Examination of spacer effects on stereochemical recognition of a remote sterically hindered chiral center in lipase-catalyzed acylation

Article abstract of DOI:10.1016/j.molcatb.2012.05.006

To date, the enzyme-catalyzed kinetic resolution of the secondary alcohol [Ar-C*H(CH₃)OH, Ar = 2′,4′,6′- triisopropylphenyl] has not been available, due to high steric hindrance around the hydroxy group. To achieve resolution, the reaction site was extended by the introduction of two kinds of spacers, [-C(=O)CH₂] and [-C(=O)C**HCN]. In the first substrate, the recognition of remote chirality [Ar-C*H(CH₃)O-C(=O)CH₂OH] by acylation with Burkholderia cepacia lipase was examined by changing reaction conditions and acyl donors. An E = 22 in the preference of (1′R)-isomer, was recorded with vinyl acetate as an acyl donor at 25 °C. In the second substrate, there was a matched enantiomeric pair [stereoselective ratio at C-1′ = 15, in the preference of (1′R)-isomer] and a mismatched pair [stereoselective ratio at C-1′ = 2.5, in the preference of (1′S)-isomer] based on the relative stereochemistry between the two chiral centers [Ar- C*H(CH₃)O-C(=O)C**HCN-OH].

Full text of DOI:10.1016/j.molcatb.2012.05.006

Journal of Molecular Catalysis B: Enzymatic 81 (2012) 52–57
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Examination of spacer effects on stereochemical recognition of a remote
sterically hindered chiral center in lipase-catalyzed acylation
Ryohei Kobayashia, Hanghang Huanga,b, Manabu Hamadaa, Toshinori Higashia,
Mitsuru Shojia, Takeshi Sugaia,∗
a Department of Pharmaceutical Science, Keio University, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512, Japan
b Department of Chemical and Biological Engineering, Zhejiang University, Road 38, Hangzhou, Zhejiang, China
a r t i c l e i n f o
a b s t r a c t
To date, the enzyme-catalyzed kinetic resolution of the secondary alcohol [Ar-C*H(CH3)OH, Ar = 2ꢀ,4ꢀ,6ꢀ-
triisopropylphenyl] has not been available, due to high steric hindrance around the hydroxy group.
To achieve resolution, the reaction site was extended by the introduction of two kinds of spac-
ers, [ C( O)CH2 ] and [ C( O)C**HCN ]. In the first substrate, the recognition of remote chirality
[Ar C*H(CH3)O C( O)CH2 OH] by acylation with Burkholderia cepacia lipase was examined by chang-
ing reaction conditions and acyl donors. An E = 22 in the preference of (1ꢀR)-isomer, was recorded with
vinyl acetate as an acyl donor at 25 ◦C. In the second substrate, there was a matched enantiomeric pair
[stereoselective ratio at C-1ꢀ = 15, in the preference of (1ꢀR)-isomer] and a mismatched pair [stereoselec-
tive ratio at C-1ꢀ = 2.5, in the preference of (1ꢀS)-isomer] based on the relative stereochemistry between
the two chiral centers [Ar C*H(CH3)O C( O)C**HCN OH].
Article history:
Received 18 February 2012
Received in revised form 20 April 2012
Accepted 8 May 2012
Available online 16 May 2012
Keywords:
Lipase
Hydrolysis
Acylation
Kinetic resolution
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
( )-2a was used as the substrate. Compound 2a has a primary
alcohol, which is highly susceptible to the enzyme-catalyzed reac-
tion. In 2007, Gotor and co-workers reported an approach to
resolve sterically hindered tertiary alcohol by means of lipase-
catalyzed acylation, after derivation to hydroxyacetate esters [4].
Some successful examples have also been reported in which a
hydroxymethyl group was introduced on the nitrogen atom of lac-
tam substrates for the recognition of remote chirality [5–9].
As primary alcohols are often excessively active toward
lipase-catalyzed acylation [10,11], we designed another sub-
strate 3a (Fig. 1) with an electron-withdrawing cyano group.
Extensive studies have already been devoted to the enzyme-
catalyzed kinetic resolution of cyanohydrin-type substrates
[12].
