Chemoenzymatic synthesis of the C-13 side chain of paclitaxel (Taxol) and docetaxel (Taxotere)

Article abstract of DOI:10.1016/S0957-4166(00)00418-3

Reduction of methyl 3-chloro-2-oxo-3-phenylpropanoate with various reducing agents gave syn- and anti-3-chloro-2-hydroxy-3-phenylpropanoates 3, which underwent an efficient lipase-catalyzed resolution. All four diastereomers were subsequently converted to N-benzoyl-(2R,3S)-3-phenylisoserine methyl ester, C-13 side chain analogues of paclitaxel (Taxol).

Full text of DOI:10.1016/S0957-4166(00)00418-3

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 11 (2000) 4485–4497
Chemoenzymatic synthesis of the C-13 side chain of
paclitaxel (Taxol) and docetaxel (Taxotere)
Hiromi Hamamoto,a Vakhid A. Mamedov,b,† Makiko Kitamoto,b Nobuyuki Hayashib and
Sadao Tsuboib,*
aDepartment of Applied Chemistry, Faculty of Engineering, Okayama University, Tsushima,
Okayama 700-8530, Japan
bDepartment of Environmental Chemistry and Materials, Faculty of Environmental Science and Technology,
Okayama University, Tsushima, Okayama 700-8530, Japan
Received 31 August 2000; accepted 16 October 2000
Abstract
Reduction of methyl 3-chloro-2-oxo-3-phenylpropanoate with various reducing agents gave syn- and
anti-3-chloro-2-hydroxy-3-phenylpropanoates 3, which underwent an efficient lipase-catalyzed resolution.
All four diastereomers were subsequently converted to N-benzoyl-(2R,3S)-3-phenylisoserine methyl ester,
C-13 side chain analogues of paclitaxel (Taxol). © 2000 Elsevier Science Ltd. All rights reserved.
1. Introduction
Paclitaxel 1a (Taxol), an antimicrotubule agent isolated from the bark of Taxus brevifolia,1
has recently attracted much attention because of its efficacy in the treatment of various types of
cancer.2 One major impediment to the wide use of Taxol in cancer chemotherapy is its extremely
limited availability. Also, chemical complexity has prohibited the production of paclitaxel 1a by
total synthesis,3 and the chemical complexity of paclitaxel dictates that its commercial produc-
tion by total synthesis is not likely to be economical. The naturally derived 10-deacetylbaccatin
III is obtained in fairly high yield (1 g/kg) from the leaves of the European yew (Taxus
baccata).4 It is important to recognize that the leaves are quickly regenerated and hence, a large
amount of 10-deacetylbaccatin III can be supplied continuously without threatening the survival
of the yew species. Thus, a viable approach for the preparation of paclitaxel 1a is to utilize more
accessible baccatin III or 10-deacetylbaccatin III as precursors via semi-synthetic routes.5 The
structures of compounds 1a, 1b, and III are described in Fig. 1.
* Corresponding author. Tel/fax: 81-86-251-8898; e-mail: stsuboi6@cc.okayama-u.ac.jp
†
Visiting researcher of JSPS, on leave from the present address: A. E. Arbuzov Institute of Organic and Physical
Chemistry, Russian Academy of Sciences, Arbuzov str. 8, Kazan 420088, Russia.
0957-4166/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(00)00418-3

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Figure 1.
As the role of the (2R,3S)-3-phenylisoserine moiety in the biological activity of paclitaxel and
docetaxel became evident,6 the development of short and practical synthetic routes for phenyl
isoserine derivatives has become very important. Numerous papers concerning the preparation
of the enantiomerically pure or enriched C-13 side chain of paclitaxel have been published.7
Among these methods, a route employing enzyme is one important way. Although some papers
describe lipase-catalyzed synthesis of the C-13 side chain of paclitaxel,7d,g it was difficult to
obtain the compound with high enantiomeric excess. Here, we describe the preparation of
3-chloro-2-hydroxy-3-phenylpropanoate with high enantiomeric excess via lipase-catalyzed
kinetic resolution of the reduction products of 3-chloro-3-phenylpyruvate obtained by Darzens-
type reaction and its application to the synthesis of the C-13 side chain of paclitaxel.
The Darzens condensation has been widely used to prepare glycidic esters. The Darzens
condensation of dichloroacetate with aromatic aldehydes gives 2-chloro-2,3-epoxypropanoate or
its rearranged products, 3-chloro-2-oxopropanoate, which will become important key intermedi-
ates because it has reactive functional groups such as chlorine, carbonyl, and ester groups. In
this paper, we describe a convenient preparation of anti-3-chloro-2-hydroxyester from Darzens
reaction products and its lipase-catalyzed resolution, as summarized in Fig. 2. The C-13 side
chain of paclitaxel was prepared from chiral anti-3-chloro-2-hydroxyester in a few steps.
Figure 2.

