Synthesis and antigenic property of a novel Sialyl 6-O-Sulfo Lewis X neo-glycolipid containing lactamized neuraminic acid

Article abstract of DOI:10.1081/CAR-200030039

Synthesis and antigenic property of a novel 6-O-sulfated sLe^x neo-glycolipid containing lactamized neuraminic acid are described. Coupling of methyl (methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-trifluoroacetamido-D- glycero-α-D-galacto-2-nonulo

Full text of DOI:10.1081/CAR-200030039

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Synthesis and Antigenic Property
of a Novel Sialyl 6‐O‐Sulfo Lewis X
Neo‐glycolipid Containing Lactamized
Neuraminic Acid
Masanori Yamaguchi a , Hideharu Ishida a , Akiko Kanamori b , Reiji
Kannagi b c & Makoto Kiso a c
a Department of Applied Bio‐organic Chemistry , Gifu University ,
Gifu, 501‐1193, Japan
b Department of Molecular Pathology, Research Institute , Aichi
Cancer Center , Nagoya, Japan
c CREST , Japan Science and Technology Agency (JST) , Japan
Published online: 17 Aug 2006.
To cite this article: Masanori Yamaguchi , Hideharu Ishida , Akiko Kanamori , Reiji Kannagi & Makoto
Kiso (2004) Synthesis and Antigenic Property of a Novel Sialyl 6‐O‐Sulfo Lewis X Neo‐glycolipid
Containing Lactamized Neuraminic Acid, Journal of Carbohydrate Chemistry, 23:4, 201-215, DOI:
10.1081/CAR-200030039
To link to this article: http://dx.doi.org/10.1081/CAR-200030039
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JOURNAL OF CARBOHYDRATE CHEMISTRY
Vol. 23, No. 4, pp. 201–215, 2004
Synthesis and Antigenic Property of a Novel Sialyl
6-O-Sulfo Lewis X Neo-glycolipid Containing
Lactamized Neuraminic Acid#
2
Masanori Yamaguchi,1 Hideharu Ishida,1, Akiko Kanamori,
*
Reiji Kannagi,2,3 and Makoto Kiso1,3,
*
1Department of Applied Bio-organic Chemistry, Gifu University, Gifu, Japan
2Department of Molecular Pathology, Research Institute,
Aichi Cancer Center, Nagoya, Japan
3CREST, Japan Science and Technology Agency (JST), Japan
CONTENTS
ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
I. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
II. RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . 203
III. EXPERIMENTAL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
A. General Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
#Synthetic Studies on Sialoglycoconjugates, Part 136. For part 135, see Ref.[1]
.
Correspondence: Hideharu Ishida and Makoto Kiso, Department of Applied Bio-organic
*
Chemistry, Gifu University, Gifu 501-1193, Japan; E-mail: ishida@cc.gifu-u.ac.jp or kiso@cc.
gifu-u.ac.jp.
201
DOI: 10.1081/CAR-200030039
0732-8303 (Print); 1532-2327 (Online)
www.dekker.com
Copyright # 2004 by Marcel Dekker, Inc.

202
Yamaguchi et al.
ABSTRACT
Synthesis and antigenic property of a novel 6-O-sulfated sLex neo-glycolipid contain-
ing lactamized neuraminic acid are described. Coupling of methyl (methyl 4,7,8,9-
tetra-O-acetyl-3,5-dideoxy-5-trifluoroacetamido-D-glycero-a-D-galacto-2-nonulopyr-
anosylonate)-(2 ! 3)-4,6-di-O-acetyl-2-O-benzoyl-1-thio-b-D-galactopyranoside (3)
with 2-(tetradecyl)hexadecyl (2,3,4-tri-O-benzyl-a-L-fucopyranosyl)-(1 ! 3)-2-acet-
amido-2-deoxy-6-O-4-methoxyphenyl-b-D-glucopyranoside (7) gave
a protected
sLex tetrasaccharide glycolipid (8). Removal of all the acyl protecting groups and
subsequent lactamization afforded the lactamized sLex derivative(10), which was
converted to the target compound (14) by selective removal of the 4-methoxyphenyl
group and 6-O-sulfation of the GlcNAc residue, and removal of all protective groups
under the basic conditions furnished the target molecule. The antigenic property of
the synthesized neo-glycolipid was examined by TLC-immunostaining with G159
monoclonal antibody.
Key Words: Selectin; Sialic acid; Sialyl Lewis X; Glycolipid.
INTRODUCTION
L-selectin mediates the attachment of lymphocytes to specialized high endothelial
venules (HEV) in the course of their migration from the blood to lymphoid tissues.[2]
We have shown that the sialyl 6-O-sulfo Lewis X serves as the major endogenous
ligand for L-selectin on HEV in human lymph nodes,[3,4] and proposed the ligand proces-
sing pathway involving “activation” by de-N-acetylation of sialic acid and “inactivation”
by lactamization (Fig. 1). The G159 mAb has newly been cloned as the monoclonal
antibody which specifically recognizes the lactamized-sialyl 6-O-sulfo Lex ganglioside.[5]
Figrure 1. Proposed ligand processing pathway and binding reactivity for human L-selectin.

Property of Sialyl 6-O-Sulfo Lewis X Neo-glycolipid
203
To investigate the structure required for recognition by G159 monoclonal antibody, we
have synthesized lactamized GM4[6] and sLex[7] analogs containing lactamized neuraminic
acid. However, these compounds were not recognized by G159mAb, suggesting that the
antibody may specifically recognize the structure involving the 6-O-sulfated GlcNAc
residue. We here describe the synthesis of lactamized-sialyl 6-O-sulfo Lewis X tetrasaccharide
glycolipid (14) containing both lactamized neuraminic acid and the 6-O-sulfated GlcNAc
residue being expected as the minimal structure defined by G159 mAb (Fig. 2).
RESULTS AND DISCUSSION
The main problems to be solved in the synthesis of the title compound 14 are (i) suit-
able protection of the amino group at C-5 of neuraminic acid and 6-OH of the GlcNAc
residue, which undergo lactamization and sulfation, respectively, and (ii) efficient intro-
duction of the lipid part and fucose. To solve the first problem, we designed the suitably
protected sialyl Lewis X tetrasaccharide glycolipid 8 (Sch. 1) as the key intermediate,
Figure 2. Lactamized-sialyl Lex analog.