Substituted arylethanols and their corresponding esters are
good substrates for achieving high enantiomerically selective
hydrolytic enzyme-catalyzed kinetic resolution [1]. In most
cases, (R)-forms are “fast” enantiomers while (S)-forms are
“slow” enantiomers (Fig. 1). The introduction of
a bulky
group at the ortho-position suppresses the reactivity. For
example, the enzyme-catalyzed kinetic resolution of 1-(2ꢀ,4ꢀ,6ꢀ-
triisopropylphenyl)ethanol (Fig. 1, 1a) failed [2], due to the very low
reactivity of both enantiomers. The resolution was attempted using
the corresponding chloroacetate 1b, a more reactive substrate due
to the introduction of the electron-withdrawing halogen atom.
However, no reaction was observed by applying four lipases, i.e.,
Candida antarctica lipase B (Novozymes, Novozym 435), Burkholde-
ria cepacia lipase (Amano, PS-IM), Candida rugosa lipase (Meito,
OF), and pig pancreatic lipase (Sigma), under hydrolytic conditions,
even at temperatures as high as 60 ◦C. It was also reported that in
a protease-catalyzed hydrolysis of the same substrate (Fig. 1, 1b),
there was no reaction [3].
2. Experimental
All mps are uncorrected. IR spectra were measured as films for
oils or as ATR on a Jeol FT-IR SPX60 spectrometer. 1H NMR spec-
tra were measured in CDCl3 at 400 MHz, on a VARIAN 400-MR
spectrometer. HPLC data were recorded on SHIMADZU SPD-M20A
multi-channel detector. Optical rotation values were recorded on
a Jasco P-1010 polarimeter. Silica gel 60 N (spherical and neutral,
100-210 ␮m, 37560-79) of Kanto Chemical Co. was used for column
chromatography. Preparative TLC was performed with Merck Silica
Gel 60 F254 plates (0.5 mm thickness, No. 5744).
In this paper, we report the introduction of two kinds of spac-
ers to alleviate the steric interaction between the enzyme catalytic
site and bulky substituents. As a first approach, hydroxyacetate
∗
Corresponding author. Tel.: +81 3 5400 2665; fax: +81 3 5400 2665.
E-mail address: sugai-tk@pha.keio.ac.jp (T. Sugai).
1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2012.05.006

R. Kobayashi et al. / Journal of Molecular Catalysis B: Enzymatic 81 (2012) 52–57
53
J = 6.8 Hz, 6H, CH(CH3)2], 1.22 [d, J = 6.8 Hz, 6H, CH(CH3)2], 1.27 [d,
ꢀ
ꢀ
ꢀ
J = 6.8 Hz, 6H, CH(CH3)2], 1.65 (d, J1 ,2 = 6.8 Hz, 3H, H2 ), 2.84 [hept,
1H, CH(CH3)2], 3.45 [br, 2H, CH(CH3)2], 4.06 (d, J2a,2b = 17.2 Hz, 1H,
H2a), 4.16 (d, 1H, H2b), 6.61 (q, 1H, H1), 7.00 (s, 2H, Ar H); 13C
NMR: ı 22.2, 23.8, 23.9, 24.2, 24.9, 29.4, 34.1, 60.8, 70.1, 131.3,
148.4, 172.7; IR: 3367, 2958, 2870, 1740, 1458, 1377, 1207, 1092,
1053 cm−1. Anal. Calcd for C19H30O3: C 74.47, H 9.87; found: C
74.41, H 9.83.
2.3. 1ꢀ-(2ꢀꢀ,4ꢀꢀ,6ꢀꢀ-Triisopropylphenyl)ethyl 1,1-dihydoxyacetate
(4)
To
a solution of 2a (150 mg, 0.489 mmol) in anhydrous
CH2Cl2 (3.6 mL) were added DMSO (5.4 mL), triethylamine (409 ␮L,
2.93 mmol, 6.0 equiv.) and SO3-pyridine (311 mg, 1.96 mmol,
4.0 equiv.) with stirring. After stirring for 5 h at room tempera-
ture, saturated aqueous Na2S2O3 solution was added to the reaction
mixture. The mixture was extracted twice with AcOEt, and the com-
bined organic layer was washed with brine, dried over Na2SO4,
and concentrated in vacuo. The residue was purified by silica gel
column chromatography (10 g). Elution with hexane/AcOEt = 3:1
afforded a mixture of 4 (101 mg, 65%) and its aldehyde form as
amorphous solid. 1H NMR: ı 1.22 [d, J = 6.9 Hz, 6H, CH(CH3)2], 1.22
[d, J = 6.8 Hz, 6H, CH(CH3)2], 1.28 [d, J = 6.8 Hz, 6H, CH(CH3)2], 1.69
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
(d, J1 ,2 = 6.8 Hz, 2.25H, H2 ), 1.74 (d, J1 ,2 = 6.8 Hz, 0.75H, H2 ), 2.84
[hept, 1H, CH(CH3)2], 3.47 [br, 2H, CH(CH3)2], 5.18 (s, 0.75H, H2),
6.58 (q, 0.75H, H1ꢀ), 6.65 (q, 0.25H, H1ꢀ), 7.00 (s, 2H, Ar H), 9.36
(s, 0.25H, aldehyde). This was employed for the next step without
further purification.
