Synthetic studies on glycosphingolipids from protostomia phyla: Synthesis of glycosphingolipids from the parasite Schistosoma mansoni

Article abstract of DOI:10.1248/cpb.58.811

Synthetic access to three neutral glycosphingolipids from the parasite Schistosoma mansoni adult worm has been achieved. These structures differ significantly from those of other parasites and exhibit a unique structural motif termed "schisto-core" consis

Full text of DOI:10.1248/cpb.58.811

June 2010
Regular Article
Chem. Pharm. Bull. 58(6) 811—817 (2010)
811
Synthetic Studies on Glycosphingolipids from Protostomia Phyla:
Synthesis of Glycosphingolipids from the Parasite Schistosoma mansoni
Takayuki KANAYA,a Satomi YAGI,a Frank SCHWEIZER,b Tadahiro TAKEDA,a Fumiyuki KIUCHI,a and
,a
*
Noriyasu HADA
a Faculty of Pharmacy, Keio University; 1–5–30 Shibakoen, Minato-ku, Tokyo 105–8512, Japan: and b Department of
Chemistry, University of Manitoba; Winnipeg, Manitoba, R3T 2N2, Canada.
Received December 28, 2009; accepted March 15, 2010; published online March 16, 2010
Synthetic access to three neutral glycosphingolipids from the parasite Schistosoma mansoni adult worm has
been achieved. These structures differ significantly from those of other parasites and exhibit a unique structural
motif termed “schisto-core” consisting of GalNAcb1→4Glcb1→sequence. We have synthesized glycosphin-
golipids, b-D-GalNAcp-(1→4)-b-D-Glcp-(1→1)Cer (1), b-D-GlcNAcp-(1→3)-b-D-GalNAcp-(1→4)-b-D-Glcp-(1→
1)Cer (2) and b-D-Galp-(1→4)-b-D-GlcNAcp-(1→3)-b-D-GalNAcp-(1→4)-b-D-Glcp-(1→1)Cer (3).
Key words glycosphingolipid; Schistosoma mansoni; schisto-core; chemical synthesis
Investigation of the functional roles of mammalian carbo- but also will provide useful information how changes in the
hydrates in biological processes have been an area of intense glycan structure relate to different stages in the parasite de-
study in recent years.1) In contrast, much less time has been velopment. Based on this background, we have initiated
devoted to structures of glycosphingolipids (GSLs) from in- synthetic research efforts to prepare GSLs (A—F). In this
vertebrates that differ significantly from mammalian glycans. paper we report on the synthesis of GSLs (A—C; 1—3) this
Due to these differences in the glycan structure we have been time. Disaccharide 1 contains the schisto-core sequence Gal-
interested in the relationships between the structure and bio- NAcb1→4Glcb1→Cer while trisaccharide 2 and tetrasac-
logical activity of GSLs from invertebrate species and have charide 3 are elongated glycan sequences that contain a ter-
synthesized oligosaccharides found in various Protostomia minal schisto-core. Oligosaccharides 1—3 serve as molecu-
phyla.2—9) In the course of our studies, we paid attention to lar probes to explore glycosphingolipid-mediated interactions
the presence of unique GSLs found in the parasite, Schisto- containing the schisto core in S. mansoni. The syntheses of
soma mansoni.10) Makaaru et al. identified a novel glycan di- and trisaccharide derivatives were conducted by stepwise
core structure termed “schisto-core” in the GSLs isolated synthesis of suitably protected monosaccharide-based glyco-
from S. mansoni adult worm and they can be extended to syl donors and acceptors. The tetrasaccharide derivative was
other unique glycan sequences (Fig. 1: A—F).11) Moreover, conducted by block synthesis of a disaccharide acceptor and
the parasite egg and the cercariae are a rich source of highly disaccharide donor.
antigenic, multifucosylated GSLs.10,12,13) Studies on the gly-
cobiology of parasitic helminthes can contribute to a better Results and Discussion
understanding of glycolipid-mediated host–parasite interac-
The synthetic scheme for the synthesis of target com-
tions including species specific- and organ-specific infections pounds 1—3 are shown in Charts 1—3. Initially, the per-O-
acylated 2-(trimethylsilyl)ethyl glycoside derivatives 8, 16
and 27 were prepared that serve as synthetic intermediates
Reagents and conditions: (a) TMSOTf, MS 4 Å, CH2Cl2, 97%; (b) Zn, Ac2O, AcOH,
85%; (c) 1), Pd–C/H2, THF–MeOH 2), Ac2O, Pyr., 95%; (d) 1), TFA, CH2Cl2 2),
CCl3CN, DBU, CH2Cl2, 73%; (e) TMSOTf, MS 4 Å, CH2Cl2, 57%; (f) MeONa, diox-
ane/MeOH, 67%.
Fig. 1. Structures of Glycosphingolipids from the Parasite S. mansoni and
Chart 1
Target Compounds 1—3
© 2010 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: hada-nr@pha.keio.ac.jp

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Reagents and conditions: (a) TsOH, CHCl3–MeOH, 60%; (b) guanidine nitrate, NaOMe, CH2Cl2/MeOH, 89%; (c) BDA, NaHSO4·SiO2, CH3CN, 89%; (d) TMSOTf, MS 4 Å,
CH2Cl2, 85%; (e) 1), Zn, Ac2O, AcOH 2), Pd–C/H2, MeOH, AcOH 3) Ac2O, Pyr., 62% (3 steps); (f) 1), TFA, CH2Cl2 2), CCl3CN, DBU, CH2Cl2, 78%; (g) TMSOTf, MS 4 Å,
CH2Cl2, 52%; (h) MeONa, dioxane/MeOH, 86%.
Chart 2
Reagents and conditions: (a) 1) Et3N, MeOH 2) BDA, CSA, CH3CN, MS 3 Å, 72% (2 steps) 3) ClAcCl, CH2Cl2–Pyr., 75% 4) NaBH3CN, MS 3 Å, HCl–Et2O, 86%; (b) TM-
SOTf, MS 4 Å, CH2Cl2, 86%; (c) thiourea, Pyr., EtOH, 85%; (d) Ac2O, Pyr., 82%; (e) 1), TFA, CH2Cl2 2), CCl3CN, DBU, CH2Cl2, 74%, (f) TMSOTf, MS 4 Å, CH2Cl2, 83%; (g)
1), Zn, Ac2O, AcOH 2), Pd–C/H2, MeOH, AcOH 3), Ac2O, Pyr., 42% (3 steps); (h) 1), TFA, CH2Cl2 2), CCl3CN, DBU, CH2Cl2, 61%; (i) TMSOTf, MS 4 Å, CH2Cl2, 34%; (j)
MeONa, dioxane/MeOH, 55%.
Chart 3
for the installation of the ceramide moiety. Glycosylation of accharide derivative 6 was selected as precursor for the syn-
414) with 515) in the presence of trimethylsilyl trifluoro- thesis of trisaccharide 16. Two methods were studied for the
methanesulfonate (TMSOTf)16) and 4 Å molecular sieves selective deacetylation of 6 in the presence of benzoyl- and
(MS 4 Å) in CH2Cl2 gave the desired disaccharide (6) after Troc-protecting groups. Initially, we attempted to selectively
purification in 97% yield. The anomeric proton of the saponify the acetyl groups with p-toluenesulfonic acid17) in
GalNAc unit appeared as a doublet at d 4.30 (d, Jꢀ8.5 Hz). CHCl3–MeOH to provide disaccharide 12 in 60% yield.
Subsequently, the 2,2,2-trichloroethoxycarbonyl (Troc) group However, the yield could be significantly improved by using
was converted to an acetamido moiety by reduction and N- guanidine/guanidinium nitrate reagent (G/GHNO3)18) that
acetylation with Zn–Ac2O–AcOH to afford 7 in 85% yield. provided 12 in 89% yield. Benzylidenation19) of disaccharide
Removal of benzyl group from 7 by catalytic hydrogenolysis 12 produced acceptor 13, which was subjected to glycosyla-
over 10% Pd/C in MeOH and O-acetylation provided 8. Dis- tion with donor 1420) using TMSOTf as promoter to afford

June 2010
813
UV fluorescence and by charring with 10% H2SO4. Column chromatography
was carried out on Silica Gel 60 (E. Merck). 2-(Trimethylsilyl)ethyl 2,3-di-
O-benzoyl-6-O-benzyl-b-D-glucopyranoside (4),14) 3,4,6-tri-O-acetyl-2-deoxy-
2-(2,2,2-trichloroethoxycarbonylamino)-1-O-D-galactopyranosyl
trichloroacetimidate (5),15) 3,4,6-tri-O-acetyl-2-deoxy-2-(2,2,2-trichloroethoxy-
the desired trisaccharide 15 in 85% yield. The b-glycosidic
linkage was assigned on the basis of homonuclear coupling
constants (H-1 of GlcNAc, dꢀ4.96 ppm, JH1,H2ꢀ8.0 Hz). Re-
moval of the Troc-protecting group was achieved with Zn in
a mixture containing Ac2O and AcOH, followed by catalytic carbonylamino)-1-O-
D
-glucopyranosyl trichloroacetimidate (14),19) 2-
(trimethylsilyl)ethyl 3,4,6-tri-O-acetyl-2-deoxy-2-(2,2,2-trichloroethoxycar-
bonylamino)-b-D-glucopyranoside (19)20) and 2,3,4,6-tetra-O-acetyl-1-O-D-
galactopyranosyl trichloroacetimidate (21)21) were prepared as reported.