204
Yamaguchi et al.
Scheme 1. (a) DMTST/CH2Cl2, MS4A, 08C, 57%; (b) NaOMe/MeOH, then H2O, 458C, 95%; (c)
1, HBTU, HOBt/DMF, 658C, 2, Ac2O/Pyr, 76.2% (two steps); (d) 1, H2, Pd(OH)2/EtOH, 2, Ac2O/
Pyr, 93% (two steps).
in which the amino group of neuraminic acid and 6-OH of the GlcNAc residue are pro-
tected by the trifluoroacetyl (TFAc) and 4-methoxyphenyl (MP) groups, respectively.
The TFAc protected sialic acid is equivalent to the N-deacetylated and lactamized sialic
acids, and the MP group can be chemoselectively cleaved by ceric ammonium nitrate
(CAN). For the second problem, we examined the efficient construction of 2-(tetrade-
cyl)hexadecyl (2,3,4-tri-O-benzyl-a-L-fucopyranosyl)-(1 ! 3)-2-acetamido-6-O-4-meth-
oxyphenyl-2-deoxy-b-D-glucopyranoside (7) (Sch. 2).

Property of Sialyl 6-O-Sulfo Lewis X Neo-glycolipid
205
Scheme 2. (a) 1, NIS, TfOH/benzene, 78C; 2, 80% AcOHaq, 458C, 51%, two steps and (b) MPOH,
PPh3, DEAD/THF, 808C, 88%.
The suitably protected sialyl galactose donor 3 was prepared by treatment of 1[8] with
boron trifluoride etherate in dichloromethane–acetic anhydride, giving the acetate 2 (97%,
a/b ¼ 5/1),[9] and successive treatment of 2 with methylthiotrimethylsilane (TMSSMe)
and trimethylsilyl trifluoromethanesulfonate (TMSOTf ) in dichloroethane for 72 hr at
508C (Sch. 3). For the synthesis of acceptor 7, compound 4 was first synthesized by
using the established method,[10] and then coupled with the fucose donor 5.[11] The result-
ing crude disaccharide was treated with 80% AcOH at 458C to afford 6 in 51% yield in two
steps (Sch. 2). The low yield of this reaction was owing to a cleavage of the tri-O-benzy-
1
lated fucoside during the acidic hydrolysis of the benzylidene acetal. In the H NMR
spectrum of 6, the anomeric proton of fucose appeared at d 4.94 (d, J1,2 ¼ 3.8 Hz, H-1b), indi-
cating the newly formed glycoside linkage to be a. The regioselective 4-methoxyphenylation
.
Scheme 3. (a) BF3 OEt2, Ac2O/CH2Cl2, 97% and (b) TMSSMe, TMSOTf/CH2ClCH2Cl, 508C, 89%.

206
Yamaguchi et al.
at O-6 of 6 was carried out by treatment with p-methoxyphenol (MPOH), PPh3, and
diethylazodicarboxylate (DEAD) in THF,[12,13] to give 7 in 88% yield.
Coupling of 3 and 7, promoted by dimethyl(methylthio)sulfonium triflate
(DMTST),[14,15] gave the desired tetrasaccharide glycolipid 8 in 57% yield (Sch. 1).
The partial N,O-deacylation of 8 with sodium methoxide in methanol for 72 hr at 458C,
and subsequent saponification of the methylester group afforded N-deacetylated sLex
derivative 9 in 95% yield, which upon treatment with HBTU[16] and HOBt in DMF at
658C, followed by acetylation to afford the desired 10 in good yield (76%, two steps).
The 1H NMR spectrum of 10 showed signals at d 2.32 (dd, 1H, J3b,4 ¼ 10.1 Hz,
Jgem ¼ 14.6 Hz, H-3cb), and d 2.41 (dd, 1H, J3a,4 ¼ 5.5 Hz, Jgem ¼ 14.6 Hz, H-3ca),
which are characteristic of the B5,2 boat conformation of sialic acid.[6] Hydrogenolytic
removal of the benzyl (Bn) groups in 10 and the following acetylation gave 11 (93%,
two steps). Selective cleavage of the MP group in 11 and the subsequent 6-O-sulfation
.
of 12 with a sulfur trioxide–pyridine (SO3 Pyr.) complex in DMF, followed by an
addition of triethylamine to stabilize the sulfate group during the column chromatography,
gave 13 in good yield. Removal of all protective groups in 13 under alkaline conditions
furnished the target compound 14 (GSC-534) in high yield (Sch. 4).
As shown in Fig. 3, the compound 14 (GSC-534) was not stained with G159 antibody
in TLC-immunostaining, in contrast with the lactamized-sialyl 6-O-sulfo Lex ganglioside
.
Scheme 4. (a) CAN/CH3CN, H2O, 08C, 75%; (b) SO3 Pyr complex/DMF, then Et3N, r.t., 78%;
and (c) NaOMe/MeOH, r.t., 98%.

Property of Sialyl 6-O-Sulfo Lewis X Neo-glycolipid
207
Figure 3. TLC-immunostaining patterns of synthesized glycolipids. Lanes 1, 3, and 5 are Orcinol/
H2SO4 staining. Lanes 2, 4, and 6 are G159 mAb staining. Lactamized-sialyl 6-O-sulfo Lewis X
ganglioside (GSC-535) and lactamized-sialyl 6-O-sulfo paragloboside (GSC-550) were detected
with G159 mAb, but compound 14 was not.
(GSC-535)[1] as well as the corresponding paragloboside derivative (GSC-550)[1] (Fig. 2).
These results suggest that the lactose moiety could be critical for the recognition by
G159 mAb either as the essential component of the G159-defined determinant or as a
spacer to keep the determinant apart from the lipid moiety adsorbed on TLC sheets.
The difference in the structures of the ceramide and the artificial ceramide (B30) also
may affect the recognition by G159 mAb. A further study to elucidate the details of the
recognition mapping defined by G159 mAb is now under investigation.
In summary, we have successfully synthesized a novel 6-O-sulfated sLex neo-
glycolipid, which was designed as a probe to determine the minimum structure required
for the recognition by G159 mAb. Utilizing this synthetic probe, the significance of the
lactose moiety for the recognition by G159 mAb was suggested.
EXPERIMENTAL
General Methods
TLC was conducted on E. Merck silica gel 60 F-254 aluminum plate. Compounds were
visualized by exposure to UV light or by spraying with a solution of 10% H2SO4 in ethanol.
Column chromatography on silica gel (Fuji Silysia Co., 300 mesh) was performed with the
solvent systems (v/v) specified. Specific rotations were determined with a Horiba SEPA-
1
300 high-sensitive polarimeter at 258C. H NMR and 13C NMR spectra were recorded

