Synthesis of the tetrasaccharide cap domain of the antigenic lipophosphoglycan of Leishmania donovani parasite

Article abstract of DOI:10.1016/S0040-4020(00)00609-8

In this paper we report a new synthesis of the immunologically important tetrasaccharide terminal cap domain [Galp(1-4)-β-[Manp-(1-2)-α-Manp-(1-2)-α]-Manp] of lipophosphoglycan (LPG); the major cell surface GPI molecule and key virulence factor of the protozoan parasite Leishmania donovani. The synthetic approach provided a short convergent route for the LPG cap motif from lactose and mannose starting materials. The synthesis was then applied for the preparation of a radiolabelled cap epitope for macrophage receptor binding and immunological studies. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00609-8

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 6577±6584
Synthesis of the Tetrasaccharide Cap Domain of the Antigenic
Lipophosphoglycan of Leishmania donovani Parasite
*
Mani Upreti, Dipali Ruhela and Ram A. Vishwakarma
Bio-organic Chemistry Lab, National Institute of Immunology, Aruna Asaf Ali Marg, J.N.U Complex, New Delhi 110 067, India
Received 4 May 2000; revised 12 June 2000; accepted 29 June 2000
AbstractÐIn this paper we report a new synthesis of the immunologically important tetrasaccharide terminal cap domain [Galp(1-4)-b-
[Manp-(1-2)-a-Manp-(1-2)-a]-Manp] of lipophosphoglycan (LPG); the major cell surface GPI molecule and key virulence factor of the
protozoan parasite Leishmania donovani. The synthetic approach provided a short convergent route for the LPG cap motif from lactose and
mannose starting materials. The synthesis was then applied for the preparation of a radiolabelled cap epitope for macrophage receptor
binding and immunological studies. q 2000 Elsevier Science Ltd. All rights reserved.
Introduction
anchor domain. The unique features of the structure include
phosphoglycan repeats and a cryptic tetrasaccharide cap
with 1,4-b linkage between galactose and mannose resi-
dues, unique among eukaryotic carbohydrates. Structure±
activity relationships have been proposed1,3 for each of the
domains; the neutral oligosaccharide cap contains a signal
for termination of phosphoglycan assembly1b and is thought
to attach the parasite to the digestive tract of the sand¯y and
human macrophage and may also contain an epitope for
recognition by macrophage receptors,6 the phosphoglycan
repeats form a helical structure5a providing resistance1d to
hydrolases and antibodies, and the GPI core functions as the
anchor7 to the plasma membrane.
The protozoan parasite Leishmania donovani causing fatal
visceral leishmaniasis (kala azar) has a remarkable ability to
survive and proliferate in extreme environments during its
digenetic life cycle in sand¯y vector and human host. Upon
deposition of the promastigote form of the parasite into
human tissue after a sand¯y bite, rapid attachment and
entry into the macrophage and transformation to the
amastigote form are critical for the parasite. The major
parasite cell surface molecule lipophosphoglycan (LPG)
expressed1 abundantly on the promastigote has been impli-
cated in the binding of the parasite in epithelial cells of
sand¯y midgut and receptor mediated phagocytosis by
macrophage via direct interaction with carbohydrate bind-
ing sites. There is substantial evidence1 that LPG is an anti-
genic and multifunctional virulence factor essential for
infectivity and survival of the parasite by inhibition2 of
protein kinase C mediated signal transduction and related
gene transcription3 of the host. LPG de®cient mutants of
Leishmania cannot survive in the sand¯y vector or infect
mammalian macrophages but both the functions can be
restored on insertion of exogenous LPG into the plasma
membrane of de®cient strains. The intriguing structure4,5
of LPG (Fig. 1) consists of four distinct domains: (i) a
neutral oligosaccharide cap at the terminal reducing end;
(ii) variable [6Galp-b1,4-Manp-a1-phosphate]n phospho-
glycan repeat units; (iii) a conserved phosphosaccharide
core with an internal galactofuranose residue and (iv) a
1-O-alkyl(24/26:0)-2-lysoglycosylphosphatidylinositol (GPI)
The structure of LPG and its role in host±parasite inter-
action has led to signi®cant interest and its biosynthetic
pathway8 is viewed as a new target for chemotherapeutic
and vaccine design. However, the synthetic efforts towards
LPG and its structural domains have been minimal so far
and reported work includes synthesis9 of phosphoglycan
fragments and the ®rst synthesis of the tetrasaccharide cap
reported by Fraser±Reid10 from monosaccharide building
blocks using the n-pentenyl glycosidation strategy. In con-
tinuation to our work11±14 on the chemistry and biosynthesis
of cell surface GPI molecules of L. donovani parasite, we
recently reported14 a synthesis of the phosphodisaccharide
repeat of LPG. Keeping in view the immunological impor-
tance of the tetrasaccharide cap of the LPG as a potential
epitope for designing synthetic antigens and to study recep-
tor binding on the macrophage, the access to suf®cient
quantity of structurally de®ned cap was required. Herein
we report a new and ef®cient synthesis of this tetrasacchar-
ide cap of LPG using lactose and mannose as starting mate-
rials. The synthesis has been designed to enable preparation
of a radiolabelled cap. The important features of this
approach include gluco!manno transformation via glycal
Keywords: carbohydrates; glycolipids; lipophosphoglycan; Leishmania
donovani.
