 ꢁC; retention
time, 15 (R) and 20 (S) min.
1
1449, 1411, 1377, 1261, 1103, 1036, 797 cmꢂ1; H NMR
(300 MHz, CDCl3) d 1.22 (d, J¼6.0 Hz, 3H), 1.75 (dt,
J1¼J2¼6.0 Hz, 2H), 2.69 (br s, 1H), 3.39 (s, 3H), 3.68–
3.85 (m, 2H), 3.93 (tq, J1¼J2¼6.0 Hz, 1H), 4.63 (d,
J¼7.0 Hz, 1H), 4.72 (d, J¼7.0 Hz, 1H); 13C NMR
(75 MHz, CDCl3) d 20.2, 39.1, 55.4, 59.9, 72.0, 94.9.
Under an argon atmosphere, to a suspension of NaH (60% in
oil, 337 mg, 8.43 mmol) in THF (5 mL) were added a solu-
tion of (S)-6 (501 mg, 3.74 mmol) in THF (10 mL) and ben-
zyl bromide (0.44 mL, 3.74 mmol) at 0 ꢁC. The mixture was
stirred for 4 h at room temperature and the reaction was
quenched with 0.1 M phosphate buffer (pH 6.5). The prod-
ucts were extracted with AcOEt (ꢃ3), and the organic layer
was washed with brine and dried over Na2SO4. After evap-
oration under reduced pressure, the residue was purified by
flash column chromatography (hexane/AcOEt¼10/1/5/1)
to give (S)-1-benzyloxy-3-(methoxymethoxy)butane as
a colorless oil (7, 541 mg, 65%); IR (neat) 2930, 2882,
4.6.4. 5-Benzyloxy-1-hepten-3-ol (9c). Compound (R)-9c,
[a]2D8 +5.5 (c 0.40, MeOH) (80% ee); IR (neat) 3417,
2862, 2359, 1454, 1366, 1277, 1099, 1028, 993, 922,
1
737 cmꢂ1; H NMR (300 MHz, CDCl3) d 1.67–1.97 (m,
2H), 2.90 (br s, 1H), 3.59–3.76 (m, 2H), 4.30–4.40 (m,
1H), 4.52 (s, 2H), 5.10 (td, J1¼1.5 Hz, J2¼10.5 Hz, 1H),
5.26 (td, J1¼1.5 Hz, J2¼17.0 Hz, 1H), 5.87 (ddd, J1¼
5.5 Hz, J2¼10.5 Hz, J3¼17.0 Hz, 1H), 7.25–7.39 (m, 5H);
13C NMR (75 MHz, CDCl3) d 36.2, 68.1, 71.6, 73.2,
114.3, 127.6, 128.4, 137.8, 140.5; MS m/z (EI, rel intensi-
ties) 192 (M+, 9.3%), 107 (93), 91 (100), 68 (100); HRMS
m/z (EI) 192.1177 (calcd for C12H16O2: 192.1150, M+).
The ee of 9c was determined by 1H NMR analysis of
the corresponding (+)-methoxytrifluoromethylphenylace-
tate (MTPA) ester, which was converted from 9c.
1
1452, 1375, 1207, 1103, 1040, 918, 737, 698 cmꢂ1; H
NMR (300 MHz, CDCl3) d 1.19 (d, J¼6.0 Hz, 3H), 1.70–
1.89 (m 2H), 3.35 (s, 3H), 3.50–3.64 (m, 2H), 3.87 (tq,
J1¼J2¼6.5 Hz, 1H), 4.50 (s, 3H), 4.60 (d, J¼7.0 Hz, 1H),
4.67 (d, J¼7.0 Hz, 1H), 7.22–7.39 (m, 5H); 13C NMR
(75 MHz, CDCl3) d 20.6, 37.2, 55.3, 66.9, 70.6, 73.0, 95.1,
127.5, 127.7, 128.3, 138.4.
1H NMR of the MTPA ester (500 MHz, CDCl3) d 4.40
(d, J¼6.5 Hz, 1H, OCHHPh) and 4.42 (d, J¼6.5 Hz, 1H,
OCHHPh)(S), 4.48 (s, 2H, OCHHPh)(R). The absolute
configuration was determined by comparing the NMR
signal pattern of the MTPA ester with that of the authentic
sample.
