Synthetic studies on glycosphingolipids from protostomia phyla: synthesis of amphoteric glycolipid analogues from the porcine nematode, Ascaris suum.

Article abstract of DOI:10.1248/cpb.50.600

A novel amphoteric glycosphingolipid, cholinephosphoryl-(-->6)-beta-D-GlcpNAc-(1-->3)-beta-D-Manp-(1-->4)-beta-D-Glcp-(1-->)-Cer, isolated from the porcine parasitic nematode, Ascaris suum, may be expected to be involved in host-parasite interactions. Thi

Full text of DOI:10.1248/cpb.50.600

600
Chem. Pharm. Bull. 50(5) 600—604 (2002)
Vol. 50, No. 5
Synthetic Studies on Glycosphingolipids from Protostomia Phyla:
Synthesis of Amphoteric Glycolipid Analogues from the Porcine
Nematode, Ascaris suum
Isao OHTSUKA,a Noriyasu HADA,a Hiroko OHTAKA,a Mutsumi SUGITA,b and Tadahiro TAKEDA*,a
a Kyoritsu College of Pharmacy; 1–5–30 Shibakoen, Minato-ku, Tokyo 105–8512, Japan: and b Department of Chemistry,
Faculty of Liberal Arts and Education, Shiga University; 2–5–1 Hiratsu, Otsu, Shiga 520–0862, Japan.
Received December 4, 2001; accepted February 7, 2002
A novel amphoteric glycosphingolipid, cholinephosphoryl-(→6)-b-D-GlcpNAc-(1→3)-b-D-Manp-(1→4)-b-D-
Glcp-(1→)-Cer, isolated from the porcine parasitic nematode, Ascaris suum, may be expected to be involved in
host-parasite interactions. This glycosphingolipid analogue containing octyl residue in place of ceramide was
synthesized as follows: The key reaction of this synthetic procedure is the formation of a intramolecular aglycon
delivery (IAD) approach for b-selective mannosylation. Then, a coupling of phosphocholine group at the position
C-6؆ of 16 was attempted using 2-chloro-2-oxo-1,3,2-dioxaphospholane, followed by reaction of the resulting
cyclic phosphate intermediate with anhydrous trimethylamine to give 17. Subsequent debenzylation and deben-
zylidenation afforded target compound (2).
Key words amphoteric glycosphingolipid; Ascaris suum; chemical synthesis; b-mannosidic linkage; phosphocholine
Glycosphingolipids are components of cell membranes the major component of amphoteric glycolipid, a-D-Galp-
and are thought to play important roles in a variety of biolog- (1→3)-b-D-GalpNAc-(1→4)-[cholinephosphoryl-(→6)]-b-D-
ical events.1) Especially sialic acid-containing glycosphin- GlcpNAc-(1→3)-b-D-Manp-(1→4)-b-D-Glcp-(1→)-Cer
golipids, such as gangliosides have been recognized life phe- from A. suum, having many biological activities, namely
nomenon, for example extracellular recognition, cell–cell in- stimulation-associated cytokines, tumor necrosis factor a
teraction, differentiation, oncogenesis and immunity. A num- (TNF-a), interleukin 1 (IL-1) and interleukin 6 (IL-6). We
ber of sialyloligosaccharides and mimetics have been synthe- applied intramolecular aglycon delivery (IAD) approach for
sized, researched systematic understanding of structure-func- b-selective mannosylation22) to the synthesis of the glyco-
tion at molecular level.2—4) On the other hand, the structure lipid analogue. The glycolipid was the target for the synthetic
of glycosphingolipids from invertebrate animal species differ studies described herein as part of our investigation into syn-
greatly from these of mammals.5—8) We have been interested thetic oligosaccharides of structural and biological interest.