* Corresponding author. Tel.: 191-11-6174899; fax: 191-11-6162125;
e-mail: ram@nii.res.in
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00609-8

6578
M. Upreti et al. / Tetrahedron 56 (2000) 6577±6584
Figure 1. Structural domains of lipophosphoglycan (LPG) of Leishmania donovani (nˆ16±32).
and dimethyldioxirane chemistry to convert protected
lactose (Gal-b1!4-Glu) into a suitably protected Gal-
b1!4-Man intermediate with a free C2±OH group
available for stereoselective coupling with a mannobiose
donor. This avoided several protection/deprotection/
glycosidation steps required in the synthesis from mono-
saccharide building blocks, and provided an opportunity
to radiolabel the cap.
strated the unusual catalytic ef®ciency of the Vitamin B12
radical in zinc mediated reductive elimination reactions.
This provided an easy and multigram access to the required
hexa-O-acetyl-lactal (3) with straightforward work up
without any chromatographic puri®cation. The deacetyl-
ation of 3 to lactal (4) followed by benzylation led to
hexa-O-benzyl-lactal (5) in high yield; this replacement of
participatory protecting groups (i.e. acetyl) by the non-
participatory protecting benzyl groups was necessitated17
for the control of the stereochemistry during epoxidation
in the forthcoming step. This stereoselective a-epoxidation
of hexa-O-benzyl-lactal (5) was carried out by freshly
prepared 2,2-dimethyldioxirane18 soltution in acetone at
08C leading exclusively to the 1a,2a-oxirane (6). The
methanolysis of 6 with excess of anhydrous MeOH at
room temperature led to epoxide ring opening to provide
the corresponding b-glucoside (7) in quantitative yield.
Since this selective epoxide ring opening generated a unique
free C-2 hydroxyl group in the b-glucoside (7), we exploited
Result and Discussion
The key intermediate hexa-O-acetyl-lactal (3)15 was
prepared from lactose (2) in three steps involving acetyl-
ation, anomeric bromination and reductive elimination
mediated by zinc and Vitamin-B12 (Scheme 1). It is worth
mentioning here that high yield (more than 90%) could be
obtained in the last step of this sequence by the application
of a recent method16 of Forbes and Franck who demon-
Scheme 1. Reagents and conditions: (a) (i) Ac2O, NaOAc, 1008C; (ii) HBr±AcOH, 48C, 16 h; (iii) Zinc, Vitamin B12, NH4Cl, MeOH, 10 min 87% for three
steps; (b) Na2CO3, MeOH, rt, 2 h, 97%; (c) NaH, BnBr, TBAI, DMF, rt, 3 h 57%; (d) dimethyl dioxirane, CH2Cl2, 08C, 2 h, 90%; (e) MeOH, rt, 4 h, 96%; (f) (i)
(COCl)2, DMSO, CH2Cl2, 2788C; (ii) NaBH4, rt, 4 h, 64% for two steps.

M. Upreti et al. / Tetrahedron 56 (2000) 6577±6584
6579
Scheme 2. Reagents and conditions: (a) Me2NH, CH3CN, 2208C, 1 h, quant.; (b) CCl3CN, DBU, CH2Cl2, 08C, 70%; (c) TMSOTf, CH2Cl2, 4A MS, 45 min,
2308C, 60%; (d) Me2NH, CH3CN, 2208C, 5.5 h, 91%; (e) CCl3CN, DBU, CH2Cl2, 60%.
Danishefsky's approach19 to convert b-glucoside to
b-mannoside. For this the compound 7 was ®rst oxidised
by Swern oxidation to give a 2-ulose intermediate which
was directly reduced with NaBH4 in CH2Cl2±MeOH to
provide key lower half intermediate methyl O-(2,3,4,6-
tetra-O-benzyl-b-d-galactopyranosyl)-(1-4)-3,6-di-O-benzyl-
b-d-mannopyranoside (8) in 65% yield with free C-2
hydroxyl group in the mannosyl moiety ready for coupling
with a suitable mannobiosyl donor. To con®rm complete
gluco!manno transformation during the oxidation±reduc-
tion sequence, a small part of compound 8 was converted to
its C-2 acetate, the 1H NMR spectrum of which showed C2
a-proton as doublet (3 Hz) at 5.5 ppm.
prepared 1,3,4,6-tetra-O-acetyl-b-d-mannopyranose (12)
at 2308C led to the fully protected mannobiose octaacetate
(13); the coupling conditions led to formation of the major
1!2a isomer which could be isolated by silica column in
60% yield. Selective anomeric deacetylation of 13 with
dimethylamine at 2208C led to the corresponding manno-
biose heptaacetate (14) with free C-1a hydroxyl group; the
a-stereochemistry con®rmed by NMR showing a doublet at
d 5.36 (Jˆ1.5 Hz). The required dimannan a-trichloro-
acetimidate donor 15 was prepared from 14 by the Schmidt
method (CCl3CN, DBU, CH2Cl2, 2308C, 75 min) in 60%
yield after column puri®cation.
