A simple synthesis of 8-(methoxycarbonyl)octyl 3,6-di-O-(α-D-mannopyranosyl)-α-D-mannopyranoside and derivatives and their use in the preparation of neoglycoconjugates

Article abstract of DOI:10.1016/S0008-6215(98)00330-9

8-(Methoxycarbonyl)octyl 3,6-di-O-(α-D-mannopyranosyl)-α-D-mannopyranoside (10) was synthesized in 54% yield by regioselective diglycosylation of unprotected mannoside 4, employing the trichloroacetimidate donor 1, followed by debenzoylation. Derivatives

Full text of DOI:10.1016/S0008-6215(98)00330-9

Carbohydrate Research 315 (1999) 148–158
A simple synthesis of 8-(methoxycarbonyl)octyl
3,6-di-O-(a-
D
-mannopyranosyl)-a-
D
-mannopyranoside
and derivatives and their use in the preparation of
neoglycoconjugates
Bernd Becker a, Richard H. Furneaux a,*, Folkert Reck a, Oleg A. Zubkov b
a Industrial Research Ltd, Gracefield Road, PO Box 31-310, Lower Hutt, New Zealand
b Chemistry Department, Victoria Uni6ersity of Wellington, PO Box 600, Wellington, New Zealand
Received 23 October 1998; accepted 9 December 1998
Abstract
8-(Methoxycarbonyl)octyl 3,6-di-O-(a-
D
-mannopyranosyl)-a- -mannopyranoside (10) was synthesized in 54%
D
yield by regioselective diglycosylation of unprotected mannoside 4, employing the trichloroacetimidate donor 1,
followed by debenzoylation. Derivatives of compounds 4 and 10 were used to prepare conjugates containing
fluorochromes for the study of carbohydrate–lectin interactions, as well as conjugates with phospholipids for the
preparation of liposomes. © 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Mannotrioside; Neoglycoconjugates; Glycolipid; Synthesis
rather specific geometry and spatial orienta-
tion for strong binding [4]. In contrast, the
monomeric mannose receptors appear to bind
1. Introduction
Terminal a- -mannopyranosyl residues are
D
some relatively simple mannose-containing
oligosaccharides quite effectively [5]. For this
reason, there has been interest in the potential
of using synthetic mannose-containing neogly-
coconjugates of drugs and antigens as vehicles
for their delivery to the mannose receptors on
macrophages and dendritic cells [6–8]. For
example, the present enzyme replacement
therapy for Gaucher’s disease relies upon this
concept for specific cellular uptake of the en-
zyme [9].
ubiquitous features of glycoproteins of bacte-
rial pathogens, parasites, yeasts and virus en-
velopes as well as certain endogenous
glycoproteins such as lysosomal enzymes.
They are recognized by various C-type lectins,
particularly the mannose receptors of
macrophages [1] and dendritic cells [2,3], and
the collectins, soluble circulating proteins such
as the ‘mannose-binding proteins’ and the
lung surfactant proteins [4]. The collectins are
oligomeric proteins that require complex mul-
tidentate mannose-containing ligands of
The
mannose
receptor
of
human
macrophages binds branched mannose
oligosaccharide derivatives such as methyl 3,6-
di-O-(a-
D
-mannopyranosyl)-b-
D
-mannopyran-
* Corresponding author. Tel.: +644-566-6919; fax: +644-
566-6004.
oside specifically and with relatively high
0008-6215/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(98)00330-9

B. Becker et al. / Carbohydrate Research 315 (1999) 148–158
149
2. Results and discussion
affinity, in comparison with more simple
multivalent structures, such as a permanno-
sylated lysyl–serine decapeptide, which car-
ries an array of single mannosyl residues [5].
Under reaction conditions favouring the
formation of the thermodynamic products, the
synthesis of mannopyranosides normally pro-
ceeds with high a-stereoselectivity; formation
of b-glycosides is not observed, even with
non-participating protecting groups at O-2,
because of a strong anomeric effect [19,20].
Nevertheless, participating ester protecting
groups in the glycosylating donors are often
preferred, because protection and deprotec-
tion can be achieved readily and efficiently.
However, if 2-O-acetyl-protected donors are
employed, orthoesters may be obtained rather
than the desired glycosides [12,15,21]. In some
cases, with careful control of reaction condi-
tions, such orthoesters may be converted in
situ to glycosides, but in other instances the
orthoesters have to be isolated and converted
in a separate reaction step. Rearrangement of
orthoesters to glycosides is often accompanied
by side reactions such as hydrolysis, acetyl
migration from O-2 of the donor to a free
hydroxy group on the acceptor, or formation
of isomeric glycosides [22,23], and it is there-
fore often expedient to avoid their participa-
tion in the reactions.
The 3,6-di-O-(a-
D
-mannopyranosyl)- -man-
D
nopyranosyl structure is part of the common
core structure of N-glycans and is found as
a constituent of microbial polysaccharides,
such as mannans in yeast cell walls [10]. The
specificity of the mannose receptor on
dendritic cells has not yet been studied in
detail.
An efficient synthesis of neoglycoconju-
gates based on 3,6-di-O-(a-
D-mannopyran-
osyl)- -mannopyranosides was required in
D
connection with biological studies. Several
formal routes to glycosides of this trisaccha-
ride have been reported, depending on step-
wise glycosylation of a mannopyranoside
derivative protected at all positions except
O-3 [11], or diglycosylation of 2,4-di-O-benz-
yl- [12,13] or 2,4-di-O-benzoyl- [14]
mannopyranosides or of partially
D-
a
stannylated mannoside [15]. Additionally,
and of particular relevance to the present
work, a direct approach to glycosides of the
mannotriose has been applied by Kaur and
Hindsgaul using octyl b-
[16] and Verez-Bencomo and co-workers us-
ing 5-azido-3-oxapentyl -manno-
pyranoside [17,18], who obtained 17 and
34% yields, respectively, of 3,6-linked man-
notriosides with tetra-O-acetyl-a- -mannopy-
ranosyl bromide as glycosylating agent. In
the former work, the 2,6- and 4,6-linked
trisaccharides, formed together with the re-
quired product, were elegantly destroyed by
periodate oxidation, and thus the isolation
of the required trimer derivative was specifi-
cally facilitated.
We now report a simple and efficient syn-
thesis of novel a-glycosidic derivatives of the
(1“3),(1“6)-linked mannotriose suitable
for the preparation of neoglycoconjugates
which employs a minimal O-protection strat-
D-mannopyranoside
O-Benzoyl-protected donors seem to be less
prone to orthoester formation, and especially
a-D
the
donor
2,3,4,6-tetra-O-benzoyl-a- -
D
mannopyranosyl bromide [24] has been used
for the high-yielding preparations of a-man-
nosides without formation of orthoesters
[14,25]. This advantage, however, is offset by
the requirement for stoichiometric amounts of
silver salts for its activation, as these are ex-
pensive and inconvenient to use in large-scale
preparations.