in the relationships between the structure and biological
function of glycolipid derived from them and have so far Results and Discussion
synthesized oligosaccharides were isolated from various Pro-
Synthesis of Monosaccharide Derivatives Syntheses of
tostomia phyla.9—16) In the previous paper,17) we reported the the glucopyranosyl and 2-deoxy-2-phthalimido-glucopyra-
synthesis of neutral glycosphingolipid, of which core struc- nosyl building blocks 4 and 10 were carried out as depicted
ture was a b-D-GlcpNAc-(1→3)-b-D-Manp-(1→4)-b-D-Glcp- in Chart 1. Reductive ring-opening of the benzylidene acetal
(1→)-Cer, in larvae of the green-bottle fly, Lucilia cae- in 3 with sodium cyanoborohydride-hydrogen chloride in dry
sar.18,19) Sugita and co-workers.20) isolated novel amphoteric diethylether afforded compound octyl 2,3,6-tri-O-benzyl-b-
glycosphingolipid, cholinephosphoryl-(→6)-b-D-GlcpNAc- D-glucopyranoside (4).23) On the other hand, imidate donor
(1→3)-b-D-Manp-(1→4)-b-D-Glcp-(1→)-Cer (1), from the 3,4-di-O-benzyl-6-O-pivaloyl-2-deoxy-2-phthalimido-b-D-
porcine parasite nematode, Ascaris suum. This structure has glucopyranosyl trichloroacetimidate (10) was obtained from
a b-D-mannopyranosidic linkage and a phosphocholine at the 2-(trimethylsilyl)ethyl 2-deoxy-2-phthalimido-b-D-glucopy-
C-6 position of the glucosamine residue (Fig 1). Further- ranoside (5)24) by the following four step procedures. Regio-
more, Lochnit et al.21) have been published the structure of selective tritylation of the starting material with trityl chlo-
Fig. 1. Structure of an Amphoteric Glycosphingolipid from Ascaris suum (1) and Analogue (2)
To whom correspondence should be addressed. e-mail: takeda-td@kyoritsu-ph.ac.jp
© 2002 Pharmaceutical Society of Japan

May 2002
601
Reagents: (a) HCl–Et2O, NaBH3CN, THF, 65%; (b) trityl chloride, pyr., 77%; (c) BnBr, NaH, n-Bu4NBr, DMF, 88%; (d) TsOH, CH2Cl2–MeOH, quant.; (e) PivCl, pyr., quant.;
(f) (1) TFA, CH2Cl2, (2) DBU, CCl3CN, CH2Cl2, quant.
Chart 1
Reagents: (a) DDQ, CH2Cl2; (b) MeOTf, DTBMP, CH2Cl2, 35% (2 steps); (c) BnBr, NaH, DMF, 72%; (d) TBAF, THF, 83%.
Chart 2
ride (6), followed by benzylation (7) and subsequent acid butyl diphenylsilyl-2-O-p-methoxybenzyl-1-thio-a-
-mannopy-
D
hydrolysis of the trityl group (8) and then pivaloylation, ranoside (11)18,19) and 2,3-dichloro-5,6-dicyano-p-benzo-
gave compound 9. For selective removal of the 2- quinone (DDQ) in dichloromethane in the presence of 4A
(trimethylsilyl)ethyl (SE) group, compound 9 was treated24,25) molecular sieves (4A MS) afforded the mixed acetal in an es-
with trifluoroacetic acid in dichloromethane for 1 h at 0 °C to sentially quantitative yield. A stereochemical assignment of
give the 1-hydroxy compound, which, on further treatment26) this intermediate was mentioned by Ito and co-workers.27)
with trichloroacetonitrile in the presence of 1,8-diazabicy- Subsequent IAD was effected by methyl trifluoromethane-
clo[5.4.0]undec-7-ene (DBU) in dichloromethane for 30 min sulfonate (MeOTf) and 2,6-di-tert-butyl-4-methylpyridine
at 0 °C, gave only the corresponding b-trichloroacetimidate (DTBMP) in dichloromethane to afford the desired b-man-
10 (Chart 1).