2.4. 1ꢀ-(2ꢀꢀ,4ꢀꢀ,6ꢀꢀ-Triisopropylphenyl)ethyl
2-cyano-2-hydroxyacetate (3a)
Fig. 1. Attempts to introduce spacers for enzyme-catalyzed kinetic resolution of a
highly sterically hindered arylethanol.
To a solution of 4 (218 mg, 0.716 mmol) in anhydrous CH2Cl2
(4.5 mL) were added molecular sieves 3A (378 mg), ZnI2 (50 mg,
0.157 mmol, 0.2 equiv.) and trimethylsilyl cyanide (TMSCN, 510 ␮L,
4.08 mmol, 5.7 equiv.) with stirring. After stirring for 2 h at room
temperature, phosphate buffer solution (pH 7.5, 0.1 M) was added
to the reaction mixture, and the mixture was filtered to remove
insoluble materials including molecular sieves. The resulting mix-
ture was extracted twice with AcOEt. The combined organic layer
was washed with brine, dried over Na2SO4, and concentrated
in vacuo. The residue was purified by silica gel column chro-
matography (10 g). Elution with hexane/AcOEt = 10:1 afforded a
diastereomeric mixture of 3a (128 mg, 54%) as white solid. The
mixture was separated by preparative HPLC [column, Kanto Chem-
ical Co. Inc. Mightysil Si 60, 1.0 cm × 25 cm; hexane/AcOEt = 10:1;
flow rate 5.0 mL/min, detected at 267 nm]: less polar isomer tR
(min) = 17.1 (100%); more polar isomer tR (min) = 20.7 (100%).
(2R*, 1ꢀS*)-3a (less polar isomer). white solid, m.p. 124 ◦C. 1H
NMR: ı 1.24 [d, J = 6.8 Hz, 6H, CH(CH3)2], 1.24 [d, J = 6.8 Hz, 6H,
2.1. 1ꢀ-(2ꢀꢀ,4ꢀꢀ,6ꢀꢀ-Triisopropylphenyl)ethyl acetoxyacetate (2b)
To
a solution of 1a (200 mg, 0.805 mmol) in pyridine
(4.0 mL) were added acetoxyacetyl chloride (121 mg, 0.886 mmol,
1.1 equiv.) and DMAP (9.8 mg, 0.081 mmol, 0.1 equiv.) with stir-
ring. After stirring for 4 h at room temperature, ice and AcOEt
were added to the mixture. The mixture was extracted three times
with AcOEt. The combined organic layer was washed with water,
hydrochloric acid (1 M), saturated aqueous NaHCO3 solution and
brine, dried over Na2SO4, and concentrated in vacuo. The residue
was purified by silica gel column chromatography (10 g). Elution
with hexane/AcOEt = 10:1 afforded 2b (168 mg, 60%) as white solid,
m.p. 85–86 ◦C. 1H NMR: ı 1.21 [d, J = 6.8 Hz, 6H, CH(CH3)2], 1.22 [d,
J = 7.1 Hz, 6H, CH(CH3)2], 1.25 [d, J = 6.7 Hz, 6H, CH(CH3)2], 1.63 (d,
ꢀ
ꢀ
ꢀ
J1 ,2 = 6.8 Hz, 3H, H2 ), 2.13 (s, 3H, Ac), 2.83 [hept, 1H, CH(CH3)2],
3.44 [br, 2H, CH(CH3)2], 4.51 (d, J2a,2b = 15.9 Hz, 1H, H2a), 4.64 (d,
1H, H2b), 6.59 (q, 1H, H1ꢀ), 6.99 (s, 2H, Ar H); 13C NMR: ı 20.5,
22.2, 23.9, 23.9, 24.2, 24.8, 29.4, 34.1, 60.9, 69.8, 131.4, 148.4, 167.2,
ꢀ
ꢀ
CH(CH3)2], 1.30 [d, J = 6.8 Hz, 6H, CH(CH3)2], 1.75 (d, J1 ,2 = 6.8 Hz,
3H, H2ꢀ), 2.85 [hept, 1H, CH(CH3)2], 3.33 [d, J = 6.1 Hz, 1H, OH],
3.40 [br, 2H, CH(CH3)2], 4.80 (d, 1H, H2), 6.67 (q, 1H, H1ꢀ), 7.02
(s, 2H, Ar H); 13C NMR: ı 21.9, 23.8, 23.9, 24.2, 24.9, 29.5, 34.1,
60.0, 73.9, 114.8, 129.9, 149.1, 165.7. Other signals attributed to
aromatic carbons were not clearly observed by broadening, due to
some interference of free rotation between C-1ꢀ and C-1ꢀꢀ; IR: 3332,
2958, 2870, 2357, 1759, 1207, 1115, 1049 cm−1. HRMS (FAB+): m/z
331.2174 (M)+; calc. for C20H29NO3: 331.2147.