D
Our experience correlates with that of oth-
ers regarding the merits of glycosyl imidates as
donors
compared
with
corresponding
halogenoses [20]: their higher shelf stability yet
greater reactivity on activation with catalytic
amounts of a simple Lewis acid, and their
efficiency confer on them real advantages. The
syrupy imidate 1 was obtained in 68% yield
egy with 8-(methoxycarbonyl)octyl a- -
mannopyranoside (4) as acceptor and the
previously unreported 2,3,4,6-tetra-O-ben-
D
from
D
-mannose by reaction of its 2,3,4,6-te-
trabenzoate [26] with trichloroacetonitrile in
dichloromethane in the presence of potassium
carbonate. Reaction with the ‘Lemieux spacer’
zoyl-a-
D
-mannopyranosyl
trichloroacetimi-
date (1) as glycosyl donor.

150
B. Becker et al. / Carbohydrate Research 315 (1999) 148–158
indicated that other products present were
eluted appreciably earlier or later than the
required mannotrioside 8. From earlier eluting
fractions the tetrasaccharide glycoside 8-
alcohol 8-(methoxycarbonyl)octanol (2) [27–
30], with Me3SiOTf as catalyst afforded the
a-mannoside 3 in 86% yield. The formation of
orthoesters as intermediates or as byproducts
was not observed. The acceptor 2 was made
(21%) in one step from 1,9-nonanediol by
calcium hypochlorite-mediated oxidation in
the presence of acetic acid in methanol [31].
Deprotection of benzoate 3 with potassium
carbonate in methanol gave crystalline gly-
coside 4 in 85% yield [20,25,30,32].
On the grounds that the primary group can
confidently be expected to be the most reactive
of the hydroxyl functions of a-mannopyra-
nosides and that those at C-2 and C-4 will be
least reactive (axial and inherently unreactive,
respectively [33]), it can be predicted that dis-
ubstitution will lead preferentially to the 3,6-
linked products. Accordingly, unprotected
mannoside 4, treated with two equiv of
trichloroacetimidate 1 with Me3SiOTf as cata-
lyst, afforded the 3,6-branched trisaccharide
glycoside 8, which was isolated very readily in
55% yield on a 5-g scale by silica gel column
chromatography (Scheme 1). The elution
profile from gel-filtration chromatography on
lipophilic Sephadex LH-20 size-exclusion gel
(methoxycarbonyl)octyl 3,4,6-tri-O-(a-
D
-man-
nopyranosyl)-a- -mannopyranoside dodeca-
D
benzoate was obtained in 5% yield, while the
4,6- and 2,6-linked trisaccharides derivatives
were not detected; neither were orthoesters. It
was important to use only two equivalents of
trichloroacetimidate 1. Otherwise, substantial
proportions of higher glycosylated products
were obtained. Activation of the glycosylating
agent 1 by zinc triflate–triflic acid again gave
compound 8 as the major product in similar
yield to that obtained with Me3SiOTf, but the
reaction mixture was more heterogeneous
(TLC), and purification of the main product
was consequently more difficult.
A sample of mannotrioside 8 was acetylated
to give the diacetate 9 for characterisation by
NMR methods. The specific movements to
1
lower field positions of the H NMR signals
for H-2 and H-4 of compound 9 proved that
the glycosidic linkages were at O-3 and O-6.
1
The H-coupled 13C NMR spectrum showed
C-1–H-1 coupling constants of 168–172 Hz
Scheme 1.

B. Becker et al. / Carbohydrate Research 315 (1999) 148–158
151
the preparation of liposomes to be used for
drug delivery [38–40].
In summary, a short and efficient synthesis
of novel 3,6-di-O-(a-
D
-mannopyranosyl)-a- -
D
mannopyranosides and the preparation from
them, and from the corresponding a- -
mannopyranosides, of neoglycoconjugates
have been demonstrated.
D
3. Experimental
Scheme 2.
General methods.—TLC was performed on
Silica Gel 60 F254 (E. Merck) with detection
by UV absorption and/or by charring after
spraying with EtOH–H2O–H2SO4 (14:4:1, v/
v). Column chromatography was performed
on Silica Gel S32–63 mm (230–400 mesh,
Riedel-de Hae¨n) at medium pressure (4–15
bar) and for reversed-phase work on LiChro-
prep RP-18 25–40 mm (E. Merck) at normal
pressure. Lipophilic Sephadex LH-20 (Sigma)
was used for gel filtration. NMR spectra were
for the three anomeric carbon atoms, which is
indicative of a-glycosides, b-anomers giving
values that are 10 Hz smaller [34–36].
Treatment of product 8 with potassium car-
bonate in methanol gave the unprotected
mannotrioside 10 quantitatively, and reaction
of compounds 4 or 10 with 1,2-diaminoethane
[37] afforded amides 5 and 11, respectively,
which are suitable for the preparation of con-
jugates with amine-reactive probes. Alkaline
hydrolysis of the ester groups of compounds 4
and 10, followed by acidification, afforded the
free acids 6 and 12, which were converted to
the N-acyloxysuccinimide compounds 7 and
13. Coupling of amides 5 and 11 with fluores-
cein isothiocyanate and eosin isothiocyanate
gave the fluorochromes 14, 15 and 16, 17,
respectively (Scheme 2), which were required
for the study of carbohydrate–lectin interac-
tions, especially with the mannose receptor.
Coupling the activated compounds of 7 or 13
with dipalmitoyl-l-a-phosphatidyl ethano-
lamine (DPPE) afforded the neoglycolipids 18
and 19 (Scheme 3), which were required for
1
recorded for H at 300 or 500 MHz and for
13C at 75.5 or 125 MHz, using a Bruker
AC300E or a Varian Unity 500 spectrometer.
Me4Si was used as internal standard, unless
1
otherwise stated. Assignments of H and 13C
resonances were based on 2D-experiments
(1H–1H COSY, 1H–1H TOCSY, 1H–13C
1
HMQC, H–13C HMBC) and polarisation-
transfer experiments (DEPT). HMBC experi-
ments were optimized for the detection of
long-range couplings (5–7 Hz). High-resolu-
tion FABMS was performed in the positive-
ion mode for compounds 16 and 17 in a 1:5
dithioerythritol–dithiothreitol (‘magic bullet’)
matrix on a VG70-250S mass spectrometer
(VG Analytical) equipped with a standard
liquid secondary caesium ion gun, by Dr John
Allen (Hort Research Ltd, Palmerston North,
New Zealand). All other samples were exam-
ined in a 3-nitrobenzyl alcohol matrix (except
for amide 5: glycerol matrix) on an MS 80
RFA spectrometer (Kratos), equipped with a
TECH ZN11 NF FAB ion gun with xenon as
bombarding atom by Dr Bruce Clark, Univer-
sity of Canterbury, New Zealand. Optical ro-
tations were determined with a Perkin–Elmer
241 polarimeter. Fluorescein isothiocyanate
isomer I was purchased from Aldrich Chemi-
Scheme 3.