Benzoylceramide 10 was prepared from phytosphingosine, which was pur-
chased from Degussa (The Netherlands) by the conventional four-step pro-
cedure.24)
hydrogenation over 10% Pd–C in MeOH–AcOH and acetyla-
tion to provide trisaccharide intermediate 16. Tetrasaccharide
intermediate 26 was prepared by block synthesis of a disac-
charide acceptor 13 and disaccharide donor 25. Glycosyl ac-
ceptor 20 was obtained from 1921) by deacetylation, benzyli-
denation, chloroacetylation, and reductive ring-opening of
the benzylidene acetal as previously described.3) TMSOTf-
promoted glycosylation of acceptor 20 with donor 2122) was
carried out in the presence of 4 Å MS in CH2Cl2 and afforded
the desired disaccharide 22 as the sole product in 86% yield.
2-(Trimethylsilyl)ethyl 3,4,6-tri-O-acetyl-2-deoxy-2-(2,2,2-trichloro-
ethoxycarbonylamino)-b-D-galactopyranosyl-(1→4)-2,3-di-O-benzoyl-6-
O-benzyl-b-D-glucopyranoside (6) A solution of 4 (1.77 g, 3.06 mmol)
and 5 (2.85 g, 4.57 mmol) containing activated 4 Å MS (5.0 g) in dry CH2Cl2
(25.0 ml) was stirred under an atmosphere of argon for 18 h at room temper-
ature, then cooled to ꢁ40 °C. TMSOTf (62.3 ml, 0.34 mmol) was added, and
the mixture was stirred for 3 h at ꢁ40 °C, then neutralized with Et3N. The
solids were filtrated off and washed with CHCl3. The combined filtrate and
washings were successively washed with brine, dried (MgSO4) and concen-
trated. The product was purified by silica gel column chromatography using
2 : 1 hexane–ethyl acetate as eluent to give 6 as an amorphous powder
(3.10 g, 97%). [a]D25 ꢁ16.2 (cꢀ1.2, CHCl3). 1H-NMR (500 MHz, CDCl3) d:
8.01—7.40 (15H, m, 3Ph), 5.64 (1H, t, J2,3ꢀJ3,4ꢀ9.8 Hz, H-3 of Glc), 5.46
(1H, dd, J1,2ꢀ7.9 Hz, H-2 of Glc), 5.10 (1H, d, J3,4ꢀ3.7 Hz, H-4 of GalN),
4.94, 4.49 (2H, each d, benzylmethylene), 4.78—4.67 (4H, m, H-1 of Glc,
H-3 of GalN, CH2CCl3), 4.30 (1H, d, J1,2ꢀ8.5 Hz, H-1 of GalN), 4.21 (1H, t,
1
The b-glycosidic linkage in 22 was confirmed by H-NMR
spectroscopy. The anomeric proton of the galactose moiety
appeared as a doublet with a homonuclear coupling constant
of 7.9 Hz. Deblocking of the 3-O-chloroacetyl group in 22
was achieved with thiourea and the resulting alcohol 23 was
converted into ester 24 by acetylation. Selective removal of
the 2-(trimethylsilyl)ethyl group in 24 was achieved with tri-
fluoroacetic acid in CH2Cl2, and treatment with CCl3CN in
the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)23)
to provide the corresponding a-trichloroacetimidate 25 in
74% yield. Tetrasaccharide derivative 26 was synthesized by
coupling of donor 25 with disaccharide acceptor 13 in 83%
yield. Deblocking of 26 was achieved using the same condi-
tions as described for trisaccharide 16 produced the per-O-
acylated tetrasaccharide derivative 27.
J4,5ꢀ9.8 Hz, H-4 of Glc), 4.11—4.06 (2H, m, NH, CH2CH2Si(CH3)3), 3.86
(1H, dd, H-6a of Glc), 3.75 (1H, d, H-6b of Glc), 3.66—3.61 (3H, m, H-5 of
Glc, H-2 of GalN, CH2CH2Si(CH3)3), 3.50 (1H, t, J5,6ꢀ8.9 Hz, H-5 of
GalN), 3.54 (2H, d, H-6 of GalN), ꢁ0.01 (9H, s, Si(CH3)3). 13C-NMR
(125 MHz, CDCl3) d: 170.0ꢂ2, 165.3, 165.1, 153.7, 137.5, 132.9ꢂ3, 130.1,
129.7ꢂ3, 129.6ꢂ3, 129.2ꢂ3, 129.0, 128.7ꢂ3, 128.2, 128.1ꢂ3, 100.7 (C-1
of Glc), 100.2 (C-1 of GalN), 95.6, 74.9, 74.4, 74.3, 73.6, 73.2, 71.8, 70.1,
69.9, 67.4, 67.2, 65.9, 60.4, 52.3, 20.6, 20.44, 20.36, 17.9, 14.1, ꢁ1.52.
MALDI-TOF-MS: Calcd for C47H56Cl3NO17SiNa: ([MꢃNa]ꢃ) m/z 1062.2.
Found 1062.3. HR-FAB-MS: Calcd for C47H56Cl3NO17SiNa: ([MꢃNa]ꢃ)
m/z 1062.2280. Found 1062.2239.
Next, for the selective removal of the 2-(trimethylsi-
lyl)ethyl group, the fully acylated oligosaccharides 8, 16 and
27 were converted to the glycosyl imidate 9, 17 and 28. TM-
SOTf-promoted glycosylation of phytoceramide acceptor
1024) with glycosyl donors 9, 17 and 28 afforded the desired
b-glycosides 11 (57%), 18 (52%) and 29 (34%) yields, re-
spectively. Finally, standard deacetylation and purification by
column chromatography on Sephadex LH-20 furnished gly-
colipids 1—3.
2-(Trimethylsilyl)ethyl
2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-b-D-
galactopyranosyl-(1→4)-2,3-di-O-benzoyl-6-O-benzyl-b-D-glucopyra-
noside (7) To a solution of 6 (1.0 g, 0.18 mol) in Ac2O (10 ml) and AcOH
(5 ml) was added zinc powder (400 mg). The reaction mixture was stirred for
18 h at 40 °C. After completion of the reaction, the mixture was filtered and
extracted with CHCl3. The solution was washed with water, dried (MgSO4),
and concentrated. The filtrate was concentrated and purified by silica gel
column chromatography using 3 : 1 toluene–acetone as the eluent to give 7
1
as syrup (737 mg, 85%). [a]D25 ꢁ1.0 (cꢀ9.9, CHCl3). H-NMR (500 MHz,
CDCl3) d: 7.96—7.33 (15H, m, 3 Ph), 5.60 (1H, t, J2,3ꢀJ3,4ꢀ9.2 Hz, H-3 of
Glc), 5.39 (1H, dd, J1,2ꢀ7.9 Hz, H-2 of Glc), 5.06 (1H, d, J3,4ꢀ3.7 Hz, H-4
of GalN), 4.92 (1H, dd, J2,3ꢀ10.0 Hz, H-3 of GalN), 4.85—4.83 (2H, m,
Conclusion
In summary, a systematic and integrated approach for the
synthesis of three glycosphingolipids 1—3 found in the para- NH, benzylmethylene), 4.67 (1H, d, H-1 of Glc), 4.55 (1H, d, J1,2ꢀ7.9 Hz,
H-1 of GalN), 4.51 (1H, d, benzylmethylene), 4.17 (1H, t, J4,5ꢀ9.2 Hz, H-4
of Glc), 4.04—3.98 (1H, m, CH2CH2Si(CH3)3), 3.77—3.66 (4H, m, H-5, 6
of Glc, H-2 of GalN), 3.60—3.55 (1H, m, CH2CH2Si(CH3)3), 3.48 (1H, t,
site, Schistosoma mansoni has been accomplished. The syn-
thetic strategy described may also find use for conjugation of
the glycan portion of S. mansoni to other non-lipid-based
J5,6ꢀ7.3 Hz, H-5 of GalN), 3.40—3.32 (2H, m, H-6 of GalN), 0.95—0.82
(m, 2H, CH2CH2Si(CH3)3), ꢁ0.07 (9H, s, Si(CH3)3). 13C-NMR (125 MHz,
CDCl3) d: 170.1, 170.0, 169.8, 165.3, 165.1, 137.9, 132.9, 129.9, 129.7ꢂ3,
129.6ꢂ3, 128.7ꢂ2, 128.6ꢂ2, 128.3ꢂ3, 128.2ꢂ2, 128.1ꢂ3, 100.5 (C-1 of
Glc), 99.9 (C-1 of GalN), 74.8, 74.5, 73.6, 73.3, 71.9, 70.2, 69.8, 67.5, 67.4,
66.0, 60.5, 51.4, 23.2, 20.6, 20.5, 20.4, 17.9, ꢁ1.55. MALDI-TOF-MS:
Calcd for C46H57NO16SiNa, ([MꢃNa]ꢃ) m/z 930. Found 930. HR-FAB-MS:
Calcd for C46H57NO16SiNa, ([MꢃNa]ꢃ) m/z 930.3525. Found 930.3539.
carrier molecules. Biological testing for schistosomasis using
GSLs 1—3 is currently in progress and results will be re-
ported in detail elsewhere. It is expected that the prepared
glycosphingolipids will find use in future studies designed to
reveal the relationships between the structure and biological
activity for specific antibody detection in patients with schis-
tosomasis.
