Lipase-catalyzed enantiomeric resolution of ceramides 1

Article abstract of DOI:10.1021/jo980727u

Lipase-catalyzed enantiomeric kinetic resolution of ceramides related to C₁₆-sphinganine and C₁₈-sphingenine is described. Two hydroxy groups in readily available racemic N-stearoyl-erythroC₁₆-sphinganine were acetylated, and several kinds of lipases were screened for the hydrolysis of this substrate. Among them, a Burkholderia cepacia lipase (SC lipase A, Sumitomo Chemical Co., Ltd.) showed the highest reactivity and enantioselectivity. The rate of hydrolysis and selectivity were greatly affected by some additives. Especially, the combined use of a detergent, Triton X-100, and the solid support, Florisil, for immobilization showed the highest enantioselectivity (E = ca. 170), although the reaction rate turned low. Introduction of a double bond into the substrate (N-stearoyl-erythro-Cis-sphingenine) also retarded the hydrolysis. By utilizing the preferential hydrolysis of the acetate on the primary hydroxy group, another advantageous feature of this enzyme-catalyzed reaction, the resulting product could directly be used as the glycosyl acceptor for cerebroside synthesis.

Full text of DOI:10.1021/jo980727u

J . Org. Chem. 1998, 63, 6929-6938
6929
Lip a se-Ca ta lyzed En a n tiom er ic Resolu tion of Cer a m id es†
Mikio Bakke, Masahiro Takizawa, Takeshi Sugai,* and Hiromichi Ohta
Department of Chemistry, Keio University, 3-14-1, Hiyoshi, Yokohama 223-8522, J apan
Received April 20, 1998
Lipase-catalyzed enantiomeric kinetic resolution of ceramides related to C16-sphinganine and C18-
sphingenine is described. Two hydroxy groups in readily available racemic N-stearoyl-erythro-
C16-sphinganine were acetylated, and several kinds of lipases were screened for the hydrolysis of
this substrate. Among them, a Burkholderia cepacia lipase (SC lipase A, Sumitomo Chemical Co.,
Ltd.) showed the highest reactivity and enantioselectivity. The rate of hydrolysis and selectivity
were greatly affected by some additives. Especially, the combined use of a detergent, Triton X-100,
and the solid support, Florisil, for immobilization showed the highest enantioselectivity (E ) ca.
170), although the reaction rate turned low. Introduction of a double bond into the substrate (N-
stearoyl-erythro-C18-sphingenine) also retarded the hydrolysis. By utilizing the preferential
hydrolysis of the acetate on the primary hydroxy group, another advantageous feature of this
enzyme-catalyzed reaction, the resulting product could directly be used as the glycosyl acceptor
for cerebroside synthesis.
In tr od u ction
A wide range of physiological activity as well as the
requirement for biochemical studies and medicinal ap-
plications of sphingoids1,2 involving sphingosines, cera-
mides (N-acylsphingenines, e.g. 1, N-acylsphinganines,
e.g. 2, Figure 1), and cerebrosides has prompted the
development of chemical syntheses of these compounds
in diastereomerically3-5 and enantiomerically6-12 well-
defined forms.
In this paper, we present a new entry to enantiomeri-
cally enriched forms of ceramides: the lipase-catalyzed
kinetic resolution of the racemic forms of ceramides and
the precursors, readily available by straightforward
F igu r e 1. Ceramides: N-acylsphingenines and N-acylsphin-
ganines.
syntheses.3,4 This approach provides both enantiomers,
which would potentially be subjected to a further wide
range of biological evaluation.
† The experimental part of this work was taken from the forthcoming
M.S. thesis of M. B. (1999) and the B.S. thesis of M. T. (1996).
* Address correspondence to this author: Fax +81-45-563-5967;
sugai@chem.keio.ac.jp.
Resu lts a n d Discu ssion
(1) Reviews: Shapiro, D. In Chemistry of Sphingolipids; Herman-
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Chem., Int. Ed. Engl. 1997, 36, 1955-1959.
Our first attempt was a lipase-catalyzed enantioselec-
tive acylation of a secondary amine.13 The racemic
substrate (()-(4R*,5S*)-4a with a sphinganine (satu-
rated) side-chain was obtained by a catalytic hydrogena-
tion of (()-(4R*,5S*)-3, which was prepared according to
Grob’s nitroaldol synthesis3a,cf.10g (Scheme 1). To our
disappointment, a Candida antarctica lipase-catalyzed
acylation13 (lipase B, Novo Nordisk Co., SP525) of the
amino group of 4a , however, was very slow. Only under
forced conditions, at elevated temperature and reduced
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S0022-3263(98)00727-0 CCC: $15.00 © 1998 American Chemical Society
Published on Web 09/09/1998

6930 J . Org. Chem., Vol. 63, No. 20, 1998
Bakke et al.
pressure,14 the desired C-N bond formation occurred and
the acylated product 4b was obtained in a 22% yield. In
the absence of lipase, the reaction did not proceed. This
lipase-catalyzed amide formation is an interesting tool
for ceramide synthesis, because an N-acyl group could
be introduced under neutral conditions without any
condensing reagent other than the lipase; however, the
product 4b was racemic and further enantiomeric resolu-
tion was required.
Sch em e 1. P r ep a r a tion of Ra cem ic Su bstr a tes
for Lip a se-Ca ta lyzed Rea ction s a n d Attem p ted
En a n tioselective N-Acyla tion
Then we switched to another approach, an enantiose-
lective hydrolysis of the primary O-acyl group, discrimi-
nating the stereochemistry on the adjacent carbon atom.15
Indeed, in the case of the O,O′-diacetylated substrate 2b
(Scheme 1), its structure is closely related to a triacyl
glycerol, and the problem in enantioselective hydrolysis
is looked upon as the site (sn-1 or sn-3) specificity of
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42, 5961-5968. (b) Yamanoi, T.; Akiyama, T.; Ishida, E.; Abe, H.;
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lipases,16 if we see the two long side-chains and the
primary acetyl group as the three fatty acid ester
moieties of triacyl glycerol.
From the screening of several kinds of lipases on
hydrolyzing 2b, a Burkholderia cepacia lipase (SC lipase
A, Sumitomo Chemical Co., Ltd.)17 was found as the most
(15) Terao, Y.; Murata, M.; Achiwa, K.; Nishio, T.; Akamatsu, M.;
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I.; Nozawa, M.; Nagumo, S. Tetrahedron: Asymmetry 1993, 4, 757-
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metry 1994, 5, 785-788. Sih, J . C.; Gu, R. L. Tetrahedron: Asymmetry
1995, 6, 357-360. Ferraboschi, P.; Casati, S.; Manzocchi, A.; San-
taniello, E. Tetrahedron: Asymmetry 1995, 6, 1521-1524. Akita, H.;
Chen, C. Y.; Uchida, K. Tetrahedron: Asymmetry 1995, 6, 2131-2134.
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1256. Ikeda, K.; Achiwa, K. BioMed. Chem. Lett. 1997, 7, 225-228.
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165-168. Ota, Y.; Minesaki, T.; Oshima, A. Biosci. Biotech. Biochem.
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Biosci. Biotech. Biochem. 1996, 60, 2059-2063.

Lipase-Catalyzed Enantiomeric Resolution of Ceramides
J . Org. Chem., Vol. 63, No. 20, 1998 6931
Sch em e 2. Hyd r olysis w ith Na tive SC Lip a se A
Sch em e 3. Hyd r olysis w ith Im m obilized SC
Lip a se A
etate (2R,3S)-2c, and subsequently a part of (2R,3S)-2c
was slowly hydrolyzed to diol (2R,3S)-2a , as shown in
Table 1. On the other hand, the hydrolysis of a natural
(2S,3R) form of 2b stopped at the stage of monoacetate
(2S,3R)-2c. In both cases, it was noteworthy that no
regioisomer 2e was observed in the mixture. A sequen-
tial kinetic resolution19 provided both enantiomers of
ceramides: natural enantiomer as diacetate (87% ee),
and nonnatural enantiomer as diol (98% ee).
Although the resolution worked well as above, there
remained a serious technical problem in this lipase-
catalyzed reaction. Due to the crystalline and hydro-
phobic nature of the substrate 2b, the hydrolysis only
proceeds in a two-phase system between a buffer and a
hydrophobic solvent, decane.cf.20 As the hydrolysis took
place and the products formed, the mixture turned into
an emulsion caused by lipase protein as well as the
products themselves, which resulted in a seriously in-
tractable situation at the step of extractive workup.
To avoid this laborious isolation procedure, the use of
an immobilized form of lipase14 was attempted. As
expected, the extraction was very much facilitated after
the enzyme protein was readily removed by simple
filtration at the beginning of workup. Moreover, the
distribution of the products dramatically changed com-
pared with the results obtained by using unimmobilized
(native) SC lipase A. The hydrolysis stopped at the stage
of monoacetate: (+)-2b (58% yield, 69% ee) and (-)-2c
(41% yield, 98% ee) were obtained, as shown in Scheme
3.
