Synthesis of novel ganglioside GM4 analogues containing N-deacetylated and lactamized sialic acid: Probes for searching new ligand structures for human L-selectin

Article abstract of DOI:10.1016/S0008-6215(00)00278-0

Novel ganglioside GM4 analogues, which contain N-deacetylated or lactamized sialic acid instead of usual N-acetylneuraminic acid, were synthesized in a highly efficient manner. (Methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-trifluoroacetamido-D-glycero-α- D-galacto-2-nonulopyranosylonate)-(2 → 3)-4,6-di-O-acetyl-2-O-benzoyl-D-galactopyranosyl trichloroacetimidate was coupled with 2-(tetradecyl)hexadecanol to give the desired β-glycoside in high yield. Successive O- and N-deacylation, and saponification of the methyl ester group afforded the N-deacetylated sialyl derivative that was converted by treatment with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in Me₂SO into the lactamized sialic acid-containing ganglioside GM4 analogue.

Full text of DOI:10.1016/S0008-6215(00)00278-0

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Carbohydrate Research 330 (2001) 1–5
Synthesis of novel ganglioside GM4 analogues
containing N-deacetylated and lactamized sialic acid:
probes for searching new ligand structures for human
L-selectinꢀ
Nobumasa Otsubo, Hideharu Ishida,*1 Makoto Kiso*2
Department of Applied Bioorganic Chemistry, Gifu Uni6ersity, Gifu 501-1193, Japan
Received 1 July 2000; accepted 16 October 2000
Abstract
Novel ganglioside GM4 analogues, which contain N-deacetylated or lactamized sialic acid instead of usual
N-acetylneuraminic acid, were synthesized in a highly efficient manner. (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-D-galacto-
pyranosyl trichloroacetimidate was coupled with 2-(tetradecyl)hexadecanol to give the desired b-glycoside in high
yield. Successive O- and N-deacylation, and saponification of the methyl ester group afforded the N-deacetylated
sialyl derivative that was converted by treatment with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
in Me2SO into the lactamized sialic acid-containing ganglioside GM4 analogue. © 2001 Elsevier Science Ltd. All
rights reserved.
Keywords: Novel ganglioside GM4 analogues; Lactamized sialic acid; N-deacetylated sialic acid
1. Introduction
considerable molecular heterogeneity is noted
in sLex-like determinants. We have demon-
Selectins (E-, P-, and L-)2 are a family of
carbohydrate-binding proteins implicated in
lymphocyte homing, leukocyte recruitment to
sites of inflammation, thrombosis, cancer
metastasis, and so on. Sialyl Lewis X (sLex)
has been assumed to be a common carbohy-
drate ligand for the three selectins,3,4 but a
strated
with
chemically
synthesized
gangliosides5 that sLex sulfated at C-6 of the
N-acetylglucosamine residue (6-O-sulfo sLex)
serves as the major ligand for L-selectin on
high endothelial venules (HEV) in human
lymph nodes.6,7 Very recently, it has been
shown that a novel N-deacetylated sialyl
derivative of 6-O-sulfo sLex is a superior lig-
and for human L-selectin,8,9 raising a new
regulation mechanism of ligand activity based
on the heterogeneity of sialic acid in the sLex
structures9 (Fig. 1). The lactamized form of
the N-deacetylated sialyl derivative may func-
ꢀ Synthetic studies on sialoglycoconjugates, Part 117. For
part 116, see Ref. 1.
1 *Corresponding author. Tel.: +81-58-2932918; e-mail:
ishida@cc.gifu-u.ac.jp
2 *Corresponding author. Tel.: +81-58-2932916; fax: +
81-58-2932840; e-mail: kiso@cc.gifu-u.ac.jp
0008-6215/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(00)00278-0

