Synthesis of some aglycon analogs of globotriosylceramide

Article abstract of DOI:10.1016/S0008-6215(99)00230-X

Seven aglycon analogs of globotriosylceramide were synthesized by glycosylation of suitable functionalized alcohols with peracetylated globotriose trichloroacetimidate, followed by further transformations of the aglycon and removal of the protecting groups. Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0008-6215(99)00230-X

www.elsevier.nl/locate/carres
Carbohydrate Research 322 (1999) 181–189
Synthesis of some aglycon analogs of
globotriosylceramide
Henrik C. Hansen, Go¨ran Magnusson *
Organic Chemistry 2, Center for Chemistry and Chemical Engineering, Lund Uni6ersity, PO Box 124,
S-221 00 Lund, Sweden
Received 6 May 1999; accepted 13 August 1999
Abstract
Seven aglycon analogs of globotriosylceramide were synthesized by glycosylation of suitable functionalized
alcohols with peracetylated globotriose trichloroacetimidate, followed by further transformations of the aglycon and
removal of the protecting groups. © 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Globotriosylceramide aglycon analogs; Protection groups; Functionalized alcohols
microcalorimetry [5]), globotriosides with al-
ternative aglycons might do so. The objectives
of the present investigation were to find simple
analogs of Gb3 that inhibit verotoxin binding
at low concentrations and that are more solu-
ble than Gb3 in aqueous solution. We report
here the synthesis of some novel globo-
triosides (1–7, Fig. 1), the structural design
being based on the preliminary observation [6]
that semisynthetic 2 is an efficient inhibitor of
verotoxin binding.
1. Introduction
Globotriosylceramide [Gal-a-(1“4)Gal-b-
(1“4)Glc-b-(1“Cer, Gb3] is present on the
surface of virtually all mammalian cells, where
it functions inter alia as receptor for microbes
and toxins [1]. Of special interest here is the
binding of Escherichia coli-derived verotoxin
b subunits to Gb3 prior to toxin action by the
a subunit [2]. In the preceding paper [3] we
described the synthesis of some aminodeoxy
globotriosides, designed to fit the receptor site
of the verotoxin b subunit. However, since
simple globotriosides do not bind verotoxin
strongly enough to inhibit its binding to im-
mobilized Gb3 in the microwell and thin-layer
plate assays [4] (although its binding to the
verotoxin b subunit could be demonstrated by
The exploitation of 1–7 as inhibitors of
globotriose-binding proteins will be reported
in due course.
2. Results and discussion
Synthesis of compounds 1–7.—The synthe-
ses of 1 and 3 were based on glycosylation of
the corresponding alcohols with peracetylated
globotriosyl trichloroacetimidate, whereas 2,
4, 5, and 6 were obtained by N-acylation of
2-aminoethyl globotriosides with suitably acti-
vated carboxylic acids.
* Corresponding author. Tel.: +46-46-222-8215; fax: +
46-46-222-8209.
E-mail address: goran.magnusson@orgk2.lth.se (G. Mag-
nusson)
0008-6215/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(99)00230-X

182
H.C. Hansen, G. Magnusson / Carbohydrate Research 322 (1999) 181–189
Synthesis of aglycon alcohols.—1-Adaman-
product, probably due to low solubility in
dimethyl formamide (DMF) of the initially
formed cesium salt of di-1,3-mercaptomethyl-
benzene; the solubility of the monoalkylated
product is higher, and consequently, it reacts
faster with methyl acrylate. Compound 9 was
then S-alkylated with 3-bromopropionic acid
in the presence of Cs2CO3, to yield the ester–
acid 10 (58%). Condensation of 10 with N-hy-
droxysuccinimide (NHS) in the presence of
EDC furnished the NHS–ester 11 (79%), suit-
able for N-acylation of compounds 17 and 19
(Scheme 3).
taneacetic acid was activated by reaction with
N-(3-dimethylaminopropyl)-N%-ethylcarbodi-
imide (EDC), and then treated with
ethanolamine to furnish the crystalline com-
pound 8 in 75% yield (Scheme 1).
Treatment of 1,3-bis-mercaptomethylben-
zene with methyl acrylate in the presence of
triethylamine gave the mono-S-alkylated ester
9 (70%), together with 10–15% of the corre-
sponding di-S-alkylated product. Attempted
alkylation with Cs2CO3 as base instead of
triethylamine gave mainly the dialkylated
Fig. 1. Inhibitors of globotriose-binding proteins.

H.C. Hansen, G. Magnusson / Carbohydrate Research 322 (1999) 181–189
183
hydroxide then furnished the carboxylic acid 7
(98%).
An alternative route to compound 6 was
also investigated. Catalytic hydrogenation of
the azido group of the known [8] globotrioside
18 in a mixture of ethanol and one equivalent
of hydrochloric acid, furnished the amine 19
in 78% yield. The presence of hydrochloric
acid is crucial in such hydrogenations because
the amine must be protonated as formed in
order to avoid poisoning of the catalyst [3].
N-Acylation of 19 with the NHS–ester 11,
followed by O-deacetylation of the crude ma-
terial with sodium methoxide–methanol, gave
compound 6 in 79% yield.
The yield in N-acylations with the NHS–es-
ter 11 was lower for the unprotected com-
pound 17 than for O-acetylated 19 (62 versus
79%). The difference may be attributable to
the difference in solubility, which is especially
pronounced for larger saccharides. We there-
fore suggest that N-acylations of larger sugars
with moderate solubility should be performed
with the O-protected derivatives whenever this
is convenient.