noside 12 in 35% yield and the a-mannoside was not de-
Synthesis of Oligosaccharide Derivatives b-Mannosyl- tected. The anomeric hydrogen atom of the mannose unit ap-
1
ation was attempted in the intramolecular aglycon delivery peared as a signal at d 4.41 (s, H-1Ј) by the H-NMR spec-
approach. Thus, treatment of octyl 2,3,6-tri-O-benzyl-b-D- trum. The JCH coupling constant of C-1 (159.3 Hz) confirmed
glucopyranoside (4) with ethyl 4,6-O-benzylidene-3-O-tert- the configuration of the b-mannoside linkage in the 13C-

602
Vol. 50, No. 5
Table 1. 13C-NMR Data (d) for Selected Compounds
NMR spectrum.28) Compound 12 was subjected to O-benzy-
lation, followed by removal of tert-butyldiphenylsilyl group
with tetrabutylammonium fluoride (TBAF), to give the disac-
charide acceptor 14 (83%) (Chart 2). Glycosylation of 14
with the glycosyl donor 10 in the presence of trimethylsilyl
trifluoromethane sulfonate (TMSOTf) and 4A MS in
dichloromethane for 2 h at Ϫ25 °C gave the desired trisac-
charide 15 in 94% high yield.29) The 13C-NMR spectrum
showed three anomeric carbon atom signals at d 103.42 (C-
1), 102.12(C-1Ј) and 97.22 (C-1Љ). The 1H-NMR spectrum of
15 showed an anomeric proton doublet for a b-glucosamine
unit at d 5.41 (d, J1,2ϭ7.9 Hz, H-1Љ) indicating the newly
formed glycosidic linkage to be b-D configuration (Table 1).
Removal of the pivaroyl and phthalimido group from 15 by
NH2NH2–H2O, then only N-acetylation gave 16 in 81% yield.
Synthesis of Trisaccharide Containing Phosphocholine
Coupling of a phosphocholine group at the position C-6Љ of
16 was attempted using 2-chloro-2-oxo-1,3,2-dioxaphospho-
lane, followed by reaction of the resulting cyclic phosphate
intermediate with anhydrous trimethylamine at 65 °C in a
sealed vessel to give 17 in 69% yield for two steps.30) Com-
pound 17 revealed an [MϩH]ϩ ion peak at m/z 1451 in the
time of flight mass spectrometer (TOF-MS) spectrum. Re-
moval of the benzyl groups from 17 by catalytic hydrogenol-
ysis over 10% Pd–C gave the target compound 2 (Chart 3).
In conclusion, the analogue synthesis of novel amphoteric
glycosphingolipid 1 was achieved by employing highly ster-
eocontrolled glycosylation methods.
Compound
Carbon atom
12
13
14
15
C-1 (Glc)
103.7
(157.2)
81.8
82.8
78.2
103.5
(153.1)
82.3
84.7
78.1
103.6
(155.7)
82.3
84.7
78.2
103.4
(159.3)
82.3
84.8
78.0
74.7
68.9
102.1
(159.3)
79.7
79.5
78.1
67.8
68.4
97.2
(167.5)
55.8
73.3
77.4
76.7
62.7
101.3
74.4
74.9
75.0
75.1
75.7
76.5
(JC,H
)
2
3
4
5
6
74.5
68.5
74.5
68.9
74.6
69.3
C-1 (Man)
100.1
(159.3)
71.4
72.6
78.2
101.7
(163.5)
79.5
73.4
78.5
101.9
(159.2)
79.2
70.7
78.0
(JC,H
)
2
3
4
5
6
66.6
68.5
67.3
68.5
67.0
68.5
C-1 (GlcNAc)
(JC,H
)
2
3
4
5
6
PhCHϭ
PhCH2–
101.1
75.4
74.9
74.7
102.3
75.8
75.4
74.9
74.4
102.7
75.7
75.2
74.8
74.7
OCH2–
70.2
69.9
70.1
69.9
Reagents: (a) TMSOTf, CH2Cl2, 94%; (b) (1) NH2NH2–H2O, EtOH, (2) Ac2O, pyr., 81%; (c) (1) 2-chloro-2-oxo-1,3,2,-dioxaphospholane, Et3N, benzene, (2) Me3N, CH3CN,
69%; (d) Pd–C, AcOH, MeOH, 90%.
Chart 3

May 2002
603
Experimental
CH2Cl2 (1 ml), cooled to 0 °C, was added CF3COOH (1 ml), and the mixture
1
Optical rotations were determined with a JASCO digital polarimeter. H- was stirred for 1 h at room temperature and concentrated. Ethyl acetate and
and 13C-NMR spectra were recorded on a JNM A 500 FT NMR spectrome- toluene (1 : 2) were added and evaporated to give the 1-hydroxy compound.
