Chemoenzymatic synthesis of an α-substituted serine derivative

Article abstract of DOI:10.1016/S0957-4166(99)00270-0

An asymmetric synthesis of an α-substituted serine derivative was achieved by employing PLE-catalyzed hydrolysis of a prochiral malonate derivative, and the absolute configuration of the hydrolyzed product was unambiguously determined by the X-ray analysis of a camphorsulfonic acid derivative.

Full text of DOI:10.1016/S0957-4166(99)00270-0

Pergamon
Tetrahedron: Asymmetry 10 (1999) 2703–2712
Chemoenzymatic synthesis of an α-substituted serine derivative
Toshio Honda,∗ Tatsuya Koizumi, Yuki Komatsuzaki, Rie Yamashita, Kazuo Kanai and
Hiromasa Nagase
Faculty of Pharmaceutical Sciences, Hoshi University, Ebara 2-4-41, Shinagawa-ku, Tokyo 142-8501, Japan
Received 26 May 1999; accepted 16 June 1999
Abstract
An asymmetric synthesis of an α-substituted serine derivative was achieved by employing PLE-catalyzed
hydrolysis of a prochiral malonate derivative, and the absolute configuration of the hydrolyzed product was
unambiguously determined by the X-ray analysis of a camphorsulfonic acid derivative. © 1999 Elsevier Science
Ltd. All rights reserved.
1. Introduction
Development of synthetic procedures for chiral α,α-disubstituted α-amino acids are of continuing
interest,1 since these compounds are needed for the synthesis of enzyme inhibitors and peptidomimetics.
Among these amino acids, an α-substituted serine structure is often observed as a subunit of many
biologically active natural products, such as conagenin 1, lactacystin 2, myriocins 3, and mycestericins 4
(Fig. 1).2
Recently we have been involved in the asymmetric synthesis of biologically active natural products by
utilization of PLE-catalyzed asymmetric hydrolysis of the pro-chiral malonate derivatives, and reported
the preparation of the key intermediates for aphanorphine3 and furanosesquiterpenes.4 As an extension
of this work, we are interested in the synthesis of an α-substituted serine derivative for the preparation
of myriocin and its congeners.
As a means of investigating the scope and limitation of the PLE-catalyzed asymmetric hydrolysis of
pro-chiral malonates, we chose the structurally simple cyclohexene derivatives as the starting materials,
in which the discriminative sites for the enzyme lay between the sp2- and sp3-carbons. To the best of
our knowledge for PLE-catalyzed asymmetric hydrolyses of malonate derivatives, no applications of
compounds having such a small functional difference have appeared. In this paper, we would like to report
our results for the PLE-catalyzed asymmetric hydrolysis of a malonate bearing very similar substituents.
∗
Corresponding author. E-mail: honda@hoshi.ac.jp
0957-4166/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(99)00270-0

2704
T. Honda et al. / Tetrahedron: Asymmetry 10 (1999) 2703–2712
Figure 1.
2. Results and discussion
The starting pro-chiral diethyl malonate 7 was prepared from 2,2-dicarboethoxycyclohexanone 54 in
two steps involving reduction of the carbonyl group and dehydration of the alcohol 6 as described in
Scheme 1, in 82% overall yield. The corresponding dimethyl malonate 8 was prepared by treatment of 7
with sodium methoxide, whereas dipentyl malonate 9 was obtained by treatment of 7 with pentyl alcohol
in refluxing toluene in the presence of p-toluenesulfonic acid.
Scheme 1. Reagents and conditions: (i) NaBH4, MeOH, room temp.; (ii) TFMSA, pyridine, −15°C to room temp.; (iii) NaOMe,
MeOH, 0°C; (iv) p-TSA, 1-pentanol, 80°C
Incubation of the diethyl malonate derivative 7 in a 0.2 M phosphate buffer solution with 5% acetone
between 15–20°C resulted in the desired acid-ester 10 in 92% yield (Table 1). Although the absolute
configuration at the newly generated stereogenic center of 10 could not be determined at this stage, it
was assumed to be R based on the proposed models for PLE hydrolysis of malonate derivatives5 and
also on our earlier work3,4 (Scheme 2). Because of the relative instability of 10 at room temperature,
the enantiomeric excess from the enzymatic hydrolysis was determined after reduction of 10 to the
corresponding alcohol-ester 13. Thus, reduction of the acid 10 was carried out using sodium borohydride
via the mixed anhydride, prepared from the acid with ethyl chloroformate, to give the (S)-alcohol-ester
13, [α]D +50.4 (c 0.4, CHCl3), in 65% yield. The enantiomeric excess of 13 was determined to be 64% by
HPLC analysis [Chiralcel OD (Daicel Chemical Industries) (solvent: 5% isopropanol–hexane; retention

T. Honda et al. / Tetrahedron: Asymmetry 10 (1999) 2703–2712
2705
Table 1
Chemoenzymatic hydrolysis of the diester with PLE
time of the major isomer: 45.07 min and minor isomer: 53.17 min; detection: UV 210 nm)]. Similarly,
the enzymatic hydrolysis of the dimethyl ester 8 and dipentyl ester 9 afforded the corresponding acids
(11 and 12) in 99 and 65% yields, respectively, which were also converted into the acid-esters (14 and
15) by adopting the same procedure as described for the preparation of 13. HPLC analysis [Chiralcel OD
(Daicel Chemical Industries)] indicated that the enantiomeric excess of alcohols 14 and 15 were 59 and
25%, respectively. Based on these results, it was found that the bulky ester group (run 3) did not seem to
be suitable for the enzymatic hydrolysis of malonate derivatives in terms of yield and enantioselectivity,
probably due to the presence of steric hindrance, and the mode of enantioselectivity seemed to be the
same in the hydrolysis of these esters as expected. Since we presumed that further modifications of the
ester groups would not improve the enantiomeric excess of the products, we focused our attention on the
conversion of the hydrolyzed compound to an α-substituted serine derivative.