2-(Trimethylsilyl)ethyl
2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-b-D-
galactopyranosyl-(1→4)-6-O-acetyl-2,3-di-O-benzoyl-b-D-glucopyra-
noside (8) To a solution of 7 (340 mg, 0.37 mmol) in MeOH (4.0 ml) was
Experimental
General Methods Optical rotations were measured with a Jasco P-1020 hydrogenolysed under hydrogen in the presence of 10% Pd/C (400 mg) for
digital polarimeter. 1H- and 13C-NMR spectra were recorded with a JMN 18 h at room temperature, then filtered and concentrated. The residue was
A500 FT NMR spectrometer and a JEOL JNM-ECP600 with Me4Si as acetylated with Ac2O (3.0 ml) in pyridine (4.5 ml). The reaction mixture was
the internal standard for solutions in CDCl3. Matrix assisted laser desorp-
poured into ice H2O and extracted with CHCl3. The extract was washed se-
tion/ionization-time of flight (MALDI-TOF)-MS was recorded on an Ap- quentially with 5% HCl, aq NaHCO3, and brine, dried (MgSO4), and con-
plied Biosystems Voyager RP mass spectrometer. High-resolution mass centrated. The residue was purified by silica gel column chromatography
spectra were recorded on a JEOL JMS-700 under FAB conditions. TLC was using 2 : 1 toluene–acetone as eluent to give 8 as syrup (305 mg, 95%). [a]D25
performed on Silica Gel 60 F254 (E. Merck) with detection by quenching of
ꢃ16.3 (cꢀ7.5, CHCl3). 1H-NMR (500 MHz, CDCl3) d: 8.04—7.36 (15H,

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Vol. 58, No. 6
NaOMe (15.0 mg) at 45 °C. The mixture was stirred for 5 h and then neutral-
ized with Amberlite IR 120 [Hꢃ]. The mixture was filtered and concen-
trated. The product was purified by Sephadex LH-20 column chromatogra-
phy in MeOH to give 1 as white solid (14.5 mg, 67%). [a]D25 ꢁ26.4 (cꢀ0.4,
1 : 1 CHCl3-MeOH). mp 128—129 °C. 1H-NMR (600 MHz, 1 : 1 CDCl3-
CD3OD) d: 4.48 (1H, d, Jꢀ8.0 Hz, H-1 of GalN), 4.30 (1H, d, Jꢀ7.7 Hz, H-
1 of Glc). MALDI-TOF-MS: Calcd for C48H92N2O14Na: ([MꢃNa]ꢃ) m/z
944. Found 944. HR-FAB-MS: Calcd for C48H93N2O14: ([MꢃH]ꢃ) m/z
921.6446. Found 921.6465.
2-(Trimethylsilyl)ethyl 2-Deoxy-2-(2,2,2-trichloroethoxycarbony-
lamino)-b-D-galactopyranosyl-(1→4)-2,3-di-O-benzoyl-6-O-benzyl-b-D-
glucopyranoside (12) (a) To a solution of 6 (1.75 g, 1.68 mmol) was
added TsOH (0.97 g, 3.36 mmol, 2.0 eq) in CHCl3/MeOH (18.0 ml, 9 : 1).
The reaction mixture was stirred for 48 h at room temperature then neutral-
ized with Et3N. The filtrate was washed with brine, dried (MgSO4), and con-
centrated. The filtrate was concentrated and purified by silica gel column
chromatography using 20 : 1 chloroform–methanol as the eluent to give 12
as syrup (0.93 g, 60.4%).
m, 3Ph), 6.01 (1H, d, NH), 5.74 (1H, t, J2,3ꢀJ3,4ꢀ9.2 Hz, H-3 of Glc), 5.43
(1H, t, J1,2ꢀ7.9 Hz, H-2 of Glc), 5.24—5.21 (2H, m, H-3, 4 of GalN), 4.83
(1H, d, J1,2ꢀ7.9 Hz, H-1 of GalN), 4.78 (1H, d, H-1 of Glc), 4.57 (1H, br d,
H-6a of Glc), 4.36 (1H, m, H-6b of Glc), 4.09—4.03 (2H, m, H-4 of Glc,
CH2CH2Si(CH3)3), 3.95—3.89 (2H, m, H-5 of Glc, H-2 of GalN), 3.69—
3.64 (2H, m, H-5 of GalN, CH2CH2Si(CH3)3), 3.51—3.42 (2H, m, H-6 of
GalN), 1.02—0.88 (2H, m, CH2CH2Si(CH3)3), ꢁ0.01 (9H, s, Si(CH3)3). 13C-
NMR (125 MHz, CDCl3) d: 171.0, 170.5, 170.2, 170.0, 169.8, 165.3, 165.1,
133.0, 129.7ꢂ3, 129.6ꢂ3, 129.4, 128.3ꢂ2, 128.2ꢂ3, 100.4 (C-1 of Glc),
100.3 (C-1 of GalN), 76.1, 73.1, 72.9, 71.9, 70.3, 69.7, 67.5, 66.0, 62.4,
60.3, 51.8, 23.1, 20.9, 20.5ꢂ2, 20.4, 17.8, ꢁ1.59. MALDI-TOF-MS: Calcd
for C41H53NO17SiNa ([MꢃNa]ꢃ) m/z 882.3. Found 882.8. HR-FAB-MS:
Calcd for C41H53NO17SiNa ([MꢃNa]ꢃ ) m/z 882.2980. Found 882.2968.
2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-b-D-galactopyranosyl-(1→4)-
6-O-acetyl-2,3-di-benzoyl-1-O-a-D-glucopyranosyl Trichloroacetimidate
(9) To a solution of 8 (300 mg, 0.35 mmol) in CH2Cl2 (3.0 ml), cooled to
0 °C was added CF3CO2H (1.5 ml), and the mixture was stirred for 3 h at
room temperature and concentrated. EtOAc and toluene (1 : 2) were added
and evaporated to give the reducing sugar. To a solution of the residue in
CH2Cl2 (3.0 ml) cooled at 0 °C were added DBU (53.1 ml, 0.35 mmol) and
CCl3CN (0.36 ml, 3.49 mmol). The reaction mixture was stirred for 6 h at
room temperature. After completion of the reaction, the mixture was con-
centrated. The residue was purified by silica gel column chromatography
using 15 : 1 chloroform–methanol as eluent to give 9 as an amorphous pow-
der (230 mg, 73%). [a]D25 ꢃ9.5 (cꢀ1.0, CHCl3). 1H-NMR (500 MHz,
CDCl3) d: 8.61 (1H, s, C(NH)CCl3), 8.54—8.13 (10H, m, 2Ph), 6.71 (1H, d,
(b) To a solution of 6 (1.08 g, 1.04 mmol) was added guanidinium nitrate
(622 mg) and MeONa (54.2 mg) in MeOH/CHCl3 (50 ml, 9 : 1). The reaction
mixture was stirred for 1 h at room temperature then neutralized with Am-
berlite IR 120 [Hꢃ]. The filtrate was washed with brine, dried (MgSO4), and
concentrated. The filtrate was concentrated and purified by silica gel column
chromatography using 20 : 1 chloroform–methanol as the eluent to give 12
1
as syrup (0.85 g, 89%). [a]D25 ꢃ13.6 (cꢀ1.5, CHCl3). H-NMR (500 MHz,
CDCl3) d: 7.93—7.32 (15H, m, 3Ph), 5.57 (1H, t, J2,3ꢀJ3,4ꢀ9.2 Hz, H-3 of
Glc), 5.39 (1H, t, J1,2ꢀ7.9 Hz, H-2 of Glc), 4.87 (1H, d, NH), 4.80, 4.49
(2H, each d, 2 benzylmethylene), 4.72 (1H, d, CH2CCl3), 4.65 (1H, d, H-1 of
Glc), 4.60 (1H, d, CH2CCl3), 4.21 (1H, d, J1,2ꢀ7.9 Hz, H-1 of GalN), 4.07
(1H, t, J4,5ꢀ10.0 Hz, H-4 of Glc), 4.03—3.98 (2H, m, CH2CH2Si(CH3)3),
3.85 (1H, br d, H-6a of Glc), 3.77—3.69 (2H, m, H-6b of Glc, H-4 of GalN),
3.65 (1H, br d, H-5 of Glc), 3.59—3.54 (2H, m, CH2CH2Si(CH3)3), 3.49
(1H, dd, J2,3ꢀ11.0 Hz, H-2 of GalN), 3.28, (1H, br s, H-3 of GalN), 3.12,
(2H, br s, H-5 of GalN, OH), 3.02 (2H, br s, H-6a of GalN, OH), 2.93 (2H,
br s, H-6 of GalNb, OH), 0.94—0.81 (2H, m, CH2CH2Si(CH3)3), ꢁ0.07 (9H,
s, Si(CH3)3). 13C-NMR (125 MHz, CDCl3) d: 166.3, 165.2, 155.2, 137.6ꢂ3,
133.4, 133.0ꢂ2, 129.7, 129.5ꢂ3, 129.0, 128.8ꢂ3, 128.5, 128.2ꢂ3, 100.9
(C-1 of GalN), 100.3 (C-1 of Glc), 95.6, 76.6, 74.6, 74.5, 74.4, 74.0, 73.6,
71.9ꢂ2, 68.2, 68.0, 67.4, 61.1, 55.1, 17.9ꢂ3, ꢁ1.51. MALDI-TOF-MS:
Calcd for C41H50Cl3NO14SiNa: ([MꢃNa]ꢃ) m/z 936.2. Found 937.3. HR-
FAB-MS: Calcd for C41H50Cl3NO14SiNa: ([MꢃNa]ꢃ) m/z 936.1964. Found
936.1919.