Having secured access to both the lower half Gal-Man
acceptor (8) and mannobiose trichloroacetimidate donor
(15) in high yielding steps, ®nal glycoside coupling of the
two intermediates was carried out using TMSOTf at 2308C
which led to the fully protected tetrasaccharide cap (methyl
O-(2,3,4,6-tetra-O-acetyl-a-d-manno pyranosyl)-(1-2)-O-
(3,4,6-tri-O-acetyl-a-d-mannopyranosyl)-(1-2)-O-[(2,3,4,6-
tetra-O-benzyl-b-d-galactopyranosyl)-(1-4)]-3,6-di-O-benzyl-
a-d-mannopyranoside, 16) in 63 % yield after repeated
silica column chromatography. This was followed by global
deprotection in two steps of debenzylation (palladium
The upper half mannobiose donor 15 was prepared from
mannose (Scheme 2) by the following steps. For this the
known mannose trichloroacetimidate (11)20 was prepared
from mannose pentaacetate (9) by selective anomeric
deacetylation with dimethylamine at 2208C followed by
reaction with trichloroacetonitrile/DBU using Schmidt
method21 to obtain corresponding trichloroacetimidate
(11) in 70% yield; the a-mannosyl stereochemistry was
established by NMR analysis. The trimethylsilyltri¯ate
(TMSOTf) mediated coupling of this donor with freshly
Scheme 3. Reagents and conditions: (a) TMSOTf, DCM, 45 min, 2308C, 67%; (b) (i) Pd(OH)2, H2, 4 h, (ii) NaOMe, MeOH, quant; (c) (i) Ac2O/AcOH/
H2SO4, rt, (ii) Na2CO3, MeOH, rt, 41% for two steps.

6580
M. Upreti et al. / Tetrahedron 56 (2000) 6577±6584
hydroxide, H2) and deacetylation (NaOMe, MeOH) to
provide the methyl glycoside of the tetrasaccharide cap
(17) in quantitative yield (Scheme 3). Finally the acetolysis
(Ac2O/AcOH/H2SO4) and decetylation (NaOMe, MeOH)
led to the fully deprotected tetrasaccharide Cap domain
(41% yield) of the LPG of L. donovani parasite.
reagents and anhydrous solvents were introduced by
syringes through septa-isolated ¯asks under nitrogen atmo-
sphere. The NMR spectra (1H, 13C, two-dimensional 1H±1H
COSY, DQFCOSY, TOCSY, gradient-HMQC) were
obtained on a Bruker 300 MHz spectrometer (Avance
DRX-300); the spectra recorded in CDCl3 unless stated
otherwise. Chemical shifts are expressed in d (ppm) relative
to residual solvent resonance, and coupling constants J
expressed in Hz. Electrospray ionisation mass spectra
were obtained on a quadrupole mass spectrometer (VG Plat-
form-II) using acetonitrile±water (1:1) mobile phase. High-
resolution FAB mass spectra were performed at the Indian
Institute of Chemical Technology, Hyderabad. Optical rota-
tions (Na D line) were measured on a Perkin±Elmer 241
polarimeter.
For the radiochemical synthesis of the Cap, the protected
disaccharide 7 was oxidised by Swern oxidation to obtain
the corresponding 2-ulose intermediate which was then
reduced with radiolabelled NaB3H4 (speci®c activity
600 mCi/mmol) in diglyme solution to give labelled
[2-3H]-methyl-O-(2,3,4,6-tetra-O-benzyl-b-d-galactopyrano-
syl)-(1-4)-3,6-di-O-benzyl-b-d-mannopyranoside (8). This
labelled reduced material was coupled with protected
mannobiose trichloroacetimidate (15) to provide lebelled
protected tetrasaccharide 16 in 67% yield. The rest of the
deprotection steps were carried out essentially as described
above for unlabelled synthesis. The labelled materials were
visualised on TLC plates by autoradiography and analysed
by scintillation counting. This provided labelled LPG cap of
high speci®c activity (600 mCi/mmol) which is now being
used by us for macrophage receptor binding and epitope
recognition experiments.
Hexa-O-acetyl lactal (3). A solution of Vitamin B12
(310 mg, 0.228 mmol) in anhyd MeOH (80 mL) was thor-
oughly purged with nitrogen for 30 min and zinc powder
(17.5 g, 267.6 mmol) and ammonium chloride (14.2 g,
266.25 mmol) were added to the solution. The reaction
mixture was stirred for another 45 min and heptaacetyl
lactosyl bromide (9.4 g, 13.5 mmol), freshly prepared
from lactose by a known method,15 dissolved in MeOH
(30 mL) was added. Immediately after addition of the bro-
mide, the dark red solution changed to reddish yellow and
then back to dark red in 5 min. The solution was ®ltered
through celite to remove zinc, the celite pad washed with
MeOH and the ®ltrate was concentrated to give a white and
red solid. This was dissolved in water (100 mL) and
extracted with CH2Cl2 (3£60 mL). Organic extracts were
combined, dried over Na2SO4, and concentrated to provide
hexa-O-acetyl lactal (3, 7.2 g, 87%) as amorphous solid, mp
1138 (lit.15 mp 1148); [a]Dˆ2188 (c 1.0, CHCl3) (lit.15
2188, c 1.0, CHCl3).
Conclusion
The biologically important terminating cap domain of LPG
has been synthesised from lactose and mannose in a high
yielding convergent approach. This synthesis was designed
to enable radio isotope labelling of the cap structure for its
application in macrophage receptor binding and immunolo-
gical studies. The current approach provided an opportunity
for tritium labelling at non-exchangeable C-2 position of the
lower mannose residue by using labelled NaB3H4 in the
reduction of 7 to 8 step to obtain a labelled acceptor.