H. Hamamoto et al. / Tetrahedron: Asymmetry 11 (2000) 4485–4497
4487
This efficient synthesis can also be readily modified to give the side chain of docetaxel 1b, a
synthetic Taxol derivative, which was found to have significantly greater cancer chemothera-
peutic potential than paclitaxel itself.8
2. Results and discussion
Base-catalyzed condensation of aldehydes with dichloroacetate has been previously reported.9
This Darzens condensation reaction of dichloroacetates with aromatic aldehydes sometimes
gives 3-chloro-2-oxoester as a rearrangement product of a-chloroglycidates.9 In this report
benzaldehyde was used as an aromatic aldehyde, and methyl 3-chloro-2-oxopropanoate 2 was
obtained in high yield (Scheme 1).
Scheme 1. Reagents and conditions: NaOMe, THF, −50°C, 3 h, then rt overnight, 91%
Various reducing reagents were examined to obtain anti-3-chloro-2-hydroxyester 3. Reduction
with sodium borohydride gave a syn-selective product. On the other hand, L-Selectride®,
K-Selectride®, KS-Selectride®, and lithium aluminum hydride in the presence of a small amount
of silica gel10 gave an anti-selective product predominantly. According to the dipole-type and
Cram-type of the Felkin–Ahn model, the reduction with sodium borohydride is effective in the
syn-selectivity of this reduction under influence of dipole, and the reduction with bulky reducing
reagents is effective in the anti-selectivity under the influence of steric effect between the phenyl
group and bulkiness of the reagent (Fig. 3). In this reduction, we found the best anti-selectivity
can be obtained by KS-Selectride® in THF at −78°C (Table 1). These syn- and anti-isomers were
separated by silica gel column chromatography.
Figure 3. Felkin–Ahn model

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Table 1
Reduction of methyl 3-chloro-2-oxo-3-phenylpropanoate
Entry
Conditions
Product 3
Reagent
Solvent
Temp. (°C)
Time (min)
Yielda (%)
syn/antib
1
2
3
4
5
6
NaBH4
NaBH4
LiAlH4–SiO2
L-Selectride®
K-Selectride®
KS-Selectride®
MeOH/THF
iso-PrOH
Et2O
THF
THF
0
0
Rt
−78
−78
−78
10
10
120
120
120
120
88
70
48
75
86
81
76/24
75/25
31/69
34/66
17/83
13/87
THF
a Isolated yield by SiO2 column chromatography.
b Ratios of syn and anti were determined by analysis of GC–MS. Some of the products were analyzed by GC and
the same results were obtained.
The kinetic resolution of racemic methyl syn- and anti-3-chloro-2-hydroxyester 3 was
achieved by lipase-mediated enantioselective transesterification in organic media. The yield and
enantiomeric excesses are listed in Table 2.
Table 2
The lipase catalyzed transesterifications of syn- and anti-3
Entry
Conditions
Yield and eea
(2R)-4 or -5
Eb
3
R
Lipase
Solvent Temp. (°C) Time (h) (2S)-3
1
2
3
4
5
6
7
(9)-syn Ac Amano PS iPr2O
(9)-syn Bz Amano PS iPr2O
35
35
35
60
35
35
35
25
240
240
28
27
46
50%, 95% ee 45%, 93% ee
32%, 18% ee 6%, 80% ee
41%, 61% ee 12%, \99% ee \100
49%, 78% ee 31%, 80% ee
46%, \99% ee 45%, 93% ee
42%, 86% ee 31%, 97% ee
25%, 33% ee 8%, 96% ee
\100
11
(9)-syn Bz Meito SL
(9)-syn Bz Meito SL
iPr2O
iPr2O
22
\100
\100
63
(9)-anti Ac Amano PS iPr2O
(9)-anti Ac Meito SL
(9)-anti Ac TOYOBO
iPr2O
iPr2O
25
a Ee was determined by analysis of chiral GC.
b For E value, see Ref. 13.

H. Hamamoto et al. / Tetrahedron: Asymmetry 11 (2000) 4485–4497
4489
Lipase Amano PS in the presence of vinyl acetate was suited for stereospecific transesterifica-
tion of both ( )-syn- and ( )-anti-alcohol 3 with high enantiomeric excesses and chemical yields.
Vinyl benzoate was also used to obtain benzoate product (2R,3R)-5 from ( )-syn-3 for the
synthesis of C-13 side chains from (2S,3S)-7. Unfortunately we could not obtain (2S,3S)-methyl
3-azide-2-benzoyl-3-phenylpropanoate by reaction of (2R,3R)-5 with sodium azide in DMF. The
absolute configuration of the a-carbon of anti-4 was determined to be (S) from that of known
compounds 6 or 7, which were prepared as shown in Scheme 2. On the other hand, the absolute
configuration of the a-carbon of anti-3 was also determined to be (S) after conversion to the
epoxide. Lipase-catalyzed kinetic resolution of 3-hydroxy esters was reported by several groups.
These reports show that the reaction of the (S)-isomer proceeds much faster than that of the
(R)-isomer in the acylation of the hydroxy group as well as in the hydrolysis of its acetate. The
present kinetic resolution also gives the same result as that of the literature.11
Scheme 2. Reagents and conditions: (a) NaN3, DMF, 60°C, 42 h, 82%; (b) BzCl, DMAP, CH2Cl2, rt, 5 h, 100%; (c)
H2, Pd/C, MeOH, rt, 48 h, 71%; (d) (Boc)2O, H2, Pd/C, EtOAc, rt, 48 h, 70%; (e) NaOMe, MeOH, 0°C, 8 h, 27%;
(f) NaN3, NH4Cl, acetone/water, reflux, 24 h, 75%
An alternative enantioselective synthesis of paclitaxel 1a and docetaxel 1b side chain from
enzymatically resolved 3-chloro-2-hydroxyester 3 and 3-acetoxy-2-chloroester 4 was achieved as
shown in Schemes 2 and 4.7k
Azide displacement of (2S,3R)-3 with sodium azide in DMF at 60°C led to the C-3 inverted
product (2R,3S)-6 in 82% yield. The hydroxy azide (2R,3S)-6 was also obtained from (2S,3S)-3
via two steps, but epoxidation of (2S,3S)-3 gave (2R,3R)-glycidate 7 in only 27% yield. The
hydroxy azide (2R,3S)-6 was then subjected to benzoylation, followed by hydrogenation with
10% Pd/C under 1 atm of hydrogen at room temperature in methanol,7a furnishing the paclitaxel
side chain (methyl ester, (2R,3S)-8) in 71% yield (two steps) after recrystallization from
methanol. Catalytic hydrogenation of the hydroxy azide 6 in ethyl acetate in the presence of 1.2
equivalent of di-tert-butyl dicarbonate furnished directly the docetaxel side chain (methyl ester,
(2R,3S)-9) in 70% yield.
The resolution of ( )-syn-hydroxy azide 6 by lipase-mediated transesterification was also
carried out by lipase Amano PS in the presence of vinyl acetate at 35°C, which gave the best
selectivity for the resolution of syn- and anti-3-chloro-2-hydroxyester 3 (Scheme 3). The