J1,2ꢀ3.7 Hz, H-1 of Glc), 6.07 (1H, t, J2,3ꢀJ3,4ꢀ9.2 Hz, H-3 of Glc), 5.88
(1H, d, NH), 5.47 (1H, dd, H-2 of Glc), 5.27 (1H, dd, J2,3ꢀ11.0 Hz,
J
J
3,4ꢀ3.1 Hz, H-3 of GalN), 5.16 (1H, d, H-4 of GalN), 4.97 (1H, d,
1,2ꢀ8.5 Hz, H-1 of GalN), 4.52 (1H, br d, H-6a of Glc), 4.35—4.28 (1H, m,
H-5, 6b of Glc), 4.15—4.11 (1H, t, J4,5ꢀ9.8 Hz, H-4 of Glc), 3.78—3.72
(1H, dd, H-2 of GalN), 3.60 (1H, t, J5,6ꢀ7.3 Hz, H-5 of GalN), 3.52—3.41
(2H, m, H-6 of GalN). 13C-NMR (125 MHz, CDCl3) d: 170.8, 170.6, 170.1,
170.0, 169.8, 165.4, 165.2, 160.5, 133.5, 133.4, 129.9ꢂ2, 129.8ꢂ2, 129.6,
129.51, 129.45ꢂ2, 128.7, 128.4, 128.3, 100.1 (C-1 of GalN), 92.9 (C-1 of
Glc), 77.2, 75.5, 71.2, 70.5, 70.4, 70.1, 69.4, 66.0, 61.9, 60.2, 52.3, 23.1,
20.8, 20.5, 20.4. MALDI-TOF-MS: Calcd for C38H41Cl3N2O17Na:
([MꢃNa]ꢃ) m/z 925.1. Found 925.8.
2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-b-D-galactopyranosyl-(1→4)-
6-O-acetyl-2,3-di-O-benzoyl-b-D-glucopyranosyl-(1→1)-(2S,3S,4R)-3,4-
di-O-benzoyl-2-palmitoylamidooctadecane-1,3,4-triol (11) A solution of
9 (37.5 mg, 44.2 mmol) and (2S,3S,4R)-3,4-di-O-benzoyl-2-palmitoylami-
dooctadecane-1,3,4-triol 10 (47.1 mg, 66.2 mmol) containing activated 4 Å
MS (200 mg) in dry CH2Cl2 (0.4 ml) was stirred under an atmosphere of
argon for 18 h at room temperature, then cooled to 0 °C. TMSOTf (97 ml,
35.4 mmol) was added, and the mixture was stirred for 3 h at room tempera-
ture, then neutralized with Et3N. The solids were filtrated off and washed
with CHCl3. The combined filtrate and washings were successively washed
with brine, dried (MgSO4) and concentrated. The product was purified by
flash silica gel column chromatography using 100 : 1 chloroform–methanol
as eluent to give 11 as an amorphous powder (35.6 mg, 57%). [a]D25 ꢃ23.5
(cꢀ1.0, CHCl3). 1H-NMR (500 MHz, CDCl3) d: 8.06—7.36 (20H, m, 4 Ph),
6.01 (1H, d, ceramide-NH), 5.69—5.66 (1H, m, NH of GalN), 5.60 (1H, t,
2-(Trimethylsilyl)ethyl 4,6-O-Benzylidene-2-deoxy-2-(2,2,2-trichloro-
ethoxycarbonylamino)-b-D-galactopyranosyl-(1→4)-2,3-di-O-benzoyl-6-
O-benzyl-b-D-glucopyranoside (13) To
a solution of 12 (756 mg,
0.83 mmol) in CH3CN (10 ml) was added NaHSO4·SiO2 (1.5 g) and ben-
zaldehyde dimethyl acetal (BDA) (0.25 ml, 1.66 mmol, 2.0 eq). The reaction
mixture was stirred for 2 h at room temperature, then neutralized with Et3N.
The solids were filtrated off and the filtrated was concentrated. The product
was purified by silica gel column chromatography using 10 : 1 toluene–ace-
tone as eluent to give 13 as white solid (734 mg, 89%). [a]D25 ꢁ13.0 (cꢀ7.7,
1
CHCl3). mp 164—165 °C H-NMR (CDCl3) d: 8.03—7.25 (20H, m, 4Ph),
5.75 (1H, t, J2,3ꢀJ3,4ꢀ8.8 Hz, H-3 of Glc), 5.41 (1H, dd, J1,2ꢀ7.9 Hz, H-2 of
Glc), 5.33 (1H, s, PhCH), 5.14 (1H, d, NH), 4.87 (1H, d, benzylmethylene),
4.78—4.66 (3H, m, H-1 of Glc, CH2CCl3, benzylmethylene), 4.46 (1H, d,
J2,3ꢀJ3,4ꢀ9.1 Hz, H-3 of Glc), 5.34—5.32 (1H, m, ceramide-H-4), 5.28 (1H,
t, J1,2ꢀ8.2 Hz, H-2 of Glc), 5.11 (1H, d, J3,4ꢀ3.3 Hz, H-4 of GalN), 5.06
(1H, dd, J2,3ꢀ11.0 Hz, H-3 of GalN), 4.64 (1H, d, H-1 of Glc), 4.61 (1H, d,
J1,2ꢀ7.9 Hz, H-1 of GalN), 4.21 (1H, t, J4,5ꢀ8.8 Hz, H-4 of Glc), 4.10—4.03
(2H, m, H-6a of Glc, CH2CH2Si(CH3)3), 3.94 (1H, d, J3,4ꢀ1.8 Hz, H-4 of
GalN), 3.86 (1H, dd, H-6b of Glc), 3.77—3.75 (1H, m, H-5 of Glc), 3.74—
3.50 (4H, m, H-2, 3, 6a of GalN, CH2CH2Si(CH3)3), 3.46 (1H, d, H-6b of
GalN), 2.95 (1H, s, H-5 of GalN), 1.01—0.88 (1H, m, CH2CH2Si(CH3)3),
ꢁ0.01 (9H, s, Si(CH3)3) . 13C-NMR (125 MHz, CDCl3) d: 165.3, 154.8,
137.9, 137.5, 132.9, 132.7, 129.8ꢂ2, 129.7ꢂ2, 129.6, 129.0ꢂ2, 128.6ꢂ3,
128.5ꢂ2, 128.2ꢂ2, 128.1ꢂ2, 128.0ꢂ2, 126.5ꢂ2, 101.0 (C-1 of GalN),
100.5 (C-1), 95.5, 74.6ꢂ2, 74.5, 73.7, 73.6, 72.4, 71.0, 68.3, 68.0, 67.4,
66.4, 55.6, 17.9, ꢁ1.49. MALDI-TOF-MS: Calcd for C48H54Cl3NO14SiNa:
([MꢃNa]ꢃ) m/z 1024. Found 1024. HR-FAB-MS: Calcd for
C48H54Cl3NO14SiNa: ([MꢃNa]ꢃ) m/z 1024.2277. Found 1024.2218.
J
1,2ꢀ7.1 Hz, H-1 of GalN), 4.60—4.57 (1H, m, ceramide-H-2), 4.22—4.20
(1H, m, H-6a of Glc), 4.01—3.95 (3H, m, H-4, 6b of Glc, ceramide-H-1a),
3.86 (1H, dd, H-2 of GalN), 3.73—3.71 (1H, m, H-5 of Glc), 3.61 (1H, dd,
ceramide-H-1b), 3.52 (1H, t, J5,6ꢀ7.3 Hz, H-5 of GalN), 3.44 (1H, dd,
J6a,6bꢀ7.8 Hz, H-6a of GalN), 3.34 (1H, dd, H-6b of GalN), 1.95—0.86
(62H, m, alkyl). 13C-NMR (125 MHz, CDCl3) d: 173.3, 172.9, 170.9, 170.5,
170.3, 170.1, 169.9, 166.3, 165.3, 165.2, 165.1, 163.5, 133.8, 133.43,
133.39, 133.2, 133.0, 130.1, 129.9, 129.8, 129.73, 129.66, 129.6, 129.5,
128.9, 128.7, 128.5, 128.3, 100.8 (C-1 of Glc), 99.9 (C-1 of GalN), 91.9,
76.0, 73.9, 73.7, 72.9, 72.7, 72.2, 72.1, 70.4, 69.9, 67.0, 65.9, 62.5, 61.6,
60.3, 51.4, 49.9, 47.8, 36.8, 36.4, 31.9, 29.69, 29.66, 29.6, 29.5, 29.3, 29.2,
25.8, 25.7, 25.5, 25.4, 23.1, 22.7, 20.7, 20.6, 20.5, 14.1. MALDI-TOF-MS:
Calcd for C84H116N2O22Na: ([MꢃNa]ꢃ) m/z 1528. Found 1528. HR-FAB-
MS: Calcd for C84H116N2O22Na: ([MꢃNa]ꢃ) m/z 1527.7917. Found
1527.7946.