Since the radiolabel come at a fairly late stage of the synth-
esis, the access to deprotected cap required only three more
steps. Considering the obvious dif®culties encountered in
handling labelled intermediates in a multistep synthesis, it
was desirable to incorporate radiolabel at the ®nal steps of
synthesis. Therefore, the present approach is practically
suitable for preparation of the labelled cap of LPG. The
work on the macrophage receptor binding using radiola-
belled cap will be published in due course.
Hexa-O-benzyl lactal (5). A solution of hexa-O-acetyl
lactal (3, 7.26 g, 13.0 mmol) and freshly dried sodium car-
bonate (9.0 g, 85 mmol) in anhyd MeOH (150 mL) was
stirred at room temperature for 90 min. The suspension
was ®ltered to remove excess Na2CO3 and the ®ltrate was
concentrated under reduced pressure to give deacetylated
lactal 4 as an amorphous solid (3.87 g, 98%); mp 191±
1938; Rfˆ0.2 in 7:3 CHCl3±MeOH; [a]Dˆ1278 (c 1.6,
H2O) (lit.15 1278, c 1.6, H2O). The compound 4 (500 mg,
1.62 mmol) dissolved in anhyd DMF (5 mL) was added
dropwise at 08C to a suspension of NaH (1.3 g, 60% disper-
sion in paraf®n) in DMF (5 mL), followed by addition of
benzyl bromide (2 mL, 16.8 mmol) and few crystals of
tetrabutyl ammonium iodide. The reaction mixture was
brought to room temperature and stirred for 3 h. After
completion of the reaction, the mixture was cooled to 08C
and quenched with MeOH (5 mL), diluted with cold water
(50 mL) and extracted with diethyl ether (3£30 mL). The
ethereal layer was dried over Na2SO4 and concentrated to
give a crude product which was ¯ash chromatographed
using 5% ethyl acetate in hexane to provide compound 5
as colourless oil (792 mg, 58%); Rfˆ0.6 in 50% ethyl ace-
tate±hexane; [a]Dˆ22.1 (c 0.73, CHCl3); IR nmax (®lm)
Experimental
General methods
The reagents used for synthesis were from Aldrich and
Fluka, and organic solvents were of analytical purity. The
radiolabelled NaB3H4 (5±10 Ci/mmol) was obtained from
Amersham and was diluted to the speci®c activity of
0.6 mCi/mmol for synthetic experiments. Thin layer chro-
matography was performed on Merck Kieselgel 60 F254
plates, compounds visualised by ammonium molybdate±
ceric sulfate developing reagent. The spots of radioactive
compounds were observed by exposure of TLC plates to X-
ray ®lms and ¯uorography. Flash chromatography was
carried out with silica gel 60 (200±400 mesh). Liquid
1
2910, 2850, 1450, 1375, 1250, 1210, 1150, 1100 cm21; H
NMR (CDCl3, 300 MHz) d 7.39±7.27 (m, 30H, Ph), 6.43
(dd, Jˆ6.2, 1.1 Hz, 1H, H-1), 4.92 (d, Jˆ10.8 Hz, 1H), 4.86
(dd, Jˆ6.3, 3.6 Hz, 1H, H-2), 4.82 (d, Jˆ11.7 Hz, 1H), 4.71

M. Upreti et al. / Tetrahedron 56 (2000) 6577±6584
6581
(d, Jˆ9.6 Hz, 1H), 4.70 (brs, 2H), 4.55 (m, 7H), 4.35 (dd,
Jˆ4.2, 1.0 Hz, 2H), 4.29 (m, 1H), 4.14 (m, 2H), 3.86 (brs,
1H), 3.83±3.74 (m, 2H), 3.65 (m, 1H), 3.54±3.41 (m, 5H);
13C NMR (CDCl3, 75 MHz) d 138.76±137.85 (6 ipso C),
128.3±127.4 (30 ArC's), 102.79, 99.75, 82.26, 79.4, 75.12,
75.85, 75.54, 73.47, 73.43, 73.25, 73.12, 72.92, 72.31,
70.30, 68.57, 67.97; HRMS(FAB): calcd for (M1Na)1
C54H56O9Na 871.382204, found 871.386586.