Scheme 2. Reagents and conditions: (i) PLE, 5% acetone–phosphate buffer, 20°C; (ii), ClCO2Et, NEt3, THF, 0°C then NaBH4,
EtOH, 0°C
In order to determine the absolute configuration, the alcohol-ester 14 was treated with (+)-10-
camphorsulfonyl chloride to give the sulfonate ester 16, which, after purification by silica gel column
chromatography, was recrystallized from ethyl acetate to provide the fine crystals (Scheme 3). The X-ray
analysis of the sulfonate 16 unambiguously demonstrated that the newly generated stereogenic center of
the enzymatic hydrolysis product was R as expected (Fig. 2).
Scheme 3. Reagents and conditions: (i) NEt3, DMAP, CH2Cl2

2706
T. Honda et al. / Tetrahedron: Asymmetry 10 (1999) 2703–2712
Figure 2. The ORTEP drawing of the sulfonate 16
Although the absolute configuration of the half-ester was opposite to the natural myriocins, we
attempted to establish the route for the conversion of the carboxyl function into an amino group with
the retention of configuration, and decided to employ the Curtius rearrangement.6 Thus, the reaction
of the acid 10 with diphenylphosphoryl azide (DPPA) afforded the relatively stable isocyanate 17, in
64% yield, which on treatment with benzyl alcohol in refluxing benzene gave the N-benzyloxycarbonyl
derivative 18 in 49% yield (Scheme 4). Finally, ozonolysis of the olefin 18, followed by reduction with
sodium borohydride furnished the desired α-substituted serine derivative 19, in 64% yield, in the correct
enantiomeric series for the myriocins.
Scheme 4. Reagents and conditions: (i) DPPA, NEt3, CH2Cl2, reflux; (ii) PhCH2OH, benzene, reflux; (iii) O3, CH2Cl2, −78°C
then NaBH4, EtOH, 0°C
In summary, we have disclosed an alternative procedure for the preparation of an α-substituted serine
derivative in relatively few steps via asymmetric enzymatic hydrolysis of a malonate derivative, in which
the difference between the sp2 and sp3 carbons was discriminated by the enzyme. The enantiomeric
excess of the hydrolyzed product obtained here was not high enough for the synthesis of natural
compounds; however, the skeletal modification, such as the functionalization of the starting material at
the 2- and 6-positions of the cyclohexene ring, would be able to increase the enantioselectivity. Moreover,
the synthesis of chiral compounds having either R- or S-configuration at the quaternary stereogenic
center might also be possible by employing the functional exchange reaction, although attempting such
transformation under various reaction conditions could not be achieved at the present time.

T. Honda et al. / Tetrahedron: Asymmetry 10 (1999) 2703–2712
2707
3. Experimental
3.1. General procedures
Melting points were measured with a Yanagimoto MP apparatus and are uncorrected. IR spectra were
1
recorded for thin films on a JASCO FT/IR-200 Fourier transform infrared spectrophotometer. H and
13C NMR spectra were obtained for solutions in CDCl3 on a JEOL PMX 270 instrument (270 MHz),
and chemical shifts are reported in ppm on the δ-scale from internal Me4Si. Mass spectra were measured
with a JEOL JMS D-300 spectrometer. Optical rotations were taken with a JASCO DIP-360 polarimeter.
All new compounds described in the Experimental section were homogeneous on TLC. Enantiomeric
excess (ee) determinations were carried out using a 5% isopropanol in n-hexane mobile phase with a
Chiralcel OD column (Daicel Chemical Industries) on a Hitachi HPLC instrument.