208
Yamaguchi et al.
at 300 K with a Varian Unity Inova 500 (500 MHz) or Varian Unity Inova 400 (100.6 MHz)
spectrometer, respectively. The values (ppm) are given relative to Me4Si as the internal stan-
dard. MALDI-TOF mass spectra were recorded on a BRUKER Daltonics Autoflex-G
MALDI-TOF Mass spectrometer instrument using a 2,5-dihydroxybenzoic acid matrix.
˚
Dichloromethane, methanol, ethanol, benzene, and DMF were kept dry over 4 A MS,
˚
while pyridine and acetonitrile were kept dry over 3 A MS.
Methyl
(methyl
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-trifluoroacetamido-D-
glycero-a-D-galacto-2-nonulopyranosylonate)-(2 ! 3)-4,6-di-O-acetyl-2-O-benzoyl-
1-thio-b-D-galactopyranoside (3). To a solution of 1 (108 mg, 0.118 mmol) in Ac2O
.
(0.5 mL) and dry CH2Cl2 (1 mL) was added BF3 OEt2 (130 mL, 0.47 mmol), and the
mixture was stirred for 24 hr at room temperature and extracted with chloroform.
The extract was successively washed with NaHCO3 and water, dried (Na2SO4), and
concentrated. Column chromatography (70 : 1 CHCl3–MeOH) of the residue on silica
gel gave 2 (108.1 mg, 97.3%) as an amorphous mass (a/b ¼ 5/1).
To a solution of 2 (108.1mg, 0.12mmol) in dry CH2ClCH2Cl (1mL) were added
TMSSMe (57.4 mL, 0.45mmol) and TMSOTf (77.8 mL, 0.4 mmol). The mixture was
stirred for 72hr at 508C. After dilution with chloroform, the extract was successively
washed with 1 M sodium carbonate and water, dried (Na2SO4), and concentrated. Column
chromatography (100: 1 CHCl3–MeOH) of the residue on silica gel afforded 3 (94.5 mg,
1
88.6%) as an amorphous mass; [a]D þ 31.28 (c 0.18, CHCl3); H NMR (CDCl3) d 1.42
(s, 3H, MeS), 1.71 (t, 1H, Jgem ¼ J3ax,4 ¼ 12.5Hz, H-3bax), 1.94, 2.05, 2.056, 2.12, 2.16,
2.18 (6s, 18H, 6AcO), 2.57 (dd, 1H, J3eq,4 ¼ 4.5 Hz, Jgem ¼ 12.5Hz, H-3beq), 3.84
(s, 3H, COOMe), 4.34 (dd, 1H, J8,9 ¼ 2.8 Hz, Jgem ¼ 12.5Hz, H-9b), 4.69 (d, 1H,
J1,2 ¼ 9.8 Hz, H-1a), 4.76 (dd, J2,3 ¼ 9.6 Hz, J3,4 ¼ 3.2 Hz, H-3a), 4.96 (m, 1H, H-4b),
5.03 (d, 1H, H-4a), 5.18 (dd, J6,7 ¼ 2.0, J7,8 ¼ 8.9 Hz, H-7b), 5.35 (t, 1H, H-2a), 5.52
(m, 1H, H-8b), 6.48 (d, 1H, J5,NH ¼ 9.1 Hz, NH of b), 7.46–8.14 (m, 5 H, 1Ph).
13C NMR (CDCl3) d 11.40, 11.99, 20.45, 20.70, 20.74, 20.79, 21.48, 37.29, 49.63,
53.29, 62.10, 62.24, 66.30, 67.95, 68.48, 68.62, 71.10, 72.20, 74.36, 83.22, 96.84,
128.46, 130.06, 130.26, 133.53, 157.67, 165.53 (C55O), 167.82 (C55O), 169.77
(C55O), 170.22 (C55O), 170.52 (C55O), 170.73 (C55O), 170.74 (C55O), 170.79 (C55O).
Anal. Calcd for C38H46F3NO20S (925.83): C, 49.30; H, 5.01; N, 1.51. Found: C,
49.12; H, 4.83; N, 1.25.
2-(Tetradecyl)hexadecyl (2,3,4-tri-O-benzyl-a-L-fucopyranosyl)-(1 ! 3)-2-acet-
amido-2-deoxy-b-D-glucopyranoside (6). To a solution of 4 (105 mg, 0.14 mmol) and
˚
5 (113.6 mg, 0.21 mmol) in dry benzene (5 mL) was added molecular sieves 4 A
(300 mg), and the mixture was stirred for 6 hr at room temperature, then cooled to 08C.
N-Iodosuccinimide (NIS; 145 mg, 0.64 mmol) and TMSOTf (8.3 mL, 42 mmol) were
added to the mixture, and the resultant mixture was stirred for 2 hr at 78C and neutralized
with Et3N. After dilution with chloroform, the precipitate was filtered off, and washed with
chloroform. The filtrate and washings were combined, and successively washed with 1 M
sodium carbonate and sodium thiosulfate, dried (Na2SO4), and concentrated.
The residue was dissolved in AcOH (8 mL) and water (2 mL), and the reaction
mixture was stirred for 48 hr at 458C, and then concentrated. Column chromatography
(200 : 1 CHCl3–MeOH) of the residue on silica gel gave 6 (82.8 mg, 51%, two steps) as
1
an amorphous mass; [a]D 226.88 (c 0.4, CHCl3); H NMR (CDCl3) d 0.87 (t, 6H,
J ¼ 6.6 Hz, 2CH3), 1.16 (d, 3H, J5,6 ¼ 6.4 Hz, H-6b), 1.22–1.52 (m, 53H, 26CH2
and CH), 1.58 (s, 3H, AcN), 3.25 (dd, 1H, J1,2 ¼ 7.8 Hz, J2,3 ¼ 9.2 Hz, H-2a), 3.31