ter in CDCl3 with Me4Si as the internal standard. Matrix-assisted laser des- To a solution of the residue in CH2Cl2 (2 ml) cooled at 0 °C were added
orption ionization-time of flight mass spectrometer (MALDI-TOF-MS) was trichloroacetonitlile (161 ml, 1.2 mmol) and DBU (6.9 ml, 0.05 mmol). The
recorded on a Perceptive Voyager RP mass spectrometer. TLC was per- mixture was stirred for 30 min at 0 °C. After completion of the reaction, the
formed on Silica gel 60 F254 (E. Merck) with detection by quenching of UV mixture was concentrated. Column chromatography of the residue on silica
fluorescence and by spraying with 10% H2SO4. Column chromatography gel (3 : 1 hexane–ethyl acetate) gave 10 (28 mg, quant.); [a]D24 ϩ10.3°
1
was carried out on silica gel 60 (E. Merck). Octyl 4,6-O-benzylidene-2,3-di- (cϭ0.7, CHCl3); H-NMR (CDCl3) d: 8.54 (1H, s, NH), 7.67—6.85 (14H,
O-benzyl-b-D-glucopyranoside (3), 2-(trimethylsilyl)ethyl 2-deoxy-2-phthal- m, Phϫ3), 6.41 (1H, d, J1,2ϭ8.5 Hz, H-1), 4.90, 4.83, 4.67, 4.50 (4H, each d,
imido-b-D-glucopyranoside (5) and ethyl 4,6-O-benzylidene-3-O-tert-butyl
diphenylsilyl-2-O-p-methoxybenzyl-1-thio-a-D-mannopyranoside (11) were
prepared by literature method.25—27)
Jgemϭ10.9 Hz, benzyl methyleneϫ2), 4.45—4.43 (3H, m, H-2, H-3, H-6a),
4.32 (1H, dd, H-6b), 3.89 (1H, q, H-5), 3.77 (1H, t, H-4), 1.24 (9H, t,
C(CH3)3).
Octyl 2,3,6-Tri-O-benzyl-b-D-glucopyranoside (4) To a solution of 3
(1.82 g, 3 mmol) and NaBH3CN (1.64 g, 24 mmol) in dry tetrahydrofuran
(THF) (10 ml) was added 3A MS, and the mixture was stirred for 2 h at
room temperature, then cooled to 0 °C. HCl–Et2O was added until the solu-
Octyl 4,6-O-Benzylidene-3-O-tert-butyldiphenylsilyl-b-D-mannopyra-
nosyl-(1→4)-2,3,6-tri-O-benzyl-b-D-glucopyranoside (12) To a stirred
mixture of DDQ (248 mg, 1.1 mmol) and 4A MS in dry CH2Cl2 (3 ml) were
added 11 (550 mg, 0.82 mmol), 4 (340 mg, 0.6 mmol) and 4A MS in dry
tion was acidic (pH paper, gas evolution). After 1 h, the reaction mixture was CH2Cl2 (11 ml) at 0 °C. The mixture was stirred for 10 min at 0 °C and for
poured into ice-water and extracted with CHCl3. The extract was washed se- 3 h at room temperature. The mixture was quenched with a solution of
quentially with saturated aqueous NaHCO3, dried (Na2SO4) and concen- ascorbic acid (0.7%) and NaOH (0.9%) in water (5 ml), diluted with ethyl
trated. The product was purified by silica gel column chromatography using acetate and filtered off. The filtrate was successively washed with water, sat-
6 : 1 hexane–ethyl acetate as eluent to give 4 (1.1 g, 66%).