152
B. Becker et al. / Carbohydrate Research 315 (1999) 148–158
stirred for 30 min at rt over molecular sieves
(4A, pearled). A solution of Me3SiOTf (50 mL,
cal Co., eosin isothiocyanate from Molecular
Probes, and DPPE from Sigma Chemical Co.
˚
0.26 mmol) in CH2Cl2 (2 mL) was added
dropwise. After 2 h the reaction mixture was
treated with triethylamine (0.3 mL) and evap-
orated in vacuo. The residue was dissolved in
CH2Cl2 (100 mL) and petroleum ether (100
mL) was added. Precipitated trichloroac-
etamide was removed by filtration, and the
filtrate was evaporated in vacuo. Chromatog-
raphy on silica gel with 5:1 petroleum ether–
EtOAc afforded glycoside 3 (8.0 g, 10.4 mmol,
86%) as a syrup: [h]2D0 −45.3° (c 1.0, CHCl3);
1H NMR (300 MHz, CDCl3): l 8.07 (m, 4 H,
Bz), 7.96 (m, 2 H, Bz), 7.83 (m, 2 H, Bz),
7.63–7.23 (m, 12 H, Bz), 6.11 (dd, 1 H, J3,4 ꢀ
J4,5 10.0 Hz, H-4), 5.93 (dd, 1 H, J2,3 3.3 Hz,
H-3), 5.71 (dd, 1 H, J1,2 1.7 Hz, H-2), 5.10 (d,
1 H, H-1), 4.72 (dd, 1 H, J5,6a 2.4 Hz, J6a,6b
12.0 Hz, H-6a), 4.51 (dd, 1 H, J5,6b 4.5 Hz,
H-6b), 4.44 (ddd, 1 H, H-5), 3.83 (m, 1 H,
CH2O-spacer), 3.66 (s, 3 H, CH3), 3.58 (m, 1
H, CH2O-spacer), 2.32 (t, 2 H, J 7.5 Hz,
CH2COO), 1.75–1.50 (m, 4 H, CH2), 1.45–
1.25 (m, 8 H, CH2); 13C NMR (75.5 MHz,
CDCl3): l 97.664 (d, JC-1,H-1 168.8 Hz, C-1);
FABMS: m/z Calcd for C44H47O12 (M+H)+:
767.3068. Found: 767.3144.
2,3,4,6-Tetra-O-benzoyl-h-
osyl trichloroacetimidate (1).—A solution of
2,3,4,6-tetra-O-benzoyl-a,b- -mannopyranose
D
-mannopyran-
D
[26] (30 g, 50.3 mmol) in CH2Cl2 (200 mL)
was treated with KCO3 (10 g) and
trichloroacetonitrile (20 mL, 199 mmol) under
vigorous stirring at room temperature (rt)
overnight. The reaction mixture was filtered
and the volatiles were evaporated in vacuo.
Chromatography on silica gel with 4:1
petroleum ether–EtOAc afforded compound
1 (28 g, 37.8 mmol, 81%) as a syrup: [h]2D0−
1
28.2° (c 1.0, CHCl3); H NMR (300 MHz,
CDCl3): l 8.86 (s, 1 H, NH), 8.08 (m, 4 H,
Bz), 7.97 (m, 2 H, Bz), 7.84 (m, 2 H, Bz),
7.65–7.23 (m, 12 H, Bz), 6.57 (br s, 1 H, H-1),
6.23 (dd, 1 H, J3,4ꢀJ4,5 10.0 Hz, H-4), 5.98
(dd, 1 H, J2,3 3.3 Hz, H-3), 5.94 (dd, 1 H, J1,2
1.8 Hz, H-2), 4.73 (dd, 1 H, J5,6a 2.4 Hz, J6a,6b
12.2 Hz, H-6a), 4.63 (ddd, 1 H, J5,6b 4.1 Hz,
H-5), 4.52 (dd, 1 H, H-6b); 13C NMR (75.5
MHz, CDCl3): l 94.757 (d, JC-1,H-1 180.5 Hz,
C-1). Anal. Calcd for C36H28Cl3NO10: C,
58.35; H 3.80. Found: C, 58.18; H 3.87.
8-(Methoxycarbonyl)octanol (2).—See Refs.
[27–30]. 1,9-Nonanediol (12 g, 75 mmol) was
dissolved in MeOH (80 mL) and MeCN (300
8-(Methoxycarbonyl)octyl h- -mannopyran-
D
˚
oside (4).—See Refs. [25,30,32]. Compound 3
(5.9 g, 7.7 mmol) in MeOH (100 mL) was
stirred with KCO3 (500 mg) overnight then
neutralized with ion-exchange resin (Amber-
lite IR 120, H+-form). Removal of the resin
and the solvents gave a solid (2.7 g, 100%).
Chromatography on silica gel with 10:1
CHCl3–MeOH afforded glycoside 4 (3.0 g, 8.6
mmol, 85%): mp 84 °C (petroleum ether–
EtOAc) (lit. [25] 81–82 °C), [h]2D0 +51.5° (c
1.0, MeOH) (lit. [32] +52.7°, c 0.4, MeOH);
mL). Molecular sieves (4A, powdered, 30 g),
calcium hypochlorite (44 g, 0.31 mol) and
acetic acid (36 mL), were added, and the
mixture was mechanically stirred overnight in
the dark at rt [41]. The mixture was filtered,
evaporated in vacuo, and the residue was dis-
solved in CH2Cl2. The solution was washed
with aq sodium thiosulfate (50 g/200 mL),
then brine, dried (MgSO4), and the solvent
was evaporated in vacuo. Kugelrohr distilla-
tion of the residue afforded a mixture of 1,9-
nonanediol and product, which was separated
by chromatography on silica gel with 4:1
petroleum ether–EtOAc to afford ester 2 (3 g,
1
the H NMR (D2O) spectrum was identical to
that reported [30].
8-[N-(2-Aminoethyl)carboxamido]octyl h- -
D
1
16 mmol, 21%) as an oil: H NMR data were
mannopyranoside (5).—Compound 4 (107 mg,
0.31 mmol) was dissolved in 1,2-di-
aminoethane (2 mL) and heated to 60 °C for
36 h [37]. The solution was evaporated in
vacuo, and the residue was dissolved in water
and loaded on a C-18 SepPak cartridge. Ab-
sorbed amide 5 was washed with water, eluted
identical to those reported [28].