Scheme 1. (a) HOCH2CH2NH2, EDC, CH2Cl2, N2, 22 °C, 14
h. (b) Et3N, DMF, 22 °C, 2.5 h. (c) Cs2CO3, DMF, 22 °C, 14
h. (d) NHS, EDC, CH2Cl2, N2, 22 °C, 14 h.
Synthesis of compounds 1–3.—Treatment of
3-O-benzoylated azidosphingosine (13) [7]
with the trisaccharide trichloroacetimidate 12
[8] in the presence of BF3·OEt2 gave the gly-
coside 14 in 66% yield (Scheme 2). The synthe-
sis of 14 has been reported previously, but
without full experimental details [9]. Similarly,
treatment of compound 8 with trichloroace-
timidate 12 in the presence of BF3·OEt2 gave
globotrioside 16 in 70% yield.
Reduction of the azido group of 14 by
hydrogen sulfide gave the corresponding crude
amine, which was then acylated with 1-
adamantaneacetic acid in the presence of
EDC, to give 15 in 79% yield.
O-Deacylation of 14, 15, and 16 with
sodium methoxide–methanol gave com-
pounds 1–3 in 95, 90, and 85% yield,
respectively.
Synthesis of compounds 4–7.—The known
aminoethyl globotrioside 17 [8] (Scheme 3)
was N-acylated with the NHS ester of t-butyl-
acetic acid to give compound 4 (60%), with
acetic anhydride to give 5 (100%), and with
the NHS–ester 11 to give 6 (62%). Hydrolysis
of the methyl ester 6 with aqueous sodium
3. Experimental
General experimental procedures and meth-
ods were as described previously [3]. The com-
pounds 12 [3,10], 13 [7], 14 [11], 17 [12], and
18 [3] have been reported.
(2S,3R,4E)-2-Azido-3-hydroxyoctadec-4-
enyl (h -
gal-actopyranosyl)-(1“4)-i-
D
- galactopyranosyl) - (1“4) - (i -
D
-
D
-glucopyranoside
(1).—Compound 14 (7 mg, 0.005 mmol) was
treated with 0.5 M NaOMe–MeOH for 16 h.
The reaction mixture was neutralized with
Duolite C436 (H+) resin, filtered, and concd.
The residue was chromatographed on a re-
versed-phase column (Varian Mega Bond Elut
C18 (water–MeOH 1:0“4:1“3:2“2:3“
1:4“0:1, 5 mL of each) to give 1 (4 mg, 95%);
[h]2D2 +29° (c 0.4, MeOH); 1H NMR
(CD3OD–D2O 3:1): l (assignments of aglycon
protons are shown in italic) 5.73–5.83
(m, 1 H, H-5), 5.50 (dd, 1 H, J 7.7, 15.4 Hz,
H-4), 4.93 (br s, 1 H, H-1¦), 4.46 (d, 1 H, J
7.6 Hz, H-1%), 4.36 (d, 1 H, J 7.8 Hz,

184
H.C. Hansen, G. Magnusson / Carbohydrate Research 322 (1999) 181–189
H-1), 4.30 (br t, 1 H, J 6.3 Hz, H-5¦), 4.17 (br
t, 1 H, J 6.7 Hz, H-3), 4.00 (d, 1 H, J 2.8 Hz,
H-4%), 3.79–3.97 (m, 8 H, incl. H-2¦), 3.51–
3.77 (m, 9 H, incl. H-2%,3%,3), 3.42–3.49 (m, 1
H), 3.30 (H-2 hidden under solvent peak),
2.00–2.11 (m, 2 H, CH2CHꢀCH), 1.20–1.45
(m, 24 H, CH2ꢁ), 0.89 (br t, 3 H, J 6.8 Hz,
(2S,3R,4E)-2-(1-Adamantaneacetamido)-3-
hydroxy - octadec - 4 - enyl (h - - galactopyran -
osyl)-(1“4)-(i- -galactopyranosyl)-(1“4)-
i- -glucopyranoside (2).—Compound 15 (18
D
D
D
mg, 0.012 mmol) was treated with 0.5 M
NaOMe–MeOH for 16 h. The mixture was
neutralized with Duolite C436 (H+) resin,
filtered, and concd. The residue was chro-
matographed on a reversed-phase column
(Varian Mega Bond Elut C18, water–MeOH
4:1“3:2“1:1“2:3“3:7“1:4“0:1, 4 mL
of each) to give 2 (10.4 mg, 90%); [h]2D2 +36°
13
CH3); C NMR (CD3OD–D2O 3:1): l 136.6,
129.3, 105.1, 104.1, 102.4, 80.6, 79.4, 76.7,
76.4, 76.2, 74.6, 74.3, 73.5, 72.6, 71.1, 70.8,
70.4, 70.3, 67.1, 62.4, 61.6, 33.3, 33.0, 30.6,
30.5, 30.4, 30.1, 30.0, 23.7, 14.6; HRMS calcd
for C36H65N3NaO17 [M+Na]: 834.4212.
Found: 834.4207.
1
(c 1.0, MeOH); H NMR (CD3OD–D2O 2:1):
l (assignments of aglycon protons are shown
Scheme 2. (a) Molecular sieves, CH2Cl2, 30 min, then BF3·OEt2, N2, 22 °C, 1.5 h. (b) NaOMe, MeOH. (c) H2S, pyridine, water,
0 °C, 1 h, then 22 °C, 48 h, then EDC, 1-adamantaneacetic acid, CH2Cl2, 22 °C, 8 h. (d) Molecular sieves, CH2Cl2, 20 min, then
BF3·OEt2, 22 °C, 1.5 h.