2-(Trimethylsilyl)ethyl 6-O-Trityl-2-deoxy-2-phthalimido-b-
urated aqueous NaHCO3, dried (Na2SO4), and concentrated to give mixed
acetal. To a solution of this acetal, 2,6-di-tert-butyl-4-methyl-pyridine
D
-glucopy-
ranoside (6) To a solution of 5 (500 mg, 1.2 mmol) in pyridine (5 ml) was (DTBMP: 683 mg, 3.3 mmol) and 4A MS in dry CH2Cl2 (20 ml) were stirred
added trityl chloride (375 mg, 1.3 mmol) for 4 h at 50 °C, then concentrated. for 2 h at room temperature, then cooled to 0 °C. Methyl trifluoromethane-
The product was purified by silica gel column chromatography using 20 : 1 sulfonate (MeOTf: 340 ml, 3.1 mmol) was added to the mixture, which was
CHCl3–MeOH as eluent to give 6 (600 mg, 77%); [a]D24 Ϫ16.5° (cϭ1.0, stirred for 10 min at 0 °C and for 24 h at room temperature, then neutralized
CHCl3); 1H-NMR (CDCl3) d: 7.85—7.24 (19H, m, Phϫ4), 5.24 (1H, d, with Et3N. The mixture was filtered off and washed with water, dried
J
1,2ϭ7.9 Hz, H-1), 4.30 (1H, t, H-3), 4.13 (1H, t, H-2), 3.94 (1H, q, (Na2SO4) and concentrated. The product was purified by silica gel column
–OCH2–), 3.62 (1H, t, H-4), 3.61 (1H, t, H-6a), 3.53—3.48 (2H, m, H-5, chromatography using 8 : 1 hexane–ethyl acetate as eluent to give 12 (222
–CH2–), 3.44 (1H, dd, H-6b), Ϫ0.08 (9H, s, Si(CH3)3). MALDI-TOF-MS: mg, 35%); [a]D24 ϩ11.2° (cϭ1.0, CHCl3); H-NMR (CDCl3) d: 7.66—7.14
Calcd for C38H41NO7Si: m/z 651. Found: m/z 674 [MϩNa]ϩ.
(30H, m, Phϫ6), 5.34 (1H, s, benzylidene methyne), 4.95, 4.87, 4.75, 4.68,
2-(Trimethylsilyl)ethyl 3,4-Di-O-benzyl-6-O-trityl-2-deoxy-2-phthal- 4.57, 4.30 (6H, each d, Jgemϭ10.4 Hz, benzyl methyleneϫ3), 4.41 (1H, s, H-
imido-b-D-glucopyranoside (7) To a solution of 6 (580 mg, 0.9 mmol), 1Ј), 4.33 (1H, d, 1,2ϭ7.9 Hz, H-1), 4.04 (1H, dd, 5,6aϭ10.4 Hz,
tetrabutyl ammonium bromide (88 mg, 0.27 mmol) and BnBr (0.44 ml, 3.6 6a,6bϭ4.9 Hz, H-6aЈ), 3.92 (1H, q, –OCH2–), 3.90 (1H, t, J4,5ϭ9.2 Hz, H-4Ј),
1
J
J
J
mmol) in N,N-dimethylformamide (DMF) (10 ml) was added NaH (66 mg, 3.85 (1H, t, J3,4ϭ9.2 Hz, H-3), 3.72 (1H, dd, J2,3ϭ3.7 Hz, J3,4ϭ9.8 Hz, H-3Ј),
0.27 mmol) for 2 h at 0 °C. The mixture was diluted with ethyl acetate 3.60—3.59 (2H, m, H-4, H-6b), 3.58 (1H, d, J2,3ϭ3.7 Hz, H-2Ј), 3.56 (1H, t,
(10 ml), then washed with water, dried (Na2SO4) and concentrated. The
J6a,6bϭ10.4 Hz, H-6bЈ), 3.48—3.45 (2H, m, H-5, –OCH2–), 3.38—3.35 (2H,
product was purified by silica gel column chromatography using 10 : 1 m, H-2, H-6b), 2.89 (1H, q, H-5Ј), 2.69 (1H, s, OH), 1.64 (2H, q,
hexane–ethyl acetate as eluent to give 7 (666 mg, 88%); [a]D24 ϩ5.50° –OCH2CH2–), 1.25 (10H, s, –CH2–ϫ5), 1.03 (9H, s, CH3ϫ3), 0.88 (3H, t,
(cϭ1.0, CHCl3); 1H-NMR (CDCl3) d: 7.81—6.84 (34H, m, Phϫ6), 5.20
–CH2CH3). 13C-NMR (CDCl3): d 103.7 (JCHϭ157.2 Hz, C-1), 101.1 (ben-
(1H, d, J1,2ϭ7.9 Hz), 4.80, 4.69, 4.44, 4.42 (4H, each d, Jgemϭ11.2 Hz, ben- zylidene methyne), 100.1 (JCHϭ159.3 Hz, C-1Ј), 82.8 (C-3), 81.8 (C-2), 78.1
zyl methyleneϫ2), 4.28 (1H, t, H-3), 4.26 (1H, t, H-2), 4.05—3.98 (2H, m, (C-4Ј), 78.1 (C-4), 75.4, 74.9, 74.7 (benzyl methylene), 74.3 (C-5), 72.6 (C-
H-6a, –OCH2–), 3.63 (1H, t, H-4), 3.60—3.54 (2H, m, H-5, H-6b), 3.28 3Ј), 71.4 (C-2Ј), 70.2 (–OCH2–), 68.6 (C-6), 68.5 (C-6Ј), 66.6 (C-5Ј), 31.8
(1H, q, –OCH2–), Ϫ0.07 (9H, s, Si(CH3)3). MALDI-TOF-MS: Calcd for (–OCH2CH2–), 29.8, 29.7, 29.4, 29.3 (–CH2–), 26.8 (CH3ϫ3), 26.2
C52H53NO7Si: m/z 831. Found: m/z 854 [MϩNa]ϩ.