8-(Methoxycarbonyl)octyl 2,3,4,6-tetra-O-
benzoyl-h-
D
-mannopyranoside
(3).—Tri-
chloroacetimidate 1 (9 g, 12.1 mmol) and
8-(methoxycarbonyl)octanol 2 (2.5 g, 12.4
mmol) were dissolved in CH2Cl2 (100 mL) and

B. Becker et al. / Carbohydrate Research 315 (1999) 148–158
153
CH2COO), 1.78 (m, 2 H, CH2CH2COO),
1.66–1.50 (m, 2 H, CH2CH2O), 1.50–1.20
(m, 8 H, CH2).
with 1:1 H2O–MeCN, and the solvent was
evaporated to afford amide 5 (110 mg, 0.29
mmol, 95%) as a syrup: [a]2D0+40.0° (c 1.0,
1
8-(Methoxycarbonyl)octyl (2,3,4,6-tetra-O-
H2O); H NMR (300 MHz, D2O, HDO=
benzoyl-h-
6-tetra-O-benzoyl-h-
6)]-h- -mannopyranoside (8).—Acceptor
D
-mannopyranosyl)-(1“3)-[(2,3,4,
4.8 ppm): l 4.91 (br s, 1 H, H-1), 3.98 (m, 1
H, H-2), 3.95–3.65 (m, 6 H, H-3, 4, 5, 6a,
6b, CH2O-spacer), 3.59 (m, 1 H, CH2O-
spacer), 3.33 (t, 2 H, J 6.2 Hz, CH2NH),
2.82 (t, 2 H, J 6.2 Hz, CH2NH2), 2.31 (t, 2
H, J 7.3 Hz, CH2CON), 1.72–1.57 (m, 4 H,
CH2), 1.45–1.30 (m, 8 H, CH2); FABMS:
m/z Calcd for C17H35N2O7 (M+H)+:
379.2444. Found: 379.2453.
D
-mannopyranosyl)-(1“
D
4
(1.0 g, 2.85 mmol), after dissolution in
toluene and removal of the solvent (×3),
was dissolved in CH2Cl2 (60 mL) and stirred
˚
for 1 h at rt over molecular sieves (4 A,
pearled). The solution was cooled to
−40 °C and Me3SiOTf (180 mL, 0.9 mmol)
was added. Stirring was applied for 5 min,
and trichloroacetimidate 1 (4.22 g, 5.7
mmol) in CH2Cl2 (15 mL) was added drop-
wise with stirring over 30 min. The solution
was stirred for a further 60 min, during
which time the temperature rose to −18 °C.
NaHCO3 (0.50 g) was added, and the mix-
ture was stirred for 30 min. The solids were
filtered off, and the solvents were evapo-
rated. Chromatography on silica gel with 5:1
toluene–EtOAc afforded mannotrioside 8
(2.36 g, 1.57 mmol, 55%), as a syrup: [h]2D0−
9-(h- -Mannopyranosyloxy)nonanoic acid
D
(6).—Compound 4 (109 mg, 0.31 mmol) was
dissolved in 2:1 oxolane–H2O (4.5 mL).
Aqueous NaOH (15%, 50 mL) was added,
and the reaction mixture was left for 2 days
at rt. Strongly acidic ion-exchange resin
(Amberlite IR 120, H+-form) was added
with stirring and the pH was brought to 3.8.
The mixture was filtered, evaporated in
vacuo, and the crude product was freeze-
dried to afford acid 6 (100 mg, 0.30 mmol,
96%) as a syrup: [a]2D0+67.3° (c 1.0, MeOH);
1H NMR (300 MHz, MeOD): l 4.63 (d, 1
H, J1,2 1.3 Hz, H-1), 3.72 (dd, 1 H, J5,6a 2.4
Hz, J6a,6b 11.8 Hz, H-6a), 3.73–3.55 (m, 4
H, H-2, 3, 6b, CH2O-spacer), 3.51 (dd, 1 H,
J3,4 ꢀJ4,5ꢀ9.2 Hz, H-4), 3.42 (ddd, 1 H,
J5,6b 5.5 Hz, H-5), 3.31 (m, 1 H, CH2O-
spacer), 2.18 (t, 2 H, J 7.4 Hz, CH2COO),
1.55–1.45 (m, 4 H, CH2), 1.35–1.17 (m, 8
H, CH2); FABMS: m/z Calcd for C15H29O8
(M+H)+: 337.1862. Found: 337.1855.
1
22.8° (c 1.0, CHCl3); H NMR (300 MHz,
CDCl3; the assignments of the Man% and
Man%% residue resonances could be inter-
changed): l 8.17–7.80 (m, 16 H, Bz), 7.60–
7.15 (m, 24 H, Bz), 6.17 (dd, 1 H, J3%,4% 10.3
Hz, J4%,5% 9.7 Hz, H-4%), 6.15 (dd, 1 H, J3%%,4%%
10.1 Hz, J4%%,5%% 9.8 Hz, H-4%%), 6.06 (dd, 1 H,
J2%,3% 2.8 Hz, H-3%), 6.00 (dd, 1 H, J2%%,3%% 3.2
Hz, H-3%%), 5.91 (br s, 1 H, H-2%), 5.81 (br s,
1 H, H-2%%), 5.49 (br s, 1 H, H-1%), 5.36 (br
s, 1 H, H-1%%), 4.96 (m, 1 H, H-5%%), 4.78–
4.50 (m, 6 H, H-1, 5%, 6a%, 6b%, 6a%%, 6b%%),
4.30–4.18 (m, 3 H, H-2, 4, 6a), 4.09–3.99
(m, 2 H, H-3, 6b), 3.90 (m, 1 H, H-5), 3.73
(m, 1 H, CH2O-spacer), 3.62 (s, 3 H,
CH3O), 3.44 (br s, 1 H, OH), 3.38 (m, 1 H,
CH2O-spacer), 2.96 (br s, 1 H, OH), 2.23 (t,
2 H, J 7.6 Hz, CH2COO), 1.60–1.47 (m, 4
H, CH2), 1.40–1.18 (m, 8 H, CH2); 13C
NMR (75.5 MHz, CDCl3, selective data,
coupling constants from HMBC): l 99.973
(d, JC-1,H-1 168 Hz, C-1), 99.823 (d, JC-1%,H-1%
172 Hz, C-1%), 97.557 (d, JC-1%%,H-1%% 172 Hz,
C-1%%); FABMS: m/z Calcd for C84H82O26 Cs
(M+Cs)+: 1639.4149. Found: 1639.4182.