H.C. Hansen, G. Magnusson / Carbohydrate Research 322 (1999) 181–189
185
13
1.77–1.85 (m, 3 H), 1.42–1.61 (m, 12 H); C
NMR (D2O): l 175.3, 103.6, 102.5, 100.7,
79.1, 77.7, 75.8, 75.1, 74.7, 73.3, 72.5, 71.24,
71.15, 69.5, 69.3, 68.9, 68.8, 60.8, 60.7, 60.5,
51.0, 42.5, 39.4, 36.5, 32.8, 28.7; HRMS calcd
for C32H53NNaO17 [M+Na]: 746.3211.
Found: 746.3243.
2-(t-Butylacetamido)ethyl (h-
anosyl)-(1“4)-(i- -galactopyranosyl)-(1“4)-
i- -glucopyranoside (4).—t-Butylacetic acid
D
-galactopyr-
D
D
(0.064 mL, 0.5 mmol) was dissolved in CH2Cl2
(6 mL), N-hydroxysuccinimide (NHS, 288 mg,
2.5 mmol) and N-(3-dimethylaminopropyl)-
N%-ethylcarbodiimide (EDC, 480 mg, 2.5
mmol) were added, and the reaction mixture
was stirred at room temperature (rt) for 14 h.
The mixture was washed with water, dried,
and concd. The residue was chromatographed
(SiO2, 2:1 EtOAc–heptane) to give the NHS–
Scheme 3. (a) Me3CCH2COOH, NHS, EDC, CH2Cl2, 22 °C,
14 h. (b) Ac2O, MeOH, 22 °C, 14 h. (c) DMF, pyridine,
60 °C, 4.5 h. (d) NaOH, water, 22 °C, 5.5 h. (e) H2, PdꢁC,
EtOH, aq HCl, 2 h. (f) Et3N, CH2Cl2, 22 °C, 3 h, then
NaOMe–MeOH, 22 °C, 6 h.
1
ester of t-BuCH2CO (102 mg, 96%); H NMR
in italic) 7.82 (d, 1 H, J 9.0 Hz, NH), 5.69–
5.78 (m, 1 H, H-5), 5.46 (br dd, 1 H, J 7.8,
15.3 Hz, H-4), 4.45 (d, 1 H, J 7.7 Hz, H-1%),
4.35 (d, 1 H, J 7.8 Hz, H-1), 4.30 (br t, 1 H,
J 6.6 Hz, H-5¦), 4.17 (dd, 1 H, J 4.9, 10.4 Hz),
4.09 (t, 1 H, J 8.4 Hz, H-3), 3.79–4.02 (m, 8
H), 3.44–3.75 (m, 9 H), 1.90–2.05 (m, 7 H),
1.59–1.75 (m, 12 H), 1.20–1.41 (m, 24 H),
0.88 (t, 3 H, J 6.8 Hz, CH3); 13C NMR
(CD3OD–D2O 2:1): l 174.1, 136.1, 131.0,
105.1, 104.3, 102.3, 80.7, 79.4, 76.7, 76.3, 76.0,
74.7, 74.2, 72.6, 71.0, 70.7, 70.3, 62.3, 61.6,
54.9, 52.1, 43.7, 37.9, 34.0, 33.3, 33.0, 30.6,
30.3, 30.1, 30.0, 23.7, 14.6; HRMS calcd for
C48H83NNaO18 [M+Na]: 984.5508. Found:
984.5485.
(CDCl3): l 2.80 (m, 4 H, COCH2CH2CO),
2.44 (s, 2 H, t-BuCH2CO), 1.09 (s, 9 H,
CMe3); 13C NMR (CDCl3): l 169.7, 167.3,
44.9, 31.6, 29.8, 26.0. The NHS–ester (8.2 mg,
0.038 mmol) and 17 (7 mg, 0.013 mmol) were
dissolved in dry pyridine (2 mL) at 65 °C. The
mixture was stirred for 12 h and concd.
The residue was chromatographed on a re-
versed-phase column (Varian Mega Bond Elut
C18, 1:0“9:1“4:1“7:3“3:2“1:1 water–
MeOH, 5 mL of each) to give 4 (5 mg, 60%);
1
[h]2D2 +35° (c 0.4, water); H NMR (D2O): l
4.81 (d, 1 H, J 3.9 Hz, H-1¦), 4.36, 4.35 (d, 1
H each, J 7.7 and 8.0 Hz, H-1 and H-1%), 4.22
(t, 1 H, J 6.6 Hz, H-5¦), 3.15–3.93 (m, 21 H),
2.00 (s, 2 H, CH2CO), 0.85 (s, 9 H, CMe3); 13C
NMR (D2O): l 176.1, 103.6, 102.5, 100.7,
79.0, 77.7, 75.8, 75.1, 74.7, 73.2, 72.5, 71.23,
71.15, 69.5, 69.3, 68.9, 60.8, 60.7, 60.4, 49.7,
39.4, 30.6, 29.4; HRMS calcd for
C26H47NNaO17 [M+Na]: 668.2742. Found:
668.2722.