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H. Hamamoto et al. / Tetrahedron: Asymmetry 11 (2000) 4485–4497
enantiomeric excesses of the acetylated enantiomers showed 76% ee and that of unreacted
enantiomers was 80% ee. However, this enantioselectivity is not as high as the resolution of syn-
and anti-3-chloro-2-hydroxyester 3. From this result, we can conclude that the resolution of
( )-syn-hydroxy azide 6 by lipase is not suitable for this side-chain synthesis. The absolute
configuration of the a-carbon of acetate syn-10 was determined to be (S), based on the sign of
the optical rotation of unreacted (2R,3S)-6.
Scheme 3. The lipase catalyzed transesterification of 6
On the other hand, enzymatically resolved 3-chloro-2-acetoxyester 4 was also applied to the
synthesis of the paclitaxel side chain. (2R,3R)-Methyl 3-chloro-2-hydroxyester 3 was obtained in
90% yield from (2R,3R)-3-chloro-2-acetoxyester 4 by treatment of HCl. As shown in Scheme 4,
Scheme 4. Reagents and conditions: (g) K2CO3, MeOH, −20 to 0°C, 2 h, 93%; (h) NaN3, NH4Cl, acetone/H2O, reflux,
23 h, 96%; (i) HCl, acetone/MeOH, rt, 24 h, 90%; (j) NaN3, DMF, 60°C, 48 h, 83%; (k) BzCl, DMAP, CH2Cl2, rt,
4 h; (l) H2, Pd/C, MeOH, rt, 48 h, 70% (over two steps); (m) PPh3, DEAD, benzene, rt, 20 h, 90%; (n) 1N HCl,
MeOH, reflux, 2 h, 90%; (o) PPh3, DEAD, PhCO2H, benzene, rt, 12 h, 66%; (p) (Boc)2O, H2, Pd/C, MeOH, rt, 72
h, 97%