(–CH2CH3), 14.1 (–CH2CH3). MALDI-TOF-MS: Calcd for C64H78O11Si:
2-(Trimethylsilyl)ethyl 3,4-Di-O-benzyl-2-deoxy-2-phthalimido-b-D-
glucopyranoside (8) To a solution of 7 (315 mg, 0.34 mmol) in CH2Cl2
(3 ml) and MeOH (1.5 ml) was added p-toluene sulfonic acid (146 mg,
0.68 mmol) for 20 h at room temperature. The mixture was added Et3N, and
m/z 1050. Found: m/z 1073 [MϩNa]ϩ.
Octyl 2-O-Benzyl-4,6-O-benzylidene-3-O-tert-butyldiphenylsilyl-b-D-
mannopyranosyl-(1→4)-2,3,6-tri-O-benzyl-b-D-glucopyranoside (13) To
a solution of 12 (437 mg, 0.41 mmol) and BnBr (0.10 ml, 0.8 mmol) in DMF
concentrated. The product was purified by silica gel column chromatography (5 ml) was added NaH (14.9 mg, 0.62 mmol) for 2 h at 0 °C. The mixture
using 20 : 1 benzene–acetone as eluent to give 8 (293 mg, quant.); [a]D24
was diluted with ethyl acetate (10 ml), washed with water, dried (Na2SO4)
1
ϩ1.40° (cϭ1.0, CHCl3); H-NMR (CDCl3) d: 7.67—6.86 (14H, m, Phϫ3), and concentrated. The product was purified by silica gel column chromatog-
5.19 (1H, d, J1,2ϭ7.9 Hz), 4.38, 4.80, 4.73, 4.45 (4H, each d, Jgemϭ11.0 Hz, raphy using 10 : 1 hexane–ethyl acetate as eluent to give 13 (341 mg, 72%);
benzyl methyleneϫ2), 4.35 (1H, t, J3,4ϭ8.8 Hz, H-3), 4.20 (1H, t, H-2), 3.93 [a]D24 Ϫ8.71° (cϭ1.0, CHCl3); 1H-NMR (CDCl3) d: 7.65—7.12 (35H, m,
(1H, dd, H-6a), 3.87 (1H, q, –OCH2–), 3.80—3.72 (2H, m, H-4, H-6b), 3.56 Phϫ7), 5.31 (1H, s, benzylidene methyne), 4.95, 4.93, 4.92, 4.77, 4.76, 4.71,
(1H, q, H-5), 3.43 (1H, q, –OCH2–), Ϫ0.08 (9H, s, Si(CH3)3). MALDI-TOF- 4.68, 4.43 (8H, each d, Jgemϭ11.6 Hz, benzyl methyleneϫ4), 4.30 (1H, d,
MS: Calcd for C33H59NO7Si: m/z 589. Found: m/z 612 [MϩNa]ϩ.