3.2. Diethyl 2-hydroxycyclohexane-1,1-dicarboxylate 6
To a stirred solution of the ketone 5 (100 mg, 0. 41 mmol) in methanol (2.5 ml) was added sodium
borohydride (15.6 mg, 0.41 mmol) portionwise at −15°C, and the resulting mixture was stirred for a
further 5 h at the same temperature. After quenching the reaction by addition of a saturated ammonium
chloride solution, the mixture was extracted with ethyl acetate. The organic layer was washed with brine
and dried over Na2SO4. Evaporation of the solvent gave a residue, which was subjected to column
chromatography on silica gel. Elution with hexane:ethyl acetate (6:1, v/v) afforded the alcohol 6 (96.3
mg, 96%) as a colorless oil; νmax (thin film)/cm−1 3525, 2943, 1730; δH: 1.27 and 1.30 (each 3H, each t,
J=7.1, 2×CH3), 1.49–2.00 (7H, m, methylene protons), 2.18–2.36 (1H, m, methylene proton), 3.69 (1H,
d, J=9.1, OH), 4.01 (1H, dt, J=3.7 and 9.1, 2-H), 4.14–4.34 (4H, m, 2×OCH2); δC: 13.4, 21.5, 22.1, 29.3,
30.7, 59.5, 60.8, 71.6 and 170.7; (found: C, 59.00; H, 8.35; calcd for C12H20O5: C, 59.00; H, 8.25%).
[Found: (M+), 244.1335; calcd for C12H20O5: (M+), 244.1311].
3.3. Diethyl 2-cyclohexene-1,1-dicarboxylate 7
To a stirred solution of the alcohol 6 (5.16 g, 21.1 mmol) in pyridine (50 ml) was added portionwise
trifluoromethanesulfonic anhydride (TFMSA) (10.67 ml, 63.3 mmol) at −15°C, and the resulting mixture
was allowed to warm up to room temperature over a period of 20 h. The solution was cooled to 0°C
and treated with 10% potassium hydrogen sulfate solution, and extracted with ethyl acetate. The extract
was washed with a saturated sodium hydrogen carbonate solution and brine, and dried over Na2SO4.
Evaporation of the solvent gave a residue, which was subjected to column chromatography on silica
gel. Elution with hexane:ethyl acetate (20:1, v/v) afforded the olefin 7 (4.09 g, 86%) as a colorless oil;
νmax (thin film)/cm−1 2980, 1732, 1257; δH: 1.18 (6H, t, J=7.1, 2×CH3), 1.58–1.71 (2H, m, methylene
protons), 1.90–2.10 (4H, m, methylene protons), 4.12 (4H, q, J=7.1, 2×OCH2), 5.81 (1H, dt, J=1.8 and
10.1, 2-H), 5.92 (1H, dt, J=3.6 and 10.1, 3-H); δC: 13.7, 18.9, 24.1, 28.5, 54.3, 61.0, 123.9, 130.9 and
170.7; (found: C, 63.50; H, 8.05; calcd for C12H18O4: C, 63.70; H, 8.00%). [Found: (M+), 226.1207;
calcd for C12H18O4: (M+), 226.1205].
3.4. Dimethyl 2-cyclohexene-1,1-dicarboxylate 8
To a stirred solution of the diethyl ester 7 (293 mg, 1.30 mmol) in methanol (10 ml) was slowly added
sodium methoxide (175 mg, 3.24 mmol) at 0°C and the resulting mixture was stirred for 5 min at the

2708
T. Honda et al. / Tetrahedron: Asymmetry 10 (1999) 2703–2712
same temperature and for 15 h at room temperature. After treatment with a saturated ammonium chloride
solution, the organic solvent was removed and the residue was extracted with ethyl acetate. The extract
was washed with a saturated sodium hydrogen carbonate solution and brine, and dried over Na2SO4.
Evaporation of the solvent gave a residue, which was subjected to column chromatography on silica gel.
Elution with hexane:ethyl acetate (20:1, v/v) afforded the dimethyl ester 8 (189 mg, 74%) as a colorless
oil; νmax (thin film)/cm−1 2955, 1738, 1436; δH: 1.58–1.71 (2H, m, methylene protons), 1.92–2.11 (4H,
m, methylene protons), 3.67 (6H, s, 2×Me), 5.81 (1H, dt, J=1.8 and 10.1, 2-H), 5.93 (1H, dt, J=3.6 and
10.1, 3-H); δC: 19.6, 24.2, 28.7, 52.5, 54.4, 123.8, 131.2 and 171.4; (found: C, 60.90; H, 7.40; calcd for
C10H14O4: C, 60.60; H, 7.10%). [Found: (M+), 198.0881; calcd for C10H14O4: (M+), 198.0892].