N - [9 - (h -
D
- Mannopyranosyloxy)nonanoyl-
oxy]succinimide (7).—Carboxylic acid 6 (30
mg, 89 mmol), N,N%-dicyclohexylcarbodiimide
(26 mg, 126 mmol) and N-hydroxysuccin-
imide (12 mg, 104 mmol), were dissolved in
2:1 oxolane–CHCl3 (3 mL) and stirred
overnight at rt. Chromatography on silica
gel with 10:1 CHCl3–MeOH afforded com-
pound 7 (30 mg, 69 mmol, 78%) as a syrup:
1
[h]2D0+43.6° (c 1.0, oxolane); H NMR (300
MHz, 10:1 CDCl3–MeOD): l 4.80 (br s, 1
H, H-1), 3.92–3.72 (m, 5 H, H-2, 3, 4, 6a,
6b), 3.67 (m, 1 H, CH2O-spacer), 3.57 (m, 1
H, H-5), 3.41 (m, 1 H, CH2O-spacer), 2.85
(s, 4 H, CH2CON), 2.62 (t, 2 H, J 7.4 Hz,

154
B. Becker et al. / Carbohydrate Research 315 (1999) 148–158
CH3O), 3.63 (m, 1 H, CH2O-spacer), 2.46
8-(Methoxycarbonyl)octyl 2,4-di-O-acetyl-
(2,3,4,6-tetra-O-benzoyl-h- -mannopyranosyl)-
(1“3)-[(2,3,4,6-tetra-O-benzoyl-h- -manno-
pyranosyl)-(1“6)]-h- -mannopyranoside (9).
(t, 2 H, J 7.5 Hz, CH2COO), 1.75–1.64 (m, 4
H, CH2CH2O, CH2CH2COO), 1.50–1.36 (m,
8 H, CH2); 13C NMR (125 MHz, D2O,
CH3O=52.9 ppm, selective data, coupling
constants from HMBC): l 103.175 (d, JC-1%,H-1%
172 Hz, C-1%), 100.681 (d, JC-1,H-1 172 Hz,
C-1), 100.244 (d, JC-1%%,H-1%% 171 Hz, C-1%%),
79.443 (C-3), 74.150 (C-2%), 73.513 (C-2%%),
71.928 (C-2); FABMS: m/z Calcd for
C28H50O18Na (M+Na)+: 697.2895. Found:
697.2895.
D
D
D
—Compound 8 (50 mg, 0.03 mmol) was dis-
solved in 3:1 pyridine–Ac2O (4 mL) and left
for 1 day at rt in the dark. The solution was
diluted with CH2Cl2 (20 mL), washed with aq
NaHCO3, M HCl, phosphate buffer (pH 7),
and water, dried (MgSO4), and evaporated in
vacuo to afford diacetate 9 (50 mg, 0.03
mmol, 95%) as a syrup: [h]2D0 −19.3° (c 1.0,
CHCl3); 1H NMR (300 MHz, CDCl3): l
8.15–7.80 (m, 16 H, Bz), 7.63–7.12 (m, 24 H,
Bz), 6.19 (dd, 1 H, J3%%,4%%ꢀJ4%%,5%% 10.1 Hz, H-
4%%), 6.14 (dd, 1 H, J3%,4%ꢀJ4%,5% 10.0 Hz, H-4%),
5.94 (dd, 1 H, J2%%,3%% 3.2 Hz, H-3%%), 5.82 (dd, 1
H, J2%,3% 3.2 Hz, H-3%), 5.76 (dd, 1 H, J1%%,2%% 1.7
Hz, H-2%%), 5.53 (dd, 1 H, J1%,2% 1.7 Hz, H-2%),
5.47–5.41 (m, 2 H, H-2, 4), 5.38 (d, 1 H,
H-1%), 5.16 (d, 1 H, H-1%%), 4.87 (d, 1 H, J1,2 1.7
Hz, H-1), 4.76–4.46 (m, 6 H, H-5%, 5%%, 6a%, 6b%,
6a%%, 6b%%), 4.39 (dd, 1 H, J2,3 3.3 Hz, J3,4 9.7
Hz, H-3), 4.07–3.97 (m, 2 H, H-5, 6a), 3.83
(m, 1 H, CH2O-spacer), 3.72 (m, 1 H, H-6b),
3.61 (s, 3 H, CH3O), 3.51 (m, 1 H, CH2O-
spacer), 2.34 (s, 3 H, CH3CO), 2.28 (s,
3 H, CH3CO), 2.22 (t, 2 H, J 7.7 Hz,
CH2COO), 1.65–1.47 (m, 4 H, CH2), 1.43–
1.18 (m, 8 H, CH2); FABMS: m/z Calcd for
C88H86O28Cs (M+Cs)+: 1723.4360. Found:
1723.4409.
8-[N-(2-Aminoethyl)carboxamido]octyl (h-
mannopyranosyl) - (1“3) - [(h - -mannopyran-
osyl)-(1“6)]-h- -mannopyranoside (11).—
D
-
D
D
Compound 10 (100 mg, 0.148 mmol) was dis-
solved in 1,2-diaminoethane (5 mL) and
heated to 60 °C for 36 h [38]. The solution was
then evaporated in vacuo, diluted with phos-
phate buffer (pH 6), and desalted by gel-filtra-
tion chromatography on a Sephadex G-10
column with water as eluant. The eluate was
passed through a C-18 SepPak cartridge, and
absorbed amide 11 was washed with water,
eluted with a 3:1“1:1 gradient of H2O–
MeCN and the solvent was evaporated to
afford amide 11 (80 mg, 0.113 mmol, 77%) as
1
a syrup: [h]2D0+73.2° (c 1.0, H2O); H NMR
(500 MHz, D2O, HDO=4.8 ppm): l 5.16
(d, 1 H, J1%,2% 1.7 Hz, H-1%), 4.95 (d, 1 H,
J1%%,2%% 1.7 Hz, H-1%%), 4.88 (d, 1 H, J1,2 1.5 Hz,
H-1), 4.14 (m, 1 H, H-2), 4.12 (dd, 1 H,
J2%,3% 3.5 Hz, H-2%), 4.06–4.02 (m, 1 H, H-6a),
4.03 (dd, 1 H, J2%%,3%% 3.2 Hz, H-2%%), 3.97–3.69
(m, 15 H, H-3, 3%, 3%%, 4, 4%, 4%%, 5, 5%, 5%%, 6b,
6a%, 6b%, 6a%%, 6b%%, CH2O-spacer), 3.61 (m, 1 H,
CH2O-spacer), 3.40 (t, 2 H, J 6.0 Hz,
CH2NH), 2.94 (t, 2 H, J 6.0 Hz, CH2NH2),
2.32 (t, 2 H, J 7.6 Hz, CH2CON), 1.65 (m, 4
H, CH2CH2O, CH2CH2CON), 1.46–1.34 (m,
8-(Methoxycarbonyl)octyl (h-
osyl)-(1“3)-[(h- -mannopyranosyl)-(1“6)]-
h- -mannopyranoside (10).—Compound
D
-mannopyran-
D
D
8
(2.11 g, 1.40 mmol) was dissolved in MeOH
(50 mL) and stirred with KCO3 (500 mg)
overnight at rt. The mixture was neutralized
with strongly acidic ion-exchange resin (Am-
berlite IR 120, H+-form), filtered, and evapo-
rated in vacuo. The residue was dissolved in
water and freeze-dried. Freeze-drying was re-
peated once to afford mannotrioside 10 (936
mg, 1.39 mmol, 99%) as an amorphous solid:
8
H, CH2); FABMS: m/z Calcd for
C29H55N2O17 (M+H)+: 703.3501. Found:
703.3510.