was stirred for 40 min followed by addition of triethylamine
(5 mL) to give a clear solution. The mixture was brought to
room temperature, diluted with cold water (30 mL) and
extracted with CH2Cl2. The organic layer was dried over
Na2SO4 and concentrated under reduced pressure to yield
the oxidised 2-ulose intermediate (Rfˆ0.53 in 50% ethyl
acetate in hexane). This product was dissolved in 50%
CH2Cl2 in MeOH (4 mL) and NaBH4 (150 mg,
3.96 mmol) was added at 08C. The reaction mixture was
brought to room temperature and after 4 h it was diluted
with CH2Cl2 and washed with cold water. The organic
layer was collected, dried over Na2SO4 and concentrated
to give a crude product which was puri®ed by ¯ash chro-
matography using 20% ethyl acetate in hexane to yield
methyl O-(2,3,4,6-tetra-O-benzyl-b-d-galactopyranosyl)-
(1!4)-3,6-di-O-benzyl-b-d-mannopyranoside 8 as colour-
less gum (149 mg, 64.5%); Rfˆ0.27 in 50% ethyl acetate±
hexane; [a]Dˆ13.89, (c 0.26, CHCl3); IR nmax (®lm) 3500±
3300 (br), 2910, 2850, 1490, 1450, 1370, 1210, 1100,
Hexa-O-benzyl-lactal-1,2a-epoxide (6). A solution of 2,2-
dimethyl dioxirane in acetone was freshly prepared by the
reported method18 from potassium monoperoxy sulphate
(Oxone, DuPont), and 15 mL of this was added dropwise
to a solution of compound 5 (250 mg, 0.3 mmol) in dry
CH2Cl2 (15 mL) at 08C. After 2 h the reaction mixture
was concentrated with a stream of nitrogen gas, and further
dried under vacuum to provide a semisolid hexa-O-benzyl
lactal 1,2a-epoxide 6 (232 mg, 90%); Rfˆ0.16 in 50% ethyl
acetate±hexane; [a]Dˆ121.8 (c 0.24, CHCl3); IR nmax
(®lm) 3022, 2900, 1500, 1450, 1375, 1210, 1100,
1
1075 cm21; H NMR (CDCl3, 300 MHz, COSY, TOCSY)
1
1070 cm21; H NMR (CDCl3, 300 MHz, COSY) d 7.36±
d 7.30±7.21 (m, 30H, Ph), 4.94 (d, Jˆ11.4 Hz, 1H), 4.78 (d,
Jˆ11.4 Hz, 1H), 4.74 (d, Jˆ6.6 Hz, 1H), 4.68 (m, 3H),
4.54±4.41 (m, 4H), 4.46 (d, Jˆ5 Hz, 1H, H-10), 4.35 (d,
Jˆ2.1 Hz, 1H, H-1), 4.30 (d, Jˆ11.7 Hz, 1H), 4.08 (t,
Jˆ8.7 Hz, 1H), 4.03 (bs, 1H), 3.90 (d, Jˆ2.4 Hz, 1H),
3.84±3.80 (dd, Jˆ2.5, 8.1 Hz, 1H), 3.78±3.73 (dd, Jˆ5.4,
10.4 Hz, 1H), 3.58±3.39 (m, 6H), 3.52 (s, 3H, OMe); 13C
NMR (CDCl3, 75 MHz) d 138.85, 138.63, 138.39, 138.33,
137.88, 135.52 (6 ipso C), 128.30±127.29 (30 ArC's),
103.07, 100.65, 82.48, 79.75, 79.15, 75.27, 75.06,
74.49, 73.39, 73.07, 72.94, 72.54, 72.33, 68.52, 68.29,
56.87; ESMS m/z 919.5 (M1Na)1; HRMS(FAB):
calcd for (M1Na)1 C55H60O11Na 919.403333, found
919.400521.
7.23 (m, 30H, Ph), 5.03 (d, Jˆ11.8 Hz, 1H), 4.99 (d,
Jˆ1.96 Hz, 1H, H-1), 4.90 (t, Jˆ12 Hz, 1H), 4.76 (m,
5H), 4.70±4.52 (m, 3H), 4.45±4.32 (m, 4H), 3.90 (m,
3H), 3.89±3.76 (m, 3H), 3.65 (m, 2H), 3.51±3.37 (m,
3H), 3.11 (d, Jˆ1.97 Hz, 1H, H-2); 13C NMR (CDCl3,
75 MHz) d 138.8±138.9 (6 ipso C), 128.3±127.4 (30
ArC's), 102.51, 82.4, 79.7, 76.6, 75.2, 74.6, 73.5, 73.47,
73.41, 73.0, 72.69, 72.61, 69.17, 68.36; HRMS(ESMS):
calcd for (M1Na)1 C54H56O10Na 887.3771, found
887.3761.
Methyl O-(2,3,4,6-tetra-O-benzyl-b-d-galactopyrano-
(7).
syl)-(1!4)-3,6-di-O-benzyl-b-d-glucopyranoside
The a-epoxide (6, 232 mg, 0.268 mmol) was dissolved in
dry MeOH (150 mL) and allowed to stir at room tempera-
ture for 4 h. The solvent was evaporated and the residue
dried under vacuum to yield b-methyl lactoside 7
(231 mg, 96%) as semisolid material; Rfˆ0.37 in 50%
ethyl acetate in hexane; [a]Dˆ112 (c 0.20, CHCl3); IR
nmax (CHCl3) 3500±3300 (br), 2900, 2850, 1490, 1450,
2,3,4,6-Tetra-O-acetyl-a-d-mannopyranosyl-trichloro-
acetimidate (11). The 1,2,3,4,6-penta-O-acetyl-a-d-manno-
pyranose (9, 500 mg, 1.28 mmol) was dissolved in dry
CH3CN saturated with dimethylamine (35 mL) and stirred
at 2208C for 1 h, TLC con®rmed complete disappearance
of the starting material. Extra dimethylamine was removed
under reduced pressure at room temperature and the reaction
mixture was concentrated. Flash column chromatography
(25:75 ethyl acetate±hexane) provided 2,3,4,6-tetra-O-ace-
tyl-a-d-mannopyranose (10, 445 mg) in quantitative yield.