2-(1-Adamantaneacetamido)ethyl (h-
actopyranosyl)-(1“4)-(i- -galactopyranosyl)-
(1“4)-i- -glucopyranoside (3).—Compound
D
-gal-
D
D
16 (20 mg, 0.018 mmol) was treated with 0.5
M NaOMe–MeOH for 14 h. The mixture was
neutralized with Duolite C436 (H+) resin,
filtered, and concd. The residue was chro-
matographed on a reversed-phase column
(Varian Mega Bond Elut C18, water–MeOH
1:0“9:1“4:1“7:3“3:2“1:1“2:3, 5 mL
of each) to give 3 (10.8 mg, 85%); [h]2D5 +51°
2-(Acetamido)ethyl (h-
D
-galactopyranosyl)-
(1“4)-(i- -galactopyranosyl)-(1“4)-i- -
D
D
glucopyranoside (5).—To a solution of 17 (3
mg, 0.006 mmol) in MeOH (2 mL) was added
Ac2O (0.1 mL). The mixture was stirred at rt
for 14 h, and the mixture was concd. The
residue was chromatographed on a reversed-
phase column (Varian Mega Bond Elut C18,
1
(c 1.0, water); H NMR (D2O): l 4.81 (d, 1 H,
J 3.9 Hz, H-1¦), 4.36 (d, 2 H, J 7.9 Hz,
H-1,1%), 4.22 (br t, 1 H, J 6.5 Hz, H-5¦),
3.15–3.92 (m, 21 H), 1.88 (s, 2 H, CH2CO),
water–MeOH
1:0“9:1“4:1“7:3“3:2“

186
H.C. Hansen, G. Magnusson / Carbohydrate Research 322 (1999) 181–189
1:1, 5 mL of each) to give 5 (3.2 mg, 100%);
35.3, 34.2, 26.9, 26.0; HRMS calcd for
C35H56NO19S2 [M+H]: 858.2888. Found:
858.2900.
1
[h]2D3 +35° (c 0.3, water); H NMR (D2O): l
4.79 (d, 1 H, J 3.8 Hz, H-1¦), 4.35, 4.34 (d, 1
H each, J 7.8 and 8.0 Hz, H-1 and H-1%), 4.20
(br t, 1 H, J 6.5 Hz, H-5¦), 3.39–3.90 (m, 19
H), 3.24–3.30 (m, 1 H, CH2NH), 3.13–3.19
5 - [3 - (4 - Carboxy - 2 - thiabutyl) - phenyl] - 4 -
pentanecarboxamidoethyl (h -
osyl)-(1“4)-(i-
i- -glucopyranoside (7).—To a solution of 6
D
- galactopyran -
D
-galactopyranosyl)-(1“4)-
13
D
(m, 1 H, CH2NH), 1.84 (s, 3 H, CH3CO); C
(6 mg, 0.007 mmol) in water (1 mL) was
added aq 0.5 M NaOH (0.07 mL, 0.035
mmol). The mixture was stirred at rt for 5.5 h
and the mixture was neutralized with Duolite
C436 (H+) resin, filtered, and concd to give 7
NMR (D2O): l 174.5, 103.6, 102.5, 100.6,
78.9, 77.7, 75.8, 75.1, 74.7, 73.2, 72.5, 71.2,
71.1, 69.4, 69.2, 68.9, 60.8, 60.7, 60.3, 39.7,
23.6, 22.2; HRMS calcd for C22H39NNaO17
[M+Na]: 612.2116. Found: 612.2122.
1
5 - [3 - (4 - Methoxycarbonyl - 2 - thiabutyl) -
(5.8 mg, 98%); [h]2D3 +16° (c 0.7, water); H
phenyl]-4-pentanecarboxamidoethyl (h-
actopyranosyl)-(1“4)-(i-
(1“4)-i- -glucopyranoside (6).—(a) The
D-gal-
NMR (D2O): l 7.11–7.25 (m, 4 H, Ar), 4.79
(d, 1 H, J 3.9 Hz, H-1¦), 4.33, 4.32 (d, 1 H
each, J 7.7 and 8.0 Hz, H-1 and H-1%), 4.20 (br
t, 1 H, J 6.4 Hz, H-5¦), 3.12–3.91 (m, 21 H),
2.48–2.61 (m, 6 H), 2.30–2.39 (m, 6 H); 13C
NMR (D2O): l 175.0, 139.3, 139.2, 129.7,
129.5, 128.1, 128.0, 103.6, 102.5, 100.7, 79.0,
77.7, 75.8, 75.1, 74.6, 73.2, 72.5, 72.4, 71.2,
71.1, 68.4, 69.2, 68.9, 68.8, 62.8, 60.8, 60.7,
39.7, 35.7, 35.3, 35.2, 27.1, 26.9; HRMS calcd
for C34H54NO19S2 [M+H]: 844.2731. Found:
844.2715; HRMS calcd for C34H53NNaO19S2
[M−H+Na]: 866.2551. Found: 866.2539.
(2-Hydroxyethyl)-1-adamantaneacetamide
D
-galactopyranosyl)-
D
NHS–ester 11 (7.4 mg, 0.016 mmol) and com-
pound 17 (10 mg, 0.018 mmol) were dissolved
in a mixture of freshly distilled DMF (1 mL)
and pyridine (1 mL). The reaction mixture
was stirred at 60 °C for 4.5 h and the mixture
was concd. The residue was chromatographed
on a reversed-phase column (Varian Mega
Bond Elut C18, 1:0“9:1“4:1“7:3“3:2“
1:1 water–MeOH, 5 mL of each) to give 6
(8.5 mg, 62%).