170.2; IR: 2958, 2870, 1743, 1427, 1381, 1203, 1084, 1057 cm−1
Anal. Calcd for C21H32O4: C 72.38, H 9.26; found: C 72.49, H 9.28.
.
2.2. 1ꢀ-(2ꢀꢀ,4ꢀꢀ,6ꢀꢀ-Triisopropylphenyl)ethyl hydroxyacetate (2a)
To a solution of 2b (20.0 mg, 0.057 mmol) in cyclopentanol
(0.4 mL) was added C. antarctica lipase B (Novozymes, Novozym
435, 40.0 mg), and the mixture was stirred for 31 h at room tem-
perature. After removal of insoluble materials, the filtrate was
concentrated in vacuo. The residue was purified by silica gel column
chromatography (9 g). Elution with hexane/AcOEt = 10:1 afforded
2a (14.3 mg, 81%) as white solid, m.p. 118 ◦C. 1H NMR: ı 1.20 [d,
(2R*,1ꢀR*)-3a (more polar isomer). white solid, m.p. 121 ◦C. 1H
NMR: ı 1.24 [d, J = 6.8 Hz, 6H, CH(CH3)2], 1.25 [d, J = 6.8 Hz, 6H,
ꢀ
ꢀ
CH(CH3)2], 1.30 [d, J = 6.4 Hz, 6H, CH(CH3)2], 1.72 (d, J1 ,2 = 6.5 Hz,
3H, H2ꢀ), 2.85 [hept, 1H, CH(CH3)2], 3.34 (s, 1H, OH), 3.47 [br, 2H,
CH(CH3)2], 4.92 (s, 1H, H2), 6.67 (q, 1H, H1ꢀ), 7.02 (s, 2H, Ar H).
13C NMR: ı 22.2, 23.8, 23.9, 24.2, 24.9, 29.5, 34.1, 60.1, 74.2, 114.6,

54
R. Kobayashi et al. / Journal of Molecular Catalysis B: Enzymatic 81 (2012) 52–57
129.6, 149.1, 165.6. For the number of signals, see the comment as
( )-2a (20.0 mg, 0.065 mmol). HPLC for 1a from 2a: tR (min) = 11.0
(21.9%), 13.3 (78.1%); 1a from 2d: tR (min) = 11.0 (85.0%), 13.3
(15.0%); conv. 45% with E = 10.
above; IR: 3433, 2958, 2870, 2357, 1743, 1292, 1099, 1049 cm−1
.
HRMS (FAB+): m/z 331.2181 (M)+; calc. for C20H29NO3: 331.2147.
In the similar manner as described above, a solution of 4
(105.2 mg, 0.346 mmol) in anhydrous CH2Cl2 (5.0 mL) was treated
with molecular sieves 3A (150 mg), ZnI2 (25 mg, 0.079 mmol,
0.1 equiv.) and TMSCN (210 ␮L, 1.68 mmol, 4.9 equiv.). After stir-
ring for 1 h at room temperature, the mixture was cooled to 0 ◦C
and pyridine (0.5 mL) and Ac2O (0.5 mL) were added. The stir-
ring was continued for 3 h at 0 ◦C, and the reaction was quenched
and worked-up as in the same manner as described before. The
crude residue was purified by silica gel column chromatography
(10 g). Elution with hexane/AcOEt = 15:1 afforded diastereomeric
mixture of 3b (68.7 mg, 53%) as colorless oil. 1H NMR: ı 1.23 [d,
J = 7.1 Hz, 12H, CH(CH3)2], 1.28 [d, J = 6.6 Hz, 6H, CH(CH3)2], 1.69 (d,
2.6. B. cepacia lipase-catalyzed acetylation of (2R*,1ꢀR*)-3a
To a solution of 3a (20.0 mg, 0.060 mmol) and vinyl acetate
(24 ␮L) in toluene (376 ␮L) was added B. cepacia lipase (150.0 mg),
and the mixture was stirred for 6.5 h at 30 ◦C. After removal of
insoluble materials, the filtrate was concentrated in vacuo. The
residue was purified by preparative TLC. Development with hex-
ane/AcOEt = 4:1 afforded 3a (8.1 mg, 41%) and 3b (5.1 mg, 23%).