Property of Sialyl 6-O-Sulfo Lewis X Neo-glycolipid
209
(dd, 1H, OCH2C of alkyl part), 3.41 (dd, 1H, H-4a), 3.68 (s, 1H, H-4b), 3.74–3.79 (m, 3H,
H-5a, H-60a and OCH2C of alkyl part), 3.86–3.90 (m, 2H, H-3a and H-6a), 3.94 (dd, 1H,
J2,3 ¼ 10.5 Hz, J3,4 ¼ 2.1 Hz, H-3b), 4.09 (dd, 1H, J1,2 ¼ 3.8 Hz, J2,3 ¼ 10.5 Hz, H-2b),
4.84 (d, 1H, J1,2 ¼ 7.8 Hz, H-1a), 4.94 (d, 1H, J1,2 ¼ 3.8 Hz, H-1b), 5.52 (d, 1H,
JNH,2 ¼ 7.3 Hz, NH), 7.25–7.40 (m, 15H, 3Ph).
13C NMR (CDCl3) d 14.14, 16.71, 22.69, 23.05, 26.63, 26.79, 29.37, 29.66, 29.71,
30.10, 30.16, 30.87, 31.13, 31.92, 38.09, 56.12, 62.73, 68.14, 70.98, 72.86, 73.08,
74.08, 74.72, 75.00, 75.94, 76.73, 79.06, 83.96, 99.27, 100.62, 127.46, 127.61, 127.67,
127.75, 127.98, 128.21, 128.28, 128.35, 128.46, 128.57, 138.22, 138.28, 138.43, 170.58
(C55O).
Anal. Calcd for C65H103NO10 (1057.76): C, 73.75; H, 9.81; N, 1.32. Found: C, 73.60;
H, 9.58; N, 1.23.
2-(Tetradecyl)hexadecyl (2,3,4-tri-O-benzyl-a-L-fucopyranosyl)-(1 ! 3)-2-acet-
amido-2-deoxy-6-O-4-methoxyphenyl-b-D-glucopyranoside (7). To a solution of 6
(82.8 mg, 0.078 mmol) in THF (5 mL) were added PPh3 (102.1 mg, 0.39 mmol), DEAD
(123.1 mL, 0.28 mmol), and MPOH (58.8 mg, 0.47 mmol), and the mixture was stirred
under reflux for 17 hr. After completion of the reaction, the mixture was concentrated.
Column chromatography (200 : 1 CHCl3–MeOH) of the residue on silica gel afforded 7
(80.1 mg, 88%) as an amorphous mass; [a]D 224.08 (c 0.86, CHCl3); 1H NMR
(CDCl3) d 0.87 (t, 6H, J ¼ 6.9 Hz, 2CH3), 1.15 (d, 3H, J5,6 ¼ 6.4 Hz, H-6b), 1.21–1.52
(m, 53H, 26CH2 and CH), 1.59 (s, 3H, AcN), 3.27 (dd, 1H, Jgem ¼ 9.6 Hz,
Jvic ¼ 6.6 Hz, OCH2C of alkyl part), 3.32 (dd, 1H, J2,3 ¼ 10.0 Hz, H-2a), 3.49 (t, 1H,
H-4a), 3.61 (m, 1H, H-5a), 3.69 (s, 1H, H-4b), 3.75 (s, 3H, MeOPh), 3.79
(dd, 1H, OCH2C of alkyl part), 3.95 (m, 2H, H-3a and H-3b), 4.09 (dd, 1H,
J1,2 ¼ 3.6 Hz, J2,3 ¼ 10.2 Hz, H-2b), 4.09 (m, 1H, H-5b), 4.10–4.14 (m, 2H, H-6a and
H-60a), 4.86 (d, 1H, J1,2 ¼ 8.5 Hz, H-1a), 4.98 (d, 1H, J1,2 ¼ 3.6 Hz, H-1b), 5.51 (d, 1H,
JNH,2 ¼ 7.1 Hz, NH), 6.79–7.40 (m, 19H, MeOPh, 3Ph).
13C NMR (CDCl3) d 11.34, 14.38, 20.39, 20.83, 24.34, 24.47, 27.07, 27.37, 27.42,
27.83, 27.88, 28.62, 28.87, 29.63, 35.73, 53.37, 54.08, 65.75, 66.28, 68.03, 70.52,
70.60, 71.66, 71.80, 72.68, 73.62, 74.41, 74.73, 74.94, 75.05, 76.78, 81.68, 96.80,
97.94, 112.17, 113.89, 125.18, 125.37, 125.44, 125.62, 125.97, 126.06, 126.16, 126.24,
135.95, 136.02, 136.15, 150.94, 151.62, 168.20 (C55O).
Anal. Calcd for C72H109NO11 (1163.80): C, 74.25; H, 9.43; N, 1.20. Found: C, 74.09;
H, 9.18; N, 1.01.
2-(Tetradecyl)hexadecyl (methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-trifluoro-
acetamido-D-glycero-a-D-galacto-2-nonulopyranosylonate)-(2 ! 3)-(4,6-di-O-acetyl-
2-O-benzoyl-b-D-galactopyranosyl)-(1 ! 4)-[(2,3,4-tri-O-benzyl-a-L-fucopyrano-
syl)-(1 ! 3)]-2-acetamido-2-deoxy-6-O-4-methoxyphenyl-b-D-glucopyranoside
(8). To a solution of 3 (44.8 mg, 0.048 mmol) and 7 (37 mg, 0.031 mmol) in dry
˚
CH2Cl2 (0.6 mL) was added molecular sieves 4 A (38 mg), and the mixture was stirred
for 3 hr at room temperature, then cooled to 08C. DMTST (89.5 mg, 0.18 mmol) was
added to the mixture, and the resultant mixture was stirred for 3 days at 08C
and neutralized with Et3N. After dilution with chloroform, the precipitate was filtered
off, and washed with chloroform. The filtrate and washings were combined, and succes-
sively washed with 1 M sodium carbonate and water, dried (Na2SO4), and concentrated.
Column chromatography (80 : 1 CHCl3–MeOH) of the residue on silica gel afforded 8
(37.1 mg, 57.2%) as an amorphous mass; [a]D þ10.58 (c 0.7, CHCl3); 1H NMR

