A facile and novel route to the antigenic branched phosphoglycan of the protozoan Leishmania major parasite

Article abstract of DOI:10.1016/j.tetlet.2004.01.155

A novel approach for the iterative synthesis of the antigenic branched phosphoglycan of the protozoan parasite Leishmania major is presented that exploits remarkable dual selectivity in the functionalization of D-lactal, providing flexibility to extend th

Full text of DOI:10.1016/j.tetlet.2004.01.155

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 2589–2592
A facile and novel route to the antigenic branched phosphoglycan of
the protozoan Leishmania major parasiteq
Dipali Ruhela and Ram A. Vishwakarma*
Bio-organic Chemistry Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, JNU Complex, New Delhi 110067, India
Received 17 October 2003; revised 20 January 2004; accepted 30 January 2004
Abstract—A novel approach for the iterative synthesis of the antigenic branched phosphoglycan of the protozoan parasite Leish-
mania major is presented that exploits remarkable dual selectivity in the functionalization of
the PG chain either towards the reducing or nonreducing end.
ꢀ 2004 Elsevier Ltd. All rights reserved.
D-lactal, providing flexibility to extend
The protozoan parasite Leishmania, responsible for
multiple diseases ranging from cutaneous to fatal vis-
ceral leishmaniasis, survives in extremely hostile milieus
throughout its life cycle in the sand-fly vector and the
human host. For this, all Leishmania species synthesize1
a unique class of molecules termed phosphoglycans,
including the membrane-bound lipophosphoglycan
(LPG) expressed by the infectious promastigote form,
and the proteophosphoglycan (PPG) secreted by the
intracellular amastigote form of the parasite. Their
role(s) in parasite virulence has been a topic of intense
debate2 in recent years but now it has been established3
that the principal virulent determinant in Leishmania
major consists of the PG repeat units, independent of
their molecular platform (lipid linked in LPG and pep-
tide linked in PPG). The intriguing structure4 of LPG
(Fig. 1) consists of four distinct functional domains; the
alkyllysophosphatidylinositol lipid anchor, the con-
served glycan core with an internal Galf residue, vari-
able phosphoglycan (PG) repeats, and a neutral
oligomannose cap. The most distinct feature of LPG is
the dynamic PG domain made of [-6Galp-b1,4-Manp-
a1-phosphate]n repeats (n ¼ 2–32), which is unique
among all the eukaryotic carbohydrates. Although LPG
is expressed by all Leishmania species, there is one
remarkable difference between the structure of LPG
from L. major and that from other species; the 3-OH
position of the galactose residue of PG repeats is highly
substituted with 1-3b-galactosyl branches in L. major (as
shown in Fig. 1), whereas this position remain totally
unsubstituted in Leishmania donovani where the PGs are
linear.
OH O
OH OH
O
OH
O
HO
O
O
O
HO
HO
OH
OH
O
O P O-
O
n
REPEATING PHOSPHOGLYCAN
HEXASACCHARIDE CORE
1-O-alkyl-lyso-PI LIPID
CAP
-6-Galα1,6-Galα1,3-Galf,β1,3-Manα1,3-Manα1,4GlcNα1,6-
Manα1,2-Manα1-PO4-
Figure 1. Structure of lipophosphoglycan of L. major.
q Supplementary data associated with this article can be found in the online version, at doi:10.1016/j.tetlet.2004.01.155
* Corresponding author. Tel.: +91-11-26717178; fax: +91-11-26162125; e-mail: ram@nii.res.in
0040-4039/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.01.155