2-(Trimethylsilyl)ethyl 3,4,6-Tri-O-acetyl-2-deoxy-2-(2,2,2-trichloro-
ethoxycarbonylamino)-b-D-glucopyranosyl-(1→3)-4,6-O-benzylidene-2-
deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-b-D-galactopyranosyl-
(1→4)-2,3-di-O-benzoyl-6-O-benzyl-b-D-glucopyranoside
(15) Com-
pound 15 was prepared from 13 (1.42 g, 1.42 mmol) and 14 (1.30 g,
2.13 mmol) by the same method described for preparation of 6. The product
was purified by silica chromatography using 3 : 2 hexane–ethyl acetate as
2-Acetamido-2-deoxy-b-D-galactopyranosyl-(1→4)-b-D-glucopyra-
nosyl-(1→1)-(2S,3S,4R)-2-palmitoylamidooctadecane-1,3,4-triol (1) To
a solution of 10 (35.6 mg) in MeOH (1.5 ml) was added dioxane (1.5 ml) and

June 2010
815
1 : 1 CHCl3–CD3OH). mp 145—146 °C. 1H-NMR (600 MHz, 1 : 1
CDCl3–CD3OD) d: 4.89 (1H, d, J1,2ꢀ8.5 Hz, H-1 of GlcN), 4.60 (1H, d,
eluent to give 15 as an amorphous powder (1.75 g, 85%). [a]D25 ꢃ35.7
(cꢀ4.4, CHCl3). 1H-NMR (500 MHz, CDCl3) d: 4.96 (1H, d, J1,2ꢀ8.0 Hz,
H-1 of GlcN), 4.80 (1H, d, J1,2ꢀ9.3 Hz, H-1 of GalN), 4.72 (1H, d,
J
1,2ꢀ8.0 Hz, H-1 of GalN), 4.57 (1H, d, J1,2ꢀ7.7 Hz, H-1 of Glc). MALDI-
TOF-MS: Calcd for C56H105N3O19Na: ([MꢃNa]ꢃ) m/z 1146.7. Found
1146.1. HR-FAB-MS: Calcd for C56H105N3O19Na: ([MꢃNa]ꢃ) m/z
1146.7240. Found 1146.7185.
J
1,2ꢀ9.3 Hz, H-1 of Glc). 13C-NMR (125 MHz, CDCl3) d: 170.4, 170.2,
169.3, 165.4, 165.2, 153.8, 138.3, 137.5, 134.4, 132.84, 132.76, 129.7ꢂ2,
129.6ꢂ2, 128.94, 128.87, 128.6, 128.5ꢂ2, 128.4, 128.1ꢂ2, 128.0ꢂ2,
127.9, 127.8, 127.7, 126.3, 100.7, 100.4 (C-1 of Glc), 100.4 (C-1 of GalN),
99.1 (C-1 of GlcN), 95.5, 95.4, 75.5, 75.10, 74.99, 74.8, 74.4, 74.2, 73.8,
73.2, 72.4, 71.8, 68.4, 68.3, 67.3, 66.2, 61.9, 56.1, 53.8, 20.7, 20.52, 20.48,
17.9ꢂ2, ꢁ1.68. MALDI-TOF-MS: Calcd for C63H72Cl6N2O23SiNa:
([MꢃNa]ꢃ) m/z 1485.2. Found 1486.3.
2-(Trimethylsilyl)ethyl 6-O-Benzyl-3-O-chloroacetyl-2-deoxy-2-(2,2,2-
trichloroethoxycarbonylamino)-b-D-glucopyranoside (20) To a solution
of compound 19 (3.0 g, 5.16 mmol) in MeOH (30.0 ml) was added Et3N
(6.0 ml). The reaction mixture was stirred for 18 h at room temperature and
concentrated to give the reducing sugar. To a solution of the residue in
CH3CN (16.0 ml) was added Camphor-10-sulfonic acid (CSA) (369 mg,
1.55 mmol, 0.3 eq), 3 Å MS (2.5 g) and BDA (1.6 ml, 10.3 mmol, 2.0 eq).
The reaction mixture was stirred under an atmosphere of argon for 5 h at
room temperature. The solids were filtrated off and washed with CHCl3. The
combined filtrate was concentrated. The product was purified by silica gel
column chromatography using 4 : 1 hexane–ethyl acetate as eluent to give
2-(trimethylsilyl)ethyl 4,6-O-benzylidene-2-deoxy-2-(2,2,2-trichloroethoxy-
carbonylamino)-b-D-glucopyranoside as white solid (2.05 g, 72%). To a so-
lution of this compound (1.62 mg, 2.98 mmol) in CH2Cl2 (30.0 ml) was
added pyridine (6.0 ml) then cooled to 0 °C. ClAcCl (0.48 ml, 5.97 mmol,
2.0 eq) was added, and the mixture was stirred for 5 h at room temperature.
The mixture was diluted with CHCl3, washed with aq. 5% HCl, aq. NaHCO3
and brine, dried (MgSO4) and concentrated. The product was purified by sil-
ica gel column chromatography using 3 : 1 hexane–ethyl acetate as eluent to
give 2-(trimethylsilyl)ethyl 4,6-O-benzylidene-3-O-chloroacetyl-2-deoxy-2-
(2,2,2-trichloroethoxycarbonylamino)-b-D-glucopyranoside as an amor-
phous powder (1.39 g, 75%). To a solution of this compound (474 mg,
0.77 mmol) in THF (8.0 ml) was added 3 Å MS (500 mg) and NaBH3CN
(386 mg, 6.16 mmol, 8.0 eq). The reaction mixture was stirred under an
atmosphere of argon for 3 h at room temperature, then cooled to 0 °C.
HCl–Et2O (12.0 ml) was added. The addition was discontinued when the so-
lution became acidic, and the mixture was stirred for 30 min at 0 °C. The
solids were filtrated off and washed with CHCl3. The combined filtrate and
washings were successively washed with aq. NaHCO3 and brine, dried
(MgSO4) and concentrated. The product was purified by silica gel column
chromatography using 3 : 1 hexane–ethyl acetate as eluent to give 20 as
syrup (410 mg, 86%). [a]D25 ꢁ26.3 (cꢀ5.67, CHCl3). 1H-NMR (500 MHz,
CDCl3) d: 7.35—7.25 (5H, m, Ph), 5.43 (1H, d, NH), 5.20 (1H, t,
2-(Trimethylsilyl)ethyl 2-Acetamido-O-3,4,6-tri-O-acetyl-2-deoxy-b-D-
gluctopyranosyl-(1→3)-2-acetamide-4,6-di-O-acetyl-2-deoxy-b-D-galac-
topyranosyl-(1→4)-6-O-acetyl-2,3-di-O-benzoyl-b-D-glucopyranoside
(16) To a solution of 15 (1.75 g, 0.13 mmol) in Ac2O (10.0 ml) and AcOH
(20.0 ml) was added zinc powder (3.0 g). The reaction mixture was stirred
for 18 h at 45 °C. After completion of the reaction, the mixture was filtered
and the filtrate was diluted with CHCl3, washed with water, dried (MgSO4)
and concentrated to give the intermediate. To a solution of the residue in
MeOH (10.0 ml) was added AcOH (5.0 ml) and hydrogenolysed under hy-
drogen in the presence of 10% Pd/C (500 mg) for 18 h at room temperature,
then filtered and concentrated. The residue was acetylated with acetic anhy-
dride (20.0 ml) in pyridine (30.0 ml). The reaction mixture was added
toluene and concentrated. The residue was purified by silica gel column
chromatography using 3 : 1 toluene–acetone as eluent to give 16 as syrup
(855 mg, 62%). [a]D25 ꢃ31.3 (cꢀ2.5, CHCl3). H-NMR (600 MHz, CDCl3)
1
d: 4.91 (1H, d, J1,2ꢀ8.2 Hz, H-1 of GlcN), 4.88 (1H, d, J1,2ꢀ8.2 Hz, H-1 of
GalN), 4.79 (1H, d, J1,2ꢀ7.7 Hz, H-1 of Glc). 13C-NMR (150 MHz, CDCl3)