As already described, the hydrolysis proceeds in two
steps with native SC lipase A. The enantiomeric ratio21
of each step (E1, E2) was 9 and 31, respectively (Table 1,
entry 1). A Pseudomonas cepacia lipase (lipase PS,
Amano pharmaceutical Co., entry 2) showed a marked
contrast, a larger E1 value (30) compared with the
selectivity in the second step (E2 ) 4). The use of
immobilized lipase not only caused lowering of the
reaction rate, but also a great change in E1 from 9 to 168
(entry 3); thus, we became interested in this change of
selectivity.22 The immobilized form14,23 of SC lipase A
consists of an enzyme protein, a detergent, Triton X-100
(poly(ethylene glycol) octylphenyl ether), and a solid
support, Florisil (magnesium silicate). The separate use
of two additives (entries 4 and 5) revealed the selectivity
mostly depended upon the detergent (entry 5, E1 ) 170).
potent candidate. The treatment of 2b with the lipase
gave a mixture of three products, unreacted (+)-2b (38%),
(-)-monoacetate 2c (54%), and (-)-diol 2a (7%) (Scheme
2). The enantiomeric excesses (ee’s) of the products were
determined after conversion to the bis-MTPA esters18 2d ,
by the HPLC analyses as well as 1H NMR measurements.
The absolute configuration of (-)-2a was determined as
(2R,3S), by comparing its sign of rotation of a derivative
(-)-5a with that of an analogue, (-)-5b, whose absolute
configuration was unambiguously established.9e From
these results, the stereochemical course of the reaction
was figured out as depicted in Scheme 2. SC lipase A
preferentially hydrolyzed a nonnatural (2R,3S) form of
ceramide diacetate. In this case, the primary acetate of
(2R,3S)-2b was predominantly hydrolyzed to give monoac-
(17) A patent on this enzyme has been applied for by Sumitomo
Chemical Co., Ltd.; J apanese Patent Application Number: 08-344076.
For correspondence, Dr. Satoshi Mitsuda, Biotechnology Laboratory,
Sumitomo Chemical Co., Ltd., 4-2-1 Takatsukasa, Takarazuka 665-
0051, J apan.
(18) Dale, J . A.; Dull, D. L.; Mosher, H. S. J . Org. Chem. 1969, 34,
2543-2549. Dale, J . A.; Mosher, H. S. J . Am. Chem. Soc. 1973, 95,
512-519.
(19) Sugai, T.; Ikeda, H.; Ohta, H. Tetrahedron 1996, 52, 8123-
8134.
(20) Linear saturated hydrocarbons such as decane and dodecane
have been utilized as the diffuser of ceramides into aqueous phase:
see J i, L.; Zhang, G.; Uematsu, S.; Akahori, Y.; Hirabayashi, Y. FEBS
Lett. 1995, 358, 211-214.
(21) Chen, C.-S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J . J . Am. Chem.
Soc. 1982, 104, 7294-7299.

6932 J . Org. Chem., Vol. 63, No. 20, 1998
Ta ble 1. Effect of Ad d itives on th e Selectivity
Bakke et al.
entry
substrate
lipase
additive
time (d)
E1
E2
1
2
3
4
5
6
7
8
9
2b
2b
2b
2b
2b
1b
1b
1b
1b
B. cepacia (SC lipase A)
P. cepacia (Amano PS)
SC lipase A
none
none
3a
9
31
4
5.5b
30
168
58
170
5
Florisil, Triton X-100
Florisil
Triton X-100
none
Florisil
Triton X-100
Florisil, Triton X-100
4a
-
-
-
62
-
3a
4a
SC lipase A
4c
4c
12
no reactionc
no reactionc
Enzyme/substrate ) 1/10 (w/w). Enzyme/substrate ) 4/1 (w/w). c Enzyme/substrate ) 1/3 (w/w).
a
b
Sch em e 4. Tr a n sester ifica tion w ith Im m obilized
SC Lip a se A
As mentioned above, the treatment of 2b with lipase
afforded the nonnatural enantiomer of diol 2a , and this
result suggested to us a possibility of the lipase-catalyzed
kinetic resolution of the intermediate, monoacetate (()-
2c itself. To our surprise, it was revealed that the
hydrolysis 2c was extremely slow, and almost no diol 2a
was observed. This result suggested that the reactivity
and the selectivity depended on the aggregation and
orientation of the substrate under the reaction interface,
which was greatly affected by the addition of detergent
(Triton X-100). The successful enzymatic hydrolyses
proceeded in a two-phase solution as a complex micelle
consisting of the starting diacetate, monoacetate, and
diol.
Next, our attention was directed to the lipase-catalyzed
reaction in a homogeneous organic solution. The acyla-
tion of hydroxy groups was attempted in a solvent system
of vinyl acetate-tetrahydrofuran, which had successfully
been applied in lipase-catalyzed transesterification of
long-chain R-hydroxy acid.24 SC lipase A-catalyzed acyl-
ation proceeded smoothly to provide a completely regio-
selective acylation on the primary hydroxy group (Scheme
4); however, the product 2e was racemic. Even worse,
lipase PS lost such regioselectivity: the product was a
mixture of 1-O-acetylated form 2e and its regioisomer,
3-O-acetylated form 2c (87:13).
The results obtained so far encouraged us to attempt
the kinetic resolution of ceramide diacetate of sphinge-
nine type 1b which was prepared as shown in Scheme
5.4a,b,25 The introduction of a double bond to the substrate
surprisingly retardedcf.26 the progress of hydrolysis. The
native SC lipase A exhibited similar E1 and E2 values to
this substrate compared with 2b, respectively (entries 1
and 6). The addition of Florisil enhanced the value of
E1 to some extent (12, entry 7). On the other hand,
Triton X-100 completely suppressed the hydrolysis (entry
8). The distribution of the products starting from (()-
1b is described in Scheme 5.
(22) Guo, Z.-W.; Sih, C. J . J . Am. Chem. Soc. 1989, 111, 6836-6841.
Kitaguchi, H.; Itoh, I.; Ono, M. Chem. Lett. 1990, 1203-1206. Itoh,
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Soc. J pn. 1991, 64, 624-627. Okabe, M.; Sun, R.-C.; Zenchoff, G. B. J .
Org. Chem. 1991, 56, 4392-4397. Nakamura, K.; Takebe, Y.; Ki-
tayama, T.; Ohno, A. Tetrahedron Lett. 1991, 32, 4941-4944. Tsukube,
H.; Betchaku, A.; Hiyama, Y.; Itoh, T. J . Chem. Soc., Chem. Commun.
1992, 1751-1752. Brevet, J .-L.; Mori, K. Synthesis 1992, 1007-1012.;
Hirose, Y.; Kariya, K.; Sasaki, I.; Kurono, Y.; Ebiike, H.; Achiwa, K.
Tetrahedron Lett. 1992, 33, 7157-7160. Secundo, F.; Riva, S.; Carrea,
G. Tetrahedron: Asymmetry 1992, 3, 267-280. Moore, A. N. J .; Dutton,
P. J .; Zahalka, H. A.; Burton, G. W.; Ingold, K. U. J . Am. Chem. Soc.
1995, 117, 5677-5686. Hansen, T. V.; Waagen, V.; Partali, V.;
Anthonsen, H. W.; Anthonsen, T. Tetrahedron: Asymmetry 1995, 6,
499-504. Kinoshita, M.; Ohno, A. Tetrahedron 1996, 52, 5397-5406.
Ohtsuka, Y.; Katoh, O.; Sugai, T.; Ohta, H. Bull. Chem. Soc., J pn. 1997,
70, 483-491. Review: Kvittingen, L. Tetrahedron 1994, 50, 8253-
8274. Itoh, T.; Takagi, Y.; Tsukube, H. J . Mol. Catal. B: Enzymol. 1997,
3, 259-270.
(25) Nakagawa, Hino and co-workers reported the synthesis3c of
racemic ceramide 1b via Grob’s nitroaldol synthesis. In our case,
however, the contamination of threo-isomer was observed at the stage
of the reduction of nitro group to amino group with LiAlH4, probably
due to the epimerization caused by basicity of the reagent prior to the
reduction. From this reason, we synthesized 1b essentially according
to the Schmidt’s procedure,4a,b with some modifications as described
in the Experimental Section.
(23) Yamada, H.; Sugai, T.; Ohta, H.; Yoshikawa, S. Agric. Biol.
Chem. 1990, 54, 1579-1580.
(24) Sugai, T.; Ohta, H. Tetrahedron Lett. 1991, 32, 7063-7064.
(26) An example of the low reactivity of the sphingenine-related
substance was reported very recently.12c.

Lipase-Catalyzed Enantiomeric Resolution of Ceramides
J . Org. Chem., Vol. 63, No. 20, 1998 6933
Sch em e 5. Hyd r olysis of Cer a m id e Dia ceta te
w ith Dou ble Bon d w ith SC Lip a se A
Sch em e 6. Both En a n tiom er s of Mon oa ceta te a n d
th e Syn th esis of Ga la ctosyl Cer a m id e
problematic; indeed, the diacetate 2b was obtained in
45% as the major byproduct. Our achievement in devel-
oping the regioselective hydrolysis using SC lipase A,
however, cleanly solved this problem, as 2b was readily
converted back to the glycosyl acceptor, monoacetate 2c.