H. Hamamoto et al. / Tetrahedron: Asymmetry 11 (2000) 4485–4497
4491
(2R,3R)- and (2R,3S)-3-chloro-2-hydroxyester 3 were applied to the synthesis of (2S,3S)-N-ben-
zoylphenylisoserine 8 and (2S,3S)-N-tert-butoxycarbonylphenylisoserine 9, the side chain of
epi-paclitaxel and epi-docetaxel, respectively, by the same method as shown in Scheme 2.
Treatment of (2S,3S)-6, which was prepared from (2R,3R)-4, under the Mitsunobu reaction
conditions12 gave (4S,5R)-11 in 66% yield, which can also be obtained from (2S,3S)-8 under the
same conditions in 90% yield. Treatment of (4S,5R)-11 with 1N HCl under reflux afforded the
paclitaxel side chain (2R,3S)-8 in 90% yield. The spectral (1H and 13C NMR) data of synthetic
(2R,3S)-8 were fully identical with those of the authentic paclitaxel side chain. Physical
properties of synthetic 8 (mp 179–180°C) (lit.7a 183–184°C), and specific rotation {[h]D20 −50.5 (c
1.00, MeOH)}{lit.7a [h]2D0 −49.6 (c 1.00, MeOH)} were also in good agreement with those
reported previously.
Hydrogenation of (2S,3S)-6 in methanol in the presence of 10% Pd/C and di-tert-butoxycar-
bonate gave (2S,3S)-9 in 97% yield.
In conclusion, this work has demonstrated the preparation of optically active 3-chloro-2-
hydroxy-3-phenylpropanoate via lipase-catalyzed kinetic resolution, whose starting material can
be easily obtained by Darzens condensation with dichloroacetate in large scale. Two of the four
diastereomers were obtained in high enantiomeric excess. Each diastereomer was applied to the
synthesis of C-13 side chains of paclitaxel and docetaxel.
3. Experimental
3.1. General
Melting points were obtained in open capillary tubes on a Mel-Temp-II hot-stage microscope
1
and are uncorrected. H NMR spectra were recorded on a Varian Gemini 200 spectrometer or
Varian 500 spectrometer in CDCl3 solution. 13C NMR spectra (50 MHz) were recorded on a
Varian Gemini 200 spectrometer in CDCl3 solution. IR spectra were recorded on a JASCO
FT/IR-5000. Optical rotations were recorded on a SEPA-300 HORIBA polarimeter (Lamp
u=589 nm) in CHCl3 or MeOH solutions. Enantiomeric excess was determined by Chiral-GC,
GC-380 GL Science fitted with Chiral-DEXCB (0.25×25 mm). The ratio of syn and anti was
determined by GC–MS, GC-17A SHIMADZU (sr. Phase TC-5, dt=0.25 mm, 0.25×30 m),
GC–MS-QP5000 SHIMADZU.
Reagents and solvents were purified by standard means. Tetrahydrofuran was distilled from
sodium wire/benzophenone and stored under a nitrogen atmosphere. Dichloromethane, benzene,
N,N-dimethylformamide, and methanol were distilled from calcium hydride. All other chemicals
were used as received.
3.2. Preparation of methyl 3-chloro-2-oxo-3-phenylpropanoate 2
Sodium (6.94 g, 302 mmol) was treated with anhydrous MeOH (100 ml). After evaporation
of MeOH, the residue was cooled to −50°C in THF (400 ml), then methyl dichloroacetate (43.20
g, 302 mmol) and benzaldehyde (32.0 g, 302 mmol) were added under an atmosphere of
nitrogen. Stirring was continued for 3 h and then allowed to stand overnight at room
temperature. After evaporation of THF, the reaction mixture was extracted three times with
EtOAc. The organic layer was dried over MgSO4 and the solvent was evaporated. The crude

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H. Hamamoto et al. / Tetrahedron: Asymmetry 11 (2000) 4485–4497
product was purified by vacuum distillation (bp 105°C/5 mmHg) to yield the ketoester 29 (58.40
1
g, 91%) as a yellow oil: IR: 1740, 1458, 1247, 1064, 702 cm−1; H NMR (200 MHz, CDCl3) l
3.84 (s, 3H), 6.23 (s, 1H), 7.37 (m, 5H).
3.3. Reduction of methyl 3-chloro-2-oxo-3-phenylpropanoate 2
3.3.1. Reduction with sodium borohydride
To a stirred solution of NaBH4 (1.00 g, 26 mmol) in THF (30 ml) was added dropwise MeOH
(30 ml) solution of 2 (12.6 g, 60 mmol) at 0°C. Stirring was continued for 10 min at the same
temperature, then the mixture was poured into ice water and neutralized with 1N HCl. The
organic material was extracted three times with EtOAc and washed with brine (100 ml). The
organic layer was dried over MgSO4 and evaporated. The residual crude products were purified
by column chromatography (eluent: hexane/EtOAc 8:1–5:1) to give ( )-3 (11.1 g, 88%, syn/anti
ratio=76:24). Pure ( )-syn-3 and ( )-anti-3 were isolated by column chromatography (eluent:
hexane/EtOAc 25:1–10:1). ( )-syn-3 (4.05 g, 32%) as white crystals: mp 79–80°C (lit.13 mp
77–79°C); Rf 0.17 (hexane/EtOAc 4:1). Spectral data were identical with those of an authentic
sample. ( )-anti-3 (2.34 g, 18%) as a colorless oil: Rf 0.15 (hexane/EtOAc 4:1); IR: 3426, 2958,
1744, 1495, 1456, 1286, 1120 cm−1; 1H NMR (200 MHz, CDCl3) l 2.93 (d, 1H, J=7.20 Hz), 3.75
(s, 3H), 4.66 (dd, 1H, J=4.00 and 7.20 Hz), 5.23 (d, 1H, J=4.00 Hz), 7.3–7.4 (m, 5H). A
mixture of ( )-syn-3 and ( )-anti-3 (4.83 g, 38%) was also obtained.
3.3.2. Reduction with lithium aluminum hydride–SiO2
To silica gel (3.75 g, Merck 60, for column chromatography) dried at 150°C under vacuum (5
mmHg) for 3 h was added LiAlH4 (266 mg, 7.00 mmol) and anhydrous ether (25 ml) under an
atmosphere of nitrogen. After stirring for 2 h, 2 (1.06 g, 5.00 mmol) was added to this mixture
and stirring was continued for another 3 h. The whole mixture was then heated to reflux
temperature and kept for 15 min. After cooling and quenching with a few drops of aqueous
ammonium chloride solution, the mixture was filtered and the silica gel was washed thoroughly
with ether. The filtrate and washings were combined and evaporated. The residual crude mixture
was purified by column chromatography (silica gel Merck 60 with a mixture of hexane/EtOAc
20:1–5:1) to give 3 (514 mg, 48%, syn/anti ratio=31:69).
3.3.3. Reduction with KS-Selectride®
To a stirred solution of 2 (212 mg, 1.00 mmol) in THF (5 ml) was added KS-Selectride® (1.0
M in THF, 0.710 ml, 0.710 mmol) under an atmosphere of nitrogen at −78°C. Stirring was
continued for 120 min at the same temperature, and then 30% aqueous H2O2 (0.5 ml) was added
dropwise. The reaction mixture was allowed to warm to room temperature, controlled to weak
acidity by ammonium chloride, and extracted three times with EtOAc. The organic materials
were washed with sodium bicarbonate and water, and dried over MgSO4. The residual crude
mixture was purified by column chromatography (silica gel Merck 60 with a mixture of
hexane/EtOAc 20:1–5:1) to give 3 (81.8 mg, 81%, syn/anti ratio=13:87).
3.4. Lipase-catalyzed kinetic resolution of methyl ( )-syn-3-chloro-2-hydroxy-3-phenyl-
propanoate 3
A mixture of ( )-syn-3 (3.21 g, 15.0 mmol), lipase (Amano PS, 3.21 g), vinyl acetate (3.86 g,
44.8 mmol), and diisopropyl ether (105 ml) was stirred for 25 h at 35°C. The reaction was