{9 - O - h -
D
- Mannopyranosyl - (1“3) - [h-
D
-
1
[h]2D0+93.9° (c 0.94, MeOH); H NMR (500
mannopyranosyl - (1“6)] - h -
D
- mannopyran-
MHz, D2O, HDO=4.8 ppm): l 5.18 (br s, 1
H, H-1%), 4.97 (br s, 1 H, H-1%%), 4.90 (br s, 1
H, H-1), 4.16 (br s, 1 H, H-2), 4.14 (m, 1 H,
H-2%), 4.08–4.04 (m, 2 H, H-2%%, 6a), 4.01–3.71
(m, 15 H, H-3, 3%, 3%%, 4, 4%, 4%%, 5, 5%, 5%%, 6b,
6a%, 6b%, 6a%%, 6b%%, CH2O-spacer), 3.76 (s, 3 H,
osyloxy}nonanoic acid (12).—Compound 10
(216 mg, 0.32 mmol) was dissolved in 2:1
oxolane–H2O (6 mL) and aq NaOH (0.2 mL,
15%) was added. After 3 days at rt, strongly
acidic ion-exchange resin (Amberlite IR 120,
H+-form) was added and the pH was brought

B. Becker et al. / Carbohydrate Research 315 (1999) 148–158
155
7.20–7.18 (m, 3 H, H-7fl, 1%fl, 8%fl), 6.70–6.66
(m, 4 H, H-2%f1,4%f1,5%f1,7%f1), 4.76 (d, 1 H, J1,2
1.5 Hz, H-1), 3.83 (dd, 1 H, J2,3 3.0 Hz, H-2),
3.85–3.76 (m, 4 H, H-6a, 6b, CH2NHCS),
3.75 (dd, 1 H, J3,4 9.5 Hz, H-3), 3.71 (dd, 1 H,
J4,5 9.5 Hz, H-4), 3.67 (m, 1 H, CH2O-spacer),
3.52 (ddd, 1 H, J5,6a ꢀJ5,6b ꢀ3.0 Hz, H-5),
3.46 (t, 2 H, J 6.0 Hz, CH2NH), 3.39 (m, 1 H,
CH2O-spacer), 2.20 (t, 2 H, J 7.5 Hz,
CH2CON), 1.61–1.52 (m, 4 H, CH2), 1.39–
1.24 (m, 8 H, CH2); FABMS: m/z Calcd for
C38H46N3O12S (M+H)+: 768.2802. Found:
768.2825.
to 3.8. The mixture was filtered, the filtrate
was evaporated in vacuo, and the crude
product was freeze-dried from water to afford
carboxylic acid 12 (212 mg, 0.32 mmol, 100%)
as an amorphous solid: [h]2D0+81.4° (c 1.0,
1
MeOH); H NMR (500 MHz, D2O, HDO=
4.8 ppm): l 5.18 (d, 1 H, J1%,2% 1.7 Hz, H-1%),
4.97 (d, 1 H, J1%%,2%% 1.7 Hz, H-1%%), 4.89 (d, 1 H,
J1,2 1.5 Hz, H-1), 4.15 (m, 1 H, H-2), 4.14 (dd,
1 H, J2%,3% 3.2 Hz, H-2%), 4.08–4.04 (m, 2 H,
H-2%%, 6a), 3.99–3.71 (m, 15 H, H-3, 3%, 3%%, 4,
4%, 4%%, 5, 5%, 5%%, 6b, 6a%, 6b%, 6a%, 6b%%, CH2O-
spacer), 3.63 (m, 1 H, CH2O-spacer), 2.46 (t, 2
H, J 7.6 Hz, CH2COO), 1.75–1.64 (m, 4 H,
CH2CH2O, CH2CH2COO), 1.49–1.38 (m, 8
H, CH2); FABMS: m/z Calcd for
C27H48O18Na (M+Na)+: 683.2738. Found:
683.2745.
8-{Carboxy-[2 -(2%,4%,5%,7%-tetrabromofluor-
esceinylthioureido)ethyl])amido}octyl h- -man-
D
nopyranoside (15).—Compound 5 (10 mg, 26
mmol) was dissolved in aq NaHCO3 (2 mL,
0.2M, pH 9), and eosin isothiocyanate (20 mg,
28 mmol) was added. The mixture was stirred
overnight at rt then evaporated in vacuo. Re-
versed-phase chromatography (C-18 silica)
with 9:1“4:1 H2O–MeCN, followed by chro-
matography on silica gel with 5:1 MeCN–
H2O afforded compound 15 (19 mg, 17 mmol,
N-{9-[(h-
D
-Mannopyranosyl)-(1“3)-[(h-
D
-
mannopyranosyl)-(1“6)]-h-
D
-mannopyranos-
yloxy)]nonanoyloxy}succinimide (13).—Com-
pound 12 (49 mg, 74 mmol), N,N%-dicyclo-
hexylcarbodiimide (22 mg, 107 mmol), and
N-hydroxysuccinimide (10 mg, 87 mmol) were
dissolved in 2:1:2 oxolane–CHCl3–MeOH (6
mL) and stirred overnight at rt. The solvents
were evaporated in vacuo and gel filtration of
the residue on lipophilic Sephadex LH-20 with
1:1 CHCl3–MeOH afforded compound 13 (42
mg, 55 mmol, 75%) as a syrup: [h]2D0 +64.5° (c
64%) as a syrup, [h]2D0 −16.5° (c 1.0, H2O); H
1
NMR (300 MHz, D2O, HDO=4.80 ppm): l
7.83 (br s, 1 H, H-4f1), 7.68 (m, 1 H, H-6f1),
7.55 (brs, 2 H, H-1%f1, 8%f1), 7.12 (m, 1 H,
H-7fl), 4.76 (br s, 1 H, H-1), 3.90–3.75 (m, 6
H, H-2, 3, 6a, 6b, CH2NHCS), 3.69 (dd, 1 H,
J3,4ꢀJ4,5 ꢀ9.5 Hz, H-4), 3.60–3.45 (m, 4 H,
H-5, CH2NH, CH2O-spacer), 3.25 (m, 1 H,
CH2O-spacer), 2.13 (m, 2 H, CH2CON), 1.37
(m, 2 H, CH2CH2CON), 1.27 (m, 2H,
1
0.8, 3:1 CHCl3 –MeOH); H NMR (500 MHz,
3:1 CDCl3–MeOD): l 5.09 (br s, 1 H, H-1%),
4.86 (br s, 1 H, H-1%%), 4.73 (br s, 1 H, H-1),
4.08–3.34 (m, 20 H, H-2, 2%, 2%%, 3, 3%, 3%%, 4, 4%,
4%%, 5, 5%, 5%%, 6a, 6b, 6a%, 6b%, 6a%%, 6b%%, CH2O-
spacer), 2.87 (s, 4 H, CH2CON), 2.63 (t, 2 H,
J 7.5 Hz, CH2COO), 1.79–1.72 (m, 2 H,
CH2CH2O), 1.05–0.85 (m, 8 H, CH2);
79
FABMS: m/z Calcd for C38H Br281Br2N3
40
Na2O12S (M−H+2Na)+: 1127.8821. Found:
1127.8842.