(b) Compound 19 (11 mg, 0.011 mmol) and
the NHS–ester 11 (6.4 mg, 0.015 mmol) were
dissolved in CH2Cl2 (3 mL) and Et3N (0.020
mL). The mixture was stirred at rt for 3 h and
concd. The residue was chromatographed
(3:1:1 EtOAc–heptane–MeOH) to give O-
acetylated 6 (12 mg), part of which (11 mg,
0.009 mmol) was then deacetylated with 0.5 M
NaOMe–MeOH. After 6 h, the mixture was
neutralized with Duolite C436 (H+) resin,
filtered, and concd. The residue was chro-
matographed on a reversed-phase column
(Varian Mega Bond Elut C18, 1:0“9:1“
4:1“7:3“3:2“1:1 water–MeOH, 5 mL of
each) to give 6 (6.3 mg, 79% based on 19);
(8).—1-Adamantaneacetic acid (100 mg, 0.515
mmol) was dissolved in dry CH2Cl2 (5 mL)
and EDC (200 mg, 1.03 mmol) was added at
0 °C under N2. The mixture was stirred for 1 h
and EtONH2 (0.085 mL, 0.64 mmol) was
added. The reaction mixture was stirred at rt
for 14 h, filtered, and concd. The residue was
chromatographed (10:1 EtOAc–MeOH) to
give 8 (92 mg, 75%); mp 106–109 °C (EtOAc–
heptane); 1H NMR (CDCl3): l 6.10 (br s, 1 H,
NH), 3.72 (br t, 2 H, J 4.9 Hz, CH2O), 3.42
(br q, 2 H, J 5.2 Hz, CH2NHCO), 3.32 (br s,
1 H, OH), 1.94–2.00 (m, 5 H), 1.59–1.74 (m,
13
12 H); C NMR (CDCl3): l 172.9, 63.0, 52.1,
1
[h]2D3 +34° (c 0.8, water); H NMR (D2O): l
43.0, 42.9, 37.1, 33.2, 29.0; HRMS calcd for
C14H24NO2 [M+H]: 238.1807. Found:
238.1802.
7.12–7.30 (4 H, Ar), 4.82 (d, 1 H, J 3.7 Hz,
H-1¦), 4.36, 4.35 (d, 1 H each, J 7.8 Hz, H-1
or H-1%), 4.23 (br t, 1 H, J 6.6 Hz, H-5¦),
3.15–3.93 (m, 24 H), 2.53–2.62 (m, 4 H), 2.49,
2.39 (br t, 4 H each, J 6.6 Hz); 13C NMR
(D2O): l 175.5, 174.9, 139.2, 129.6, 129.5,
128.1, 103.6, 102.5, 100.7, 79.0, 77.7, 75.8,
75.1, 74.7, 73.2, 72.5, 71.22, 71.15, 69.5, 69.2,
68.9, 68.8, 60.8, 60.7, 60.4, 52.6, 39.7, 35.7,
1-Mercaptomethyl-3-(4-methoxycarbonyl-2-
thiabutyl)-benzene (9).—A mixture of methyl
acrylate (0.048 mL, 0.53 mmol), Et3N (0.074
mL, 0.53 mmol) and DMF (0.9 mL) was
added dropwise (2 h) to a mixture of 1,3-bis-
mercaptomethylbenzene (0.087 mL, 0.587
mmol) and DMF (1 mL). The mixture was

H.C. Hansen, G. Magnusson / Carbohydrate Research 322 (1999) 181–189
187
stirred for 30 min, water (3 mL) was added,
and the reaction mixture was extracted with
Et2O (2×10 mL). The organic phase was
dried and concd and the residue was chro-
matographed (1:5 EtOAc–heptane) to give 9
(m, 4 H, Ar), 3.77, 3.74 (s, 2 H each,
ArCH2S), 3.69 (s, 3 H, OCH3), 2.75–2.88 (m,
8 H), 2.66–2.72 (m, 2 H, CH2CH2), 2.54–2.60
(m, 2 H, CH2CH2).
(2S,3R,4E)-2-Azido-3-benzoyloxyoctadec-
1
4-enyl (2,3,4,6-tetra-O-acetyl-h-
anosyl)-(1“4)-(2,3,6-tri-O-acetyl-i-
actopyranosyl)-(1“4)-2,3,6-tri-O-acetyl-i-
D
-galactopyr-
(95 mg, 70%); H NMR (CDCl3): l 7.17–7.31
D
-gal-
(m, 4 H, Ar), 3.72–3.76 (m, 4 H, ArCH2S),
3.69 (s, 3 H, OCH3), 2.70 (dt, 2 H, J 1.1, 7.4
Hz, CH2CH2), 2.57 (dt, 2 H, J 1.1, 7.2 Hz,
CH2CH2), 1.78 (t, 1 H, J 7.6 Hz, SH); 13C
NMR (CDCl3): l 172.7, 142.0, 139.0, 129.3,
128.9, 128.0, 127.3, 52.3, 36.6, 34.7, 29.3, 26.7.