3.5. Dipentyl 2-cyclohexene-1,1-dicarboxylate 9
A solution of the diethyl ester 7 (230 mg, 1.02 mmol) and p-tolenesulfonic acid (40 mg, 0.21 mmol)
in 1-pentanol (5 ml) was heated at 80°C for 2 days. After the addition of a saturated sodium hydrogen
carbonate solution, the mixture was extracted with ethyl acetate. The extract was washed with a saturated
sodium hydrogen carbonate solution and brine, and dried over Na2SO4. Evaporation of the solvent gave a
residue, which was subjected to column chromatography on silica gel. Elution with hexane:ethyl acetate
(100:1, v/v) afforded the dipentyl ester 9 (265 mg, 84%) as a colorless oil; νmax (thin film)/cm−1 2958,
1750, 1735, 1467; δH: 0.83 (6H, t, J=6.8, 2×CH3), 1.16–1.34 (8H, m, methylene protons), 1.47–1.71
(6H, m, methylene protons), 1.91–2.10 (4H, m, methylene protons), 4.04 (2H, t, J=6.6, OCH2), 4.05
(2H, t, J=6.6, OCH2), 5.81 (1H, d, J=10.1, 2-H), 5.91 (1H, dt, J=3.5 and 10.1, 3-H); δC: 13.7, 19.1, 22.0,
24.2, 27.8, 28.0, 28.7, 54.5, 65.3, 124.0, 131.0 and 170.9; (found: C, 69.45; H, 9.70; calcd for C18H30O4:
C, 69.65; H, 9.75%). [Found: (M+), 310.2141; calcd for C18H30O4: (M+), 310.2144].
3.6. (R)-1-Ethoxycarbonyl-2-cyclohexene-1-carboxylic acid 10
The diethyl ester 7 (600 mg, 2.65 mmol) was incubated with PLE (Amano, 120 mg) in a solution of
5% acetone in phosphate buffer (0.2 M, pH 7.2, 20 ml) at 20°C. The mixture was slowly stirred for 2 days
and was treated with 1 M potassium hydrogen sulfate solution. After addition of ethyl acetate, the mixture
was filtered through a pad of Celite to remove insoluble materials. The filtrate was extracted with ethyl
acetate and the extract was washed with brine and dried over Na2SO4. Evaporation of the solvent gave a
residue, which was subjected to column chromatography on silica gel. Elution with hexane:ethyl acetate
(8:1, v/v) afforded the half-ester 10 (482 mg, 92%) as a colorless oil; [α]D +9.4 (c 0.5, CHCl3); νmax
(thin film)/cm−1 3500, 3184, 2942, 1715; δH: 1.27 (3H, t, J=7.1, CH3), 1.66–1.80 (2H, m, methylene
protons), 1.99–2.19 (4H, m, methylene protons), 4.22 (4H, q, J=7.1, OCH2), 5.89 (1H, dt, J=1.9 and
10.1, 2-H), 6.03 (1H, dt, J=3.6 and 10.1, 3-H), 9.25–10.27 (1H, br s, COOH); δC: 13.9, 19.1, 24.3, 28.7,
54.6, 61.8, 123.4, 131.9, 170.7 and 177.2; (found: C, 60.40; H, 7.20; calcd for C10H14O4: C, 60.60; H,
7.10%). [Found: (M+), 198.0889; calcd for C10H14O4: (M+), 198.0892].
3.7. (R)-1-Methoxycarbonyl-2-cyclohexene-1-carboxylic acid 11
Enzymatic hydrolysis of the dimethyl ester 8 (227 mg, 1.15 mmol) was carried out under essentially
the same reaction conditions as for the preparation of the mono-ethyl ester to give the mono-methyl ester
11 (209 mg, 99%) as a colorless oil; [α]D +5.7 (c 0.8, CHCl3); νmax (thin film)/cm−1 3510, 3180, 2952,
2617, 1725, 1438; δH: 1.59–1.92 (2H, m, methylene protons), 1.92–2.14 (4H, m, methylene protons),
3.69 (3H, s, Me), 5.82 (1H, dt, J=2.0 and 10.1, 2-H), 5.96 (1H, dt, J=3.7 and 10.1, 3-H), 8.44–9.42 (1H,

T. Honda et al. / Tetrahedron: Asymmetry 10 (1999) 2703–2712
2709
br s, COOH); δC: 19.1, 24.3, 28.8, 52.9, 54.5, 123.4, 131.9, 171.2 and 176.9; (found: C, 58.65; H, 6.85;
calcd for C9H12O4: C, 58.70; H, 6.55%). [Found: (M+), 184.0750; calcd for C9H12O4: (M+), 184.0735].