CH2CH2COO),
CH2CH2O), 1.44–1.30 (m, 8 H, CH2).
8-{Carboxy-[2-( fluoresceinylthioureido)eth-
yl]amido}octyl h- -mannopyranoside (14).—
1.63–1.54
(m,
2
H,
8-{Carboxy-[2 -( fluoresceinylthioureido)eth-
yl]amido}octyl (h-
D
-mannopyranosyl)-(1“3)-
-mannopy-
D
[(h- -mannopyranosyl)-(1“6)]-h-
D
D
Amide 5 (14 mg, 37 mmol), fluorescein isothio-
cyanate (25 mg, 64 mmol), and KCO3 (10 mg,
72 mmol) were dissolved in 1:1 acetone–H2O
(2 mL). The mixture was stirred overnight at
rt, then evaporated in vacuo. Gel filtration on
Sephadex LH-20 with 5:4:1 CHCl3–MeOH–
H2O afforded compound 14 (25 mg, 33 mmol,
ranoside (16).—Compound 11 (60 mg, 85
mmol), fluorescein isothiocyanate (38 mg, 98
mmol), and KCO3 (17 mg, 123 mmol) were
dissolved in 1:1 acetone–H2O (2 mL) and
stirred overnight at rt. The solution was
evaporated in vacuo. Gel filtration on Sep-
hadex LH-20 with 4:6:1 CHCl3 –MeOH–
H2O, followed by reversed-phase chromatog-
raphy (C-18 silica) with 15:1“5:1 H2O–
MeCN, afforded compound 16 (65 mg,
1
88%) as a syrup, [h]2D0−12.5° (c 1.0, H2O); H
NMR (500 MHz, 1:1 CDCl3–MeOD): l 7.95
(br s, 1 H, H-4f1), 7.81 (br d, 1 H, H-6f1),

156
B. Becker et al. / Carbohydrate Research 315 (1999) 148–158
79
K)+; C50H Br281Br2KN3O22S (M+K)+:
60 mmol, 70%) as a syrup, [h]2D0+22.0° (c
1.0, H2O); 1H NMR (500 MHz, D2O,
HDO=4.80 ppm): l 7.79 (br s, 1 H, H-4fl),
7.60 (br d, 1 H, H-6fl), 7.28–7.26 (m, 2 H,
H-1%fl, 8%fl), 7.21 (d, 1 H, H-7fl), 6.73–6.69
(m, 4 H, H-2%fl, 4%fl, 5%fl, 7%fl, 5.13 (d, 1 H,
J1%,2% 1.5 Hz, H-1%), 4.91 (d, 1 H, J1%%,2%% 1.5
Hz, H-1%%), 4.77 (d, 1 H, J1,2 1.0 Hz, H-1),
4.12 (dd, 1 H, J2%,3% 3.0 Hz, H-2%), 4.07 (m, 1
H, H-2), 4.01 (dd, 1 H, J2%%,3%% 3.2 Hz, H-2%%),
4.03–4.00 (m, 1 H, H-6a), 3.97–3.66 (m, 16
H, H-3, 3%, 3%%, 4, 4%, 4%%, 5, 5%, 5%%, 6b, 6a%,
6b%, 6a%%, 6b%%, CH2NHCS), 3.58–3.52 (m, 3
H, CH2NCO, CH2O-spacer), 3.37 (m, 1 H,
CH2O-spacer), 2.24 (t, 2 H, J 7.0 Hz,
CH2CON), 1.53 (m, 2 H, CH2CH2CON),
1.40 (m, 2 H, CH2CH2O), 1.21 (m, 2 H,
CH2CH2CH2CON), 0.97 (m, 6 H, CH2);
FABMS: m/z Calcd for C50H66N3O22S (M+
H)+; C50H65N3NaO22S (M+Na)+: 1092. 3855;
1114.3678. Found: 1092. 3867; 1114.3717.
8 - {Carboxy - [2 - (2%,4%,5%,7% - tetrabromofluo-
61
1443.9821; 1445.9807. Found: 1443.9815;
1445.9839.
8-[Carboxy-2-(1,2-dihexadecanoyl-sn-glyc-
ero-3-sodio-phospho)ethanolamido]octyl h-
mannopyranoside (18).—1,2-Dihexadecanoyl-
sn-glycero-3-phosphoethanolamine (DPPE)
D
-
(40 mg, 58 mmol) was suspended in 8:1 ox-
olane–H2O (3 mL). NaHCO3 (25 mg, 0.3
mmol) was added, and the mixture was
warmed to ꢀ30 °C to obtain a clear solu-
tion. Compound 7 (25 mg, 58 mmol), dis-
solved in oxolane (0.83 mL), was added, and
stirring was continued for 1 h at rt, with
occasional warming as precipitation oc-
curred. After filtration, the oxolane was
evaporated in vacuo, and the residue was
freeze-dried. Chromatography on silica gel
with 30:15:1 CHCl3–MeOH–H2O, followed
by chromatography on lipophilic Sephadex
LH-20 with 2:1“6:1 CHCl3–MeOH, af-
forded compound 18 (46 mg, 45 mmol, 77%)
as a syrup: [h]2D0+17.8° (c 1.0, CHCl3,
MeOH 3:1); 1H NMR (300 MHz, 3:1
resceinylthioureido)ethyl]amido}octyl(h-
nopyranosyl)-(1“3)-[(h- -mannopyranosyl)-
(1“6)]-h- -mannopyranoside (17).—Com-
D-man-
D
D
CDCl3–MeOD):
l
5.21 (m,
1
H,
pound 11 (20 mg, 28 mmol) was dissolved in
aq NaHCO3 (2 mL, 0.2 M, pH 9) and eosin
isothiocyanate (20 mg, 28 mmol) dissolved in
N,N-dimethylformamide (1 mL) was added.