d: 100.4 (C-1 of Glc), 100.3 (C-1 of GalN), 99.2 (C-1 of GlcN). MALDI-
TOF-MS: Calcd for C53H70N2O24SiNa: ([MꢃNa]ꢃ) m/z 1169.4. Found
1170.2. HR-FAB-MS: Calcd for C53H70N2O24SiNa: ([MꢃNa]ꢃ) m/z
1169.3986. Found 1169.3945.
2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-b-D-gluctopyranosyl-(1→3)-2-
acetamido-4,6-di-O-acetyl-2-deoxy-b-D-galactopyranosyl-(1→4)-6-O-
acetyl-2,3-di-O-benzoyl-1-O-a-D-glucopyranosyl Trichloroacetimidate
(17) Compound 17 was prepared from 16 (130 mg, 0.11 mmol) by the
same method described for preparation of 9. The product was purified by sil-
ica chromatography using 40 : 1 chloroform–methanol as eluent to give 17
(105 mg, 78%). [a]D25 ꢃ42.6 (cꢀ0.5, CHCl3). H-NMR (600 MHz, CDCl3)
1
J2,3ꢀJ3,4ꢀ9.8 Hz, H-3), 4.76 (1H, d, CH2CCl3), 4.65—4.54 (3H, m,
d: 8.62 (1H, s, OC(NH)CCl3), 7.99—7.33 (10H, m, 2Ph), 6.70 (1H, d,
CH2CCl3, 2 benzylmethylene), 4.54 (1H, d, J1,2ꢀ8.5 Hz, H-1), 4.11 (2H, q,
CH2Cl), 3.97—3.92 (1H, m, CH2CH2Si), 3.80—3.72 (3H, m, H-4, 6), 3.63
(1H, dd, H-2), 3.57—3.52 (2H, m, H-5, CH2CH2Si), 3.25 (1H, d, OH),
ꢁ0.01 (9H, s, Si(CH3)3). 13C-NMR (125 MHz, CDCl3) d: 168.1, 154.32,
137.5, 128.5ꢂ3, 128.0, 127.7ꢂ2, 101.8, 100.1 (C-1), 95.4, 74.5, 73.7ꢂ2,
70.7, 70.1, 67.4, 56.0, 40.8, 18.0ꢂ2, ꢁ1.5. MALDI-TOF-MS: Calcd for
C23H33Cl4NO9SiNa, ([MꢃNa]ꢃ) 642.0, Found 642.8.
J1,2ꢀ3.6 Hz, H-1 of Glc), 6.55 (1H, d, NH of GalN), 6.35 (1H, d, NH of
GlcN), 5.98 (1H, t, J2,3ꢀJ3,4ꢀ9.9 Hz, H-3 of Glc), 5.48 (1H, dd, H-2 of Glc),
5.25 (1H, d, J3,4ꢀ9.7 Hz, H-3 of GlcN), 5.20 (1H, d, J3,4ꢀ3.2 Hz, H-4 of
GalN), 5.00 (1H, t, J4,5ꢀ9.7 Hz, H-4 of GlcN), 4.96 (1H, d, J1,2ꢀ8.2 Hz, H-1
of GalN) , 4.80 (1H, d, J1,2ꢀ8.2 Hz, H-1 of GlcN), 4.55 (1H, d, H-6a), 4.39
(1H, dd, J2,3ꢀ7.2 Hz, H-3 of GalN), 4.30—4.27 (1H, m, H-5 of Glc), 4.19—
4.05 (4H, m, H-4, 6b of Glc, H-6 of GlcN), 3.67—3.53 (4H, m, H-6a of Glc,
H-5 of GalN, H-2, 5 of GlcN), 3.34—3.31 (2H, m, H-6b of Glc, H-2 of
GalN). 13C-NMR (125 MHz, CDCl3) d: 171.5, 170.7, 170.6, 170.2, 169.4,
169.3, 165.4, 165.3, 160.5, 133.5, 133.3, 129.8ꢂ3, 129.6ꢂ3, 128.4ꢂ2,
128.33ꢂ2, 128.26ꢂ2, 100.4 (C-1 of GlcN), 98.9 (C-1 of GalN), 93.0 (C-1
of Glc), 90.6, 74.5, 74.4, 72.1, 71.5, 71.3, 71.0, 70.3, 70.1, 68.3, 68.1, 62.0,
61.5, 61.4, 55.0, 54.5, 23.4, 23.2, 20.8, 20.64, 20.59, 20.55ꢂ2, 20.5ꢂ2,
20.4. MALDI-TOF-MS: Calcd for C50H58Cl3N3O24Na: ([MꢃNa]ꢃ) m/z
1212.2. Found 1212.7.
2-(Trimethylsilyl)ethyl 2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl-
(1→4)-6-O-benzyl-3-O-chloroacetyl-2-deoxy-2-(2,2,2-trichloroethoxycar-
bonylamino)-b-D-glucopyranoside (22) Compound 22 was prepared from
20 (715 mg, 1.20 mmol) and 21 (851 mg, 1.73 mmol) by the same method
described for preparation of 6. The product was purified by silica chro-
matography using 3 : 1 chloroform–methanol as eluent to give 22 as an
amorphous powder (952 mg, 86%). [a]D25 ꢁ4.34 (cꢀ4.3, CHCl3). H-NMR
1
(500 MHz, CDCl3) d: 7.37—7.28 (5H, m, Ph), 5.28 (1H, d, NH), 5.24 (1H,
d, J3,4ꢀ3.7 Hz, H-4 of Gal), 5.16 (1H, t, J3,4ꢀ9.2 Hz, H-3 of GlcN), 4.93
(1H, t, J1,2ꢀ7.9 Hz H-2 of Gal), 4.78—4.63 (4H, m, H-3 of Gal, CH2CCl3,
benzylmethylene), 4.48 (1H, d, J1,2ꢀ8.6 Hz, H-1 of GlcN), 4.44 (1H, d, ben-
zylmethylene), 4.40 (1H, d, H-1 of Gal), 4.10—4.02 (3H, m, H-6 of Gal,
ClCH2CO), 3.96—3.88 (2H, m, H-4 of GlcN, CH2CH2Si(CH3)3), 3.70 (1H,
d, H-6 of GlcN), 3.66—3.59 (2H, m, H-2 of GlcN, H-5 of Gal), 3.54—3.49
(1H, m, CH2CH2Si(CH3)3), 3.44 (1H, br d, H-5 of GlcN), ꢁ0.03 (9H, s,
Si(CH3)3). 13C-NMR (125 MHz, CDCl3) d: 170.3, 170.2, 170.0, 168.8,
167.0, 154.2, 137.6, 128.6ꢂ2, 128.1, 128.0ꢂ2, 100.4 (C-1 of GlcN), 100.1
(C-1 of Gal), 95.4, 74.5ꢂ2, 74.3, 73.6, 70.9ꢂ2, 70.6ꢂ2, 69.1, 67.3, 67.2,
66.9, 61.1, 55.9, 40.8, 20.6, 20.5, 18.0, ꢁ1.5. MALDI-TOF-MS: Calcd for
C37H51Cl4NO17SiNa: ([MꢃNa]ꢃ) m/z 972.2. Found 972.5. HR-FAB-MS:
Calcd for C37H51Cl4NO17SiNa: ([MꢃNa]ꢃ) m/z 972.1578. Found 972.1601.
2-(Trimethylsilyl)ethyl 2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl-
(1→4)-6-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-b-D-
glucopyranoside (23) To a solution of 22 (780 mg, 0.82 mmol) in EtOH
(5.0 ml) was added pyridine (3.0 ml) and thiourea (509 mg, 6.56 mmol). The
reaction mixture was stirred for 4 h at 80 °C. The mixture was diluted with
CHCl3, washed with aq. 5% HCl, aq. NaHCO3 and brine, dried (MgSO4)
2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-b-D-gluctopyranosyl-(1→3)-2-
acetamido-4,6-di-O-acetyl-2-deoxy-b-D-galactopyranosyl-(1→4)-6-O-
acetyl-2,3-di-O-benzoyl-b-D-glucopyranosyl-(1→1)-(2S,3S,4R)-3,4-di-O-
benzoyl-2-palmitoylamidooctadecane-1,3,4-triol (18) Compound 18 was
prepared from 17 (110 mg, 93.0 mmol) and 10 (106 mg, 140 mmol) by the
same method described for preparation of 11. The product was purified by
silica chromatography using 60 : 1 chloroform–methanol as eluent to give 18
as syrup (85.8 mg, 52%). [a]D25 ꢃ28.9 (cꢀ1.0, CHCl3). 1H-NMR (600 MHz,
CDCl3) d: 5.00 (1H, d, J1,2ꢀ8.5 Hz, H-1 of GlcN), 4.77 (1H, d, J1,2ꢀ8.3 Hz,
H-1 of GalN), 4.56 (1H, d, J1,2ꢀ7.7 Hz, H-1 of Glc). 13C-NMR (150 MHz,
CDCl3) d: 100.4 (C-1 of Glc), 100.2 (C-1 of GalN), 97.7 (C-1 of GlcN).
MALDI-TOF-MS: Calcd for C96H133N3O29Na: ([MꢃNa]ꢃ) m/z 1815. Found
1815.