HPLC: 1a from 3a: tR (min) = 11.0 (31.2%), 13.3 (68.8%); 1a from 3b:
tR (min) = 11.0 (91.2%), 13.3 (8.8%); conv. 31% with stereoselective
ratio at C-1ꢀ = 15.
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
J1 ,2 = 6.8 Hz, 1.3H, H2 ), 1.72 (d, J1 ,2 = 6.9 Hz, 1.7H, H2 ), 2.21 (s, 3H,
Ac), 2.85 [hept, 1H, CH(CH3)2], 3.43 [br, 2H, CH(CH3)2], 5.74 (s, 0.6H,
H2), 5.84 (s, 0.4H, H2), 6.63 (q, 1H, H1ꢀ), 7.01 (s, 2H, Ar H). 13C NMR:
ı 20.0, 22.0, 22.1, 23.8, 23.9, 24.2, 29.4, 29.5, 34.1, 59.2, 59.4, 73.2,
73.4, 112.3, 112.5 130.0, 130.1, 148.9, 149.0, 160.8, 161.0, 168.2,
2.7. B. cepacia lipase-catalyzed acetylation of (2R*,1ꢀS*)-3a
In a similar manner as described in the Section 2.6, a solution of
(2R*,1ꢀS*)-3a (20.0 mg, 0.060 mmol) in toluene (376 ␮L) and vinyl
acetate (24 ␮L) was added B. cepacia lipase (150 mg), and the mix-
ture was stirred for 6.5 h at 30 ◦C. The same workup and purification
gave 3a (10.9 mg, 55%) and 3b (2.6 mg, 12%). HPLC: 1a from 3a:
tR (min) = 11.0 (53.6%), 13.3 (46.4%); 1a from 3b: tR (min) = 11.0
(29.9%), 13.3 (70.1%); conv. 15% with stereoselective ratio at C-
1ꢀ = 2.5.
168.3; IR: 2958, 2870, 2357, 1759, 1373, 1281, 1200, 1049 cm−1
.
The theoretical calculations for the stereoisomers of 5 were
performed by using SPARTAN (Wavefunction, Inc.). Conformers
of model compounds were surveyed by Hartree–Fock Molecular
Orbital Model at RHF/3-21G level.
2.5. B. cepacia lipase-catalyzed acetylation of ( )-2a
2.8. B. cepacia lipase-catalyzed acetylation of a diastereomeric
mixture of 3a
To a solution of 2a (100 mg, 0.327 mmol) in vinyl acetate (2.0 mL)
was added B. cepacia lipase (Amano, PS-IM, 200 mg), and the
mixture was stirred for 11 h at 25 ◦C. After removal of insoluble
materials, the filtrate was concentrated in vacuo. The residue was
purified by silica gel column chromatography (4 g). Elution with
hexane/AcOEt = 10:1 afforded 2a (37.9 mg, 38%) and 2b (50.5 mg,
44%). Hydroxyacetate 2a and acetoxyacetate 2b resulted from the
lipase-catalyzed reaction were treated with K2CO3 in refluxing
methanol to give 1a.
In a similar manner as described in the Section 2.6, a solution
of 3a (20.0 mg, 0.060 mmol) in vinyl acetate (400 ␮L) was added
B. cepacia lipase (40.0 mg), and the mixture was stirred for 2.0 h
at 30 ◦C to give 3a (13.3 mg, 67%) and 3b (2.8 mg, 12%). HPLC:
1a from 3a: tR (min) = 11.0 (46.3%), 13.3 (53.7%); 1a from 3b: tR
(min) = 11.0 (67.9%), 13.3 (32.1%); conv. 17% with stereoselective
ratio at C-1ꢀ = 2.3.
25
1a from 2a. [␣]D −38.3 (c 1.36, CHCl3); HPLC [column, Daicel
Chiralcel OD-H, 0.46 cm × 25 cm; hexane/i-PrOH = 100:1; flow rate
0.5 mL/min, detected at 220 nm]: 1a from 2a: tR (min) = 11.0 [9.7%
for (R)-], 13.3 [90.3% for (S)-].