3.8. (R)-1-Pentyloxycarbonyl-2-cyclohexene-1-carboxylic acid 12
Enzymatic hydrolysis of the dipentyl ester 9 (143 mg, 0.46 mmol) was carried out under essentially
the same reaction conditions as for the preparation of the mono-ethyl ester to give the mono-pentyl ester
12 (72 mg, 65%) as a colorless oil; [α]D +3.6 (c 0.7, CHCl3); νmax (thin film)/cm−1 3520 (sh), 3300 (sh),
2958, 2650, 1740, 1710; δH: 0.89 (3H, t, J=6.7, CH3), 1.23–1.40 (4H, m, methylene protons), 1.54–1.82
(4H, m, methylene protons), 1.96–2.24 (4H, m, methylene protons), 4.15 (2H, t, J=6.6, OCH2), 5.89
(1H, d, J=10.1, 2-H), 6.02 (1H, dt, J=3.6 and 10.1, 3-H), 7.49–8.90 (1H, br s, COOH); (found: C, 64.80;
H, 8.55; calcd for C13H20O4: C, 65.00; H, 8.40%). [Found: (M+), 240.1353; calcd for C13H20O4: (M+),
240.1361].
3.9. (R)-Ethyl 1-hydroxymethyl-2-cyclohexene-1-carboxylate 13
Ethyl chloroformate (0.05 ml, 0.52 mmol) was added dropwise to a solution of the half-ester 10 (80
mg, 0.40 mmol) and triethylamine (0.08 ml, 0.57 mmol) in dry THF (3 ml) at 0°C under argon, and
the resulting mixture was stirred for a further 30 min at the same temperature. The solution was filtered
to remove the precipitated triethylamine hydrochloride and the filtrate was diluted with ethanol (1 ml).
To this solution was added dropwise sodium borohydride (15 mg, 0.40 mmol) and the mixture was
stirred at 0°C for a further 12 h. The reaction was quenched by the addition of a saturated ammonium
chloride solution and extracted with dichloromethane. The extract was washed with brine and dried over
Na2SO4. Evaporation of the solvent gave a residue, which was subjected to column chromatography
on silica gel. Elution with hexane:ethyl acetate (4:1, v/v) afforded the alcohol 13 (60 mg, 65%) as a
colorless oil; [α]D +50.4 (c 0.4, CHCl3); νmax (thin film)/cm−1 3458, 2940, 1721; δH: 1.27 (3H, t, J=7.1,
CH3), 1.60–1.75 (2H, m, methylene protons), 1.60–1.75 (3H, m, methylene protons), 1.92–2.16 (3H, m,
methylene protons), 2.34 (1H, t, J=6.3, OH), 3.61 (1H, dd, J=6.1 and 10.9, CHHOH), 3.70 (1H, dd,
J=6.1 and 10.9, CHHOH), 4.18 (2H, q, J=7.1, OCH2), 5.64 (1H, dt, J=2.0 and 10.1, 2-H), 5.93 (1H,
dt, J=3.8 and 10.1, 3-H); δC: 14.2, 18.8, 24.8, 27.6, 49.1, 60.9, 68.2, 125.6, 130.9 and 175.8; (found: C,
64.90; H, 8.80; calcd for C10H16O3: C, 65.20; H, 8.75%). [Found: (M+), 184.1075; calcd for C10H16O3:
(M+), 184.1099].
3.10. (R)-Methyl 1-hydroxymethyl-2-cyclohexene-1-carboxylate 14
The half-ester 11 (209 mg, 1.14 mmol) was reduced with sodium borohydride (43 mg, 1.14 mmol)
via the corresponding mixed anhydride by the same procedure as for the preparation of the alcohol
13 to give the primary alcohol 14 (120 mg, 62%) as a colorless oil: [α]D +50.5 (c 1.1, CHCl3); νmax
(thin film)/cm−1 3450, 2948, 1725, 1434; δH: 1.56–1.75 (3H, m, methylene protons), 1.92–2.20 (3H,
m, methylene protons), 2.63 (1H, br s, OH), 3.62 (1H, dd, J=4.8 and 10.7, CHHOH), 3.69 (1H, dd,
J=4.8 and 10.7, CHHOH), 3.72 (3H, s, Me), 5.65 (1H, dt, J=1.8 and 10.1, 2-H), 5.93 (1H, dt, J=3.8 and
10.1, 3-H); δC: 18.8, 24.8, 27.6, 49.2, 52.1, 125.6, 130.9 and 176.2; (found: C, 63.20; H, 8.55; calcd for
C9H14O3: C, 63.50; H, 8.30%). [Found: (M++1), 171.1030; calcd for C9H14O3: (M++1), 171.1021].