Con clu sion
SC Lipase A-catalyzed enantiomeric kinetic resolution
of ceramides related to C16-sphinganine and C18-sphin-
genine was achieved. The rate of hydrolysis and the
selectivity were greatly affected by the additive, espe-
cially the detergent, Triton X-100. By combining the
enantioselective and the preferential hydrolysis of the
acetate on the primary hydroxyl group, the protected
forms of both enantiomers of ceramide acetate with free
primary alcohol became available, which is a new entry
to glycosyl ceramide synthesis.
As we stated above, an advantageous feature of this
lipase-catalyzed hydrolysis is the remarkable regioselec-
tivity. Scheme 6 illustrates the preparation of both
natural and nonnatural enantiomers of ceramide with a
liberated primary alcohol, which would be appropriate
for cerebroside syntheses.27 First, nonnatural (2R,3S)-
2c was obtained (41% yield, 98% ee) by immobilized SC
lipase A-catalyzed hydrolysis of (()-2b leaving a partially
enantiomerically enriched form of (2S,3R)-2b. The ee of
the recovered (2S,3R)-2b could be enhanced (96% ee) by
the repetition of hydrolysis with the same immobilized
lipase, while the contaminated (2R,3S)-isomer was thor-
oughly hydrolyzed to the corresponding monoacetate.
Then, native SC lipase A-catalyzed hydrolysis in a low
enantioselective but a highly regioselective manner was
applied to (2S,3R)-2b, to afford the natural form of
monoacetate (2S,3R)-2c in 85% yield. This monoacetate
was effectively glycosylated with tetra-O-acetyl-R-galac-
tosyl trichloroacetimidate to give 10 in 52% yield. In this
glycosylation reaction so far, the migration of the acetyl
group from the glycosyl donor to the acceptor had been
Exp er im en ta l Section
All mps were uncorrected. IR spectra were measured as
KBr disks on a J asco IRA-202 and FT/IR-410 spectrometer
unless otherwise stated. 1H NMR spectra were measured in
CDCl3 with TMS as the internal standard at 270 MHz on a
J EOL J NM EX-270 spectrometer unless otherwise stated.
Mass spectra were recorded on Hitachi M-80B spectrometer
at 70 eV. Optical rotations were recorded on a J asco DIP 360
polarimeter. Wako Gel B-5F and silica gel 60 K070-WH (70-
230 mesh) of Katayama Chemical Co. were used for prepara-
tive TLC and column chromatography, respectively.
er yth r o-2,2-Dim eth yl-5-n itr o-4-tr idecyl-1,3-dioxan e (()-
(4R*,5S*)-3 a n d th r eo-2,2-Dim eth yl-5-n itr o-4-tr id ecyl-1,3-
d ioxa n e (()-(4R*,5R*)-3. According to the reported proce-
dure,3f a mixture of (()-(4R*,5S*)-3 (37%) and (()-(4R*,5R*)-3
(38%) was prepared starting from 2-nitroethanol and tetra-
decanal.
(27) (a) Tanahashi, E.; Murase, K.; Shibuya, M.; Igarashi, Y.; Ishida,
H.; Hasegawa, A.; Kiso, M. J . Carbohydr. Chem. 1997, 16, 831-858.
(b) Kiso, M.; Nakamura, A.; Hasegawa, A. J . Carbohydr. Chem. 1987,
6, 411-422.

6934 J . Org. Chem., Vol. 63, No. 20, 1998
Bakke et al.
(()-(4R*,5S*)-3: Rf 0.51 [silica gel, hexanes-ethyl acetate
(10:1)], 1H NMR δ 0.88 (3H, t, J ) 6.5 Hz, 13′-CH3), 1.18 (22H,
m), 1.38 and 1.52 [total 6H, each 3H, s, O2C(CH3)2], 1.56 (2H,
m, 1′-CH2), 4.09 (1H, dd, J ) 5.6, 12.0 Hz, 6-eqH), 4.18 (1H,
m, 4-H), 4.22 (1H, dd, J ) 7.0, 12.0 Hz, 6-axH), 4.44 (1H, ddd,
J ) 5.6, 7.0, 9.0 Hz, 5-H). Its NMR spectrum was identical
with that reported previously.3f
(()-(4R*,5R*)-3: Rf 0.16 [silica gel, hexanes-ethyl acetate
(10:1)], 1H NMR δ 0.87 (3H, t, J ) 6.6 Hz, 13′-CH3), 1.25 (22H,
m), 1.44 and 1.48 [total 6H, each 3H, s, O2C(CH3)2], 1.56 (2H,
m, 1′-CH2), 4.06 (1H, dt, J ) 3.3, 6.6 Hz, 4-H), 4.19 (1H, dd, J
) 4.6, 13.0 Hz, 6-axH), 4.35 (1H, dd, J ) 3.0, 13.0 Hz, 6-eqH),
4.51 (1H, ddd, J ) 3.0, 3.3, 4.6 Hz, 5-H). A solution of (()-
(4R*,5R*)-3 (4.92 g, 14.3 mmol) in triethylamine (100 mL) was
stirred under reflux overnight.3c After confirming a satisfac-
tory conversion by TLC analysis, the reaction was quenched
by adding 2 N hydrochloric acid. The mixture was extracted
with ethyl acetate, and the organic layer was washed with
saturated aqueous sodium hydrogen carbonate solution and
brine, dried over anhydrous sodium sulfate, and concentrated
in vacuo. The residue was purified with a silica gel column
chromatography (100 g) to give the erythro-isomer of 3 (4.46
g, 91%).
er yth r o-5-Am in o-2,2-dim eth yl-4-tr idecyl-1,3-dioxan e (()-
(4R*,5S*)-4a . The acetonide (()-(4R*,5S*)-3 (2.07 g, 6.03
mmol) was dissolved in ethanol (80 mL), and palladium on
charcoal (10%, 300 mg) and ammonium formate (13 g, 0.21
mol) were added. After stirring for 3 h at room temp, the
disappearance of the starting material was confirmed by a TLC
analysis [silica gel, developed with chloroform-methanol (9:
1)]. The mixture was filtered, and the residue was washed
with a mixture of ethanol-ethyl acetate. The combined
filtrate and washings were made alkaline by adding 1 N
potassium hydroxide solution and extracted with ethyl acetate.
The extract was washed with brine, dried over sodium sulfate,
and concentrated in vacuo to give 4a (1.89 g, 99%), IR νmax
3380, 3270, 2950, 2850, 1660, 1610, 1460, 1380, 1265, 1200,
1165, 1070, 1010, 870, 815, 720, 665 cm-1; 1H NMR δ 0.87 (3H,
t, J ) 6.6 Hz, 13′-CH3), 1.25 (22H, m), 1.38 and 1.44 [total
6H, each 3H, s, O2C(CH3)2], 1.53 (2H, m, 1′-CH2), 2.65 (1H,
ddd, J ) 5.3, 9.6, 9.6 Hz, 5-H), 3.40 (1H, m, 4-H), 3.47 (1H,
dd, J ) 9.6, 11.0 Hz, 6-axH), 3.82 (1H, dd, J ) 5.3, 11.0 Hz,
6-eqH). This was employed for the next step without further
purification.
er yth r o-2,2-Dim eth yl-5-(la u r oyla m in o)-4-tr id ecyl-1,3-
d ioxa n e (()-(4R*,5S*)-4b. A mixture of 4a (46.7 mg, 0.149
mmol), methyl laurate (1.80 g), and immobilized Candida
antarctica lipase14 (100 mg) was stirred at 60 °C under a
reduced pressure (8 mmHg) for 20 h. The mixture was directly
charged on a silica gel column (5 g). Elution with hexane
afforded the recovery of methyl laurate. Further elution with
hexanes-ethyl acetate (1:2) afforded 4b (16.1 mg, 22%) with
the recovery of the starting material. Either 4b or the
recovered 4a showed no optical rotation. 4b: 1H NMR δ 0.87
(6H, t, each 3H, J ) 6.6 Hz, 13′-CH3, 12"-CH3), 1.25 (40H, m),
1.38 and 1.42 [total 6H, each 3H, s, O2C(CH3)2], 1.58 (2H, m,
1′-CH2), 2.16 (2H, t, J ) 7.4 Hz, 2"-CH2), 3.52 (2H, m, 6-axH,
4-H), 3.89 (1H, m, 5-H), 3.91 (1H, dd, J ) 5.0, 8.8 Hz, 6-eqH),
5.33 (1H, d, J ) 8.6 Hz, NHCO).
acetate gave 4c (4.62 g, 84%) as needles, mp 65.0 °C, IR νmax
3275, 3175, 2920, 2850, 1640, 1550, 1465, 1430, 1380, 1370,
1250, 1200, 1165, 1105, 1080, 1050, 1020, 980, 960, 930, 885,
860, 840, 760, 720, 700, 560, 520 cm-1; 1H NMR δ 0.88 (6H, t,
each 3H, J ) 6.6 Hz, 13′-CH3, 12"-CH3), 1.10-1.72 (54H, m),
1.38 and 1.42 [total 6H, each 3H, s, O2C(CH3)2], 2.17 (2H, t, J
) 7.4 Hz, 2"-CH2), 3.46-3.57 (2H, m), 3.82-3.96 (2H, m), 5.28
(1H, d, J ) 8.6 Hz, NHCO). Anal. Found: C, 76.31; H, 13.38;
N, 2.49. Calcd for C37H73NO3: C, 76.62; H, 12.69; N, 2.41%.