2-Acetamido-2-deoxy-b-D-gluctopyranosyl-(1→3)-2-acetamido-
2-deoxy-b-D-galactopyranosyl-(1→4)-b-D-glucopyranosyl-(1→1)-
(2S,3S,4R)-2-palmitoylamidooctadecane-1,3,4-triol (2) Compound
2
was prepared from 18 (38.2 mg) by the same method described for prepara-
tion of 1. The product was purified by Sephadex LH-20 column chromatog-
raphy in MeOH to give 2 as white solid 20.6 mg (86%). [a]D25 ꢁ8.4 (cꢀ0.16,

816
Vol. 58, No. 6
and concentrated. The product was purified by silica gel column chromatog- 9.8 Hz, H-3 of Glc), 5.55 (1H, br, NH), 5.40—5.33 (4H, m, H-2 of Glc, H-3
raphy using 3 : 2 hexane–ethyl acetate as eluent to give 23 as a white solid of GlcN, H-4 of Gal, CHPh), 5.09—5.06 (2H, m, H-2 of Gal, benzylmethyl-
(624 mg, 85%). [a]D25 ꢁ4.3 (cꢀ4.3, CHCl3). mp 125—126 °C. 1H-NMR ene), 4.97 (1H, d, J1,2ꢀ7.9 Hz, H-1 of GlcN), 4.98—4.90 (2H, m, H-3 of
(500 MHz, CDCl3) d: 7.38—7.29 (5H, m, Ph), 5.34 (1H, d, J3,4ꢀ3.7 Hz, H-4
of Gal), 5.18—5.15 (2H, m, H-2 of Gal, NH), 4.92 (1H, dd, J2,3ꢀ9.2 Hz, H-
Gal, NH), 4.82—4.67 (7H, m, J1,2ꢀ7.9 Hz, H-1 of Glc, 3 benzylmethylene,
3CH2CCl3), 4.54 (1H, d, CH2CCl3), 4.47 (2H, d, J1,2ꢀ7.9 Hz, H-1 of GalN,
3 of Gal), 4.74—4.67 (3H, m, CH2CCl3, benzylmethylene), 4.60 (1H, br, H- H-1 of Gal), 4.29 (1H, t, J4,5ꢀ9.8 Hz, H-4 of Glc), 4.12—4.04 (5H, m, H-6a
1 of GlcN), 4.50 (1H, d, benzylmethylene), 4.48 (1H, d, J1,2ꢀ10.4 Hz, H-1
of Gal), 4.14—4.07 (2H, m, H-6 of Gal), 3.99—3.88 (4H, m, H-3 of GlcN,
H-5 of Gal, CH2CH2Si(CH3)3, OH), 3.70—3.61 (2H, m, H-4, 6 of GlcN),
3.56—3.51 (1H, m, CH2CH2Si(CH3)3), 4.47 (1H, m, H-5 of GlcN), 3.32
(1H, q, J2,3ꢀ8.3 Hz, H-2 of GlcN), ꢁ0.01 (9H, s, Si(CH3)3). 13C-NMR (125
of Glc, H-4 of GlcN, H-6 of Gal, CH2CH2Si(CH3)3), 3.89—3.61 (10H, m,
H-5, 6b of Glc, H-2, 3, 4 of GalN, H-2, 6 of GlcN, H-5 of Gal,
CH2CH2Si(CH3)3), 3.56—3.42 (3H, m, H-6 of GalN, H-5 of GlcN), 2.84
(1H, s, H-5 of GalN), ꢁ0.01 (9H, s, Si(CH3)3). 13C-NMR (125 MHz, CDCl3)
d: 170.3, 170.1, 170.0, 168.7, 165.2, 153.8, 138.3, 137.5, 132.8, 132.7,
MHz, CDCl3) d: 170.4, 170.1, 169.9, 169.1, 154.1, 138.0ꢂ2, 128.5, 127.9ꢂ 129.7, 129.6, 129.0, 128.9, 128.6, 128.4, 128.2, 128.0, 127.8, 127.7, 126.3,
2, 127.8, 101.3 (C-1 of Gal), 99.9 (C-1 of GlcN), 95.5, 81.4, 74.5, 73.9, 125.2, 100.7, 100.5 (C-1), 100.3 (C-1 of GalN, C-1 of Gal), 99.1 (C-1 of
73.6ꢂ2, 71.8, 71.2, 70.7, 68.7, 67.9, 67.2, 66.9, 61.4ꢂ2, 57.8, 20.7ꢂ2, 20.6, GlcN), 95.5, 75.3, 74.8, 74.4, 74.3, 74.2, 73.82, 73.76, 73.2, 72.4, 72.2, 70.9,
20.5, 18.0, ꢁ1.4. MALDI-TOF-MS: Calcd for C35H50Cl3NO16SiNa: ([Mꢃ 70.5, 69.1, 68.4, 68.2, 67.3, 66.7, 66.2, 60.9, 56.0, 53.9, 21.4, 20.7, 20.6,
Na]ꢃ) m/z 896. Found 896. HR-FAB-MS: Calcd for C35H50Cl3NO16SiNa:
([MꢃNa]ꢃ) m/z 896.1862. Found 896.1827.
20.5, 17.9, ꢁ1.51. MALDI-TOF-MS: Calcd for C80H92Cl6N2O30SiNa:
([MꢃNa]ꢃ) m/z 1821.4. Found 1822.1.
2-(Trimethylsilyl)ethyl 2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl-
2-(Trimethylsilyl)ethyl 2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl-
(1→4)-3-O-acetyl-6-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonyl- (1→4)-2-acetamido-3,6-di-O-acetyl-2-deoxy-b-D-glucopyranosyl-(1→3)-
amino)-b-D-glucopyranoside (24) Compound 23 was acetylated with
acetic anhydride (3.0 ml) in pyridine (4.5 ml) for 18 h. The reaction mixture
was poured into ice H2O and extracted with CHCl3. The extract was washed prepared from 26 (363 mg, 0.20 mmol) by the same method described for
2-acetamido-4,6-di-O-acetyl-2-deoxy-b-D-galactopyranosyl-(1→4)-6-O-
acetyl-2,3-di-O-benzoyl-b-D-glucopyranoside (27) Compound 27 was
sequentially with 5% HCl, aq. NaHCO3, and brine, dried (MgSO4), and con-
preparation of 16. The product was purified by silica chromatography using
centrated. The residue was purified by silica gel column chromatography 2 : 1 toluene–acetone as eluent to give 27 as syrup (120 mg, 42%). [a]D25
1
using 2 : 1 hexane–ethyl acetate as eluent to give 24 as an amorphous pow-
der (263 mg, 82%). [a]D25 ꢁ8.5 (cꢀ6.6, CHCl3). 1H-NMR (500 MHz,
CDCl3) d: 7.40—7.26 (5H, m, Ph), 5.28 (1H, d, J3,4ꢀ3.7 Hz, H-4 of Gal),
5.15 (1H, d, NH), 5.09 (1H, m, J3,4ꢀ9.8 Hz, H-3 of GlcN), 5.10 (1H, dd,
ꢃ9.5 (cꢀ0.98, CHCl3). H-NMR (600 MHz, CDCl3) d: 6.07 (1H, d, NH of
GlcN), 5.87 (1H, d, NH of GalN), 4.90 (1H, d, J1,2ꢀ8.0 Hz, H-1 of GlcN),
4.69 (1H, d, J1,2ꢀ7.7 Hz, H-1 of Glc), 4.55 (1H, d, J1,2ꢀ6.9 Hz, H-1 of
GalN), 4.48 (1H, d, J1,2ꢀ8.0 Hz, H-1 of Gal). 13C-NMR (150 MHz, CDCl3)
d: 101.0 (C-1 of Gal), 100.6 (C-1 of GalN), 100.4 (C-1 of Glc), 98.9 (C-1 of
J
1,2ꢀ7.8 Hz, J2,3ꢀ9.7 Hz, H-2 of Gal), 4.82 (1H, dd, H-3 of Gal), 4.79—4.71
(2H, m, CH2CCl3, benzylmethylene), 4.66 (1H, d, CH2CCl3) 4.48 (1H, d, GlcN). MALDI-TOF-MS: Calcd for C65H86N2O32SiNa ([MꢃNa]ꢃ) m/z
benzylmethylene), 4.44 (2H, d, H-1 of GlcN, H-1 of Gal), 4.06 (1H, dd, H-6 1457.4. Found 1456.3.