2
N. Otsubo et al. / Carbohydrate Research 330 (2001) 1–5
tion as a dormant pool after activation of
selectin ligands. We report herein a highly
efficient synthesis of N-deacetylated (4) and
lactamized (5) ganglioside GM4 analogues as
probes for searching novel selectin ligands as
well as a new regulation mechanism of ligand
activity for selectins.
fluoroacetyl groups with sodium methoxide in
methanol at 50 °C, and subsequent saponifica-
tion of the methyl ester group by addition of
water afforded the desired N-deacetylated
GM4 analogue 4 in quantitative yield.
Treatment of
4
with 1-(3-dimethyl-
aminopropyl)-3-ethylcarbodiimide hydrochlo-
ride (WSC) in dimethyl sulfoxide (Me2SO) for
12 h at 60 °C gave the desired lactamized
GM4 analogue 5 in 71% yield. The compound
was resistant to alkaline treatment with
NaOMe in MeOH, which showed that the
product 5 is not a lactone (Scheme 1).
2. Results and discussion
For the synthesis of N-deacetylated GM4
analogue 4, we employed (methyl 4,7,8,9-tetra-
O - acetyl - 3,5 - dideoxy - 5 - trifluoroaceta-
In the 1H NMR spectra (500 MHz) of 4 and
5 in CD3OD, H-3 of the N-deacetylated sialic
acid moiety appeared at l 1.71 (t, Jgem =J3,4
12.1 Hz, H-3bax) and 2.87 (dd, Jgem =12.5,
J3,4 4.8 Hz, H-3beq), respectively, and H-5
was detected at l 3.07 as a one-proton triplet
mido-
D
-glycero-a-
D
-galacto-2-nonulopyran-
osylonate)-(2“3)-4,6-di-O-acetyl-2-O-benzoyl-
D
-galactopyranosyl trichloroacetimidate (1) as
a glycosyl donor.1,8 In this glycosyl donor 1,
the amino group at C-5 of neuraminic acid is
suitably protected by the trifluoroacetyl
(TFAc) group, which is stable under acidic
conditions but can be readily removed by
alkaline treatment.
2
(J4,5=J5,6 10.1 Hz), showing an ordinary C5
chair conformation (Fig. 2). In contrast, H-3
of the lactamized sialic acid moiety in 5 ap-
peared at l 2.02 (dd, Jgem 13.9, J3a,4 4.8 Hz,
H-3ba) and l 2.29 (dd, Jgem 13.9, J3b,4 10.3 Hz,
H-3bb) as a one-proton doublet of doublets,
respectively, indicating a typical B5,2 boat con-
formation. This was further supported by a
one-proton doublet of doublets of H-5b (J4,5
Glycosylation10 of 2-(tetradecyl)hexadecan-
ol (2) with 1 in dichloromethane in the pres-
ence of trimethylsilyl trifluoromethanesul-
,
A
fonate (TMSOTf) and powdered
4
molecular sieves (AW-300) gave the desired
b-glycoside 3 in 72% yield. A significant signal
1
1
3.2, J5,6 2.1 Hz) at l 3.47. These H NMR
in the H NMR spectrum of 3 was a one-pro-
data are consistent with those reported for the
fully protected sialic acid lactam.11,12 In the
FTIR (KBr) spectrum, a significant absorp-
ton doublet at l 4.68 (J1,2 8.1 Hz, H-1a),
showing the newly formed glycosidic linkage
to be b. Removal of the O-acetyl and N-tri-
Fig. 1. Hypothetical ligand-processing pathways for the selectins.8,9