To a solution of (S)-7 (400 mg, 1.79 mmol) in THF (10 mL)
was added 2 M HCl (4 mL). After the mixture was stirred
overnight at room temperature, the reaction mixture was di-
luted with water. The products were extracted with AcOEt

7306
M. Nogawa et al. / Tetrahedron 62 (2006) 7300–7306
10. Halcomb, R. L.; Huang, H.; Wong, C.-H. J. Am. Chem. Soc.
1994, 116, 11315–11322.
11. (a) Waldmann, H.; Reidel, A. Angew. Chem., Int. Ed. 1997, 36,
647–649; (b) Sauerbrei, B.; Jungmann, V.; Waldmann, H.
Angew. Chem., Int. Ed. 1998, 37, 1143–1146.
(ꢃ3), and the organic layer was washed with brine and dried
over Na2SO4. After evaporation under reduced pressure,
the residue was purified by flash column chromatography
(hexane/AcOEt¼10/1/4/1) to give the remaining (S)-7
(193 mg, 48%) and (S)-2 as colorless oils (167 mg, 52%);
(S)-2, [a]2D7 +19.0 (c 0.95, MeOH). The spectral data were
in full agreement with those of the (R)-2 obtained by the
enzymatic reaction.
12. (a) Ulijn, R. V.; Baragan˜a, B.; Halling, P. J.; Flitsch, S. L. J. Am.
Chem. Soc. 2002, 124, 10988–10989; (b) Humphrey, C. E.;
Turner, N. J.; Easson, M. A. M.; Flitsch, S. L.; Ulijn, R. V.
J. Am. Chem. Soc. 2003, 125, 13952–13953; (c) Basso, A.;
Ulijn, R. V.; Flitsch, S. L.; Margetts, G.; Brazendale, I.;
Ebert, C.; De Martin, L.; Linda, P.; Verdelli, S.; Gardossi, L.
Tetrahedron 2004, 60, 589–594; (d) Basso, A.; Ebert, C.;
Gardossi, L.; Linda, P.; Phuong, T. T.; Zhu, M.; Wessjohann,
L. Adv. Synth. Catal. 2005, 347, 963–966.
13. (a) Gravert, D. J.; Janda, K. D. Chem. Rev. 1997, 97, 489–509;
(b) Wentworth, P.; Janda, K. D. Chem. Commun. 1999, 1917–
1924; (c) Dickerson, T. J.; Reed, N. N.; Janda, K. D. Chem. Rev.
2002, 102, 3325–3344; (d) Bergbreiter, D. E. Chem. Rev. 2002,
102, 3345–3384.
Acknowledgements
We are grateful to Amano Enzyme, Inc. and Nagase & Co.,
Ltd for the gift of enzymes. M.S. was supported by a grant
from Research Fellowships of the Japan Society for the Pro-
motion of Science (JSPS) for Young Scientists. We thank
Material Science Research Center (Meisei University) for
NMR analysis.
References and notes
14. (a) Benaglia, M.; Celentano, G.; Cozzi, F. Adv. Synth. Catal.
2001, 343, 171–173; (b) Pozzi, G.; Cavazzini, M.; Quici, S.;
Benaglia, M.; Dell’Anna, G. Org. Lett. 2004, 6, 441–443; (c)
Oikawa, M.; Tanaka, T.; Kusumoto, S.; Sasaki, M. Tetrahedron
Lett. 2004, 45, 787–790; (d) Reed, N. N.; Dickerson, T. J.;
Boldt, G. E.; Janda, K. D. J. Org. Chem. 2005, 70, 1728–1731.
15. (a)Veronese, F. M.;Morpurgo, M. IlFarmaco1999,54, 497–516;
(b) Greenwald, R. B. J. Controlled Release 2001, 74, 159–171.
16. Shimojo, M.; Matsumoto, K.; Nogawa, M.; Nemoto, Y.; Ohta,
H. Tetrahedron Lett. 2004, 45, 6769–6773.
17. (a) Inada, Y.; Yoshimoto, T.; Matsushima, A.; Saito, Y. Trends
Biotechnol. 1986, 4, 68–73; (b) Inada, Y.; Takahashi, K.;
Yoshimoto, T.; Ajima, A.; Matsushima, A.; Saito, Y. Trends
Biotechnol. 1986, 4, 190–194.
18. (a) Otamiri, M.; Adlerkreutz, P.; Mattiasson, B. Biotechnol.
Bioeng. 1994, 44, 73–78; (b) Ruppert, S.; Gais, H. J.
Tetrahedron: Asymmetry 1997, 8, 3657–3664.