er yth r o-2-(Stear oylam in o)-1,3-h exadecan ediol (()-(2R*,
3S*)-2a . A mixture of 4c (4.49 g, 7.74 mmol), methanol (40
mL), and a catalytic amount of p-TsOH was stirred overnight
under Ar at room temp. The disappearance of the starting
material was confirmed by a TLC analysis [silica gel, developed
with chloroform-methanol (12:1)]. The mixture was concen-
trated in vacuo. The residue was diluted with chloroform and
neutralized by the addition of saturated aqueous sodium
hydrogen carbonate solution. After removing insoluble ma-
terials by filtration, the organic layer was washed with brine,
dried over anhydrous sodium sulfate, and concentrated in
vacuo to give 2a (4.14 g, 99%) as a solid. This was recrystal-
lized from chloroform-methanol to give 2a (4.05 g, 97%) as
needles, mp 100.5-101.5 °C, IR νmax 3450, 3375, 3300, 3100,
2940, 2860, 1640, 1550, 1465, 1380, 1280, 1260, 1220, 1200,
1180, 1130, 1110, 1070, 1050, 1030, 940, 850, 725, 700, 580
cm-1; 1H NMR δ 0.88 (6H, t, each 3H, J ) 6.6 Hz), 1.20-1.70
(54H, m), 2.23 (2H, t, J ) 7.5 Hz), 2.61 (1H, d, J ) 6.3 Hz),
2.73 (1H, dd, J ) 4.0, 6.6 Hz), 3.70-3.88 (3H, m), 4.01 (1H,
dt, J ) 11.2, 3.6 Hz), 6.35 (1H, d, J ) 7.3 Hz, NHCO). HRMS
found: 520.5056. Calcd for C34H66NO2 (M+ - 1 - H2O):
520.5089.
er yth r o-3-Acetoxy-2-(stear oylam in o)h exadecyl Acetate
(()-(2R*,3S*)-2b. A mixture of 2a (1.09 g, 2.02 mmol),
anhydrous pyridine (10 mL), acetic anhydride (5 mL), and a
catalytic amount of DMAP was heated for a while to give a
homogeneous mixture and further stirred overnight under Ar
at room temp. The disappearance of the starting material was
confirmed by a TLC analysis [silica gel, developed with
chloroform-methanol (15:1)]. The mixture was diluted with
water (40 mL), cooled with an ice-bath, and filtered. The
residue on the filter was washed successively with water,
cooled hexane, 0.1 N hydrochloric acid, and water. The solid
was dried in vacuo to give 2b (1.23 g, 98%) as a solid. To the
residue was added silica gel (2 g), and the residue was
completely dissolved with a small portion of chloroform-
methanol, subsequently adding a small portion of hexane. The
mixture was charged on a silica gel column (15 g). Elution
with hexanes-ethyl acetate (6:1 to 0:1) to chloroform-
methanol (30:1) afforded 2b (1.20 g, 95%) as a solid. This was
recrystallized from hexanes-ethyl acetate to give 2b (1.08 g,
86%) as needles, mp 90.0-91.0 °C, IR νmax 3300, 3080, 2920,
2850, 1730, 1640, 1550, 1465, 1430, 1370, 1260, 1230, 1180,
1130, 1110, 1080, 1065, 1045, 1025, 980, 960, 940, 920, 880,
1
750, 725, 690, 600 cm-1; H NMR δ 0.88 (6H, t, each 3H, J )
6.3 Hz), 1.15-1.70 (54H, m), 2.05 (3H, s), 2.07 (3H, s), 2.17
(2H, t, J ) 7.3 Hz), 4.04 (1H, dd, J ) 4.0, 11.6 Hz), 4.25 (1H,
dd, J ) 5.9, 11.6 Hz), 4.40 (1H, dddd, J ) 3.7, 5.9, 5.9, 8.9
Hz), 4.89 (1H, dt, J ) 5.9, 5.9 Hz), 5.80 (1H, d, J ) 8.9 Hz,
NHCO). HRMS found: 503.5063. Calcd for C34H65NO (M+
2AcOH): 503.5063.
-
er yth r o-2,2-Dim eth yl-5-(stea r oyla m in o)-4-tr id ecyl-1,3-
d ioxa n e (()-(4R*,5S*)-4c. To a solution of 4a (2.99 g, 9.53
mmol) in THF (50 mL) was added an aqueous sodium acetate
solutioncf.7j (30 mL, 50%) and subsequently under vigorous
stirring was added portionwise stearoyl chloride (3.73 g, 12.3
mmol, 1.3 equiv). The mixture was further vigorously stirred
overnight under Ar at room temp. The disappearance of the
starting material was confirmed by a TLC analysis [silica gel,
developed with chloroform-methanol (12:1)]. The reaction
mixture was poured into brine and extracted with THF three
times. The organic layer was successively washed with brine,
dried over anhydrous sodium sulfate, and concentrated in
vacuo. The residue was purified with a silica gel column
chromatography (270 g). Elution with hexanes-ethyl acetate
(20:1 to 1:2) and subsequently with chloroform-methanol (20:
1) afforded 4c (4.75 g, 86%). Recrystallization from ethyl
Lip a se-Ca ta lyzed Hyd r olysis of 2b (w ith n a tive SC
lip a se A). A mixture of (()-2b (298 mg, 0.478 mmol) and
decane (3 mL) was heated at 100 °C for a while to give a
homogeneous mixture. To the mixture was added 0.1 M
phosphate buffer solution (pH 7.1, 30 mL), and after cooling
to room temp, SC lipase A (Sumitomo Chemical. Co., Ltd., 30
mg)17 was added and the suspension was stirred for 72 h at
30 °C. The progress of the hydrolysis was confirmed by a TLC
analysis [silica gel, developed with chloroform-methanol (15:
1), Rf: 2b, 0.78; 2c, 0.34; 2a , 0.20]. The mixture was diluted
with chloroform. After the organic layer was separated, the
aqueous layer was thoroughly extracted with chloroform. The
combined extract was washed with brine, dried over anhydrous
sodium sulfate, and concentrated in vacuo. The NMR spec-
trum of the crude product indicated that it was a mixture of

Lipase-Catalyzed Enantiomeric Resolution of Ceramides
J . Org. Chem., Vol. 63, No. 20, 1998 6935
diacetate 2b, monoacetate 2c, and diol 2a , judging from the
signals of amide protons. To the mixture was added silica gel
(1 g), and the product was completely dissolved with a small
portion of chloroform-methanol, subsequently adding a small
portion of hexane. The mixture was charged on a silica gel
column (10 g). Elution with hexanes-ethyl acetate (8:1 to 5:1)
101.0-102.0 °C, [R]24 ) -5.92 [c 1.03, chloroform-methanol
D
(9:1)]; IR νmax 3450, 3375, 3300, 3100, 2940, 2860, 1640, 1550,
1465, 1380, 1280, 1260, 1220, 1200, 1180, 1130, 1110, 1070,
1050, 1030, 940, 850, 725, 700, 580 cm-1. Its NMR spectrum
was identical with that of racemate with an exception of the
chemical shift of OH signals. Anal. Found: C, 75.39; H, 13.47;
N, 2.71. Calcd for C34H69NO3: C, 75.64; H, 12.88; N, 2.59%.