The dialkylated diester was isolated as a
D
-
glucopyranoside (14).—To a solution of
trichloroacetimidate 12 [3,10] (30 mg, 0.028
mmol) and (2S,3R,4E)-2-azido-3-benzoyloxy-
4-octadecen-1-ol [7] (13, 12 mg, 0.028 mmol)
in dry CH2Cl2 (3 mL) was added molecular
sieves (AW-300). The mixture was stirred for
30 min, BF3·OEt2 (0.003 mL, 0.024 mmol) was
added, and the reaction mixture was stirred at
rt under N2 for 1.5 h The mixture was filtered
through Celite and the filtrate was washed
with satd aq NaHCO3, dried, and concd. The
residue was chromatographed (1:1 EtOAc–
1
byproduct in 10–15% yield. Selected data: H
NMR (CDCl3): l 7.18–7.30 (m, 4 H, Ar), 3.72
(s, 4 H, ArCH2S), 3.69 (s, 6 H, OCH3), 2.69,
2.56 (t, 4 H each, J 7.0 Hz, CH2CH2); 13C
NMR (CDCl3): l 172.7, 138.9, 129.7, 129.2,
128.1, 52.2, 36.6, 34.7, 26.7
1-(4-Carboxy-2-thiabutyl)-3-(4-methoxy-
carbonyl-2-thiabutyl)-benzene (10).—A mix-
ture of 9 (90 mg, 0.35 mmol), 3-bromopropi-
onic acid (80 mg, 0.52 mmol) and Cs2CO3
(320 mg, 0.99 mmol) in dry DMF (3 mL) was
stirred at rt for 14 h, and AcOH (1 mL) and
CH2Cl2 (20 mL) were added. The mixture was
washed with water (3×5 mL) and the organic
phase was dried and concd. The residue was
chromatographed (7:7:2 toluene–EtOAc–hep-
tane“2:1 toluene–EtOAc+2% AcOH) to
1
heptane) to give 14 [11] (25 mg, 66%). H
NMR (CDCl3): l (assignments of aglycon
protons are shown in italic) 8.03–8.08 (m, 2
H, Ar), 7.55–7.62 (m, 1 H, Ar), 7.43–7.49 (m,
2 H, Ar), 5.93 (dt, 1 H, J 6.8, 15.0 Hz, H-5),
5.50–5.63 (m, 3 H, H-4¦, H-3, H-4), 5.39 (dd,
1 H, J 3.3, 11.0 Hz, H-3¦), 5.21 (t, 1 H, J 9.0
Hz, H-3), 5.19 (dd, 1 H, J 3.6, 11.1 Hz, H-2¦),
5.11 (dd, 1 H, J 7.8, 10.8 Hz, H-2%), 4.99 (d, 1
H, J 3.6 Hz, H-1¦), 4.93 (dd, 1 H, J 7.7, 9.1
Hz, H-2), 4.74 (dd, 1 H, J 2.5, 10.9 Hz, H-3%),
4.54 (d, 1 H, J 7.6 Hz, H-1), 4.52 (d, 1 H, J
7.7 Hz, H-1%), 4.39–4.52 (m, 3 H, H-5¦,6%,6),
4.02–4.21 (m, 4 H, incl. H-6,6%), 4.01 (br d, 1
H, J 2.0 Hz, H-4%), 3.84–3.97 (m, 2 H, H-1,2),
3.83 (t, 1 H, J 9.3 Hz, H-4), 3.76 (t, 1 H, J 6.8
Hz, H-5%), 3.64 (ddd, 1 H, J 2.0, 5.1, 9.9 Hz,
H-5), 3.59 (dd, 1 H, J 5.8, 10.3 Hz, H-1),
2.135, 2.090, 2.084, 2.072, 2.066, 2.058, 2.042,
2.040, 1.99 (s, 30 H, Ac), 1.19–1.44 (m, 24 H),
0.88 (t, 3 H, J 6.9 Hz, CH3); 13C NMR
(CDCl3): l 171.1, 170.93, 170.91, 170.8, 170.5,
170.14, 170.08, 170.0, 169.3, 165.5, 139.5,
133.6, 130.3, 130.2, 128.9, 123.0, 101.6, 100.7,
100.1, 77.4, 76.8, 75.1, 73.6, 73.2, 73.0, 72.2,
72.0, 69.4, 69.3, 68.8, 68.3, 67.54, 67.50, 63.9,
62.5, 61.7, 60.7, 32.8, 32.3, 30.10, 30.08, 30.0,
29.82, 29.78, 29.6, 29.1, 23.1, 21.4, 21.2, 21.13,
21.08, 21.0, 20.9, 14.6.
1
give 10 (67 mg, 58%); H NMR (CDCl3): l
7.18–7.31 (m, 4 H, Ar), 3.75 (s, 2 H,
ArCH2S), 3.73 (s, 2 H, ArCH2S), 3.70 (s, 3 H,
OCH3), 2.66–2.72 (m, 4 H, CH2CH2), 2.54–
13
2.63 (m, 4 H, CH2CH2); C NMR (CDCl3): l
177.1, 173.0, 138.9, 138.8, 129.7, 129.3, 128.13,
128.08, 52.3, 36.6, 34.7, 34.6, 26.6, 26.3;
HRMS calcd for C15H21O4S2 [M+H]:
329.0881. Found: 329.0893.
1-(4-Succinimidoxycarbonyl-2-thiabutyl)-3-
(4-methoxycarbonyl-2-thiabutyl)-benzene (11).
—The monoester 10 (7.3 mg, 0.022 mmol)
was dissolved in dry CH2Cl2 (2 mL), and NHS
(5.7 mg, 0.05 mmol) and EDC (9.2 mg, 0.048
mmol) were added. The reaction mixture was
stirred at rt under N2 for 14 h, CH2Cl2 (10
mL) was added, and the mixture was washed
with water (3 mL). The organic phase was
dried and concd and the residue was chro-
matographed (2:1 EtOAc–heptane) to give 11
(7.4 mg, 79%); 1H NMR (CDCl3): l 7.20–7.32
(2S,3R,4E) - 2 - (1 - Adamantaneacetamido) -
3-(benzoyloxy)-octadec-4-enyl (2,3,4,6-tetra-O-

188
H.C. Hansen, G. Magnusson / Carbohydrate Research 322 (1999) 181–189
acetyl-h-
D
-galactopyranosyl)-(1“4)-(2,3,6-
-galactopyranosyl)-(1“4)-
-glucopyranoside (15).