210
Yamaguchi et al.
(CDCl3) d 0.88 (t, 6H, J ¼ 6.9 Hz, 2CH3), 1.05 (d, 3H, J5,6 ¼ 6.4 Hz, H-6d), 1.14–1.41
(m, 53H, 26CH2 and CH), 1.51 (s, 3H, AcN), 1.71 (t, 1H, Jgem ¼ J3ax,4 ¼ 12.5 Hz,
H-3cax), 1.957, 1.959, 1.96, 1.98, 2.06, 2.18 (6s, 18H, 6AcO), 2.59 (dd, 1H,
J3eq,4 ¼ 4.6 Hz, Jgem ¼ 12.5 Hz, H-3ceq), 2.82 (dd, 1H, Jgem ¼ 8.7, Jvic ¼ 6.6 Hz,
OCH2C of alkyl part), 3.34 (s, 1H, H-4d), 3.43 (dd, 1H, Jgem ¼ 9.4 Hz, Jvic ¼ 5.7 Hz,
OCH2C of alkyl part), 3.63 (dd, 1H, J2,3 ¼ 9.8 Hz, J3,4 ¼ 2.5 Hz, H-3d), 3.68 (s, 3H,
MeOPh), 3.74 (t, 1H, H-2a), 3.87 (s, 3H, COOMe), 3.95–3.98 (m, 2H, H-9c and H-5d),
4.00 (dd, 1H, H-4d), 4.36 (dd, 1H, H-90c), 4.80 (dd, 1H, J2,3 ¼ 10.0 Hz, J3,4 ¼ 3.4 Hz,
H-3b) 4.93 (d, 1H, J1,2 ¼ 8.0 Hz, H-1b), 4.99 (m, 1H, H-4c), 5.04 (d, 1H, H-4b), 5.08
(d, 1H, J1,2 ¼ 3.6 Hz, H-1d), 5.20 (dd, 1H, J6,7 ¼ 2.1 Hz, J7,8 ¼ 9.2 Hz, H-7c), 5.28 (dd,
1H, J1,2 ¼ 8.0 Hz, J2,3 ¼ 10.0 Hz, H-2b), 5.58 (m, 1H, H-8c), 6.10 (d, 1H,
JNH,5 ¼ 8.9 Hz NH of c), 6.21 (d, 1H, JNH,2 ¼ 9.15 Hz, NH), 6.71–8.15 (m, 24H, 4Ph,
MeOPh).
13C NMR (CDCl3) d 14.13, 16.54, 20.16, 20.45, 20.61, 20.59, 20.70, 21.43, 22.70,
23.12, 26.72, 26.89, 29.38, 29.67, 29.72, 29.79, 30.23, 30.92, 31.11, 31.93, 37.97,
49.74, 53.38, 55.57, 61.62, 62.06, 66.38, 66.74, 67.48, 67.61, 67.87, 68.50, 70.88,
70.93, 71.06, 71.14, 72.66, 73.08, 73.40, 74.31, 74.50, 96.89, 97.28, 99.39, 100.41,
114.51, 115.37, 127.21, 127.31, 127.43, 127.51, 127.93, 128.14, 128.27, 128.29,
128.40, 128.62, 129.93, 130.12, 133.41, 138.57, 138.76, 139.02, 152.79, 153.93, 166.10
(C55O), 167.73 (C55O), 169.54 (C55O), 169.80 (C55O), 170.11 (C55O), 170.27
(C55O), 170.54 (C55O), 170.60 (C55O), 170.69 (C55O).
Anal. Calcd for C109H151F3N2O31 (2041.03): C, 64.10; H, 7.45; N, 1.37; O, 24.28.
Found: C, 63.83; H, 7.41; N, 1.33.
2-(Tetradecyl)hexadecyl (5-amino-3,5-dideoxy-D-glycero-a-D-galacto-2-nonulo-
pyranosylonic acid)-(2 ! 3)-(b-D-galactopyranosyl)-(1 ! 4)-[(2,3,4-tri-O-benzyl-
a-L-fucopyranosyl)-(1 ! 3)]-2-acetamido-2-deoxy-6-O-4-methoxyphenyl-b-D-gluco-
pyranoside (9). To a solution of 8 (35.8 mg, 71 mmol) in methanol (4 mL) was added cat-
alytic amount of 28% sodium methoxide in methanol, and the mixture was stirred for 72 hr
at 458C. Water (0.1 mL) was added and the mixture was stirred for 48 hr at room tempera-
ture. The mixture was neutralized with Amberlite IR-120 (Hþ) resin and filtered. The resin
was washed with methanol, and the combined filtrate and washings were concentrated.
Column chromatography (MeOH) of the residue on Sephadex LH-20 gave 9 (26.3 mg,
1
95%) as an amorphous mass; [a]D 243.28 (c 0.52, CHCl3); H NMR (CD3OD) d 0.79
(t, 6H, J ¼ 6.9 Hz, 2CH3), 1.04 (d, 3H, J5,6 ¼ 6.4 Hz, H-6d), 1.10–1.42 (m, 53H,
26CH2 and CH), 1.60 (t, 1H, Jgem ¼ J3ax,4 ¼ 12.1 Hz, H-3cax), 1.87 (s, 3H, AcN of a),
2.72 (dd, 1H, H-3ceq), 2.93 (t, 1H, H-5c), 3.14 (dd, 1H, J6,7 ¼ 2.9 Hz, J7,8 ¼ 8.0 Hz
H-7c), 3.19 (dd, 1H, J ¼ 9.6 Hz, Jvic ¼ 6.2 Hz, OCH2C of alkyl part), 3.41 (dd, 1H,
J1,2 ¼ 7.8 Hz, J2,3 ¼ 9.4 Hz, H-2b), 3.42 (m, 1H, H-8c), 3.60 (m, 1H, H-4c), 3.65 (s, 3H,
MeOPh), 3.80–3.83 (m, 2H, H-6c and H-3b), 3.86 (dd, 1H, J1,2 ¼ 3.7 Hz, J2,3 ¼ 10.3 Hz,
H-2d), 4.01 (dd, 1H, J2,3 ¼ 10.3 Hz, J3,4 ¼ 2.9 Hz, H-3d), 4.25 (d, 1H, J1,2 ¼ 7.8 Hz,
H-1b), 4.71 (m, 1H, H-5d), 5.22 (d, 1H, J1,2 ¼ 3.7 Hz, H-1d), 6.76–7.31 (m, 19H, 3Ph,
MeOPh).
13C NMR (CD3OD) d 13.09, 15.51, 22.22, 22.37, 26.37, 26.54, 29.11, 29.39, 29.74,
30.68, 30.81, 31.71, 37.98, 40.55, 52.82, 54.83, 62.01, 66.41, 69.85, 71.98, 72.27, 74.01,
74.34, 75.04, 78.43, 95.62, 97.81, 99.78, 100.74, 114.48, 115.89, 123.84, 126.95, 127.07,
127.77, 127.90, 128.06, 128.23, 136.32, 138.85, 154.26, 157.04, 171.50 (C55O), 173.03
(C55O).