2710
T. Honda et al. / Tetrahedron: Asymmetry 10 (1999) 2703–2712
3.11. (R)-Pentyl 1-hydroxymethyl-2-cyclohexene-1-carboxylate 15
The half-ester 12 (770 mg, 3.21 mmol) was reduced with sodium borohydride (122 mg, 3.22 mmol) via
the corresponding mixed anhydride by the same procedure as for the preparation of the alcohol 13 to give
the primary alcohol 15 (453 mg, 63%) as a colorless oil: [α]D +15.3 (c 0.4, CHCl3); νmax (thin film)/cm−1
3460, 2958, 1720; δH: 0.91 (3H, t, J=6.8, Me), 1.20–1.44 (4H, m, methylene protons), 1.53–1.75 (5H,
m, methylene protons), 1.90–2.15 (3H, m, methylene protons), 2.68 (1H, br s, OH), 3.61 (1H, d, J=10.8,
CHHOH), 3.69 (1H, d, J=10.7, CHHOH), 4.11 (2H, t, J=6.7, OCH2), 5.64 (1H, d, J=10.1, 2-H), 5.92
(1H, dt, J=3.8 and 10.1, 3-H); δC: 13.8, 18.8, 22.1, 24.8, 27.5, 27.9, 28.1, 49.2, 64.9, 68.2, 125.7, 130.6
and 175.7. [Found: (M++1), 227.1660; calcd for C13H23O3: (M++1), 227.1647].
3.12. (R)-Methyl 1-(+)-10-camphorsulfonyloxymethyl-2-cyclohexene-1-carboxylate 16
A solution of the alcohol 14 (50 mg, 0.29 mmol), triethylamine (0.15 ml, 1.08 mmol), a catalytic
amount of 4,4-dimethylaminopyridine and (+)-10-camphorsulfonyl chloride in dichloromethane (3 ml)
was heated at reflux for 5 days. The mixture was diluted with dichloromethane and washed with brine,
and dried over Na2SO4. Evaporation of the solvent gave a residue, which was subjected to column
chromatography on silica gel. Elution with hexane:ethyl acetate (5:1, v/v) afforded the sulfonate 16
(80 mg, 71%) as colorless prisms; mp 82.5–83.0°C (ethyl acetate); δH: 0.89 (3H, s, Me), 1.12 (3H,
s, Me), 1.37–1.52 (1H, m, methylene protons), 1.57–1.76 (4H, m, methylene protons), 1.90–2.23 (6H, m,
methylene protons), 2.33–2.54 (2H, m, methylene protons), 3.00 (1H, d, J=15.1, SO2CHH), 3.60 (1H,
d, J=15.1, SO2CHH), 3.74 (3H, s, OMe), 4.29 (1H, d, J=9.4, CHHO), 4.37 (1H, d, J=9.4, CHHO), 5.62
(1H, dt, J=2.1 and 10.1, 2-H), 6.00 (1H, dt, J=3.8 and 10.1, 3-H).
3.13. X-Ray analysis of the sulfonate 16
Compound 16 was recrystallized from ethyl acetate as monoclinic crystals, mp 82.5–83.0°C;
C19H28O6S, space group P21, with a=9.005(3), b=20.244(4), c=11.371(3) Å, β=110.28(2)°, Z=2,
Dc=1.36 g cm−3. Intensity measurements were made with CuKα radiation (λ=1.5418 Å; graphite
monochromate) on a Rigaku AFC7R diffractometer in the ω–2θ mode with 4.36°<2θ<130.20°. A
total 3425 unique reflections were collected. Of those, 2486 with I>3σ(I) were judged as observed.
Accurate cell parameters were obtained by least-squares techniques from the diffractometer setting for
24 reflections. The structure was solved using MITHRIL84, and refined by full matrix least-squares with
TEXSAN. Convergence, with anisotropic thermal parameters for all non-hydrogen atoms, was reached
at R 0.069 (Rw 0.066) using all the observed reflections.
3.14. (S)-1-Ethoxycarbonyl-2-cyclohexene-1-isocyanate 17
To a stirred solution of the half-ester 13 (1.46 g, 7.37 mmol) in dry dichloromethane (15 ml) were added
successively diphenylphosphoryl azide (2.38 ml, 11.1 mmol) and triethylamine (1.54 ml, 11.1 mmol) at
ambient temperature under argon and the resulting mixture was stirred for a further 11 h at the same
temperature. The mixture was cooled to 0°C and treated with a saturated ammonium chloride solution,
and then extracted with dichloromethane. The extract was washed with brine and dried over Na2SO4.
Evaporation of the solvent gave a residue, which was subjected to column chromatography on silica gel.
Elution with hexane:ethyl acetate (20:1, v/v) afforded the isocyanate 17 (925 mg, 64%) as a colorless
oil; νmax (thin film)/cm−1 2943, 2250, 2143, 1747, 1718; δH: 1.27 (3H, t, J=7.2, CH3), 1.61–1.83 (2H,

T. Honda et al. / Tetrahedron: Asymmetry 10 (1999) 2703–2712
2711
m, methylene protons), 1.99–2.17 (4H, m, methylene protons), 4.21 (4H, q, J=7.2, OCH2), 5.83 (1H, dt,
J=2.1 and 10.1, 2-H), 6.04 (1H, dt, J=3.8 and 10.1, 3-H); δC: 14.0, 19.1, 24.3, 28.7, 56.1, 61.9, 123.0,
132.4, 170.2 and 178.3. [Found: (M+), 195.0896; calcd for C10H13NO3: (M+), 195.0896].