H. Hamamoto et al. / Tetrahedron: Asymmetry 11 (2000) 4485–4497
4493
monitored by GC–MS and continued until half of the starting material was consumed. The
reaction mixture was filtered and the solvent was evaporated. The residual crude mixture was
purified by column chromatography (silica gel Merck 60 with a mixture of hexane/EtOAc
10:1–5:1) to give (2S,3S)-3 (1.605 g, 50%, 95% ee by chiral GC) as white crystals: mp 79–80°C
(lit.14 mp 77–79°C); [h]D17 +62 (c 1.4, CHCl3). Spectral data were identical with those of an
authentic sample.14
A mixture of ( )-syn-3 (300 mg, 1.41 mmol), lipase (Meito SL, 600 mg), vinyl benzoate (966
mg, 6.52 mmol), and diisopropyl ether (15 ml) was stirred for 28 h at 60°C. The reaction was
monitored by GC–MS and continued until half of the starting material was consumed. The
reaction mixture was filtered and concentrated, and then the residual crude mixture was purified
by column chromatography (silica gel Merck 60 with a mixture of hexane/EtOAc 10:1–5:1) to
give (2S,3S)-3 (147 mg, 49%, 78% ee by chiral GC) as white crystals: [h]1D7 +47 (c 1.4, CHCl3)
and (2R,3R)-5 (136 mg, 30%, 80% ee by chiral GC) as white crystals: mp 86–87°C; [h]1D8 −131
1
(c 1.3, CHCl3); IR: 2960, 2112, 1789, 1727, 1601, 1454, 1319, 1116, 1017, 704 cm−1; H NMR
(200 MHz) l 3.73 (s, 3H), 5.55 (d, 1H, J=4.40 Hz), 5.65 (d, 1H, J=4.40 Hz), 7.3–7.6 (m, 10H).
Anal. calcd for C17H15ClO4: C, 64.06; H, 4.74. Found: C, 63.99; H, 4.70.
3.5. Lipase-catalyzed kinetic resolution of methyl ( )-anti-3-chloro-2-hydroxy-3-phenyl-
propanoate 3
A mixture of ( )-anti-3 (2.26 g, 10.5 mmol), lipase (Amano PS, 2.26 g), vinyl acetate (2.75 g,
31.9 mmol), and diisopropyl ether (75 ml) was stirred for 28 h at 35°C. The reaction was
monitored by GC–MS and continued until half of the starting material was consumed. After the
reaction mixture was filtered and the solvent was evaporated, the residual crude mixture was
purified by column chromatography (silica gel Merck 60 with a mixture of hexane/EtOAc
20:1–5:1) to give (2S,3R)-3 (1.03 g, 46%, >99% ee) as a colorless oil: [h]1D9 −107 (c 1.5, CHCl3);
1
IR: 3426, 2958, 1744, 1495, 1456, 1286, 1120 cm−1; H NMR (200 MHz, CDCl3) l 2.93 (d, 1H,
J=7.20 Hz), 3.75 (s, 3H), 4.66 (dd, 1H, J=4.00 and 7.40 Hz), 5.23 (d, 1H, J=4.00 Hz), 7.2–7.4
(m, 5H) and (2R,3S)-4 (1.26 g, 47%, 93% ee by chiral GC) as white crystals; mp 87–88°C; [h]D19
1
+77 (c 1.3, CHCl3); IR: 1750, 1220, 1096, 772, 706, 532 cm−1; H NMR (200 MHz) l 2.09 (s,
3H), 3.76 (s, 3H), 5.30 (d, 1H, J=6.00 Hz), 5.55 (d, 1H, J=6.00 Hz), 7.3–7.4 (m, 5H). Anal.
calcd for C10H11O3Cl: C, 56.16; H, 5.11. Found: C, 56.39; H, 5.11.
3.6. Preparation of methyl (2R,3S)-3-azide-2-hydroxy-3-phenylpropanoate 6
A mixture of (2S,3R)-3 (921 mg, 4.29 mmol), sodium azide (1.395 g, 21.5 mmol), and DMF
(30 ml) was stirred for 40 h at 60°C under an atmosphere of nitrogen. The solution was diluted
with ethyl acetate (60 ml) and washed three times with water. The organic layer was dried over
MgSO4 and evaporated. The residual crude product was purified by column chromatography
(silica gel Merck 60 with a mixture of hexane/EtOAc 10:1–5:1) to give (2R,3S)-6 (782 mg, 82%)
as white crystals: mp 74–75°C; [h]D17 +92 (c 2.3, CHCl3) [lit.7a [h]D25 +105 (c 2.3, CHCl3)]. Spectral
data were identical with those of an authentic sample.7a
3.7. Preparation of methyl (2R,3S)-N-benzoyl-3-phenylisoserine 8
To a solution of (2R,3S)-6 (700 mg, 3.17 mmol) and DMAP (387 mg, 3.17 mmol) in
anhydrous CH2Cl2 (30 ml) was added dropwise benzoyl chloride (446 mg, 3.17 mmol) at 0°C.