A recrystallized sample of (+)-(2S,3R)-2b was hydrolyzed
to 2a . Recrystallization the sample as stated above from
hexanes-ethyl acetate afforded (+)-(2S,3R)-2a as needles, mp
afforded (+)-(2S,3R)-2b (113 mg, 38%) as a solid, [R]22
)
D
+9.85 (c 1.20, chloroform). A small portion of this was
hydrolyzed and converted to the corresponding bis (R)-MTPA
ester (2S,3R)-2d in the following manner.28 DCC (41.2 mg,
200 µmol), (R)-MTPA acid (32.8 mg, 140 µmol), and DMAP
(8.6 mg, 35 µmol) were dissolved in dry dichloromethane (500
µL). A mixture of (+)-(2S,3R)-2a (4.0 mg, 7.4 µmol) and a half
portion of the reagent solution (250 µL) as above was stirred
for 2 h at room temp. The progress of esterification was
confirmed by TLC analyses [silica gel, developed with chloro-
form-methanol (15:1) and hexanes-ethyl acetate (4:1)]. Hex-
ane was added to the mixture, and they were charged onto a
silica gel column. Elution with hexanes-ethyl acetate (1:0 to
8:1) afforded crude 2d . Further purification by preparative
TLC (hexanes-ethyl acetate 10:1, developed five times, Rf 0.4-
0.6) afforded 2d (5.8 mg, 81%) as colorless oil. It was
confirmed that an absolute 1:1 mixture of the diastereomers
of 2d was obtained from (()-2a through this operation. The
ee of 2a [from (2S,3R)-2b] was estimated from HPLC analysis
and the 1H NMR spectrum of 2d . HPLC (column, Senshu Pak
PEGASIL silica 60-5; solvent, hexane-tetrahydrofuran ) 15:
1; flow rate 1.0 mL/min): tR ) 25.1 min [(2R,3S), 6.3%], 28.1
min [(2S,3R), 93.7%]. Accordingly, the ee of (2S,3R)-2b was
estimated to be 87%. The 1H NMR spectrum (400 MHz, J EOL
J NM R-400, CDCl3) of a diastereomeric mixture of 2d prepared
from (()-2a was as follows: δ 0.88 (6H, t, each 3H, J ) 6.6
Hz), 1.00-1.80 (54H, m), 1.99 [1H, t, J ) 7.6 Hz (2S,3R)], 2.00
[1H, t, J ) 7.6 Hz (2R,3S)], 3.47 [1.5 H, br s (2R,3S)], 3.48
[1.5 H, br s (2S,3R)], 3.52 [1.5H, br s (2S,3R)], 3.53 [1.5H, br
s (2R,3S)], 4.08 [0.5H, dd, J ) 6.9, 11.5 Hz (2R,3S)], 4.24 [0.5H,
dd, J ) 5.9, 11.5 Hz (2S,3R)], 4.31 [0.5H, dd, J ) 3.6, 11.5 Hz
(2S,3R)], 4.31 [0.5H, dd, J ) 4.0, 11.5 Hz (2R,3S)], 4.46 [0.5H,
m (2R,3S)], 4.52 [0.5H, m (2S,3R)], 5.13 [0.5H, dt, J ) 5.9, 5.9
Hz (2S,3R)], 5.13 [0.5H, dt, J ) 5.8, 5.8 Hz (2R,3S)], 5.26 [0.5H,
d, J ) 8.9 Hz (2R,3S)], 5.33 [0.5H, d, J ) 8.9 Hz (2S,3R)], 7.35-
7.55 (10H, m). J udging from the amide protons at δ 5.26 for
(2R,3S)-2d (ca. 6%) and δ 5.33 for (2S,3R)-2d (ca. 94%), the
ee of (2S,3R)-2b was estimated to be ca. 88%, and this value
was in good accordance with that which was concluded from
the HPLC analysis.
103.0-104.5 °C, [R]23 ) +6.09 [c 1.00, chloroform-methanol
D
(9:1)]. Its IR and NMR spectra were identical with those of
(-)-(2R,3S)-2a . Anal. Found: C, 75.34; H, 13.66; N, 2.71.
Calcd for C34H69NO3: C, 75.64; H, 12.88; N, 2.59%.
Hydrolysis of (()-2b with other lipases was carried out in
the same manner. Results with Pseudomonas cepacia lipase
(Amano PS) were listed in Table 1. Candida antarctica (Novo
SP525), Aspergillus niger (Amano A), and pig pancreatic
(Sigma) lipases gave no hydrolyzed products.
Lip a se-Ca ta lyzed Hyd r olysis of 2b (w ith im m obilized
SC lip a se A). A mixture of (()-2b (1.01 g, 1.61 mmol) and
decane (10 mL) was heated at 100 °C for a while to give a
homogeneous mixture. To the mixture was added 0.1 M
phosphate buffer solution (pH 7.1, 100 mL), and after cooling
to room temp, the immobilized form of SC lipase A (1.0 g)14
was added, and the suspension was stirred for 4 days at 30
°C. Filtration with suction and extraction as stated above
afforded a crude product, and its NMR spectrum showed only
the mixture of diacetate 2b and monoacetate 2c, but no diol
2a . Purification of the mixture as stated above gave (+)-
(2S,3R)-2b (580 mg, 58%) as a solid, [R]21 ) +7.66 (c 1.02,
D
chloroform). Based on the HPLC analysis and NMR spectrum
of MTPA ester, the ee of (2R,3S)-2b was estimated to be 69%.
On the other hand, monoacetate (2R,3S)-2c (381 mg, 40%)
was obtained, [R]22 ) -12.42 (c 0.99, chloroform). Based on
D
the HPLC analysis and NMR spectrum of MTPA ester, the ee
of (2R,3S)-2c was estimated to be 98%.
Recrystallization of (-)-(2R,3S)-2c as stated above from
hexanes-ethyl acetate afforded (-)-(2R,3S)-2c as needles, mp
80.5-81.0 °C, [R]24 ) -12.64 (c 0.87, chloroform); IR νmax
D
3470, 3320, 3080, 2930, 2850, 1730, 1720, 1640, 1550, 1470,
1370, 1240, 1200, 1180, 1160, 1120, 1070, 1030, 980, 960, 720,
700, 640, 600, 590, 480 cm-1. Its NMR spectrum was identical
with stated above 2c. Anal. Found: C, 74.07; H, 13.09; N,
2.65. Calcd for C36H71NO4: C, 74.30; H, 12.30; N, 2.41%.
Deter m in a tion of th e Absolu te Con figu r a tion . A mix-
ture of (-)-(2R,3S)-2a (57.9 mg, 0.11 mmol) and 2 N hydro-
chloric acid (3 mL) was heated overnight under Ar at 110 °C.
The disappearance of the starting material was confirmed by
Recrystallization from hexanes-ethyl acetate afforded (+)-
(2S,3R)-2b as needles, mp 99.5-100.5 °C, [R]22 ) +9.87 (c
D
1.01, chloroform). Its IR and NMR spectra were identical with
those of racemate. Anal. Found: C, 73.17; H, 12.56; N, 2.37.
Calcd for C38H73NO5: C, 73.14; H, 11.79; N, 2.24%.
a
TLC analysis [silica gel, developed with chloroform-
methanol (15:1)]. The mixture was neutralized by the addition
of aqueous ammonia solution and extracted with chloroform.
The organic layer was washed with brine, dried over anhy-
drous sodium sulfate, and concentrated in vacuo. To the
residue were added excess amounts of acetic anhydride,
pyridine, and a catalytic amount of DMAP, and the resulting
mixture was stirred for 5 h at room temp. The progress of
the acetylation was monitored by a TLC analysis [silica gel,
developed with hexanes-ethyl acetate (1:3)]. The conventional
workup afforded a crude product, which was purified with a
silica gel column (1.5 g). The charge of the crude sample was
stated as above. Elution with hexanes-ethyl acetate (8:1 to
Further elution of the column [hexanes-ethyl acetate (3:
1)] afforded (-)-(2R,3S)-2c (150 mg, 54%) as a solid, [R]23
)
D
1
-6.47 (c 1.13, chloroform); H NMR δ 0.88 (6H, t, each 3H, J
) 6.7 Hz), 1.18-1.78 (54H, m), 2.13 (3H, s), 2.20 (2H, t, J )
7.6 Hz), 2.95 (1H, dd, J ) 5.5, 8.1 Hz), 3.59 (2H, m), 4.04 (1H,
m), 4.81 (1H, dt, J ) 4.8, 7.7 Hz), 6.17 (1H, d, J ) 8.6 Hz,
NHCO). A small portion of this was hydrolyzed and converted
to (2R,3S)-2d , and based on the HPLC analysis and NMR
spectrum of MTPA ester, the ee of (2R,3S)-2c was estimated
to be 49%.
Further elution of the column [hexanes-ethyl acetate (1:
1:3) gave 5a (40.9 mg, 96%) as a solid, [R]23 ) -17.8 (c 0.98,
D
1), subsequently chloroform-methanol (30:1)] afforded (-)-
chloroform) [lit.9e [R]23D ) -16.5 (c 1.2, chloroform) for (2R,3S)-
(2R,3S)-2a (17.5 mg, 7%) as a solid, [R]22 ) -5.71 [c 1.15,
D
5b, [R]24 ) +16 (c 1.0, chloroform) for (2S,3R)-5b]. This was
D
chloroform-methanol (9:1)]. Based on the NMR spectrum and
HPLC analysis of MTPA ester, the ee of (2R,3S)-2a was
estimated to be 98%.
recrystallized from hexanes-ethyl acetate to give 5a (34.0 mg,
73%) as needles, mp 97.0-97.5 °C, IR νmax 3300, 3250, 2900,
2850, 1730, 1630, 1570, 1460, 1440, 1370, 1330, 1280, 1240,
Recrystallization of (-)-(2R,3S)-2a as stated above from
hexanes-ethyl acetate afforded (-)-(2R,3S)-2a as needles, mp
1130, 1120, 1070, 1050, 1020, 880, 720, 700, 550, 520 cm-1
;
1H NMR δ 0.88 (6H, t, each 3H, J ) 6.6 Hz), 1.00-1.70 (24H,
m), 2.04 (3H, s), 2.06 (3H, s), 2.07 (3H, s), 4.05 (1H, dd, J )
4.0, 11.5 Hz), 4.25 (1H, dd, J ) 5.9, 11.5 Hz), 4.40 (1H, dddd,
J ) 4.0, 5.3, 5.9, 9.2 Hz), 4.89 (1H, dt, J ) 5.3, 7.6 Hz), 5.85
(1H, d, J ) 9.2 Hz, NHCO). Anal. Found: C, 66.03; H, 10.97;
N, 3.66. Calcd for C22H41NO5: C, 66.13; H, 10.34; N, 3.51%.