2.9. Scaled-up lipase-catalyzed acetylation of ( )-2a
25
1a from 2b. [␣]D +36.0 (c 1.51, CHCl3); HPLC under the same
In a similar manner with small scale acetylation in Section 2.5,
hydroxyacetate 2a (1.0 g, 3.27 mmol) was treated with B. cepacia
lipase (2.0 g). The workup and purification gave 2a (474 mg, 47%)
and 2b (518 mg, 46%). These were hydrolyzed to give 1a. HPLC:
1a from 2a: tR (min) = 11.0 (4.4%), 13.3 (95.6%); 1a from 2b: tR
(min) = 11.0 (85.8%), 13.3 (14.2%); conv. 56% with E = 19.
conditions as above, tR (min) = 11.0 [90.0% for (R)-], 13.3 [10.0%
for (S)-]. Former (latter) peak was assigned to be R (S); by the co-
injection with an authentic sample of (S)-1a (Aldrich, Catalog No.
672483). The conversion and E value were calculated to be 50% and
22, respectively.
In the case of the reaction for 12 h at 5 ◦C, hydroxyacetate 2a
(11.2 mg, 56%) and acetoxyacetate 2b (7.7 mg, 34%) were obtained
from 2a (20.0 mg, 0.065 mmol). HPLC: 1a from 2a: tR (min) = 11.0
(26.6%), 13.3 (73.4%); 1a from 2b: tR (min) = 11.0 (92.4%), 13.3
(7.6%). The conversion and E value were calculated to be 36% and
19, respectively.
2.10. Approach to enantiomerically enhanced (S)-2a
In a similar manner as described for the acetylation of ( )-2a,
treatment of (S)-2a (133 mg, 0.435 mmol, 91.2% ee as in Section 2.9)
with B. cepacia lipase and vinyl acetate for 12 h at 15 ◦C gave (S)-2a
(102 mg, 77%) as unreacted recovery. This was hydrolyzed to give
(S)-1a. HPLC: tR (min) = 11.0 (3.7%), 13.3 (96.3%); 92.6% ee.
In the reaction for 24 h at −5 ◦C, hydroxyacetate 2a (13.0 mg,
66%) and acetoxyacetate 2b (5.1 mg, 25%) were obtained from 2a
(20.0 mg, 0.065 mmol). HPLC: 1a from 2a: tR (min) = 11.0 (36.0%),
13.3 (64.0%); 1a from 2b: tR (min) = 11.0 (92.7%), 13.3 (7.3%).; conv.
25% with E = 17.
2.11. Approach to enantiomerically enhanced (R)-2b
In vinyl dodecanoate at 5 ◦C for 36 h, alcohol 2a (13.0 mg, 3%)
and dodecanoate 2c (184.5 mg, 58%) were obtained from ( )-2a
(200 mg, 0.654 mmol). HPLC for 1a from 2a: tR (min) = 11.0 (0.5%),
13.3 (99.5%); 1a from 2c: tR (min) = 11.0 (71.3%), 13.3 (28.7%); conv.
70% with E = 12.
With succinic anhydride (32.7 mg, 0.327 mmol, 5 equiv.) in tert-
butyl methyl ether (0.4 mL) at 25 ◦C for 20 h, alcohol 2a (10.0 mg,
50%) and hemisuccinate 2d (17.8 mg, 67%) were obtained from
In a similar manner as described for the transesterification of
)-2b, treatment of (R)-2b (494 mg, 1.42 mmol, 71.6% ee as in
(
Section 2.9) with C. antarctica lipase B and cyclopentanol for 8 h
at 15 ◦C gave (R)-2a (406 mg, 94%). Treatment of (R)-2a (189 mg,
0.618 mmol) with B. cepacia lipase and vinyl acetate for 8 h at 15 ◦C
gave (R)-2b (116 mg, 54%). This was hydrolyzed to give (R)-1a.
HPLC: tR (min) = 11.0 (99.3%), 13.3 (0.7%); 98.6% ee.

R. Kobayashi et al. / Journal of Molecular Catalysis B: Enzymatic 81 (2012) 52–57
55
Scheme 1. Reagents and conditions: (a) acetoxyacetyl chloride, DMAP, pyridine
(60%); (b) Candida antarctica lipase B (Novozymes, Novozym 435), cyclopentanol;
(c) SO3-pyridine, DMSO, CH2Cl2, then Et3N (65%); and (d) TMSCN, ZnI2, CH2Cl2 (54%).