N. Otsubo et al. / Carbohydrate Research 330 (2001) 1–5
3
lactamized sialic acid in oligosaccharides is in
progress.
3. Experimental
General procedures.—Specific rotations
were determined with a Horiba SEPA-300
1
high sensitivity polarimeter at 25 °C, and H
NMR spectra were recorded on Varian Unity
Inova (400 and 500 MHz) spectrometers with
19
TMS as the internal standard. F NMR spec-
tra were recorded on a Varian Unity Inova
400 (476.5 MHz) spectrometer, and the chemi-
cal shifts were measured in ppm relative to
fluorobenzene. FAB mass spectra were
recorded on a JEOL JMS-SX 120A mass
spectrometer/JMA-DA 7000 data system.
Preparative thin-layer chromatography (TLC)
was performed on Silica Gel 60 (E. Merck),
and column chromatography on silica gel
(Fuji Silysia Co., 300 mesh) was accomplished
with the solvent systems (v/v) specified. Con-
centrations and evaporations were conducted
in vacuo.
Scheme 1.
2-(Tetradecyl)hexadecyl (methyl 4,7,8,9-
tetra-O-acetyl-3,5-dideoxy-5-trifluoroaceta-
mido-
sylonate)-(2“3)-4,6-di-O-acetyl-2-O-benzoyl-
i- -galactopyranoside (3).—To a soln of 1
D
-glycero-h- -galacto-2-nonulopyrano-
D
D
(275 mg, 0.26 mmol) and 2-(tetra-
decyl)hexadecanol (2), (289 mg, 0.66 mmol) in
dry CH2Cl2 (10 mL) were added powdered 4
2
Fig. 2. C5 (4) and B5,2 (5) conformations of the neuraminic
1
acid moiety based on H NMR assignments.
,
A molecular sieves (AW-300; 1.0 g), and the
tion band at 1690 cm−1 was clearly detected
indicating the desired lactam structure. In the
FABMS spectra of 4 and 5, the molecular
ions of [M−H]− were clearly detected at m/z
848.72 and m/z 830.69, respectively, which
gave the common fragment ion at m/z 599.6
resulting from elimination of N-deacetylated
(−250 Da) or lactamized (−232 Da) sialic
acid, providing unambiguous evidence for the
structure assigned.
The occurrence of N-deacetylated sialic acid
has been reported among gangliosides (GM3
and GD3) in certain cell lines and tumor
tissues,13–15 but the lactam derivatives of neu-
raminic acid have only been created by chemi-
cal synthesis.11,12 Further investigation on the
biological functions of N-deacetylated and
mixture was stirred for 4 h at rt, then cooled
to 0 °C. Trimethylsilyl trifluoromethanesul-
fonate (TMSOTf; 3.57 mL, 18.4 mmol) was
added to the mixture, and it was stirred for 12
h at rt. The solids were filtered off and washed
with CHCl3. The combined filtrate and wash-
ings was washed with 1 M NaHCO3 and wa-
ter, dried (Na2SO4) and concentrated. Column
chromatography (70:1 CHCl3–MeOH) of the
residue on silica gel gave 3 (245 mg, 72%) as a
white solid: [h]D +20.6° (c 1.8, CHCl3); IR
(film) 3350, 2950, 1750, 1680, 1520, 700 cm−1
;
1H NMR (CDCl3): l 0.87 (t, 6 H, J 6.9 Hz, 2
CH3), 1.06–1.36 (m, 53 H, 26 CH2 and CH),
1.41, 1.95, 2.06, 2.07, 2.13, 2.20 (6 s, 18 H, 6
AcO), 1.74 (t, 1 H, J3ax,4=Jgem 12.5 Hz, H-
3bax), 2.58 (dd, 1 H, J3eq,4 4.7 Hz, H-3beq),