To a solution of compound 10 (335 mg, 0.962 mmol) in dry
CH2Cl2 (3 mL) was added trichloroacetonitrile (Cl3CCN,
1 mL, 10.0 equiv.) and 1,8-diaza bicyclo[5.4.0]undec-7-
ene (DBU, 74.7 mL, 0.05 equiv.) at 08C. After stirring for
75 min, the solvent was evaporated under reduced pressure
and the residue puri®ed by ¯ash chromatography with 20%
ethyl acetate±hexane to give pure 11 as viscous material
(331 mg, 70%); IR nmax (®lm) 3320 (N±H), 1750, 1680
;
1370, 1210, 1100, 1075 cm21 1H NMR (CDCl3,
300 MHz, COSY) d 7.32±7.20 (m, 30H, Ph), 5.07 (d,
Jˆ11.1 Hz, 1H), 4.90 (d, Jˆ11.4 Hz, 1H), 4.78 (dd,
Jˆ11.1, 4.5 Hz, 2H), 4.70 (dd, Jˆ11.7, 1.8 Hz, 4H), 4.54
(d, Jˆ10.5 Hz, 2H), 4.44±4.25 (m, 4H), 4.20 (d, Jˆ9 Hz,
1H), 3.95 (m, 1H), 3.90 (d, Jˆ2.7 Hz, 1H), 3.83±3.70 (m,
3H), 3.54 (s, 3H, OMe), 3.48±3.36 (m, 6H); 13C NMR
(CDCl3, 75 MHz) d 138.92, 138.82, 138.68, 138.39,
138.22, 137.92 (6 ipso C), 128.29±127.35 (30 ArC's),
103.40, 102.69, 82.65, 82.38, 79.85, 76.14, 75.40, 74.62,
74.54, 73.36, 73.32, 73.06, 72.94, 72.52, 68.09, 56.87;
ESMS m/z 919.5 (M1Na)1; HRMS(FAB): calcd for
(M1Na)1 C55H60O11Na 919.4033, found 919.4023.
1
(CvN), 1430, 1375, 1210, 1080, 1050 cm21; H NMR
(CDCl3, 300 MHz, COSY) d 8.77 (s, 1H, NH), 6.26 (d,
Jˆ1.8 Hz, 1H, H-1), 5.45 (dd, Jˆ1.8, 3.0 Hz, 1H, H-2),
5.39±5.37 (dd, Jˆ2, 5 Hz, 1H, H-3), 5.42±5.33 (m, 1H,
H-4), 4.18 (m, 1H, H-5), 4.29±4.12 (m, 2H, H-6), 2.19 (s,
3H), 2.07 (s, 3H), 2.05 (s, 3H), 2.0 (s, 3H); 13C NMR
(CDCl3, 75 MHz) d 170.45, 169.68, 169.60, 169.50,
159.62, 94.39, 71.08, 68.67, 68.13, 67.73, 65.26, 61.91,
20.54; HRMS (ESMS): calcd for (M1H)1 C16H21O10NCl3
491.0153, found 491.0187.
Methyl O-(2,3,4,6-tetra-O-benzyl-b-d-galactopyrano-
syl)-(1!4)-3,6-di-O-benzyl-b-d-mannopyranoside (8).
A solution of oxalyl chloride (95.8 mL, 0.177 mmol) in
anhyd CH2Cl2 (7 mL) was cooled to 2788C, and anhyd
DMSO (154.3 mL, 0.349 mmol) was added dropwise. The
mixture was stirred at 2788C for 10 min and solution of
methyl glycoside 7 (231 mg, 0.258 mmol) in CH2Cl2
(11.5 mL) was added over 10 min. The cloudy solution

6582
M. Upreti et al. / Tetrahedron 56 (2000) 6577±6584
1,3,4,6-Tetra-O-acetyl-2-O-(2,3,4,6-tetra-O-acetyl-a-d-
mannopyranosyl)-b-d-mannopyranose (13). A solution
of mannose trichloroacetimidate donor 11 (985 mg,
2 mmol) and freshly prepared22 1,3,4,6-tetra-O-acetyl-b-d-
mannopyranose 12 (348 mg, 1 mmol) in anhyd CH2Cl2
(20 mL) was stirred with activated molecular sieves (10 g,
showed completion of the reaction. Solvent was evaporated
under reduced pressure and the residue was ¯ash chromato-
graphed (30:70, ethyl acetate±hexane) to give the disac-
charide donor 15 as viscous material (185 mg, 60%);
[a]Dˆ131.2, (c 1.40, CHCl3); IR nmax (thin ®lm) 3320,
1H NMR (CDCl3, 300 MHz, COSY, HMQC) d 8.71(s, 1H,
NH), 6.41 (d, Jˆ1.86 Hz, 1H, H-1), 5.49±5.46 (brd,
Jˆ9.9 Hz, 1H, H-4), 5.43±5.38 (dd, Jˆ3.45, 10.2 Hz, 1H,
H-30), 5.35±5.31 (dd, Jˆ3.15, 10.2 Hz, 1H, H-3), 5.28±5.23
(m, 1H, H-40), 5.28±5.26 (dd, Jˆ1.8, 3.3 Hz, 1H, H-20),
4.98 (d, Jˆ1.5 Hz, 1H, H-10), 4.29±4.27 (dd, Jˆ2.55, 5.1,
1H, H-2), 4.24±4.14 (m, 5H), 4.12±4.10 (ddd, Jˆ3, 3.6,
3.6 Hz, 1H), 2.14±2.00 (7£s, 21H, COMe); 13C NMR
(CDCl3, 75 MHz) d 170.6, 170.5, 170.2, 169.78, 169.59,
169.37, 169.10, 99.1, 95.4, 76.4, 75.4, 74.8, 71.1, 69.7,
69.5, 69.4, 68.2, 66.0, 65.2, 62.1, 61.5, 20.74±20.53;
HRMS (ESMS): calcd for (M1Na)1 C28H36O18NCl3Na
802.0896, found 802.0801.