Property of Sialyl 6-O-Sulfo Lewis X Neo-glycolipid
211
Anal. Calcd for C87H134N2O23 (1574.94): C, 66.30; H, 8.57; N, 1.78. Found: C, 66.05;
H, 8.54; N, 1.55.
2-(Tetradecyl)hexadecyl (5-acetylamino-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-
glycero-a-D-galacto-2-nonulopyranosyl 1,5-lactam)-(2 ! 3)-(2,4,6-tri-O-acetyl-b-D-
galactopyranosyl)-(1 ! 4)-[(2,3,4-tri-O-benzyl-a-L-fucopyranosyl)-(1 ! 3)]-2-
acetamido-2-deoxy-6-O-4-methoxyphenyl-b-D-glucopyranoside (10). To a solution of
9 (26.3 mg, 16.6 mmol) in DMF (3 mL) were added HBTU (19.5 mg, 50 mmol) and HOBt
(6.83 mg, 50 mmol), and the mixture was stirred for 4 hr at 658C, and then concentrated.
Column chromatography (MeOH) of the residue on Sephadex LH-20 gave the lactamized
sLex derivative. The residue was treated with acetic anhydride (1.5 mL) and pyridine
(3 mL) for 12 hr at room temperature, then cooled to 08C. Methanol (1 mL) was added
and the mixture was concentrated, and the residue was extracted with chloroform and suc-
cessively washed with cold 2 M hydrochloric acid and water, dried (Na2SO4), and concen-
trated. Column chromatography (100 : 1 CHCl3–MeOH) of the residue on silica gel gave
10 (24.1 mg, 76.2%, two steps) as an amorphous mass; [a]D 217.68 (c 0.48, CHCl3); 1H
NMR (CDCl3) d 0.88 (t, 6H, J ¼ 6.9 Hz, 2CH3), 0.98 (d, 3H, J5,6 ¼ 6.4 Hz, H-6d), 1.19–
1.50 (m, 53H, 26CH2 and CH), 1.97, 1.98, 2.06, 2.07, 2.10, 2.13, 2.14, 2.21 (8s, 7OAc,
AcN of a), 2.32 (dd, 1H, J3b,4 ¼ 10.1 Hz, Jgem ¼ 14.6 Hz, H-3cb), 2.41 (dd, 1H,
J3b,4 ¼ 5.5 Hz, Jgem ¼ 14.6 Hz, H-3ca), 2.57 (s, 3H, AcN of c), 3.28 (dd, 2H, OCH2C
of alkyl part and H-4d), 3.57 (dd, 1H, J2,3 ¼ 10.0 Hz, J3,4 ¼ 3.0 Hz, H-3d), 3.63 (dd,
1H, Jgem ¼ 9.8 Hz, Jvic ¼ 6.6 Hz, OCH2C of alkyl part), 3.68 (s, 3H, MeOPh), 3.80
(m, 1H, H-5d), 3.94 (dd, 1H, J1,2 ¼ 3.4 Hz, J2,3 ¼ 10.1 Hz, H-2d), 4.13–4.16 (m, 2H,
H-2a and H-3b), 4.19 (dd, 1H, J8,9 ¼ 5.3 Hz, Jgem ¼ 11.8 Hz, H-9c), 4.27 (dd, 1H,
J8,9 ¼ 6.4 Hz, Jgem ¼ 11.2 Hz, H-90c), 4.49 (d, 1H, J1,2 ¼ 8.0 Hz, H-1a), 4.55 (t, 1H,
0
H-3a), 4.63 (d, 1H, H-4b), 4.85 (m, 1H, H-4c), 5.03 (d, 1H, J1,2 ¼ 3.4 Hz, H-1d), 5.12
(dd, 1H, J1,2 ¼ 8.0 Hz, J2,3 ¼ 10.6 Hz, H-2b), 5.36 (m, 1H, H-5c), 5.43 (m, 1H, H-8c),
5.77 (dd, 1H, J6,7 ¼ 3.7 Hz, J7,8 ¼ 9.8 Hz, H-7c), 6.40 (d, 1H, JNH,2 ¼ 9.2 Hz, NH),
6.74–7.39 (m, 19H, 3Ph, MeOPh).
13C NMR (CDCl3) d 14.14, 16.47, 20.62, 20.67, 20.72, 20.87, 21.03, 22.69, 23.09,
26.33, 26.84, 26.95, 29.38, 29.67, 29.72, 30.26, 31.04, 31.15, 31.93, 35.69, 38.19,
47.93, 55.21, 61.29, 61.80, 66.81, 67.55, 68.59, 69.44, 69.88, 71.24, 72.58, 72.95,
73.27, 74.50, 76.02, 79.59, 95.84, 97.70, 99.64, 100.64, 114.62, 115.30, 127.13, 127.31,
127.43, 127.51, 127.78, 128.12, 128.26, 128.32, 128.46, 138.48, 138.77, 138.98,
152.46, 154.07, 164.80 (C55O), 168.96 (C55O), 169.04 (C55O), 169.56 (C55O), 169.78
(C55O), 169.84 (C55O), 170.13 (C55O), 170.36 (C55O), 170.58 (C55O).
Anal. Calcd for C103H148N2O30 (1893.01): C, 65.31; H, 7.88; N, 1.48. Found: C,
65.04; H, 7.87; N, 1.43.
2-(Tetradecyl)hexadecyl
(5-acetylamino-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-
glycero-a-D-galacto-2-nonulopyranosyl 1,5-lactam)-(2 ! 3)-(2,4,6-tri-O-acetyl-b-D-
galactopyranosyl)-(1 ! 4)-[(2,3,4-tri-O-acetyl-a-L-fucopyranosyl)-(1 ! 3)]-2-aceta-
mido-2-deoxy-6-O-4-methoxyphenyl-b-D-glucopyranoside (11). A solution of 10
(20.1 mg, 10.6 mmol) in ethanol (2 mL) was vigorously stirred over Pd(OH)2 (40 mg)
for 24 hr at room temperature under hydrogen. The catalyst was collected and washed
with methanol. The combined filtrate and washings were concentrated, and the residue
was treated with acetic anhydride (2.5 mL) and pyridine (4 mL) for 12 hr at room tempera-
ture, then cooled to 08C. Methanol (1.5 mL) was added and the mixture was concentrated,
and the residue was extracted with chloroform and successively washed with cold 2 M