J1,2ϭ7.9 Hz, H-1), 4.22 (1H, s, H-1Ј), 4.19 (1H, dd,
J5,6aϭ10.4 Hz,
2-(Trimethylsilyl)ethyl 3,4-Di-O-benzyl-6-O-pivaloyl-2-deoxy-2-phthal-
imido-b-D-glucopyranoside (9) To a solution of 8 (293 mg, 0.34 mmol) in
pyridine (4 ml) was added pivaloyl chloride (2 ml) for 5 h at 0 °C. The mix-
J
6a,6bϭ4.88 Hz, H-6aЈ), 4.10 (1H, t, H-6a), 4.05 (1H, t, J4,5ϭ9.9 Hz, H-4Ј),
3.86 (1H, dd, J2,3ϭ3.3 Hz, J3,4ϭ12.8 Hz, H-3Ј), 3.83—3.78 (2H, m, H-6b,
–OCH2–), 3.51 (1H, d, H-2Ј), 3.46—3.45 (2H, m, H-5, –OCH2–), 3.39—3.3
1
ture was diluted with MeOH (5 ml), then extracted with CHCl3, washed with (3H, m, H-2, H-4, H-6b), 3.01 (1H, q, H-5Ј), 1.55 (2H, q, –OCH2CH2–),
5% HCl, dried (Na2SO4) and concentrated. The product was purified by sil-
ica gel column chromatography using 4 : 1 hexane–ethyl acetate as eluent to
1.25 (10H, s, –CH2–ϫ5), 1.00 (9H, s, CH3ϫ3), 0.88 (3H, t, –CH2CH3). 13C-
NMR (CDCl3): d 103.5 (C-1), 102.3 (benzylidene methyne), 101.7 (C-1Ј),
give 9 (336 mg, quant.); [a]D24 ϩ0.58° (cϭ1.0, CHCl3); 1H-NMR (CDCl3) d: 84.7 (C-3), 82.3 (C-2), 79.5 (C-2Ј), 78.5 (C-4Ј), 78.1 (C-4), 75.7, 75.4, 74.9,
7.67—6.87 (14H, m, Phϫ3), 5.15 (1H, d, J1,2ϭ7.9 Hz, H-1), 4.89, 4.80,
4.64, 4.67 (4H, each d, Jgemϭ11.2 Hz, benzyl methyleneϫ2), 4.45 (1H, d, H-
74.4 (benzyl methyleneϫ4), 74.5 (C-5), 73.4 (C-2), 69.9 (–OCH2–), 68.9
(C-6), 68.5 (C-6Ј), 67.3 (C-5), 31.6 (–OCH2CH2–), 29.9, 29.7, 29.4, 29.3
6a), 4.35 (1H, t, J3,4ϭ8.5 Hz, H-3), 4.22 (1H, dd, H-6b), 4.15 (1H, t, H-2), (–CH2–), 26.8 (CH3ϫ3), 26.0 (–CH2CH3), 14.3 (–CH2CH3). MALDI-TOF-
3.86 (1H, q, –OCH2–), 3.71 (1H, q, H-5), 3.64 (1H, t, J4,5ϭ9.8 Hz, H-4), MS: Calcd for C71H84O11Si: m/z 1140. Found: m/z 1063 [MϩNa]ϩ.
3.45 (1H, q, –OCH2–), 1.26 (9H, t, C(CH3)3), Ϫ0.08 (9H, s, Si(CH3)3).
MALDI-TOF-MS: Calcd for C38H47NO8Si: m/z 673. Found: m/z 696
[MϩNa]ϩ.
Octyl 2-O-Benzyl-4,6-O-benzylidene-b-D-mannopyranosyl-(1→4)-2,3,6-
tri-O-benzyl-b-D-glucopyranoside (14) To a solution of 13 (180 mg, 0.15
mmol) in THF (3 ml) was added tetrabutylammoniumfluoride, 1.0 M solution
3,4-Di-O-benzyl-6-O-pivaloyl-2-deoxy-2-phthalimido-b-D-glucopyra- in THF (TBAF: 4 ml). The reaction mixture was stirred for 2 h at room tem-
nosyl Trichloroacetimidate (10) To a solution of 9 (26 mg, 0.04 mmol) in perature, then extracted with CHCl3, washed with 5% HCl, dried (Na2SO4)

604
Vol. 50, No. 5
and concentrated. The product was purified by silica gel column chromatog-
raphy using 3 : 1 hexane–ethyl acetate as eluent to give 14 (115 mg, 83%);
[a]D24 Ϫ12.7° (cϭ1.0, CHCl3); 1H-NMR (CDCl3) d: 7.48—7.25 (25H, m,
Phϫ5), 5.53 (1H, s, benzylidene methyne), 5.06, 4.96, 4.94, 4.86, 4.78, 4.71,