The mixture was stirred overnight at rt, then
evaporated in vacuo. Gel filtration on Sep-
hadex LH-20 with 5:4:1 CHCl3–MeOH–
CHOCOR), 4.74 (br s, 1 H, H-1), 4.38 (dd,
1 H, J 12.0 Hz, 3.1 Hz, CH2OCOR), 4.14
(dd, 1 H, J 12.0 Hz, 6.7 Hz, CH2OCOR),
3.96–3.82 (m, 6 H, 2×CH2OP, H-6a, 2),
3.77–3.60 (m, 4 H, H-4, 3, 6b, CH2O-
spacer), 3.46 (m, 1 H, H-5), 3.41 (t, 2H, J
5.0 Hz, CH2N), 3.39 (m, 1 H, CH2O-spacer),
2.35–2.22 (m, 4 H, 2×CH2COO), 2.17 (t, 2
H, J 7.7 Hz, CH2CON), 1.63–1.50 (m, 8 H,
H2O,
followed
by
reversed-phase
chromatography (C-18 silica) with 9:1“2:1
H2O–MeCN, afforded 17 (29 mg, 20 mmol,
70%) as a syrup: [h]2D0−12.6° (c 1.0, H2O);
1H NMR (500 MHz, D2O, HDO=4.80
ppm): l 7.84 (br s, 1 H, H-4fl), 7.71 (br s, 1
H, H-6fl), 7.60 (s, 2 H, H-1%fl, 8%fl), 7.18 (br s,
1 H, H-7fl), 5.13 (br s, 1H, H-1%), 4.93 (br s,
1 H, H-1%%), 4.76 (br s, 1 H, H-1), 4.12 (m, 1
H, H-2%), 4.07 (m, 1 H, H-2), 4.03 (m, 1 H,
H-2%%), 4.17–3.61 (m, 17 H, H-3, 3%, 3%%, 4, 4%,
4%%, 5, 5%, 5%%, 6a, 6b, 6a%, 6b%, 6a%%, 6b%%,
CH2CH2CON,
2×CH2CH2COO,
CH2CH2O), 1.33–1.19 (m, 56 H, CH2), 0.85
(t, 6 H, J 6.8 Hz, 2×CH3); 13C NMR (75.5
MHz, CDCl3, MeOD, 3:1): l 174.495,
173.581, 173.207 (3×CꢀO), 100.504 (C-1),
72.956 (C-5), 71.426 (C-3), 71.050 (C-2),
70.759 (CHOCO), 67.809 (CH2O-spacer),
66.541 (C-4), 64.679 (CH2CH2OP), 63.914
(CH2OP-glyc), 62.886 (CH2OCO), 60.714 (C-
6), 40.443 (CH2N), 36.498 (CH2CON),
34.522, 34.366 (2×CH2COO), 32.185,
30.448, 29.945, 29.793, 29.605, 29.409,
29.207, 28.905, 28.667, 25.954, 25.720,
25.170, 22.917 (32×CH2), 14.185 (2×CH3);
FABMS: m/z Calcd for C52H99NNa2O15P
(M+Na)+ 1054.6548. Found: 1054.6574.
CH2NHCS),
3.61–3.48
(m,
3
H,
CH2NHCO, CH2O-spacer), 3.31 (m, 1 H,
CH2O-spacer), 2.19 (m, 2 H, CH2CON), 1.43
(m, 2 H, CH2CH2CON), 1.32 (m, 2 H,
CH2CH2O), 1.07 (m,
2
H, CH2CH2-
CH2CON), 0.96 (m, 6 H, CH2); FABMS:
79
m/z Calcd for C50H Br381Br KN3O22S (M+
61

B. Becker et al. / Carbohydrate Research 315 (1999) 148–158
157
and chromatography of the crude product on
silica gel (15:10:1 CHCl3–MeOH–H2O) gave
compound 19 (0.87 g, 0.64 mmol, 44%), which
was identical to the product prepared by
method A.
8-[Carboxy-2-(1,2-dihexadecanoyl-sn-glyc-
ero-3-sodio-phospho)ethanolamido]octyl (h-
mannopyranosyl) - (1“3) - [(h - - mannopyran-
osyl)-(1“6)]-h- -mannopyranoside (19)
D
-
D
D
Method A. By use of isolated N-oxysuccin-
imide 13. DPPE (38 mg, 55 mmol) was dis-
solved in 2:1 CHCl3–MeOH (5 mL), and
NaHCO3 (25 mg, 0.3 mmol) dissolved in wa-
ter (0.2 mL) was added, followed by com-
pound 13 (35 mg, 46 mmol), dissolved in 2:1
CHCl3–MeOH (1 mL), and the mixture was
stirred for 1 h at rt. Chromatography on
Sephadex LH-20 with 3:1 CHCl3–MeOH, fol-
lowed by chromatography on silica gel with
20:16:1 CHCl3–MeOH–H2O afforded pro-
duct 19 (43 mg, 32 mmol, 69%) as a syrup:
Acknowledgements
The authors thank Dr Herbert Wong for
recording NMR spectra and for helpful dis-
cussions, Professor Robin Ferrier for assis-
tance with preparing the manuscript, and the
New Zealand Foundation for Research Sci-
ence and Technology for financial support
under contracts C08301, C08614 and C08811.
1
[h]2D0+29.0° (c 0.7, 3:1 CHCl3 –MeOH); H
NMR (500 MHz, 3:1 CDCl3 –MeOD): l
5.28–5.21 (m, 1 H, CHOCOR), 5.15 (br s, 1
H, H-1%), 4.87 (br s, 1 H, H-1%%), 4.74 (br s, 1
H, H-1), 4.46–4.37 (m, 1 H, CH2OCOR),
4.22–4.30 (m, 1 H, CH2OCOR), 4.20–3.34
(m, 26 H, H-2, 2%, 2%%, 3, 3%, 3%%, 4, 4%, 4%%, 5, 5%,
5%%, 6a, 6b, 6a%, 6b%, 6a%%, 6b%%, 2×CH2OP,
CH2O-spacer, CH2NH), 2.40–2.27 (m, 4 H,
CH2COO), 2.22 (m, 2 H, CH2CON), 1.70–
1.50 (m, 8 H, CH2CH2CON, 2×CH2-
CH2COO, CH2CH2O), 1.45–1.20 (m, 56 H,
CH2), 0.88 (t, 6 H, 2×CH3). FABMS: m/z
Calcd for C64H120NNaO25P (M+H)+;
C64H119NNa2O25P (M+Na)+: 1356.7785;
1378.7604. Found: 1356.7756; 1378.7533.
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