212
Yamaguchi et al.
hydrochloric acid and water, dried (Na2SO4), and concentrated. Column chromatography
(70 : 1 CHCl3–MeOH) of the residue on silica gel gave 10 (17.3 mg, 93%) as an amor-
1
phous mass; [a]D 217.18 (c 0.35, CHCl3), H NMR (CDCl3) d 0.88 (t, 6H, J ¼ 6.7 Hz,
2CH3), 1.09 (d, 3H, J5,6 ¼ 6.4 Hz, H-6d), 1.24–1.52 (m, 53H, 26CH2 and CH), 1.96,
1.98, 2.04, 2.06, 2.07, 2.11, 2.12, 2.13, 2.16, 2.17, 2.20 (11s, 33H, 10OAc, AcN of a),
2.24 (dd, 1H, J3b,4 ¼ 10.5 Hz, Jgem ¼ 13.9 Hz, H-3cb), 2.29 (dd, 1H, J3a,4 ¼ 5.3 Hz,
Jgem ¼ 13.9 Hz, H-3ca), 2.56 (s, 3H, AcN of c), 3.22 (dd, 1H, Jgem ¼ 9.4 Hz,
Jvic ¼ 6.2 Hz, OCH2C of alkyl part), 3.71 (dd, 1H, Jgem ¼ 9.4 Hz, Jvic ¼ 5.3 Hz,
OCH2C of alkyl part), 3.76 (s, 3H, MeOPh), 3.93 (dd, 1H, J2,3 ¼ 10.3 Hz, J3,4 ¼ 3.4 Hz,
H-3b), 4.18 (dd, 1H, J8,9 ¼ 6.2 Hz, Jgem ¼ 11.8 Hz, H-9c), 4.20 (d, 1H, H-6c), 4.30 (dd,
1H, J8,9 ¼ 5.7 Hz, Jgem ¼ 11.4 Hz, H-90c), 4.50 (d, 1H, J1,2 ¼ 8.2 Hz, H-1b), 4.84
0
(m, 1H, H-4c), 5.02 (dd, 1H, H-2d), 5.07 (dd, 1H, J1,2 ¼ 8.2 Hz, J2,3 ¼ 10.3 Hz, H-2b),
5.18 (d, 1H, J1,2 ¼ 3.4 Hz, H-1d), 5.24 (d, 1H, H-4b), 5.34–5.37 (m, 2H, H-5c and
H-3d), 5.43 (m, 1H, H-8c), 5.69 (d, 1H, JNH,2 ¼ 9.6 Hz, NH), 5.74 (dd, 1H,
J6,7 ¼ 3.7 Hz, J7,8 ¼ 9.6 Hz, H-7c), 6.85–6.93 (m, 4H, MeOPh).
13C NMR (CDCl3) d 14.14, 15.78, 20.63, 20.69, 20.77, 20.83, 20.99, 22.70, 23.31,
26.32, 26.67, 26.89, 29.37, 29.67, 29.72, 30.12, 30.19, 30.90, 31.14, 31.93, 35.51,
38.09, 47.92, 55.55, 61.33, 61.96, 64.46, 67.39, 67.65, 68.08, 68.60, 69.40, 69.98,
71.23, 71.43, 71.73, 72.16, 72.67, 73.54, 73.96, 74.47, 95.86, 96.04, 99.88, 100.85,
114.82, 115.97, 152.39, 154.31, 164.84 (C55O), 169.05 (C55O), 169.37 (C55O), 169.47
(C55O), 169.69 (C55O), 169.75 (C55O), 169.83 (C55O), 170.18 (C55O), 170.42
(C55O), 170.48 (C55O), 170.64 (C55O), 170.68 (C55O), 171.03 (C55O).
Anal. Calcd for C88H136N2O33 (1748.90): C, 60.40; H, 7.83; N, 1.60. Found: C, 60.10;
H, 7.69; N, 1.46.
2-(Tetradecyl)hexadecyl
(5-acetylamino-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-
glycero-a-D-galacto-2-nonulopyranosyl 1,5-lactam)-(2 ! 3)-(2,4,6-tri-O-acetyl-b-D-
galactopyranosyl)-(1 ! 4)-[(2,3,4-tri-O-acetyl-a-L-fucopyranosyl)-(1 ! 3)]-2-aceta-
mido-2-deoxy-b-D-glucopyranoside (12). To a solution of 11 (17.3 mg, 9.9 mmol) in
acetonitrile (1.8 mL) and water (0.2 mL) was added CAN (16.8 mg, 0.03 mmol), and the
mixture was stirred for 2.5 hr at 08C and extracted with chloroform. The extract was suc-
cessively washed with 1 M sodium carbonate and water, dried (Na2SO4), and concen-
trated. Column chromatography (100 : 1 CHCl3–MeOH) of the residue on silica gel
1
gave 12 (12.1 mg, 74.6%) as an amorphous mass; [a]D 28.28 (c 0.24, CHCl3); H
NMR (CDCl3) d 0.88 (t, 6H, J ¼ 6.6 Hz, 2CH3), 1.22–1.52 (m, 56H, 26CH2, CH and
H-6d), 1.94, 1.95, 2.02, 2.05, 2.08, 2.09, 2.10, 2.14, 2.16, 2.18, 2.21 (11s, 33H, 10OAc,
AcN of a), 2.27 (dd, 1H, J3b,4 ¼ 10.5 Hz, Jgem ¼ 14.4 Hz, H-3cb), 2.38 (dd, 1H,
J3a,4 ¼ 5.4 Hz, Jgem ¼ 14.4 Hz, H-3ca), 2.56 (s, 3H, AcN of c), 3.20 (dd, 1H,
Jgem ¼ 9.3 Hz, Jvic ¼ 6.6 Hz, OCH2C of alkyl part), 3.28 (br-d, 1H, H-6a), 3.73 (dd,
1H, OCH2C of alkyl part), 3.85 (dd, 1H, H-2a), 4.13 (dd, 1H, J2,3 ¼ 9.8 Hz,
J3,4 ¼ 3.6 Hz, H-3b), 4.18 (dd, 1H, J8,9 ¼ 5.5 Hz, Jgem ¼ 11.6 Hz, H-9c), 4.22 (br-d,
1H, H-6c), 4.26 (dd, 1H, J8,9 ¼ 6.4 Hz, Jgem ¼ 11.6 Hz, H-90c), 4.37 (d, 1H,
0
J1,2 ¼ 7.6 Hz, H-1a) 4.60 (d, 1H, J1,2 ¼ 8.2 Hz, H-1b), 4.85 (m, 1H, H-4c), 4.97–5.00
(m, 2H, H-2d and H-5d), 5.05 (dd, 1H, J1,2 ¼ 8.2 Hz, J2,3 ¼ 10.0 Hz, H-2b), 5.21(dd,
1H, J2,3 ¼ 10.9 HZ, J3,4 ¼ 3.2 Hz, H-3d), 5.29 (d, 1H, H-4b), 5.36 (m, 1H, H-5c), 5.43
(m, 2H, H-8c and H-1d), 5.76 (dd, 1H, J6,7 ¼ 3.8 Hz, J7,8 ¼ 8 Hz, H-7c).
13C NMR (CDCl3) d 14.13, 15.77, 20.45, 20.62, 20.69, 20.70, 20.77, 20.83, 20.98,
22.70, 23.30, 26.63, 26.79, 26.89, 29.37, 29.68, 29.71, 30.13, 30.18, 30.91, 31.13,