(AW-300). The mixture was stirred for 20 min
tri-O-acetyl-i-
D
and BF3·OEt2 (0.003 mL, 0.024 mmol) was
added. The mixture was stirred for 1.5 h, then
filtered through Celite, and CH2Cl2 (10 mL)
was added. The mixture was washed with a
satd aq NaHCO3, dried, and concd. The
residue was chromatographed (3:1“4:1
EtOAc–heptane) to give 16 (22.5 mg, 70%);
2,3,6-tri-O-acetyl-i-
D
—Hydrogen sufide was bubbled through a
solution of 14 (22 mg, 0.0165 mmol) in a
pyridine–water mixture (6:1, 6 mL) for 1 h at
0 °C, and the reaction mixture was kept under
H2S at 22 °C for 48 h. Residual H2S was
removed by a stream of N2 for 1 h, and the
mixture was co-concd with toluene. The
residue was dissolved in dry CH2Cl2 (2 mL)
and EDC (20 mg, 0.1 mmol) and 1-adaman-
taneacetic acid (20 mg, 0.1 mmol) were added.
The mixture was stirred at rt for 8 h and
concd. The residue was chromatographed (2:1
EtOAc–heptane) to give 15 (19.4 mg, 79%);
1
[h]2D3 +37° (c 1.0, CHCl3); H NMR (CDCl3):
l 5.82 (br t, 1 H, J 4.9 Hz, NH), 5.59 (br d, 1
H, J 3.3 Hz, H-4¦), 5.39 (dd, 1 H, J 3.4, 11.0
Hz, H-3¦), 5.20 (t, 1 H, J 9.2 Hz, H-3), 5.18
(dd, 1 H, J 3.5, 11.0 Hz, H-2¦), 5.11 (dd, 1 H,
J 7.7, 10.8 Hz, H-2%), 4.99 (d, 1 H, J 3.6 Hz,
H-1¦), 4. 89 (dd, 1 H, J 8.0, 9.5 Hz, H-2), 4.74
(dd, 1 H, J 2.8, 10.9 Hz, H-3%), 4.52 (d, 1 H, J
7.7 Hz, H-1%), 4.48 (d, 1 H, J 7.9 Hz, H-1),
4.41–4.55 (m, 3 H, incl. H-5¦), 4.06–4.20 (m,
4 H), 4.02 (br d, 1 H, J 2.3 Hz, H-4%), 3.60–
3.84 (m, 5 H, incl. H-4, OCH2CH2), 3.35–3.52
(m, 2 H, CH2NH), 1.91–2.15 (35 H), 1.58–
1.74 (m, 12 H, adamantyl–CH2); 13C NMR
(CDCl3): l 171.5, 171.1, 170.94, 170.90,
170.88, 170.86, 170.5, 170.3, 170.03, 170.02,
169.3, 101.5, 101.2, 100.1, 77.3, 76.7, 73.4,
73.2, 72.22, 72.19, 69.9, 69.4, 69.3, 68.3, 67.6,
67.5, 62.5, 61.7, 60.7, 52.0, 43.03, 42.99, 39.5,
37.2, 33.1, 29.0, 21.4, 21.3, 21.23, 21.15, 21.11,
21.08, 21.04, 20.95; HRMS calcd for
C52H73NNaO27 [M+Na]: 1166.4268. Found:
1166.4271.
[h]2D4 +35° (c 1.0, CHCl3); H NMR (CDCl3):
1
l (assignments of aglycon protons are shown
in italic) 8.00–8.05, 7.53–7.60, 7.41–7.48 (m,
5 H, Ar), 5.89 (dt, 1 H, J 6.7, 15.3 Hz, H-5),
5.66 (d, 1 H, J 9.0 Hz, NH), 5.56–5.62 (m, 2
H, H-4¦, H-3), 5.45–5.54 (m, 1 H, H-4), 5.39
(dd, 1 H, J 3.4, 11.0 Hz, H-3¦), 5.15–5.23 (m,
2 H, H-2¦,3), 5.10 (dd, 1 H, J 7.8, 10.8 Hz,
H-2%), 4.99 (d, 1 H, J 3.6 Hz, H-1¦), 4.91 (dd,
1 H, J 7.8, 9.3 Hz, H-2), 4.73 (dd, 1 H, J 2.5,
10.8 Hz, H-3%), 4.49 (d, 1 H, J 7.7 Hz, H-1%),
4.48 (d, 1 H, J 7.7 Hz, H-1), 4.40–4.53 (m, 3
H, H-5¦,6%, H-2), 4.36 (br dd, 1 H, J 1.8, 12.0
Hz, H-6), 4.07–4.21 (m, 3 H, H-6%,6¦), 3.95–
4.05 (m, 3 H, H-4%,6, H-1), 3.73–3.82 (m, 2 H,
H-4,5%), 3.56–3.68 (m, 2 H, H-5, H-1), 1.85–
2.15 (35 H), 1.56–1.72 (m, 12 H), 1.20–1.39
(m, 24 H), 0.89 (t, 3 H, J 6.8 Hz, CH3); 13C
NMR (CDCl3): l 171.1, 170.91, 170.86, 170.8,
170.5, 170.2, 170.04, 169.96, 169.3, 165.6,
137.9, 133.5, 130.7, 130.0, 128.8, 125.0, 101.6,
100.7, 100.1, 76.8, 74.3, 73.32, 73.26, 73.0,
72.3, 69.3, 68.3, 68.0, 67.6, 67.5, 62.6, 61.8,
60.7, 52.3, 51.3, 43.0, 37.1, 33.2, 32.8, 32.4,
30.1, 30.05, 29.9, 29.8, 29.7, 29.3, 29.0, 23.1,
21.4, 21.24, 21.17, 21.1, 21.0, 20.9, 14.6;
HRMS calcd for C75H107NNaO29 [M+Na]:
1508.6826. Found: 1508.6818.
2-Aminoethyl (2,3,4,6-tetra-O-acetyl-h-
galactopyranosyl)-(1“4)-(2,3,6-tri-O-acetyl-
i - - galactopyranosyl) - (1“4) - 2,3,6 - tri - O -
acetyl-i- -glucopyranoside (19).—Compound
D
-
D
D
18 [3] (14 mg, 0.014 mmol) was dissolved in
EtOH (2 mL) and a drop of aq 0.1 M HCl
was added. The mixture was hydrogenated
(H2, Pd/C, 1 atm) for 2 h, then filtered
through Celite and concd. The residue was
chromatographed (2:1 EtOAc–MeOH+5%
Et3N) to give 19 (10.7 mg, 78%); [h]2D2 +51° (c
1
1.0, MeOH); H NMR (CDCl3–CD3OD 1:1):
l 5.27 (br d, 1 H, J 3.3 Hz, H-4¦), 5.09 (dd, 1
H, J 3.4, 11.1 Hz, H-3¦), 4.85–4.93 (m, 2 H,
H-3,2¦), 4.81 (dd, 1 H, J 7.8, 10.9 Hz, H-2%),
4.73 (d, 1 H, J 3.6 Hz, H-1¦), 4.53–4.61 (m, 2
H), 4.21 (br t, 1 H, J 6.8 Hz, H-5¦), 4.16 (dd,
1 H, J 6.4, 11.1 Hz, H-6% or H-6), 3.76–3.92
(m, 4 H), 3.52–3.70 (m, 3 H), 3.40–3.46 (m, 1
H), 1.85, 1.84, 1.80, 1.79, 1.78, 1.77, 1.760,
2-(1-Adamantaneacetamido)ethyl (2,3,4,6-
tetra-O-acetyl-h-
(2,3,6 - tri - O - acetyl - i -
(1“4)-2,3,6-tri-O-acetyl-i-
D
-galactopyranosyl)-(1“4)-
- galactopyranosyl) -
-glucopyranoside
D
D
(16).—To a solution of 8 (7.3 mg, 0.031
mmol) and 12 (30 mg, 0.028 mmol) in dry
CH2Cl2 (3 mL) was added molecular sieves

H.C. Hansen, G. Magnusson / Carbohydrate Research 322 (1999) 181–189
189
13
Magnusson, Pure Appl. Chem., 68 (1996) 2119–2128. (c)
G. Magnusson, S.J. Hultgren, J. Kihlberg, Methods Enzy-
mol., 253 (1995) 105–114.
1.755, 1.69 (s, 30 H, Ac); C NMR (CD3OD):
l 172.4, 172.3, 172.1, 172.0, 171.94, 171.91,
171.8, 171.5, 171.4, 171.2, 102.1, 101.9, 100.8,
78.8, 77.3, 74.32, 74.30, 74.0, 73.5, 72.8, 70.9,
69.8, 69.5, 69.0, 68.9, 68.5, 63.7, 63.3, 61.8,
47.7, 21.2, 21.0, 20.79, 20.75, 20.7, 20.6, 20.5;
HRMS calcd for C40H57NNaO26 (M+Na):
990.3067. Found: 990.3066.
[2] (a) P.-G. Nyholm, G. Magnusson, Z. Zheng, R. Norel, B.
Binnington-Boyd, C.A. Lingwood, Chem. Biol., 3 (1996)
263–275. (b) H. Ling, A. Boodhoo, B. Hazes, M.D.
Cummings, G.D. Armstrong, J.L. Brunton, R.J. Read,
Biochemistry, 37 (1998) 1777–1788. (c) H. Shimizu, R.A.
Field, S.W. Homans, A. Donohue-Rolfe, Biochemistry, 37
(1998) 11078–11082.
[3] H.C. Hansen, G. Magnusson, Carbohydr. Res., 322 (1999)
166–180.
[4] B. Boyd, G. Magnusson, Z. Zhiuyan, C.A. Lingwood, Eur.
J. Biochem., 223 (1994) 873–878.
[5] P.M. St. Hilaire, M.K. Boyd, E.J. Toone, Biochemistry, 33
(1994) 14452–14463.
Acknowledgements
[6] M. Mylvaganam, C.A. Lingwood, Biochem. Biophys. Res.
Commun., in press.
[7] R.R. Schmidt, P. Zimmermann, Angew. Chem., Int. Ed.
Engl., 25 (1986) 725–726.
This work was supported by the Swedish
Natural Science Research Council and the
National Institutes of Health (NIH, USA).
[8] H.C. Hansen, G. Magnusson, Carbohydr. Res., 307 (1998)
243–251.
[9] D. Qiu, R.R. Schmidt, Liebigs Ann. Chem., (1992) 217–
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