of Gal), 3.98—3.90 (2H, m, H-4 of GlcN, CH2CH2Si(CH3)3), 3.74—3.63
(4H, m, H-2, 5, 6 of GlcN), 3.56—3.50 (1H, m, CH2CH2Si(CH3)3), 3.44
(1H, br s, H-5 of Gal), 0.95—0.89 (2H, m, CH2CH2Si(CH3)3), ꢁ0.01 (9H, s,
Si(CH3)3). 13C-NMR (125 MHz, CDCl3) d: 170.5, 170.3, 170.1, 170.0,
168.8, 154.2, 137.7ꢂ2, 128.5ꢂ2, 128.0ꢂ2, 100.7 (C-1 of Gal), 100.3 (C-1
2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl-(1→4)-2-acetamido-3,6-
di-O-acetyl-2-deoxy-b-D-glucopyranosyl-(1→3)-2-acetamido-4,6-di-O-
acetyl-2-deoxy-b-D-galactopyranosyl-(1→4)-6-O-acetyl-2,3-di-O-ben-
zoyl-1-O-a-D-glucopyranosyl Trichloroacetimidate (28) Compound 28
was prepared from 27 (120 mg, 0.08 mmol) by the same method described
of GlcN), 95.5, 74.9, 74.7, 74.4, 73.6, 72.3, 70.9, 70.5, 69.1, 67.4, 67.2, for preparation of 9. The product was purified by silica chromatography
66.8, 60.9, 56.1, 20.8, 20.6ꢂ2, 20.5ꢂ2, 18.0, ꢁ1.46. MALDI-TOF-MS: using 30 : 1 chloroform–methanol as eluent to give 28 as amorphous powder
Calcd for C37H52Cl3NO17SiNa: ([MꢃNa]ꢃ) m/z 938.2. Found 938.5. HR- (76 mg, 61%). [a]D25 ꢃ45.0 (cꢀ1.0, CHCl3). 1H-NMR (600 MHz, CDCl3) d:
FAB-MS: Calcd for C37H52Cl3NO17SiNa: ([MꢃNa]ꢃ) m/z 938.1968. Found 8.60 (1H, s, OC(NH)CCl3), 6.07 (1H, d, J1,2ꢀ3.9 Hz, H-1 of Glc), 6.17 (1H,
938.1988.
2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl-(1→4)-3-O-acetyl-6-O-
benzyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-1-O-a-D-glu-
d, NH of GlcN), 5.86 (1H, d, NH of GalN), 5.02 (1H, d, J1,2ꢀ8.2 Hz, H-1 of
GlcN), 4.53 (1H, d, J1,2ꢀ7.7 Hz, H-1 of GalN), 4.48 (1H, d, J1,2ꢀ7.7 Hz, H-
1 of Gal). 13C-NMR (150 MHz, CDCl3) d: 100.9 (C-1 of Gal), 100.8 (C-1 of
copyranosyl Trichloroacetimidate (25) Compound 25 was prepared from GalN), 98.9 (C-1 of GlcN), 93.0 (C-1 of Glc), 90.7 (OC(NH)CCl3).
24 (263 mg, 0.29 mmol) by the same method described for preparation of 9.
The product was purified by silica chromatography using 2 : 1 hexane–ethyl
acetate as eluent to give 25 as an amorphous powder (202 mg, 74%). [a]D25
ꢃ37.6 (cꢀ1.2, CHCl3). 1H-NMR (500 MHz, CDCl3) d: 8.74 (1H, s,
C(NH)CCl3), 7.43—7.34 (5H, m, Ph), 6.41 (1H, d, J1,2ꢀ3.7 Hz, H-1 of
GlcN), 5.29 (1H, d, J3,4ꢀ3.7 Hz, H-4 of Gal), 5.28—5.24 (2H, m, H-3 of
GlcN, NH), 5.01 (1H, dd, J1,2ꢀ7.9 Hz, J2,3ꢀ10.4 Hz, H-2 of Gal), 4.81—
4.76 (2H, m, H-3 of Gal, benzylmethylene), 4.74—4.67 (2H, m, CH2CCl3),
4.44 (1H, d, benzylmethylene), 4.43 (1H, d, H-1 of Gal), 4.20—4.05 (4H, m,
H-2, 4 of GlcN, H-6 of Gal), 3.86 (1H, br d, H-5 of Gal), 3.79 (1H, dd, H-6a
of GlcN), 3.70—3.66 (2H, m, H-5, 6b of GlcN). 13C-NMR (125 MHz,
CDCl3) d: 171.0, 170.3, 170.1, 170.0, 168.8, 160.7, 154.2, 137.4, 128.7ꢂ2,
2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl-(1→4)-2-acetamido-3,6-
di-O-acetyl-2-deoxy-b-D-glucopyranosyl-(1→3)-2-acetamido-4,6-di-O-
acetyl-2-deoxy-b-D-galactopyranosyl-(1→4)-6-O-acetyl-2,3-di-O-ben-
zoyl-b-D-glucopyranosyl-(1→1)-(2S,3S,4R)-3,4-di-O-benzoyl-2-palmitoy-
lamidooctadecane-1,3,4-triol (29) Compound 29 was prepared from 28
(76 mg, 51.1 mmol) and 10 (78 mg, 102 mmol) by the same method described
for preparation of 11. The product was purified by silica chromatography
using 30 : 1 chloroform–methanol as eluent to give 29 as syrup (35 mg,
34%). [a]D25 ꢃ11.1 (cꢀ0.3, CHCl3). 1H-NMR (600 MHz, CDCl3) d: 6.21
(1H, d, NH of ceramide), 5.99 (1H, d, NH of GlcN), 5.73 (1H, d, NH of
GalN), 4.81 (1H, d, J1,2ꢀ8.2 Hz, H-1 of GlcN), 4.56 (1H, d, J1,2ꢀ7.4 Hz, H-
1 of Glc), 4.52 (1H, d, J1,2ꢀ8.4 Hz, H-1 of GalN), 4.47 (1H, d, J1,2ꢀ7.7 Hz,
128.3, 128.2ꢂ2, 100.4 (C-1 of Gal), 95.2, 94.9 (C-1 of GlcN), 90.7, 74.5, H-1 of Gal). 13C-NMR (150 MHz, CDCl3) d: 101.0 (C-1 of Gal), 100.6 (C-1
74.0, 73.8, 72.9, 71.0, 70.5, 70.2, 69.1, 66.8, 66.7, 60.9, 54.1, 20.8, 20.7,
20.62, 20.56, 20.5.
of Glc), 100.3 (C-1 of GalN), 98.9 (C-1 of GlcN). MALDI-TOF-MS: Calcd
for C108H149N3O37Na: ([MꢃNa]ꢃ) m/z 2103. Found 2103.
2-(Trimethylsilyl)ethyl 2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl-
(1→4)-3-O-acetyl-6-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonyl-
amino)-b-D-glucopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-(2,2,2-
trichloroethoxycarbonylamino)-b-D-galactopyranosyl-(1→4)-2,3-di-O-
benzoyl-6-benzyl-b-D-glucopyranoside (26) A solution of 13 (176 mg,
b-D-Galactopyranosyl-(1→4)-2-acetamido-2-deoxy-b-D-glucopyra-
nosyl-(1→3)-2-acetamido-2-deoxy-b-D-galactopyranosyl-(1→4)-b-D-glu-
copyranosyl-(1→1)-(2S,3S,4R)-2-hexadecanamido-4-octadecane-1,3,4-
triol (3) Compound 3 was prepared from 29 (12.0 mg) by the same
method described for preparation of 1. The product was purified by
0.18 mmol) and 25 (202 mg, 0.21 mmol) containing activated 4 Å MS Sephadex LH-20 column chromatography in MeOH to give 3 as white solid
(400 mg) in dry CH2Cl2 (4.0 ml) was stirred under an atmosphere of argon (4.1 mg, 55%). [a]D25 ꢃ4.8 (cꢀ0.13, 1 : 1 CHCl3–CD3OH). mp 185—186 °C.
for 2 h at room temperature, then cooled to ꢁ40 °C. TMSOTf (3.2 ml,
0.02 mmol) was added, and the mixture was stirred for 2 h at ꢁ40 °C, then
neutralized with Et3N. The solids were filtrated off and washed with CHCl3.
1H-NMR (600 MHz, 1 : 1 CDCl3–CD3OD) d: 4.60 (1H, d, J1,2ꢀ8.5 Hz, H-1
of GlcN), 4.48 (1H, d, J1,2ꢀ8.5 Hz, H-1 of GalN), 4.40 (1H, d, J1,2ꢀ7.7 Hz,
H-1 of Gal), 4.30 (1H, d, J1,2ꢀ7.5 Hz, H-1 of Glc). MALDI-TOF-MS: Calcd
The combined filtrate and washings were successively washed with brine, for C62H115N3O24Na: ([MꢃNa]ꢃ) 1309, Found 1309. HR-FAB-MS: Calcd
dried (MgSO4) and concentrated. The product was purified by silica gel col- for C62H115N3O24Na: ([MꢃNa]ꢃ) m/z 1308.7768. Found 1308.7742.
umn chromatography using 6 : 1 toluene–acetone as eluent to give 26 as an
1
amorphous powder (262 mg, 83%). [a]D25 ꢃ10.3 (cꢀ1.6, CHCl3). H-NMR
Acknowledgments This work was supported by a Grant-in-Aid for Sci-
(500 MHz, CDCl3) d: 8.01—7.23 (25H, m, 5Ph), 5.77 (1H, t, J2,3ꢀJ3,4ꢀ entific Research (No. 19590011) from the Ministry of Education, Culture,

June 2010
817
Sports, Science and Technology of Japan (MEXT), and the (MEXT) High-
Tech Research Center project. The authors are grateful to Ms. J. Hada for
providing HR-FAB-MS data.
Chem., 267, 2251—2257 (1992).
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13) Wuhrer M., Dennis R. D., Doenhoff M. J., Lochnit G., Geyer R., Gly-
cobiology, 10, 89—101 (2000).
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