3. Results and discussion
3.1. Preparation of substrates 2a and 3a
As the direct condensation between 1a and hydroxyacetic acid
is not available, alcohol 1a was first treated with acetoxyacetyl
chloride to give 2b in 60% yield. Our previous experience on
enzyme-catalyzed hydrolysis of acetoxyacetate to hydroxyacetate
[13] prompted us to apply four kinds of lipases for the regiose-
lective removal of the acetyl protective group of 2b to prevent
damage to the secondary hydroxyacetate itself. C. antarctica lipase
B-mediated transesterification with cyclopentanol [14] was found
to be very effective to afford 2a in 81% yield (Scheme 1). No selectiv-
ity between the enantiomers of 2a was observed. The same reaction
was very slow with other enzymes such as B. cepacia lipase, C.
rugosa lipase, and pig pancreatic lipase. For example, in the case
of B. cepacia lipase, the conversion only reached 5% after 45 h.
The alcohol 2a itself works as a precursor for 3a. The oxidation of
2a and the subsequent cyanohydrin formation [15] on the hydrate
form 4 provided 3a in 35% yield. The resulting two diastereomers
of 3a were separated by means of preparative HPLC on the silica gel
stationary phase. The less polar isomer showed an upfield shift (ı
4.80) and the more polar isomer showed a downfield shift (ı 4.92) of
the H-2 signal in their respective 1H NMR spectra. The elucidation of
the more stable conformation was attempted by theoretical calcu-
lations (Section 2.4). The convergence of calculation was, however,
difficult due to so much degree of freedom in the molecule. Thus,
we performed the calculation with a simplified model compound
5 (Fig. 2), by omitting the freely rotating 4-isopropyl group in 3a.
By considering the magnetic anisotropic effect of the aromatic ring,
the stereochemistry of the less polar isomer was assigned as (2R*,
1ꢀS*) and that of the more polar isomer was (2R*, 1ꢀR*) as shown in
Fig. 2.
Fig. 2. Stereochemical assignments for the stereoisomers of the model compound
5. For detail, see Section 2.4.
lipase B showed the quickest consumption of the starting mate-
rial, whereas the reaction was very slow with C. rugosa lipase and
pig pancreatic lipase. In contrast to 2b, the corresponding alco-
hol 2a was well accepted by B. cepacia lipase, and the acetylation
with vinyl acetate proceeded in an enantioselective manner with
an E value of 22 at 25 ◦C (Scheme 2). Aiming for higher enantios-
electivity, the reaction temperature was lowered (5 and −5 ◦C),
but no large difference in E value was observed (19 for 5 ◦C and
17 for −5 ◦C). We expected some change by the addition of tert-
butyl methyl ether (TBME) or toluene as co-solvent [16], but those
slightly retarded the reaction. By replacing the acyl donor with vinyl
dodecanoate [17] or succinic anhydride [18], the enantioselectiv-
ity decreased to as low as 12 and 10, respectively. The absolute
configuration of the “fast” enantiomer was determined to be R
by comparing its chromatographic behavior on the chiral HPLC
stationary phase with an authentic sample after complete depro-
tection of the resulting 2 to 1a.
3.3. Lipase-catalyzed acylation of 3a
3.2. Lipase-catalyzed acylation of 2a
We separately submitted (2R*, 1ꢀR*)- and (2R*, 1ꢀS*)-3a to B.
cepacia lipase-catalyzed acetylation. Even under mild reaction con-
ditions, pure diastereomers were prone to epimerize. The addition
Next, we tried the kinetic resolution of ( )-2a under acylation
conditions with vinyl acetate. Among the four lipases, C. antarctica

56
R. Kobayashi et al. / Journal of Molecular Catalysis B: Enzymatic 81 (2012) 52–57
Scheme 2. Reagents and conditions: (a) Burkholderia cepacia lipase (Amano, PS-IM),
cyclopentanol; and (b) K2CO3, MeOH.
Scheme 3. Reagents and conditions: (a) B. cepacia lipase, vinyl acetate, toluene. For
the determination of stereoselective ratio, see Sections 2.6 and 2.7.
of toluene as co-solvent was effective to suppress such undesired
side reactions.
In the case of (2R*, 1ꢀR*)-3a, which is a mixture of (2R, 1ꢀR)- and
(2S,1ꢀS)-isomers, the stereoselective ratio at C-1ꢀ was calculated to
be 15 (Scheme 3), which is comparable to that observed in the B.
cepacia lipase-catalyzed acetylation of 2a. During the progress of
acetylation, the stereochemistry at C-2 in 3b promptly equilibrated
and we could not evaluate the stereochemical preference at this
position.