3.15. (S)-Ethyl 1-benzyloxycarbonylamino-2-cyclohexene-1-carboxylate 18
A stirred solution of the isocyanate 17 (120 mg, 0.62 mmol) and benzyl alcohol (0.08 ml, 0.77 mmol)
in dry benzene (5 ml) was heated at reflux for 1 h. After evaporation of the solvent, the residue was
purified by column chromatography on silica gel using hexane:ethyl acetate (10:1, v/v) as eluent to give
the carbamate 18 (92 mg, 49%) as a colorless oil; [α]D +10.7 (c 1.1, CHCl3); νmax (thin film)/cm−1 3350,
2938, 1738, 1722, 1516, 1265; δH: 1.21 (3H, t, J=6.7, CH3), 1.55–1.86 (2H, m, methylene protons),
1.95–2.26 (4H, m, methylene protons), 4.17 (1H, distorted br s, OCH2Me), 5.09 (2H, s, OCH2Ph), 5.76
(1H, d J=9.9, 2-H), 6.00 (1H, dt, J=3.8 and 9.9, 3-H), 7.28–7.39 (5H, m, aromatic protons); δC: 14.0,
18.2, 24.6, 30.7, 58.0, 61.5, 66.7, 125.7, 128.1, 128.4, 133.0, 154.8 and 173.0; (found: C, 67.25; H,
7.15; N, 4.50; calcd for C17H21NO4: C, 67.30; H, 7.00; N, 4.60%). [Found: (M+), 303.1465; calcd for
C17H21NO4: (M+), 303.1470].
3.16. (S)-Ethyl 2-benzyloxycarbonylamino-6-hydroxy-2-hydroxymethylhexanoate 19
A solution of the cyclohexene derivative 18 (316 mg, 1.04 mmol) in dichloromethane (5 ml) was
saturated with ozone at −78°C. The solution was stirred for 30 min at the same temperature and the
ozone was removed by exchange with argon. Ethanol (1 ml) was added to the solution and the mixture
was treated with sodium borohydride (40 mg, 1.06 mmol) at −78°C and then warmed up to 0°C. After the
stirring had been continued for 12 h at the same temperature, the reaction was quenched by addition of
saturated ammonium chloride solution, and the mixture was extracted with dichloromethane. The extract
was washed with brine and dried over Na2SO4. Evaporation of the solvent gave a residue, which was
subjected to column chromatography on silica gel. Elution with hexane:ethyl acetate (1:1, v/v) afforded
the diol 19 (224 mg, 64%) as a colorless oil; [α]D −1.4 (c 0.8, CHCl3); νmax (thin film)/cm−1 3418,
2940, 2870, 1715, 1506; δH: 1.28 (3H, t, J=7.1, CH3), 1.12–1.43 (3H, m, methylene protons), 1.45–1.60
(1H, m, methylene proton), 1.65 (1H, br s, OH), 1.66–1.84 (1H, m, methylene proton), 2.06–2.25 (1H,
m, methylene proton), 2.85 (1H, br s, OH), 3.59 (2H, t, J=6.3, 6-H2), 3.83 (1H, d, J=11.4, CHHOH),
4.17 (1H, d, J=11.4, CHHOH), 4.25 (4H, q, J=7.1, OCH2Me), 5.09 (2H, s, OCH2Ph), 5.89 (1H, s, NH),
7.27–7.42 (5H, m, aromatic protons); δC: 14.0, 19.6, 31.5, 32.0, 62.0, 65.2, 65.5, 66.7, 128.0, 128.1,
128.5, 136.1, 155.1 and 172.3; (found: C, 60.15; H, 7.55; N, 4.00; calcd for C17H25NO6: C, 60.15; H,
7.45; N, 4.15%). [Found: (M+), 339.1694; calcd for C17H25NO6: (M+), 339.1682].
Acknowledgements
We are grateful to the Amano Pharmaceutical Co. Ltd of Japan for the donation of enzymes. This work
was supported by the Ministry of Education, Science, Sports, and Culture of Japan.
References
1. (a) Williams, R. M. Synthesis of Optically Active α-Amino Acids; Pergamon: Oxford, 1989; Vol. 7. (b) O’Donnell, M. J.
α-Amino acid synthesis. Tetrahedron Symposia-in-print, 1988, 44, 5253. (c) Kawabata, T.; Wirth, T.; Yahiro, K.; Fuji, K.