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H. Hamamoto et al. / Tetrahedron: Asymmetry 11 (2000) 4485–4497
Stirring was continued for 6 h at room temperature. The reaction mixture was extracted three
times with ethyl acetate (50 ml), dried over MgSO4 and evaporated. The residual crude product
was purified by column chromatography (silica gel Merck 60 with a mixture of hexane/EtOAc
10:1–5:1) to give benzoate of (2R,3S)-6 (979 mg, 100%); [h]2D0 +110 (c 2.3, CHCl3) [lit.7k [h]D20 +98
1
(c 1.0, CHCl3)]; IR: 2958, 2110, 1734, 1603, 1497, 1437, 1251, 1180, 754 cm−1; H NMR (200
MHz) l 3.71 (s, 3H), 5.18 (d, 1H, J=4.76 Hz), 5.50 (d, 1H, J=4.76 Hz), 7.3–7.6 (m, 8H),
7.9–8.1 (m, 2H). Benzoate of (2R,3S)-6 (950 mg, 3.07 mmol) was hydrogenated with 10% Pd/C
(95 mg) in methanol (20 ml) under an atmosphere of hydrogen (1 atm) for 48 h at rt. The
solution was filtered, and allowed to stand at 25°C for 48 h. The solution was concentrated and
purified by recrystallization from MeOH to give (2R,3S)-8 (652 mg, 71%) as colorless crystals:
mp 179–180°C (lit.7a mp 183–184°C); [h]2D0 −50.2 (c 1.00, MeOH) [lit.7a [h]2D0 −49.6 (c 1.00,
MeOH)]. Spectral data were identical with those of an authentic sample.7a
3.8. Methyl (2R,3S)-N-tert-butoxycarbonyl-3-phenylisoserine 9
A suspension of 10% Pd/C (25 mg) in ethyl acetate (2 ml) was stirred at 20°C under an
atmosphere of hydrogen for 10 min, whereupon a solution of di-tert-butyl dicarbonate (236 mg,
1.10 mmol) and (2R,3S)-6 (200 mg, 0.905 mmol) in ethyl acetate (5 ml) was added. The stirring
was continued for 48 h under an atmosphere of hydrogen (1 atm) at room temperature. The
solution was filtered and evaporated. The residual crude product was purified by column
chromatography (silica gel Merck 60 with a mixture of hexane/EtOAc 10:1–5:1) to give
(2R,3S)-9 (187 mg, 70%) as white crystals: mp 115°C [lit.7n mp 130.5–131.5°C]; [h]D20 −6.8 (c 1.0,
CHCl3) [lit.7n [h]D20 −7 (c 1.2, CHCl3)]. Spectral data were identical with those of an authentic
sample.7n
3.9. Preparation of methyl (2R,3R)-3-phenylglycidate 7
Sodium (178 mg, 7.74 mmol) was treated with anhydrous MeOH (30 ml). After sodium
dissolved in the solvent fully, the solution was cooled to 0°C, and then a solution of (2S,3S)-3
(1.51 g, 7.03 mmol) in anhydrous MeOH was added under an atmosphere of nitrogen. After 10
h of stirring at 0°C, the reaction mixture was neutralized by 1N HCl and extracted three times
with EtOAc. The organic layer was dried over MgSO4 and evaporated. The residual crude
product was purified by column chromatography (silica gel Merck 60 with a mixture of
hexane/EtOAc 10:1–5:1) to give (2R,3R)-7 (335 mg, 27%) as a colorless oil; [h]1D7 +13 (c 1.1,
CHCl3) [lit.7a [h]D26 +11 (c 4.4, CHCl3)]. Spectral data were identical with those of an authentic
sample.7a
3.10. Preparation of methyl (2R,3S)-3-azide-2-hydroxy-3-phenylpropanoate 6 from (2R,3R)-7
A mixture of (2R,3R)-7 (42.1 mg, 0.24 mmol), sodium azide (38.4 mg, 0.14 mmol), and
NH4Cl (47.4 mg, 0.89 mmol) in acetone/H2O (4:1, 1 ml) was stirred for 24 h under reflux. The
solution was concentrated and the residue was diluted with ethyl acetate (25 ml) and washed
three times with water. The organic layer was dried over MgSO4 and concentrated. The residual
crude product was purified by column chromatography (silica gel Merck 60 with a mixture of
hexane/EtOAc 10:1–5:1) to give (2R,3S)-6 (39.3 mg, 75%) as white crystals. Spectral data were
identical with those of an authentic sample.7a