(28) For the preparation of bis MTPA esters, the use of DCC and
MTPA acid was essential. In the case of the conventional MTPA
chloride, a significant amount of byproduct whose long chain acyl group
had migrated from amino group to hydroxyl groups made the precise
estimation of ee of the products impossible.

6936 J . Org. Chem., Vol. 63, No. 20, 1998
Bakke et al.
er yth r o-3-O-Acetyl-2-(stea r oyla m in o)-1,3-h exa d eca n e-
d iol (()-(2R*,3S*)-2c. This was essentially prepared accord-
ing to the published procedure27b starting from (()-(2R*,3S*)-
2a . Intermediates: erythro-2-(stearoylamino)-1-O-trityl-1,3-
hexadecanediol [mp 82.0-83.0 °C, IR νmax 3533, 3291, 2920,
2851, 1655, 1631, 1543, 1468, 1450, 1082, 745, 706, 633, 475
was concentrated in vacuo to a small volume, subsequently
adding 2 N hydrochloric acid (90 mL). The two-phase mixture
was stirred for 1 h under Ar at room temp. The mixture was
extracted with ether, and the organic layer was successively
washed with saturated aqueous sodium hydrogen carbonate
solution and brine, dried over anhydrous sodium sulfate, and
concentrated in vacuo. The residue was purified with a silica
gel column chromatography (13 g). Elution with hexanes-
ethyl acetate (100:1 to 30:1) afforded 7 (0.970 g, 87%). 1H NMR
δ 0.87 (3H, t, J ) 6.6 Hz), 1.15-1.40 (20H, m), 1.50 (2H, tt, J
) 6.9, 6.9 Hz), 2.33 (2H, ddt, J ) 1.3, 6.9, 6.9 Hz), 6.12 (1H,
ddt, J ) 1.3, 7.9, 15.5 Hz), 6.85 (1H, dt, J ) 6.9, 15.5 Hz), 9.50
(1H, d, J ) 7.9 Hz). Its NMR spectrum was identical with
that reported previously.30
er yth r o-(E)-3-Hydr oxy-2-am in o-4-octadecen oic Acid (()-
(2R*,3S*)-8. This was prepared according to the slight
modification of the published procedure.31 To a cooled solution
(0 °C) of 1.68 N n-butyllithium in hexane (2.70 mL, 4.53 mmol,
1.1 equiv) in freshly distilled THF (11 mL) was added distilled
diisopropylamine (1.34 mL, 9.57 mmol, 2.4 equiv). The color-
less solution was then stirred for 30 min at 0 °C, cooled to
-78 °C, and treated dropwise with a solution of N,N-bis-
(trimethylsilyl)glycine trimethylsilyl ester32 (6, 1.18 g, 4.04
mmol) in THF (8 mL). The resulting light-yellow reaction
mixture was stirred for 1 h at -78 °C and subsequently treated
with 7 (1.24 g, 5.18 mmol, 1.3 equiv) in THF (9 mL). Stirring
was continued for 2 h at -78 °C and for 1 h at 0 °C.
Subsequently, the solution was acidified with saturated am-
monium chloride solution. The colorless solid was collected
by a suction filtration, and the solid was repeatedly washed
with ethyl acetate and water to give 8 (114 mg). A combined
filtrate and washings as a two-phase mixture was cooled
overnight at -10 °C. The light-yellow precipitates were
collected by a suction filtration, and the solid was repeatedly
washed with cold ethyl acetate to give 8 (1.07 g, total 1.19 g),
mp 177.0-180.0 °C [lit.4a 192-193 °C]. This was employed
for the next step without further purification.
1
cm-1; H NMR δ 0.88 (6H, t, each 3H, J ) 6.8 Hz), 1.0-1.7
(54H, m), 2.24 (2H, t, J ) 7.6 Hz), 2.97 (1H, br), 3.25 (1H, dd,
J ) 3.3, 9.9 Hz), 3.52 (1H, dd, J ) 3.0, 9.9 Hz), 3.59 (1H, m),
3.95 (1H, dt, J ) 3.8, 7.8 Hz), 6.30 (1H, d, J ) 8.3 Hz, NHCO),
7.2-7.4 (15H, m). Anal. Found: C, 81.41; H, 10.72; N, 1.79.
Calcd for C53H83NO3: C, 81.38; H, 10.69; N, 1.79%.] and
erythro-3-O-acetyl-2-(stearoylamino)-1-O-trityl-1,3-hexadec-
anediol [mp 93.0-94.0 °C, IR νmax 3280, 2919, 2850, 1741,
1639, 1542, 1467, 1449, 1372, 1229, 1079, 1035, 764, 746, 702,
1
633 cm-1; H NMR δ 0.88 (6H, t, each 3H, J ) 6.6 Hz), 1.0-
1.7 (54H, m), 1.90 (3H, s), 2.12 (2H, t, J ) 7.6 Hz), 3.16 (1H,
dd, J ) 4.3, 9.6 Hz), 3.27 (1H, dd, J ) 3.6, 9.6 Hz), 4.30 (1H,
m), 5.05 (1H, dt, J ) 4.6, 7.2 Hz), 5.73 (1H, d, J ) 9.3 Hz,
NHCO), 7.2-7.4 (15H, m). Anal. Found: C, 80.42; H, 10.36;
N, 1.71. Calcd for C55H85NO4: C, 80.14; H, 10.39; N, 1.70%.]
The final product was a mixture of 2c and 1-O-acetylated
regioisomer 2e (83:17). Purification by silica gel chromatog-
raphy and recrystallization as stated above afforded (()-2c
(53%) as needles. Mp 79.5-80.0 °C. Its IR and NMR spectra
were identical with (-)-2c as described above. Anal. Found:
C, 74.46; H, 12.57; N, 2.47. Calcd for C36H71NO4: C, 74.30;
H, 12.30; N, 2.41%. The detailed spectrum and physical
properties of the regioisomer 2e were described bellow.
Lip a se-Ca ta lyzed Acetyla tion of 2a (w ith im m obilized
SC lip a se A). A mixture of 2a (30.2 mg, 0.056 mmol), THF
(dried over molecular sieves 4A and passed through a neutral
alumina column, 1.7 mL), vinyl acetate (freshly distilled, 1.0
mL) and immobilized SC lipase A (30.0 mg) was stirred for 22
h at 30 °C. The progress of the reaction was confirmed by a
TLC analysis [silica gel, developed with chloroform-methanol
(15:1)]. Rf of the product (0.40) was slightly larger than that
of 2c (0.34) as described above. The reaction mixture was
filtered by suction, and the filtrate was concentrated in vacuo.
The NMR spectrum of the crude product showed no signals of
regioisomer 2c and diacetate 2b. The mixture was completely
dissolved with a small portion of chloroform, subsequently
adding a small portion of hexane. The mixture was charged
on a silica gel column (1.5 g). Elution with hexanes-ethyl
acetate (7:1 to 2:1) afforded 2e (30.1 mg, 93%) as a solid. This
product showed no optical rotation. This was recrystallized
from hexanes-ethyl acetate to give 2e (25.0 mg, 77%) as
needles, mp 100.0-100.5 °C, IR νmax 3282, 2918, 2849, 1737,
er yth r o-(E)-2-Am in o-4-octadecen e-1,3-diol (()-(2R*,3S*)-
9. A mixture of 8 (1.19 g), THF (35 mL), and lithium
aluminum hydride (1.40 g, 36.9 mmol, 9.8 equiv) was stirred
for 2 days under Ar at 70 °C. The reaction was quenched by
adding successively water (1.4 mL), 10% sodium hydroxide
solution (1.4 mL), and water (2.0 mL) with ice-cooling and
further stirred for 1 h at room temp. The mixture was filtered
by a suction, and the solid was repeatedly washed with
chloroform-methanol and ethyl acetate. A combined filtrate
and washings as a two-phase mixture were separated, and the
organic layer was washed with brine, dried over sodium
sulfate, and concentrated in vacuo. The residue was purified
with silica gel column chromatography (25 g). Elution with
chloroform-methanol (20:1 to 1:1) afforded crude 9 (632 mg)
as a solid. This was recrystallized from hexanes-ethyl acetate
to give 9 (469 mg, two steps 39%) as needles, mp 63.0-63.5
1
1647, 1544, 1468, 1276, 1084, 778, 455, 423 cm-1; H NMR δ
0.88 (6H, t, each 3H, J ) 6.6 Hz), 1.0-1.7 (54H, m), 2.08 (3H,
s), 2.09 (1H, br), 2.20 (2H, t, J ) 7.6 Hz), 3.63 (1H, dt, J ) 4.3,
7.6 Hz), 4.13 (1H, m), 4.18 (1H, dd, J ) 3.6, 11.2 Hz), 4.37
(1H, dd, J ) 6.4, 11.2 Hz), 5.93 (1H, d, J ) 7.9 Hz, NHCO).