2712
T. Honda et al. / Tetrahedron: Asymmetry 10 (1999) 2703–2712
J. Am. Chem. Soc. 1994, 116, 10809. (d) Horikawa, M.; Nakajima, T.; Ohfune, Y. Synlett 1997, 253. (e) Liu, W.; Ray, P.;
Benezra, S. A. J. Chem. Soc., Perkin Trans. 1 1995, 553. (f) Cativiela, C.; Diaz-de-Villegas, M. D. Tetrahedron: Asymmetry
1998, 9, 3517. (g) Hatakeyama, S.; Matsumoto, H.; Fukuyama, H.; Mukugi, Y.; Irie, H. J. Org. Chem. 1997, 62, 2275. (h)
Carda, M.; Murga, J.; Rodriguez, S.; Gonzalez, F.; Castillo, E.; Marco, J. A. Tetrahedron: Asymmetry 1998, 9, 1703. (i)
Carloni, A.; Porzi, G.; Sandri, S. ibid. 1998, 9, 2987. (j) Frutos, R. P.; Spero, D. M. Tetrahedron Lett. 1998, 39, 2475. (k)
Marco, J. A.; Carda, M.; Murga, J.; Portoles, R.; Falomir, E.; Lex, J. ibid. 1998, 39, 3237. (l) Matsushita, M.; Maeda, H.;
Kodama, M. ibid. 1998, 39, 3749. (m) Sano, S.; Hayashi, K.; Miwa, T.; Ishii, T.; Fujii, M.; Mima, H.; Nagao, Y. ibid. 1998,
39, 5571. (n) Alias, M.; Cativiela, C.; Diaz-de-Villegas, M. D.; Calvez, J.; Lapena, Y. Tetrahedron 1998, 54, 14963. (o)
Sano, S.; Takebayashi, M.; Miwa, T.; Ishii, T.; Nagao, Y. Tetrahedron: Asymmetry 1998, 9, 3611. (p) Juaristi, E.; Balderas,
M.; Ramirez-Quiros, Y. ibid. 1998, 9, 3881. (q) Wenglowsky, S.; Hegedus, L. S. J. Am. Chem. Soc. 1998, 120, 12468, and
recent references for the synthesis of α,α0-disubstituted α-amino acids cited therein.
2. For recent syntheses: (i) Conagenin: Hatakeyama, S.; Matsumoto, H.; Fukuyama, H.; Mukugi, Y.; Irie, H. Tetrahedron
Lett. 1996, 37, 4047, and references cited therein. (ii) Lactacystin: (a) Corey, E. J.; Reichard, G. A. J. Am. Chem. Soc.
1992, 114, 10677. (b) Sunazuka, T.; Nagamitsu, T.; Matsuzaki, K.; Tanaka, H.; Omura, S. ibid. 1993, 115, 5302. (c) Uno,
H.; Baldwin, J. E.; Russell, A. T. ibid. 1994, 116, 2139. (d) Chida, N.; Takeoka, J.; Tsutsumi, N.; Ogawa, S. J. Chem.
Soc., Chem. Commun. 1995, 793. (e) Ko, S. H.; Jun, H.-S. Chem. Commun. 1998, 1929. (f) Ko, S. H.; Jun, H.-S.; Youn,
J.-H. Synlett 1998, 1045. (iii) Myriocin: (a) Yoshikawa, M.; Yokokawa, Y.; Okuno, Y.; Murakami, N. Tetrahedron 1995,
51, 6209. (b) Sano, S.; Kobayashi, Y.; Kondo, T.; Takebayashi, M.; Murayama, S.; Fujita, T.; Nagao, Y. Tetrahedron Lett.
1995, 36, 2097. (iv) Mycestericins: (a) Fujita, T.; Hamamichi, N.; Matsuzaki, T.; Kitao, Y.; Kiuchi, M.; Node, M.; Hirose,
R. ibid. 1995, 36, 8599. (b) Shibata, K.; Shingu, K.; Vassil, V. P.; Nishide, K.; Fujita, T.; Node, M.; Kajimoto, T.; Wong,
C.-H. ibid. 1996, 37, 2791. (c) Fujita, T.; Hamamichi, N.; Kiuchi, M.; Matsuzaki, T.; Kitao, Y.; Inoue, K.; Hirose, R.;
Yoneta, M.; Sasaki, S.; Chiba, K. J. Antibiot. 1996, 49, 846.
3. Hallinan, K. O.; Honda, T. Tetrahedron 1995, 51, 12211.
4. Honda, T.; Ogino, T. Tetrahedron: Asymmetry 1998, 9, 2663.
5. (a) De Jeso, B.; Belair, N.; Deleuze, H.; Rascle, M.-C.; Maillard, B. Tetrahedron Lett. 1990, 31, 653. (b) Toone. E. J.;
Jones, J. B. Tetrahedron: Asymmetry 1991, 2, 1041.
6. Hodgson, D. M.; Thompson, A. J.; Wadman, S. Tetrahedron Lett. 1998, 39, 3357.