2910, 2850, 1750, 1680 (CvN), 1380, 1210, 1150 cm21
;
Ê
4 A) under nitrogen for 30 min. The reaction mixture was
cooled to 2308C and a solution of trimethylsilyltri¯ate
(TMSOTf, 220 mL, 1.2 mmol) in dry CH2Cl2 (10 mL) was
added dropwise. The temperature was maintained below
2308C for 15 min when TLC indicated completion of the
reaction. The mixture was quenched with pyridine (5 mL)
and ®ltered through celite, and the ®ltrate was co-concen-
trated with toluene. Flash chromatography of the crude
product with 50% ethyl acetate±hexane afforded pure
mannobiose octaacetate 13 as amorphous solid (0.406 g,
60%); mp 728C Rfˆ0.20 in 50% ethylacetate±hexane;
[a]Dˆ19.0 (c 0.25, CHCl3); IR nmax (CHCl3) 1750, 1430,
1380, 1210, 1140, 1090, 1050 cm21 1H NMR (CDCl3,
;
300 MHz, COSY, HMQC) d 5.78 (d, Jˆ0.9 Hz, 1H, H-1),
5.46±5.50 (dd, Jˆ3.0, 9.9 Hz, 1H), 5.39±5.32 (t, Jˆ9.6 Hz,
2H), 5.31±5.29 (dd, Jˆ3.6, 1.95 Hz, 1H), 5.13±5.09 (dd,
Jˆ9.6, 3.0 Hz, 1H), 5.00 (d, Jˆ1.8 Hz, 1H, H-10), 4.40 (m,
1H), 4.16±4.14 (dd, Jˆ3.0, 1.2 Hz, 1H), 4.34±4.01 (m, 4H),
3.80±3.76 (m, 1H), 2.15±2.01 (8£s, 24H, COMe); 13C
NMR (CDCl3, 75 MHz) d 171, 170.7, 170.4, 169.6, 169.5,
169.3, 169.1, 168.2, 98.2, 90.8, 74.5, 73.1, 72.0, 69.8, 68.7,
68.2, 66.0, 65.6, 62.1, 61.6, 20.77±20.30 (8 C); ESMS m/z
701.0 (M1Na); HRMS(FAB): Calcd. for (M1Na)1
C28H38O19Na 701.190499, found 701.187839.
Methyl O-(2,3,4,6-tetra-O-acetyl-a-d-mannopyranosyl)-
(1!2)-O-(3,4,6-tri-O-acetyl-a-d-mannopyranosyl)-(1!2)-
O-[(2,3,4,6-tetra-O-benzyl-b-d-galactopyranosyl)-(1!4)]-
3,6-di-O-benzyl-a-d-mannopyranoside (16). A solution
of the protected Gal-Man acceptor 8 (18 mg, 0.02 mmol)
and mannobiose trichloroacetamidate donor 15 (31 mg,
0.04 mmol) in dry diethyl ether (5 mL) was stirred with
Ê
freshly activated molecular sieves (4 A, 150 mg) at room
temperature under nitrogen atmosphere for 15 min, and
trimethylsilyl tri¯ate (TMSOTf, 3.66 mL) was added. The
reaction mixture was stirred for 2 h at room temperature
when TLC showed complete disappearance of the starting
materials. The mixture was quenched with NaHCO3, ®ltered
through a celite pad and concentrated under reduced
pressure. The residue was puri®ed by silica column using
ethyl acetate±hexane (38:62) to provide the fully protected
tetrasaccharide cap domain (16, 20 mg, 67%); Rfˆ0.35 in
50% ethyl acetate±hexane; [a]Dˆ112.1 (c 0.06, CHCl3);
IR nmax (®lm) 2910, 2850, 1750, 1460, 1370, 1210, 1110,
3,4,6-Tri-O-acetyl-2-O-(2,3,4,6-tetra-O-acetyl-a-d-manno-
pyranosyl)-a-d-mannopyranose (14). The mannobiose
octaacetate 13 (298 mg, 0.44 mmol) was dissolved in
anhyd acetonitrile saturated with dimethylamine (39 mL)
at 2208C and stirred for 5 h after which TLC con®rmed
disappearance of the starting material. Excess of dimethyl-
amine was removed under reduced pressure at 308C and the
reaction mixture was concentrated. Flash column chroma-
tography of the crude product with 40% ethyl acetate±
hexane resulted in pure heptaacetate 14 as viscous solid
(254 mg, 91%); Rfˆ0.09 in 50% ethylacetate±hexane;
[a]Dˆ119.54 (c 0.22, CHCl3); IR nmax (thin ®lm) 3500±
3300 (br), 2950, 2900, 1750, 1430, 1375, 1220, 1100,
1100, 1050 cm21 1H NMR (CDCl3, 300 MHz, COSY,
;
HMQC) d 7.29±7.14 (m, 30H, ArH), 5.33 (d, Jˆ1.8 Hz,
1H, H-1), 5.38±5.32 (m, 3H), 5.25 (d, Jˆ9.9 Hz, 2H),
5.18 (dd, Jˆ1.8, 1.5 Hz, 1H), 4.91 (d, Jˆ11 Hz, 2H), 4.75
(d, Jˆ2.1 Hz, 1H), 4.66±4.60 (m, 2H), 4.62 (d, Jˆ1.64 Hz,
1H), 4.51 (d, Jˆ12 Hz, 2H), 4.43 (d, Jˆ6.9 Hz, 1H, H-10),