1
°C [lit.4a 71-73 °C], H NMR δ 0.87 (3H, t, J ) 6.8 Hz), 1.0-
Anal. Found: C, 74.22; H, 12.47; N, 2.34. Calcd for C36H71
NO4: C, 74.30; H, 12.30; N, 2.41%.
-
1.5 (22H, m), 2.04 (2H, dt, J ) 6.6, 6.9 Hz), 2.86 (5H, m), 3.68
(2H, m), 4.07 (1H, dd, J ) 5.9, 6.9 Hz), 5.45 (1H, dd, J ) 6.9,
15.2 Hz), 5.74 (1H, dt, J ) 6.6, 15.2 Hz). Its NMR spectrum
was identical with that reported previously.4b
(E)-2-Hexa d ecen a l 7. This was prepared according to the
published procedure29 with a slight modification. To a suspen-
sion of Cp2Zr(H)Cl (2.00 g, 7.74 mmol, 1.6 equiv), and molec-
ular sieves 4A (400 mg) in CH2Cl2 (15 mL) was added a
solution of 1-ethoxyacetylene (Aldrich 50% in hexane, 1.34 g,
9.55 mmol, 2.0 equiv) in CH2Cl2 (2.5 mL). Stirring for 20 min
under Ar at room temp gave an orange suspension. To the
suspension was added a solution of tetradecanal (1.00 g, 4.71
mmol) in CH2Cl2 (2.5 mL) and subsequently was added AgClO4
(60.2 mg, 0.29 mmol, 6.2 mol %). The mixture was further
stirred for 30 min under Ar at room temp. The disappearance
of the starting material was confirmed by a TLC analysis
[silica gel, developed with hexanes-ethyl acetate (10:1)]. The
reaction mixture was diluted with ether and poured into a
saturated aqueous sodium hydrogen carbonate solution. The
er yth r o-(E)-2-(Stear oylam in o)-4-octadecen e-1,3-diol (()-
(2R*,3S*)-1a . N-acylation was carried out according to the
procedure as described for 4a . 1a (99%) was prepared starting
from 9. Recrystallization from hexanes-ethyl acetate gave 1a
(95%) as needles, mp 95.0-96.0 °C [lit.4a 97-97.5 °C], 1H NMR
δ 0.88 (3H, t, each 3H, J ) 6.8 Hz), 1.00-1.70 (52H, m), 2.05
(2H, dt, J ) 6.6, 6.9 Hz), 2.23 (2H, t, J ) 7.6 Hz), 2.15 (2H,
br), 3.70 (1H, dd, J ) 2.6, 10.6 Hz), 3.91 (1H, m), 3.95 (1H, dd,
J ) 4.0, 10.6 Hz), 4.32 (1H, dd, J ) 4.0, 6.3 Hz), 5.52 (1H, dd,
J ) 6.3, 15.5 Hz), 5.78 (1H, dt, J ) 6.6, 15.5 Hz), 6.26 (1H, d,
(30) Stoffel, W.; Melzner, I. Hoppe-Seyler’s Z. Physiol. Chem. 1973,
354, 1626-1632.
(31) Shanzer, A.; Somekh, L.; Butina, D. J . Org. Chem. 1979, 44,
reaction mixture was filtered through
a Celite pad and
extracted with ether three times. The combined organic layer
3967-3969.
(32) Paulsen, H.; Brieden, M.; Benz, G. Liebigs Ann. Chem. 1987,
565-575.
(29) Maeta, H.; Suzuki, K. Tetrahedron Lett. 1993, 34, 341-344.

Lipase-Catalyzed Enantiomeric Resolution of Ceramides
J . Org. Chem., Vol. 63, No. 20, 1998 6937
J
) 7.3 Hz). Its NMR spectrum was identical with that
1.60 (4H, br), 2.03 (2H, dt, J ) 6.8, 6.8 Hz), 2.10 (3H, s), 2.20
(2H, dt, J ) 2.4, 7.8 Hz), 2.77 (1H, br), 3.65 (2H, m), 4.12 (1H,
m), 5.28 (1H, dd, J ) 7.3, 7.3 Hz), 5.47 (1H, ddt, J ) 1.5, 7.8,
15.6 Hz), 5.77 (1H, dt, J ) 6.8, 15.6 Hz), 5.95 (1H, d, J ) 8.3
Hz, NHCO). Its NMR spectrum was identical with that
reported previously.27b Based on the HPLC analysis and NMR
spectrum of MTPA ester, the ee of (2R,3S)-1c was estimated
to be 17%.
reported previously.4b
er yth r o-3-Acetoxy-2-(stea r oyla m in o)-4-octa d ecen yl Ac-
eta te (()-(2R*,3S*)-1b. Acetylation was carried out according
to the procedure as described for 2a . 1b (95%) was prepared
starting from 1a . Recrystallization from hexanes-ethyl ac-
etate gave 1b (89%) as needles, mp 105.0-107.0 °C, IR νmax
3325, 2925, 2850, 1730, 1640, 1540, 1470, 1420, 1380, 1320,
1270, 1240, 1180, 1160, 1140, 1080, 1030, 980, 970, 920, 910,
Further elution of the column [hexanes-ethyl acetate (1:1)
890, 820, 800, 720, 680, 610, 600 cm-1 1H NMR (400 MHz,
;
and subsequently chloroform-methanol (30:1)] afforded (+)-
(2R,3S)-1a (16.3 mg, 12%) as a solid, mp 95.0-96.0 °C, [R]22
CDCl3) δ 0.88 (6H, t, each 3H, J ) 6.8 Hz), 1.1-1.5 (48H, m),
1.59 (4H, m), 2.02 (2H, dt, J ) 6.8, 7.3 Hz), 2.05 (3H, s), 2.06
(3H, s), 2.16 (2H, t, J ) 7.6 Hz), 4.03 (1H, dd, J ) 3.9, 11.5
Hz), 4.30 (1H, dd, J ) 5.9, 11.5 Hz), 4.45 (1H, m), 5.27 (1H,
dd, J ) 5.9, 6.4 Hz), 5.38 (1H, ddt, J ) 1.5, 5.9, 15.1 Hz), 5.60
(1H, d, J ) 9.3 Hz), 5.79 (1H, dt, J ) 6.8, 15.1 Hz). Its NMR
spectrum was identical with an authentic sample derived from
commercially available D-erythro-sphingosine. Anal. Found:
C, 73.68; H, 12.02; N, 2.25. Calcd for C40H75NO5: C, 73.91;
H, 11.63; N, 2.15%.
D
) +3.08 [c 0.81, chloroform-methanol (9:1)]; IR νmax 3450,
3375, 3300, 3100, 2940, 2860, 1640, 1550, 1465, 1380, 1280,
1260, 1220, 1200, 1180, 1130, 1110, 1070, 1050, 1030, 940, 850,
725, 700, 580 cm-1. Its NMR spectrum was identical with that
of racemate. Based on the HPLC analysis and of NMR
spectrum MTPA ester, the ee of (2R,3S)-1a was estimated to
be 98%.
A sample of (-)-(2S,3R)-1b was hydrolyzed to (-)-(2S,3R)-
1a as a solid, mp 95.0-97.0 °C [lit.9c 97-98 °C], [R]23D ) -2.78
[c 0.88, chloroform-methanol (9:1)] [lit.9c [R]25 ) -3.1 (c 1.1,
Lip a se-Ca ta lyzed Hyd r olysis of 1b (w ith n a tive SC
lip a se A). A mixture of (()-1b (152.2 mg, 0.234 mmol) and
decane (1.5 mL) was heated at 100 °C for a while to give a
homogeneous mixture. To the mixture was added 0.1 M
phosphate buffer solution (pH 7.1, 15 mL), and after cooling
to room temp, SC lipase A (30 mg) was added, and the
suspension was stirred for 3 days at 30 °C. To the mixture
was further added SC lipase A (20 mg), and the suspension
was stirred for 1 day at 30 °C. The progress of the hydrolysis
was confirmed by a TLC analysis [silica gel, developed with
chloroform-methanol (15:1)]. The mixture was diluted with
chloroform. After organic layer was separated, the aqueous
layer was thoroughly extracted with chloroform. The com-
bined extract was washed with brine, dried over anhydrous
sodium sulfate, and concentrated in vacuo. The NMR spec-
trum of the crude product indicated that it was a mixture of
diacetate 1b, monoacetate 1c, and diol 1a , judging from the
signals of amide protons. To the mixture was added silica gel
(500 mg), and the product was completely dissolved with a
small portion of chloroform, subsequently adding a small
portion of hexane. The mixture was charged on a silica gel
column (15 g). Elution with hexane-chloroform (1:1) and
subsequently with hexanes-ethyl acetate (8:1 to 5:1) afforded
(-)-(2S,3R)-1b (38.1 mg, 25%) as a solid, [R]24D ) -8.20 (c 0.84,
chloroform). A small portion of this was hydrolyzed and
converted to the corresponding bis (R)-MTPA ester (2S,3R)-
1d in the same manner as described for 2d . The ee of (2S,3R)-
1a [from (2S,3R)-1b] was estimated from HPLC analysis and
D
chloroform)]. Its NMR spectrum was identical with that
reported previously.9c
Hydrolysis of (()-1b with other lipases was attempted in
the same manner, however, P. cepacia (Amano PS), C. ant-
arctica (Novo SP525), A. niger (Amano A), pig pancreatic
(Sigma) lipases, and immobilized SC lipase A gave no hydro-
lyzed products.