4.66, 4.54 (8H, each d, Jgemϭ11.0 Hz, benzyl methyleneϫ4), 4.49 (1H, s, H-
1Ј), 4.38 (1H, d, J1,2ϭ7.9 Hz, H-1), 4.30 (1H, dd, J5,6aϭ10.4 Hz, J6a,6bϭ4.9
Hz, H-6aЈ), 4.19 (1H, d, H-6a), 3.88—3.78 (3H, m, H-2Ј, H-4Ј, H-6bЈ, H-3,
–OCH2–), 3.69—3.66 (2H, m, H-4, H-3Ј), 3.58—3.54 (2H, m, H-6b,
–OCH2–), 3.45—3.35 (2H, m, H-2, H-5), 3.28 (1H, q, H-5Ј), 2.38 (1H, s,
OH), 1.59 (2H, s, –OCH2CH2–), 1.25 (10H, s, –CH2–ϫ5), 0.87 (3H, t,
–CH2CH3). 13C-NMR (CDCl3): d 103.6 (C-1), 102.7 (benzylidene methyne),
101.9 (C-2Ј), 84.7 (C-3), 82.3 (C-2), 79.2 (C-2Ј), 78.2 (C-4), 78.0 (C-4),
75.7, 75.6, 74.8, 74.7 (benzyl methyleneϫ4), 74.6 (C-5), 70.1 (–OCH2–),
69.3 (C-6), 68.5 (C-6Ј), 67.0 (C-5Ј), 31.6 (–OCH2CH2–), 29.9, 29.7, 29.4,
29.3 (–CH2–), 26.0 (–CH2CH3), 14.3 (–CH2CH3). MALDI-TOF-MS: Calcd
for C55H66O11: m/z 903. Found: m/z 926 [MϩNa]ϩ.
(1H, d,
J
1,2ϭ7.9 Hz, H-1), 4.08 (2H, m, OCH2CH2), 3.39 (2H, m,
CH2CH2N), 3.15 (9H, s, N(CH3)3). HR-FAB-MS: Calcd for C33H64N2O19P
[MϩH]ϩ: m/z 823.3841. Found: m/z 823.3878.
Acknowledgements This work was supported by the Sasagawa Scien-
tific Research Grant from The Japan Science Society. We gratefully ac-
knowledge a Grant-in-Aid for Scientific Research (No 12672062) from the
Ministry of Education, Science, Sports and Culture of Japan. The authors
are grateful to Ms. J. Hada for providing NMR and MS data.
References
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1
give 15 (41 mg, 94%); [a]D24 Ϫ7.60° (cϭ1.0, CHCl3); H-NMR (CDCl3) d:
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TOF-MS: Calcd for C77H91NO16: m/z 1285. Found: m/z 1308 [MϩNa]ϩ.
Octyl 3,4-Di-O-benzyl-6-O-phosphocoline-2-deoxy-2-acetamido-b-D-
glucopyranosyl-(1→3)-2-O-benzyl-4,6-O-benzylidene-b-D-mannopyra-
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H2O–MeOH as eluent to give 17 (47.8 mg, 69%); [a]D24 Ϫ14.9° (cϭ1.2,
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1
CHCl3); H-NMR (CDCl3) d: 5.11 (1H, d, H-1Љ), 4.30 (1H, d, J1,2ϭ7.9 Hz,
H-1), 4.28 (1H, s, H-1Ј), 4.11 (2H, m, OCH2CH2), 3.39 (2H, m, CH2CH2N),
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Octyl 6-O-Phosphocholine-2-deoxy-2-acetamido-b-D-glucopyranosyl-
(1→3)-b-D-mannopyranosyl-(1→4)-b-D-glucopyranoside (2) A solution
of 17 (47.8 mg, 0.33 mmol) in MeOH (3 ml) and AcOH (1 ml) was hydro-
genated over 10% Pd–C (50 mg) for 24 h at room temperature, then filtered
through Celite and the residue was washed with methanol and concentrated.
Column chromatography (1 : 1 H2O : MeOH) of the residue on Sephadex
LH-20 gave 2 (24 mg, 90%); [a]D24 Ϫ11.9° (cϭ0.6, H2O); 1H-NMR (1 : 1
CD3OD : CDCl3) d: 4.58 (1H, d, J1,2ϭ8.5 Hz, H-1Љ), 4.48 (1H, s, H-1Ј), 4.18