In contrast, when the same reaction conditions were applied
to a mixture of (2S,1ꢀR)- and (2R,1ꢀS)-3a, the selectivity value was
as low as 2.5. This was the “mismatched” pair. Interestingly, the
preferred stereochemistry at C-1ꢀ was reversed. In this pair of enan-
tiomers, the (1ꢀR)-isomer, having so far been observed as the fast
isomer, has the (2S)-configuration, which is obviously disadvanta-
geous compared to the (2R, 1ꢀS)-enantiomer [19]. The suppression
of the rate of (2S,1ꢀR)-3a was supported by the longer reaction
time that was required to reach the same conversion compared
with the “matched” pair consisting of (2R,1ꢀR)- and (2S,1ꢀS)-3a. The
reaction with a mixture of four stereoisomers showed a stereose-
lective value of 2.3 at C-1ꢀ with the preference of the (1ꢀR)-absolute
configuration.
crystallization of the chiral salts, after derivation to half succinate,
has been only way for enantiomeric resolution. Due to the high
steric hindrance, the asymmetric reduction of the corresponding
ketone has not succeeded yet, and accordingly, the enantiomer-
ically pure form of 1a is very expensive. Based on our results,
B. cepacia lipase-catalyzed acetylation was applied to 2a, under
scaled-up conditions using 1.0 g of the substrate. The enantioselec-
tivity was acceptable, giving (S)-2a (91.2% ee, 47% yield) and (R)-2b
(71.6% ee, 46% yield), respectively. This enantioselectivity (E = 19)
is marginally satisfactory for practical resolution, thus repetition
of the lipase-catalyzed acetylation and subsequent deprotection
were attempted on both enantiomers to achieve high enantiomeric
purity.
First, we tried repetition of lipase-catalyzed acetylation on the
enantiomerically enriched “slow enantiomer” [(S)-2a (91.2% ee)].
To our disappointment, the extent of acetylation of the contam-
inating (R)-isomer was not sufficient. Only an increase of 1.4%
ee was observed (Section 2.9). In the case of the lipase-catalyzed
enantiomeric resolution of primary alcohols or acetates, we had
experienced some difficulty in removing traces of the “fast enan-
tiomer,” as the enzyme-catalyzed product of the “fast isomer” itself
sometimes functioned as a competitive substrate that interfered
with the progress of the reaction on the “fast substrate” [11].
In contrast, the enhancement of ee of the “fast enantiomer” (R)-
2b by means of a second treatment of the enriched substrate with
3.4. Enhancement of ee of 2a by the repetition of lipase-catalyzed
enantioselective acylation
The alcohol 1a has been named as “stericol” and is an important
chiral auxiliary in asymmetric synthesis [2]. So far, the fractional

R. Kobayashi et al. / Journal of Molecular Catalysis B: Enzymatic 81 (2012) 52–57
57
of another electron-withdrawing cyano group on the same carbon
carrying the hydroxy group of the reaction site.
Acknowledgments
This work was supported both by a Grant-in-Aid for Scien-
tific Research (No. 23580152) and formation of a research center
in cell signaling drug discovery for molecular targeting therapies:
matching fund subsidy 2009–2013 from the Ministry of Education,
Culture, Sports, Science and Technology, Japan, and acknowledged
with thanks. Amano Enzyme Inc., Novozymes Japan, and Meito
Sangyo Co., Ltd. for the generous gift of lipases were acknowledged
with thanks. H. H. thanks Professor Cai Jin, Faculty of Chemical and
Biological Engineering, Zhejiang University, and office of student
services (international) of Keio University, for “Independent Study
I and II” of Keio international program (KIP) in 2009–2010, as an
exchange student.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.molcatb.2012.05.006.
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Scheme 4. Reagents and conditions: (a) B. cepacia lipase, vinyl acetate; and (b) C.
antarctica lipase B, cyclopentanol.
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4. Conclusion
The enantiomerically enriched forms of stericol (1a), a highly
sterically hindered secondary alcohol, were obtained by an
“indirect” resolution with lipase-catalyzed acylation (E = 22). An
oxyacetyl [ C( O)CH2
O ] spacer worked well in 2a to allevi-
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ate the steric hindrance of the two adjacent isopropyl groups. The
stereochemical preference could be reversed by the introduction