1. (a) Bornscheuer, U. T.; Kazlauskas, R. J. Hydrolases in Organic
Synthesis; Wiley-VCH:Weinheim, 1999; (b) Bommarius, A. S.;
Riebel, B. R. Biocatalysis; Wiley-VCH: Weinheim, 2004;
(c) Faber, K. Biotransformations in Organic Chemistry:
A Textbook, 5th ed.; Springer: Berlin, Heidelberg, New York,
2004.
2. For a review of modern separation techniques, see: Tzschucke,
C. C.; Markert, C.; Bannwarth, W.; Roller, S.; Hebel, A.; Haag,
R. Angew. Chem., Int. Ed. 2002, 41, 3964–4000.
3. For enzymatic acylation with succinic acid, see: (a) Terao, Y.;
Tsuji, K.; Murata, M.; Achiwa, K.; Nishio, T.; Watanabe, N.;
Seto, K. Chem. Pharm. Bull. 1989, 37, 1653–1655; (b) Gutman,
A. L.; Brenner, D.; Boltanski, A. Tetrahedron: Asymmetry 1993,
4, 839–844.
4. For lipase-catalyzed transformation followed by sulfation, see:
Yamano, T.; Kikumoto, F.; Yamamoto, S.; Miwa, K.; Kawada,
M.; Ito, T.; Ikemoto, T.; Tomimatsu, K.; Mizuno, Y. Chem. Lett.
2000, 448–449.
5. For using fluorous-tagged esters with fluorous solvents, see:
(a) Beier, P.; O’Hagan, D. Chem. Commun. 2002, 1680–1681;
(b) Luo, Z.; Swaleh, S. M.; Theil, F.; Curran, D. P. Org. Lett.
2002, 4, 2585–2587.
6. For enzymatic transesterification of esters with PEG as a
nucleophile, see: (a) Wallace, J. S.; Reda, K. B.; Williams,
M. E.; Morrow, C. J. J. Org. Chem. 1990, 55, 3544–3546; (b)
Whalen, L. J.; Morrow, C. J. Tetrahedron: Asymmetry 2000,
11, 1279–1288.
´
´
19. Lopez-Pelegrın, J. A.; Wentworth, P., Jr.; Sieber, F.; Metz,
W. A.; Janda, K. D. J. Org. Chem. 2000, 65, 8527–8531.
20. Low-molecular weight PEG was used as a tag for real time
reaction monitoring in the synthesis of oligosaccharides. (a)
Manabe, S.; Ito, Y. J. Am. Chem. Soc. 2002, 124, 12638–12639;
(b) Hanashima, S.; Manabe, S.; Ito, Y. Synlett 2003, 979–982.
21. (a) Matsumoto, K.; Sato, Y.; Shimojo, M.; Hatanaka, M.
Tetrahedron: Asymmetry 2000, 11, 1965–1973; (b)
Matsumoto, K.; Nakamura, Y.; Shimojo, M.; Hatanaka, M.
Tetrahedron Lett. 2002, 43, 6933–6936.
22. Chen, C. S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J. J. Am.
Chem. Soc. 1982, 104, 7294–7299.
23. Hultin, P. G.; Jones, J. B. Tetrahedron Lett. 1992, 33, 1399.
24. (a) Casara, P.; Darwin, C.; Metcalf, B.; Jung, M. J. Chem. Soc.,
Perkin Trans. 1 1985, 2201–2208; (b) Cadot, C.; Dalko, P. I.;
Cossy, J.; Ollivier, C.; Chuard, R.; Renaud, P. J. Org. Chem.
2002, 67, 7193–7202.
7. For enzyme-mediated kinetic resolution of carboxylic acids
anchored on polystyrene based resin, see: Nanda, S.; Rao, B.;
Yadav, J. S. Tetrahedron Lett. 1999, 40, 5905–5908.
8. For lipase-catalyzed kinetic resolution with poly(acrylamide)-
ethylene glycol (PEGA)-based resin as a nucleophile, see:
Ulijn, R. V.; Bisek, N.; Flitsch, S. L. Org. Biomol. Chem.
2003, 1, 621–622.
25. Laumen, K.; Schneider, M. P. J. Chem. Soc., Chem. Commun.
1988, 598–600.
´
26. Burgess, K.; Jennings, L. D. J. Am. Chem. Soc. 1991, 113,
6129–6139.
9. Cordova, A.; Tremblay, M. R.; Clapham, B.; Janda, K. D.
J. Org. Chem. 2001, 66, 5645–5648.