Lip a se-Ca ta lyzed Hyd r olysis of (+)-(2S,3R)-2b (w ith
n a tive SC lip a se A). A mixture of (+)-(2S,3R)-2b (96% ee,
40.0 mg, 0.064 mmol) and decane (0.4 mL) was heated at 100
°C for a while to give a homogeneous mixture. To the mixture
was added 0.1 M phosphate buffer solution (pH 7.1, 4.0 mL),
and after cooling to room temp, SC lipase A (150 mg) was
added, and the suspension was stirred for 3 days at 30 °C. To
the mixture was further added SC lipase A (50 mg), and the
suspension was stirred for 3 day at 30 °C. Purification as
stated above afforded (+)-(2S,3R)-2b (10.4 mg) and (+)-
(2S,3R)-2c (23.4 mg, 85%) as a solid. Based on the HPLC
analysis and NMR spectrum of MTPA ester, the ee of (+)-
(2R,3S)-2c was enhanced to be >99%, by the hydrolytic
removal of contaminating (-)-(2R,3S)-2b into diol 2a .
Recrystallization from hexanes-ethyl acetate afforded (+)-
(2S,3R)-2c as needles, mp 79.0-81.0 °C, [R]22 ) +13.08 (c
D
1.05, chloroform). Its IR and NMR spectra were identical with
those of racemate. Anal. Found: C, 74.08; H, 12.41; N, 2.37.
Calcd for C36H71NO4: C, 74.30; H, 12.30; N, 2.41%.
(2S,3R)-1-(2,3,4,6-Tetr a-O-acetyl-â-D-galactopyr an osyl)-
2-(stea r oyla m in o)-1,3-h exa d eca n ed iol (10). Glycosylation
was essentially carried out according to the published proce-
dure.33 To a suspension of molecular sieves 4A (200 mg), (+)-
(2S,3R)-2c (33.2 mg, 0.057 mmol), and tetra-O-acetyl-R-
galactosyl trichloroacetimidate34 (55.5 mg, 0.113 mmol, 2.0
equiv) in CH2Cl2 (0.5 mL) was added a solution of BF3‚OEt2
(14.5 µL, 0.114 mmol, 2.0 equiv) in CH2Cl2 (0.5 mL). The
suspension was then stirred for 3 h under Ar at room temp.
The progress of the reaction was confirmed by a TLC analysis
[silica gel, developed with hexanes-ethyl acetate (1:1)]. The
mixture was diluted with chloroform and poured into a
saturated aqueous sodium hydrogen carbonate solution. The
organic layer was successively washed with brine, dried over
anhydrous sodium sulfate, and concentrated in vacuo. The
residue was completely dissolved with a small portion of
chloroform, and the mixture was charged on a silica gel column
(1.8 g). Elution with hexanes-ethyl acetate (7:1 to 2:1)
afforded (+)-(2S,3R)-2b (16.0 mg, 45%). Further elution of the
chromatography afforded 10 (27.1 mg, 52%), [R]23D ) +3.57 (c
0.82, chloroform). IR (film) νmax 2950, 2850, 1750, 1650, 1540,
1
the H NMR spectrum of 1d . HPLC (same condition for 2d ):
tR ) 31.3 min [(2R,3S), 7.6%], 33.0 min [(2S,3R), 92.4%].
Accordingly, the ee of (2S, 3R)-1b was estimated to be 85%.
1
The H NMR spectrum (400 MHz, CDCl3) of a diastereomeric
mixture of 1d prepared from (()-1a was as follows: δ 0.88
(6H, t, each 3H, J ) 6.6 Hz), 1.20-1.70 (52H, m), 1.98 [2H, m
(2S,3R)], 2.01 [2H, m (2R,3S)], 3.47 [1.5 H, br s (2S,3R)], 3.49
[1.5 H, br s (2R,3S)], 3.51 [1.5H, br s (2R,3S)], 3.52 [1.5H, br
s (2S,3R)], 4.19 [0.5H, dd, J ) 5.4, 11.7 Hz (2R,3S)], 4.26 [0.5H,
dd, J ) 3.7, 11.7 Hz (2R,3S)], 4.33 [0.5H, dd, J ) 4.2, 11.5 Hz
(2S,3R)], 4.38 [0.5H, dd, J ) 5.1, 11.5 Hz (2S,3R)], 4.48 [0.5H,
m (2R,3S)], 4.53 [0.5H, m (2S,3R)], 5.23 [0.5H, d, J ) 8.8 Hz
(2R,3S)], 5.27 [0.5H, d, J ) 9.3 Hz (2S,3R)], 5.28 [0.5H, dd, J
) 7.8, 15.1 Hz (2S,3R)], 5.38 [0.5H, dd, J ) 7.3, 7.8 Hz (2S,3R)],
5.39 [0.5H, dd, J ) 3.9, 8.3 Hz (2R,3S)], 5.39 [0.5H, dd, J )
8.3, 14.7 Hz (2R,3S)], 5.80 [0.5H, dt, J ) 6.8, 15.1 Hz (2S,3R)],
5.92 [0.5H, dt, J ) 6.8, 14.7 Hz (2R,3S)], 7.36-7.51 (10H, m).
J udging from the olefin protons (H-5) at δ 5.80 for (2S,3R)-1d
(ca. 93%) and δ 5.92 for (2R,3S)-1d (ca. 7%), the ee of (2S, 3R)-
1b was estimated to be ca. 86%.
(-)-(2S,3R)-1b was finally obtained as needles by recrys-
tallization of the sample as above, mp 106.0-108.0 °C. Its IR
and NMR spectra were identical with those of the racemate.
Further elution of the column [hexanes-ethyl acetate (3:
1)] afforded (+)-(2R,3S)-1c (79.7 mg, 58%) as a solid, mp 84.0-
85.0 °C, [R]25D ) +3.08 (c 1.15, chloroform); 1H NMR (400 MHz,
CDCl3) δ 0.88 (6H, t, each 3H, J ) 6.8 Hz), 1.1-1.4 (48H, m),
1460, 1370, 1230, 1050 cm-1 1H NMR (400 MHz, CDCl3) δ
;
0.87 (6H, t, each 3H, J ) 6.6 Hz), 1.2-1.7 (54H, m), 1.98 (3H,
(33) Koike, K.; Mori, M.; Ito, Y.; Nakahara, Y.; Ogawa, T. Agric. Biol.
Chem. 1990, 54, 2931-2939.
(34) Amvam-Zollo, P.-H.; Sinay, P. Carbohydr. Res. 1986, 150, 199-
212.

6938 J . Org. Chem., Vol. 63, No. 20, 1998
Bakke et al.
s), 2.05 (3H, s), 2.05 (3H, s), 2.06 (3H, s), 2.15 (3H, s), 2.16
(2H, t), 3.59 (1H, dd, J ) 4.4, 10.1 Hz), 3.91 (2H, m, Gal-6),
4.13 (2H, m, Cer-1), 4.27 (1H, m, Cer-2), 4.43 (1H, d, J ) 7.7,
Gal-1), 4.85 (1H, dt, J ) 5.1, 8.1 Hz, Cer-3), 4.99 (1H, dd, J )
3.3, 10.4 Hz, Gal-3), 5.13 (1H, dd, J ) 7.7, 10.4 Hz, Gal-2),
5.38 (1H, d, J ) 3.3 Hz, Gal-4), 5.99 (1H, d, J ) 9.2 Hz, NHCO).
MS (70 eV); m/z(%): 910 (15), 850 (25), 654 (20), 563 (40) [M+
- Gal4Ac - H2O + 1], 489 (25), 330 (100), 163 (35), 43 (40).
HRMS Found: 550.5202. Calcd for C35H68NO3 (M+ - 4AcGal
- OCH2): 550.5196.
Yasushi Takebe of Osaka University, Dr. Yutaka Ohira
of MEC Laboratory, Daikin Industries Ltd., Dr. Hirosato
Takikawa of Science University of Tokyo, and Prof.
Makoto Kiso of Gifu University for their discussion on
ceramide synthesis. Professor Nina Berova of Columbia
University is acknowledged with thanks for her valuable
suggestion on the determination of absolute configura-
tion of our samples. We are indebted to Mr. Takahiro
Sekiguchi’s help on the glycosylation experiment. M.B.
thanks J apan Scholarship Foundation, FURUKAWA
Memorial Funds and Fujiwara Scholarship Fund for
Schlorships. Part of this work was performed through
Special Coordination Funds of the Science and Technol-
ogy Agency of the J apanese Government.
Ack n ow led gm en t. The authors thank Drs. Satoshi
Mitsuda, Takeshi Ishii, and Ayumu Inoue of Sumitomo
Chemical Co., Ltd., for the generous gift of SC lipase A
and discussion. We are indebted to Amano Pharmaceu-
tical Co. and Novo Nordisk Co. for generous gift of
lipases. We are grateful to Prof. Yasuyuki Kita and Dr.
J O980727U