H. Hamamoto et al. / Tetrahedron: Asymmetry 11 (2000) 4485–4497
4495
3.11. Lipase-catalyzed kinetic resolution of methyl (2R,3S)-3-azide-2-hydroxy-3-phenyl-
propanoate 6
A mixture of ( )-syn-6 (200 mg, 0.905 mmol), lipase (Amano PS, 200 mg), vinyl acetate (390
g, 4.53 mmol), and diisopropyl ether (15 ml) was stirred for 46 h at 35°C. The reaction was
monitored by GC–MS and continued until half of the starting material was consumed. After the
reaction mixture was filtered and evaporated, the residual crude mixture was purified by column
chromatography (silica gel Merck 60 with a mixture of hexane/EtOAc 15:1–5:1) to give
(2R,3S)-6 (88 mg, 44%, 80% ee by chiral GC) as white crystals: [h]1D9 +97 (c 1.5, CHCl3) and
(2S,3R)-10 (90.4 mg, 38%, 76% ee by chiral GC) was obtained as white crystals: mp 65°C; [h]D19
−57 (c 1.3, CHCl3) [lit.7o [h]D20 −81.6 (c 2.0, CHCl3)]; IR: 2110, 1752, 1510, 1377, 1222, 1102, 704
1
cm−1; H NMR (200 MHz) l 2.14 (s, 3H), 3.69 (s, 3H), 5.07 (d, 1H, J=4.82 Hz), 5.23 (d, 1H,
J=4.82 Hz), 7.3–7.4 (m, 5H).
3.12. Preparation of methyl (2S,3R)-3-phenylglycidate 7
K2CO3 (23.2 mg, 0.167 mmol) was added to a solution of (2R,3S)-4 (34.2 mg, 0.133 mmol)
in MeOH (1.5 ml) at −20°C. The suspension was stirred vigorously between −20 and 0°C for 2
h, and then poured into saturated aqueous NH4Cl. The mixture was extracted three times with
EtOAc and the combined organic layers were washed with water, dried over MgSO4, and
concentrated. The residual crude product was purified by column chromatography (silica gel
Merck 60 with a mixture of hexane/EtOAc 10:1–5:1) to give (2S,3R)-7 (22.1 mg, 93%) as a
colorless oil: [h]1D7 +148 (c 1.7, CHCl3); IR: 2958, 1754, 1441, 1346, 1294, 1212, 1004, 897, 816,
1
696 cm−1; H NMR (200 MHz) l 3.52 (d, 1H, J=1.76 Hz), 3.83 (s, 3H), 4.10 (d, 1H, J=1.76
Hz), 7.3–7.5 (m, 5H).
3.13. Preparation of methyl (2S,3S)-3-azide-2-hydroxy-3-phenylpropanoate 6
A mixture of (2S,3R)-7 (250 mg, 1.40 mmol), sodium azide (228 mg, 3.50 mmol), and NH4Cl
(224 mg, 4.20 mmol) in acetone/H2O (4:1, 7 ml) was stirred for 23 h under reflux. The solution
was concentrated, diluted with ethyl acetate (10 ml), and washed three times with water. The
organic layer was dried over MgSO4 and evaporated. The residual crude product was purified
by column chromatography (silica gel Merck 60 with a mixture of hexane/EtOAc 10:1–5:1) to
give (2S,3S)-6 (296 mg, 96%) as a colorless oil: [h]2D0 +137 (c 2.3, CHCl3) [lit.7o [h]D25 +125 (c 1.0,
CHCl3)]. Spectral data were identical with those of an authentic sample.7o
(2R,3R)-4 (210 mg, 0.82 mmol) was treated with conc. HCl (2 ml) in acetone/MeOH (3:4, 7
ml). The mixture was stirred for 23 h at room temperature and then diluted with water (15 ml).
After repeating the extraction three times, the organic layer was dried over MgSO4 and
evaporated. The residual crude product was purified by column chromatography (silica gel
Merck 60 with a mixture of hexane/EtOAc 10:1–5:1) to give (2R,3R)-3 (158 mg, 90%) as white
crystals: [h]2D0 −69 (c 1.3, CHCl3). A mixture of (2R,3R)-3 (158 mg, 0.74 mmol), sodium azide
(143 mg, 2.21 mmol), and DMF (3.5 ml) was stirred for 48 h at 60°C under an atmosphere of
nitrogen. The solution was diluted with ethyl acetate (10 ml) and washed three times with water.
The organic layer was dried over MgSO4 and evaporated. The residual crude product was
purified by column chromatography (silica gel Merck 60 with a mixture of hexane/EtOAc
10:1–5:1) to give (2S,3S)-6 (136 mg, 83%) as a colorless oil.