4.39 (d, Jˆ6.1 Hz, 1H), 4.33 (d, Jˆ9.9 Hz, 2H), 4.25 (m,
1H), 4.29±4.17 (m, 4H), 4.14±4.07 (m, 4H), 4.04 (m, 2H),
4.00 (m, 2H), 3.89 (d, Jˆ2.7 Hz, 1H), 3.83±3.80 (m, 1H),
3.74±3.68 (m, 1H), 3.57±3.52 (m, 1H), 3.46 (s, 3H, OMe),
3.44±3.32 (m, 4H), 2.09, 2.03, 2.01, 2.00, 1.99, 1.97, 1.96
(7£s, 21H, COMe); 13C NMR (CDCl3, 75 MHz) d 170.32,
169.93, 169.67, 169.55, 169.43, 169.34, 139.16, 138.93,
138.67, 138.50, 138.40, 138.09, 128.24±127.32 (30
ArC's), 103.00, 100.61, 99.41, 98.55, 82.46, 79.85, 79.71,
77.57, 77.10, 76.79, 75.82, 75.10, 74.73, 74.53, 73.43,
73.21, 72.95, 72.65, 72.55, 70.33, 69.70, 69.07, 68.71,
68.19, 66.12, 65.90, 62.07, 61.99, 56.81, 30.05±28.85.;
ESMS m/z 1537.1 (M1Na)1; HRMS (FAB): Calcd. for
(M1Na)1 C81H94O28Na 1537.58290, found 1537.58270.
1
1050 cm21; H NMR (CDCl3, 300 MHz, COSY) d 5.42±
5.37 (dd, Jˆ3.3, 9.9 Hz, 1H), 5.36 (d, Jˆ1.5 Hz, 1H, H-1),
5.36±5.32 (m, 2H), 5.30±5.23 (m, 2H), 4.92 (d, Jˆ1.8 Hz,
1H, H-10), 4.23±4.19 (m, 2H), 4.17±4.14 (dd, Jˆ3.7,
5.1 Hz, 1H), 4.13±4.11 (m, 2H), 4.08±4.05 (ddd, Jˆ2.4,
2.7, 2.4 Hz, 1H), 3.65±3.59 (m, 1H), 2.13±1.99 (7£s,
21H, COMe); 13C NMR (CDCl3, 75 MHz) d 171.0, 170.6,
170.3, 169.7, 169.6, 169.4, 169.37, 169.3, 98.6, 92.5, 77.2,
70.4, 69.97, 69.6, 69.5, 68.97, 68.3, 66.2, 66.0, 62.27, 62.1,
20.77-20.54 (7 C); ESMS m/z 659.2 (M1Na)1;
HRMS(FAB): Calcd for (M1H)1 C26H37O18 637.197990,
found 637.200305.
3,4,6-Tri-O-acetyl-2-O-(2,3,4,6-tetra-O-acetyl-(a-d-manno-
pyranosyl)-a-d-mannopyranosyl trichloroacetimidate
(15). To a solution of heptaacetate 14 (254 mg, 0.4 mmol)
in dry CH2Cl2 (3 mL) at 08C was added successively,
trichloroacetonitrile (10.0 equiv., 400 mL) and DBU
(0.0325 equiv., 20 mL). After stirring for 1 h at 08C TLC
Methyl O-(a-d-mannopyranosyl)-(1!2)-O-a-d-manno-
pyranosyl-(1!2)-O-[b-d-galactopyranosyl-(1!4)]-a-d-
mannopyranoside (17). Solution of the fully protected

M. Upreti et al. / Tetrahedron 56 (2000) 6577±6584
6583
tetrasaccharide cap 16 (15 mg, 0.0105 mmol) in absolute
EtOH (22 mL) and palladium hydroxide (40 mg, 20 wt%)
was stirred under slight pressure of hydrogen for 4 h. The
reaction mixture was ®ltered through celite and the ®ltrate
concentrated under reduced pressure to obtain debenzylated
material. This product was dissolved in dry MeOH (12 mL)
and treated with anhyd sodium methoxide (6 mg) and the
solution was stirred for 2 h at room temperature. The reac-
tion mixture was quenched with few drops of 0.5% HCl
solution and excess of MeOH was removed under reduced
pressure, and the residue was lyophilised three times with
the addition of water (1 mL) to remove traces of HCl. This
provided pure methyl glycoside of the tetrasaccharide cap
17 as white powder in quantitative yield; Rfˆ0.53 in
n-propanol±acetone±water (9:6:5); IR nmax (KBr) 3600±
infrastructure support. The CSIR New Delhi is acknowl-
edged for a research associate fellowship to M.U.
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1
3200 (br), 2910, 1400, 1250, 1140, 1050 cm21; H NMR
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1
(br), 2900, 1500, 1450, 1300, 1100, 1000 cm21; H NMR
(D2O, 300 MHz) d 5.23 (d, Jˆ1.46 Hz, 1H, H-1000), 5.20 (d,
Jˆ1.22 Hz, 1 H, H-100), 4.80 (bs, 1H, H-1), 4.26 (d,
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The authors thank Dr Sandip K. Basu, Director NII for
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of Science and Technology (Govt of India) is acknowledged
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