Property of Sialyl 6-O-Sulfo Lewis X Neo-glycolipid
213
31.92, 35.71, 39.12, 48.29, 61.58, 62.02, 64.52, 67.68, 68.28, 68.66, 69.47, 69.82, 71.28,
71.35, 71.46, 71.77, 72.18, 72.54, 72.62, 73.58, 73.62, 73.99, 74.39, 75.62, 96.88, 97.27,
99.48, 100.72, 169.21 (C55O), 168.37 (C55O), 169.12 (C55O), 169.58 (C55O), 169.84
(C55O), 170.12 (C55O), 170.27 (C55O), 170.42 (C55O), 170.51 (C55O), 170.55
(C55O), 170.63 (C55O), 170.77 (C55O), 171.05 (C55O).
Anal. Calcd for C81H130N2O32 (1642.86): C, 59.18; H, 7.97; N, 1.70. Found: C, 59.10;
H, 7.85; N, 1.63.
2-(Tetradecyl)hexadecyl
(5-acetylamino-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-
glycero-a-D-galacto-2-nonulopyranosyl 1,5-lactam)-(2 ! 3)-(2,4,6-tri-O-acetyl-b-D-
galactopyranosyl)-(1 ! 4)-[(2,3,4-tri-O-acetyl-a-L-fucopyranosyl)-(1 ! 3)]-2-aceta-
mido-2-deoxy-6-O-sulfo-b-D-glucopyranoside triethylammonium salt (13). To a sol-
ution of 12 (12.1 mg, 7.4 mmol) in DMF (1 mL) was added sulfur trioxide pyridine
complex (7.1 mg, 44 mmol), and the mixture was stirred for 6 hr at room temperature.
Triethylamine (0.1 mL) was added and the mixture was concentrated. Column chromato-
graphy (1 : 1 CHCl3–MeOH) of the residue on Sephadex LH-20 gave the crude sulfated
product, and this was purified by column chromatography (30:1 CHCl3–MeOH) on
silica gel to afford 13 (10.5 mg, 78%) as an amorphous mass; [a]D 228.98 (c 0.04,
1
CHCl3); H NMR (CDCl3) d 0.88 (t, 6H, J ¼ 6.6 Hz, 2CH3), 1.22 (d, 3H, J5,6 ¼ 6.4 Hz,
H-6d), 1.25–1.30 (m, 52H, 26CH2), 1.40 (t, 9H, NCH2CH3), 1.45 (m, 1H, CH), 1.94,
1.95, 2.05, 2.06, 2.07, 2.091, 2.094, 2.14, 2.160, 2.165, 2.23 (11s, 33H, 10OAc, AcN of
a), 2.29 (dd, 1H, J3b,4 ¼ 10.8 Hz, Jgem ¼ 14.3 Hz, H-3cb), 2.46 (dd, 1H, J3a,4 ¼ 5.3 Hz,
Jgem ¼ 14.3 Hz, H-3ca), 2.56 (s, 3H, AcN of c), 3.16 (m, 7H, NCH2CH3 and OCH2C of
alkyl part), 3.68 (dd, 1H, Jgem ¼ 9.6 Hz, Jvic ¼ 5.5 Hz, OCH2C of alkyl part), 3.96 (t, 1H,
H-2a), 4.20 (br-d, 1H, H-6c), 4.24–4.28 (m, 3H, H-9c, H-90c and H-3b), 4.85 (m, 2H, H-4c
and H- 5d), 4.89 (d, 1H, J1,2 ¼ 8.2 Hz, H-1b), 5.02 (dd, 1H, J1,2 ¼ 3.4 Hz, J2,3 ¼ 10.5 Hz,
H-2d), 5.04 (dd, 1H, J1,2 ¼ 8.2 Hz, J2,3 ¼ 10.1 Hz, H-2b), 5.24 (dd, 1H, J2,3 ¼ 10.5 Hz,
H-3d), 5.34 (m, 1H, H-8c), 5.37 (d, 1H, J1,2 ¼ 3.2 Hz, H-1d), 5.43 (d, 1H, H-4b), 5.51
(d, 1H, JNH,2 ¼ 8.5 Hz, NH), 5.77 (dd, 1H, J6,7 ¼ 4.1 Hz, J7,8 ¼ 8.6 Hz, H-7c).
Anal. Calcd for C87H145N3O35S (1823.94): C, 57.25; H, 8.01; N, 2.30. Found: C,
56.98; H, 7.72; N, 2.25.
2-(Tetradecyl)hexadecyl (5-amino-3,5-dideoxy-D-glycero-a-D-galacto-2-nonulo-
pyranosyl 1,5-lactam)-(2 ! 3)-(b-D-galactopyranosyl)-(1 ! 4)-[(a-L-fucopyrano-
syl)-(1 ! 3)]-2-acetamido-2-deoxy-6-O-sulfo-b-D-glucopyranoside sodium salt (14).
To a solution of 13 (5.6 mg, 3.1 mmol) in methanol (3 mL) and dioxane (1 mL) was
added catalytic amount of 28% sodium methoxide in methanol, and the mixture was
stirred for 30 hr at room temperature and then concentrated. Column chromatography
(1 : 1 CHCl3–MeOH) of the residue on Sephadex LH-20 gave the target molecule 14
1
(3.9 mg, 98%) as an amorphous mass; [a]D þ3.28 (c 0.07, 1 : 1 CHCl3–MeOH); H
NMR (CD3OD) d 0.86 (t, 6H, J ¼ 7.6 Hz, 2CH3), 1.07 (d, 3H, J5,6 ¼ 6.6 Hz, H-6d),
1.19–1.43 (m, 53H, 26CH2 and CH), 1.84 (s, 3H, AcN of a), 2.01 (dd, 1H,
J3a,4 ¼ 5.0 Hz, Jgem ¼ 13.6 Hz, H-3ca), 2.30 (dd, 1H, J3b,4 ¼ 10.3 Hz, Jgem ¼ 13.4 Hz,
H-3cb), 3.43 (m, 1H, H-8c), 3.46 (m, 1H, H-5c), 3.48 (dd, 1H, J1,2 ¼ 7.5 Hz,
J2,3 ¼ 9.6 Hz, H-2b), 3.54 (dd, 1H, H-2d), 3.58 (d, 1H, J1,2 ¼ 8.0 Hz, H-1a), 3.61
(m, 2H, H-9c and H-4d), 3.64 (dd, 1H, Jgem ¼ 14.4 Hz, H-90c), 3.70 (t, 1H,
J1,2 ¼ 8.0 Hz, H-2a), 3.78 (dd, 1H, J2,3 ¼ 10.0 Hz, J3,4 ¼ 3.2 Hz, H-3d), 3.98 (m, 2H,
H-3b and H-4c), 4.10 (br-d, 1H, H-4b), 4.37 (br-d, 1H, H-6c), 4.56 (d, 1H,
J1,2 ¼ 7.5 Hz, H-1b), 4.74 (m, 1H, H-5d), 4.92 (d, 1H, J1,2 ¼ 4.1 Hz, H-1d).

214
Yamaguchi et al.
MALDI-TOF MS (positive-ion mode, DHB, MeCN) Calcd for C59H107N2NaO24S
[M þ Na]þ 1282.6832. Found: 1282.5850.
ACKNOWLEDGMENTS
This work was partly supported by Japan Society for the Promotion of Science (Grant-
in-Aid for Scientific Research No. 12306007 to M.K.) and CREST of Japan Science and
Technology Corporation (JST). We thank Ms. Kiyoko Ito for the technical assistance.
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Received February 2, 2004
Accepted March 22, 2004