4496
H. Hamamoto et al. / Tetrahedron: Asymmetry 11 (2000) 4485–4497
3.14. Preparation of methyl (2S,3S)-N-benzoyl-3-phenylisoserine 8
To a solution of (2S,3S)-6 (177 mg, 0.80 mmol) and DMAP (4.9 mg, 0.04 mmol) in dry
CH2Cl2 (2 ml) was added dropwise benzoyl chloride (0.116, 1.00 mmol) at 0°C. Stirring was
continued for 4 h at room temperature. The reaction mixture was extracted three times with
ethyl acetate (50 ml), dried over MgSO4 and evaporated. The residual crude product was
hydrogenated with 10% Pd/C (18 mg) in methanol (1.6 ml) under an atmosphere of hydrogen
(1 atm) for 48 h at room temperature. The solution was filtered and the residual crude product
was purified by column chromatography (silica gel Merck 60 with a mixture of hexane/EtOAc
10:1) to give (2S,3S)-8 (167 mg, 70%) as white crystals: mp 138–140°C; [h]2D0 +16 (c 1.00, MeOH)
[lit.7k [h]D23 −23 (c 1, CHCl3)]. Spectral data were identical with those of the authentic sample.
3.15. Preparation of (4S,5R)-2,4-diphenyl-5-methoxycarbonyl-2-oxazoline 11
To a solution of (2S,3S)-8 (60.0 mg, 0.20 mmol) in benzene (1.0 ml) were added PPh3 (131
mg, 0.50 mmol) and DEAD (0.082 ml, 0.50 mmol) in 1.0 ml of benzene. The reaction mixture
was stirred for 20 h at room temperature and then concentrated in vacuo. The residual crude
product was purified by column chromatography (silica gel Merck 60 with a mixture of
hexane/EtOAc 15:1–10:1) to give (4S,5R)-11 (50.4 mg, 90%) as a colorless oil: [h]2D1 +15 (c 2.5,
CHCl3) [lit.7k [h]D20 +13 (c 1, CHCl3)]; IR: 3066, 3034, 2960, 2498, 1760, 1657, 1582, 1439, 1375,
1
1270, 1238, 1069, 911, 696 cm−1. H NMR (200 MHz) data were identical with those of an
authentic sample.7k
To a solution of (2S,3S)-6 (33.2 mg, 0.15 mmol) in benzene (0.5 ml) were added PPh3 (197
mg, 0.75 mmol), DEAD (0.122 ml, 0.75 mmol), and benzoic acid (91.5 mg, 0.75 mmol) in 0.5
ml of benzene. The reaction mixture was stirred for 12 h at room temperature and then
concentrated in vacuo. The residual crude product was purified by column chromatography
(silica gel Merck 60 with a mixture of hexane/EtOAc 15:1–10:1) to give (4S,5R)-11 (27.8 mg,
66%) as a colorless oil.
3.16. Hydrolysis of (4S,5R)-2,4-diphenyl-5-methoxycarbonyl-2-oxazoline 11
A solution of (4S,5R)-11 (50.4 mg, 0.18 mmol) in MeOH (3 ml) and 1N HCl (1.0 ml) was
refluxed for 2 h. The solution was concentrated, and the organic layer was extracted with ethyl
acetate. The combined extracts were washed with water, and dried over anhydrous Na2SO4. The
solvent was evaporated, and the residual crude product was purified by column chromatography
(silica gel Merck 60 with a mixture of hexane/EtOAc 2:1) to give (2R,3S)-8 (48.4 mg, 90%) as
colorless crystals: [h]2D4 −49.9 (c 1.0, MeOH). Physical and spectral data were identical with those
of the sample that was described in Section 3.14.
3.17. Methyl (2S,3S)-N-tert-butoxycarbonyl-3-phenylisoserine 9
A suspension of 10% Pd/C (5 mg) in methanol (0.5 ml) was stirred at room temperature under
an atmosphere of hydrogen (1 atm) for 10 min, whereupon a solution of di-tert-butyl
dicarbonate (59.2 mg, 0.271 mmol) and (2S,3S)-6 (50.0 mg, 0.226 mmol) in methanol (0.5 ml)
was added. The stirring was continued for 72 h under an atmosphere of hydrogen (1 atm) at
room temperature. The solution was filtered and evaporated. The residual crude product was

H. Hamamoto et al. / Tetrahedron: Asymmetry 11 (2000) 4485–4497
4497
purified by column chromatography (silica gel Merck 60 with a mixture of hexane/EtOAc
10:1–5:1), giving (2S,3S)-9 (64.8 mg, 97%) as white crystals: mp 120–121°C (lit.14 mp 136–
137°C); [h]2D3 +44 (c 1.2, CHCl3) [lit.14 [h]D20 +30.1 (c 0.50, CHCl3)]; IR: 3366, 1721, 1694, 1520,
1439, 1369, 1236, 1174, 1112, 1013, 866, 737, 518 cm−1; 1H NMR (200 MHz) l 1.39 (s, 9H), 2.83
(d, 1H, J=6.88 Hz), 3.70 (s, 3H), 4.57 (dd, 1H, J=3.38, 6.88 Hz), 5.08 (dd, 1H, J=3.38, 8.06
Hz), 5.56 (d, 1H, J=8.06 Hz), 7.3–7.4 (m, 5H).
Acknowledgements
We are grateful to the SC NMR Laboratory of Okayama University for the high-field NMR
measurements.
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