4
N. Otsubo et al. / Carbohydrate Research 330 (2001) 1–5
After completion of the reaction, the mixture
was concentrated. Column chromatography
(1:1 CHCl3–MeOH) of the residue on Sep-
hadex LH-20 gave a crude product, which was
further purified by silica gel column chro-
matography (6:1 CHCl3–MeOH) to give 5
(17.4 mg, 71%) as an amorphous mass: [h]D
−9.3° (c 0.8, MeOH); IR (KBr) 3550, 3350,
3.28 (dd, 1 H, Jgem 9.1, Jvic 7.3 Hz, OCH2C),
3.81 (dd, 1 H, Jvic 5.1 Hz, OCH2C), 3.85 (s, 3
H, COOMe), 3.99 (dd, 1 H, J8,9 5.4, Jgem 12.4
Hz, H-9b), 4.34 (dd, 1 H, J8,9% 5.4 Hz, H-9%b),
4.67 (dd, 1 H, J2,3 9.9, J3,4 2.9 Hz, H-3a), 4.68
(d, 1 H, J1,2 8.1 Hz, H-1a), 4.99 (m, 1 H,
H-4b), 5.01 (d, 1 H, H-4a), 5.18 (dd, 1 H, J6,7
2.5, J7,8 9.2 Hz, H-7b), 5.27 (dd, 1 H, H-2a),
5.55 (m, 1 H, H-8b), 7.43 (t, 2 H, J 7.3 Hz,
m-H of Ph), 7.57 (t, 1 H, p-H of Ph), 8.13 (d,
2 H, o-H of Ph); 19F NMR (CDCl3, C6H5F): l
36.91 (s, 3 F, CF3CO). Anal. Calcd for
C67H104F3NO21 (1316.55): C, 61.12; H, 7.96;
N, 1.06. Found: C, 61.01; H, 7.76; N, 0.91.
1
2950, 1690 cm−1; H NMR (CD3OD): l 0.80
(t, 6 H, J 7.0 Hz, 2 CH3), 1.14–1.54 (m, 53 H,
26 CH2 and CH), 2.02 (dd, 1 H, J3a,4 4.8, Jgem
13.9 Hz, H-3ba), 2.29 (dd, 1 H, J3b,4 10.3 Hz,
H-3bb), 3.47 (dd, 1 H, J4,5 3.2, J5,6 2.1 Hz,
H-5b), 3.53 (m, 1 H, H-8b), 3.54 (dd, 1 H, J1,2
7.8, J2,3 9.8 Hz, H-2a), 3.91 (dd, 1 H, J3,4 3.2
Hz, H-3a), 4.01 (m, 1 H, H-4b), 4.07 (d, 1 H,
H-4a), 4.14 (d, 1 H, H-1a), 4.33 (dd, 1 H, J6,7
1.6 Hz, H-6b); FABMS (negative-ion mode,
triethanolamine matrix): m/z 830.69 [M−H]−
(C45H84NO12 MW, exact 830.5994, ave.
831.1615), 599.6 [M−lactamized neuraminic
acid−H]−.
2-(Tetradecyl)hexadecyl
dideoxy- -glycero-h- -galacto-2-nonulopyra-
nosylonic acid)-(2“3)-i- -galactopyranoside
(5-amino-3,5-
D
D
D
(4).—To a solution of 3 (210 mg, 0.16 mmol)
in MeOH (10 m) was added a catalytic
amount of NaOMe, and the mixture was
stirred for 96 h at 50 °C, then water (0.5 mL)
was added. After completion of the reaction
(12 h), the mixture was neutralized with Am-
berlite IR-120 (H+) resin and filtered. The
resin was washed with MeOH, and the com-
bined filtrate and washings was concentrated.
Column chromatography (1:1 CHCl3–MeOH)
of the residue on Sephadex LH-20 gave 4 (135
mg, quant) as an amorphous mass: [h]D
−15.2° (c 1.1, 1:1 CHCl3–MeOH); IR (KBr)
3550, 3350, 2950 cm−1; 1H NMR (CD3OD): l
0.89 (t, 6 H, J 7.3 Hz, 2 CH3), 1.22–1.36 (m,
53 H, 26 CH2 and CH), 1.71 (t, 1 H, J3ax,4
=Jgem 12.1 Hz, H-3bax), 2.87 (dd, 1 H, J3eq,4
4.8 Hz, H-3beq), 3.07 (t, 1 H, J4,5 =J5,6 10.1
Hz, H-5b), 3.87 (dd, 1 H, J6,7 2.7 Hz, H-6b),
4.02 (dd, 1 H, J2,3 8.8, J3,4 2.9 Hz, H-3a),
4.23 (d, 1 H, J1,2 7.8 Hz, H-1a); FABMS
(negative-ion mode, 2:2:1 diethanolamine–tet-
ramethylurea–m-nitrobenzylalcohol matrix):
m/z 848.72 [M−H]− (C45H86NO13 MW, exact
848.6099, ave. 849.1767), 599.6 [M−
Neu5NH2−H]−.
Acknowledgements
This work was supported in part by Grants-
in-Aid (No. 12306007 and No. 12045228) for
Scientific Research from the Ministry of Edu-
cation, Science and Culture of Japan, and
Japan Society for Promotion of Science. The
authors are grateful to Dr. Takao Ikami of
Sanwa Kagaku Kenkyusho for FABMS
analysis.
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