2590
D. Ruhela, R. A. Vishwakarma / Tetrahedron Letters 45 (2004) 2589–2592
These remarkable inter-species structural differences
are clearly manifested5 biologically, for example, in
L. major, the branched PG 6-[Gal(b1fi3)]Galp-b1,4-
Manp-a1-phosphate controls5 the transformation of
the noninfective procyclic form of the parasite to the
infectious metacyclic form, and also constitutes6 the
major epitope for the attachment of the virulent pro-
mastigotes to the sand-fly mid gut and human macro-
phages.
OH OH
O
HO
O
O
O
HO
Lactose
HO
OH
1
a
OH OTBS
OH OH
O
HO
HO
b
O
O
O
O
PMBO
PMBO
HO
HO
OH
OH
3
2
The unique structure and function of LPG have gener-
ated significant interest1;7 as a target for new chemo- and
immunotherapy, but to exploit this knowledge requires
efficient synthetic approaches for the construction of
phosphoglycans of defined lengths and repeats and their
neo-glycoconjugates. The synthesis of the repeating PGs
is complicated, as compared to that of nonanomeric
types such as oligonucleotides, due to presence of ano-
meric phosphodiesters between each repeat unit, which
places additional requirements8;9 of anomeric stereo-
control during the synthesis, and also leads to instability
due to the propensity of the glycosyl ring to form a
stabilized carbocation by expulsion of the anomeric
phospho monoester leaving group. The branched
L. major phosphoglycans present specific problems due
to the presence of 30-galactosyl branches, and only one
approach to the branched PG of L. major has been
reported10;11 so far that starts from monosaccharide
building blocks using multiple protection, glycosylation
and phosphorylation steps and the synthesis is extend-
able only towards the nonreducing end.
c
OAcOTBS
OAcOTBS
AcO
OAc
O
AcO
OAc
O
d
O
O
O
O
PMBO
AcO
HO
AcO
OAc
OAc
OAc
OAc
4
5
OAcOAc
O
AcO
e
AcO
O
6
HN CCl3
OAcOTBS
AcO
OAcOAc
O
OAc
O
O
O
O
AcO
AcO
OAc
OAc
OAc
7
Scheme 1. Reagents and conditions: (a) Bu2SnO, MeOH, reflux, 4 h;
PMBCl, CsF, KI, DMF, rt, 48 h, 74%; (b) NaH, 18-crown-6, TBSCl,
THF, 0 ꢁC–rt, 30 min, 65%; (c) (i) m-CPBA, ether–NaHCO3 buffer,
0 ꢁC, 3 h, (ii) Ac2O, py, rt, 16 h, 80%; (d) DDQ, CH2Cl2–H2O, rt, 12 h,
85%; (e) compounds 5 and 6, ether, TMSOTf, )20 ꢁC, 4 h, 74%.
In continuation of our efforts on the chemistry12–15 and
biology16 of LPG, we now report a facile and novel
route for the rapid construction of the branched phos-
phoglycan of L. major, with the flexibility to extend the
PG repeats on either the nonreducing or reducing ends.
Key features of our approach include (a) remarkable
dual selectivity in protection of the easily available
in favor of the 6-OH of the Gal residue over the 6-OH of
the glucal residue (product ratio 85:15) providing the
desired 30-O-PMB-60-O-TBS-
D-lactal 3 in good yield.
The placement of TBS and PMB groups was assigned by
1H–13C HMBC experiments and the 60-position of the
TBS was further confirmed18 by a silyl-induced shift of
C-60 (61.21) as compared to C-6 (62.45). The selectivity
of silylation under the above conditions was observed
only when the PMB group was present at the 30-posi-
tion, indicating that the PMB group selectively
enhanced the nucleophilicity of the proximal CH2OH
group of the galactose residue. The reverse sequence,
selective silylation at 60-OH followed by alkylation at
30-OH, failed and yielded intractable mixtures. The next
two steps involved the stereoselective gluco fi manno
transformation of 3 by a sodium bicarbonate catalyzed
m-CPBA reaction under biphasic (5% aq bicarbonate-
diethyl ether) conditions providing, in high yield, the
manno compound 30-O-PMB-60-O-TBS-Gal-(b1fi4)-
Man, which on direct per-acetylation led to the key
intermediate 1,2,3,6-tetra-O-acetyl-4-O-(2,4-di-O-acetyl-
starting material,
D-lactal, with mutually compatible
groups at strategic positions, (b) a gluco–manno trans-
formation followed by 30-galactosylation to give the
central trisaccharide intermediate ready to serve either
as the phosphate donor or acceptor for the rapid
assembly of the phosphohexasaccharide. The starting
material
D-lactal 1 is easily prepared from lactose in
four straight forward steps (acetylation, bromination,
reductive elimination and deacetylation). The next step
(Scheme 1) was to selectively protect the 30-OH and 60-
OH positions of the galactose residue with two mutually
compatible groups. This was achieved by first placing a
p-methoxybenzyl (PMB) group on the 30-OH of 1 by
dibutyltin chemistry (Bu2SnO, MeOH, reflux; PMBCl,
CsF, KI), which led exclusively to the 30-O-PMB-
D-
lactal 2; the position of the PMB group was confirmed17
by an HMBC experiment showing a correlation between
the 30-H (d 3.53 ppm) and the benzylic carbon (70.72) of
the PMB group. The next critical step that is, selective
protection of the 60-OH, was achieved by reaction of 2
with an equimolar amount of tert-butyldimethylsilyl
chloride (TBSCl) using NaH, and catalytic amounts of
the crown ether (18-crown-6). This NaH/crown-ether
mediated silylation showed remarkable regioselectivity
3-O-PMB-6-O-TBS-b-
D
-galactopyranosyl)-a-D-manno-
pyranose 419 as the major isomer. Now the PMB group
from compound 4 was removed by DDQ in acetonitrile
to obtain the disaccharide acceptor 5 in a 85% yield,
which was then coupled (DCM, TMSOTf at )20 ꢁC)
with the freshly prepared20 2,3,4,6-tetra-O-acetyl-a-
D-
galactopyranosyl-trichloroacetimidate glycosyl donor 6.

D. Ruhela, R. A. Vishwakarma / Tetrahedron Letters 45 (2004) 2589–2592
2591
This galactosylation reaction turned out to be efficient
and provided the desired major b-isomer,21 1,2,3,6-tetra-
O-acetyl-4-O-[6-O-TBS-2,4-di-O-acetyl-3O-(2,3,4,6-tetra-O-
meric H-phosphonate group [(dP ꢀ 0:13; dH 5.48 (dd, J1;2
1.9, J1;P 8.0, H-1), 6.90 (d, JH;P 642, H–P)]. The second
part of the compound 7 was converted to the PG-
acceptor 10 by HF mediated (aq HF–CH3CN, 5:95,
0 ꢁC, 2 h) removal of TBS from the 6-position of the
distal galactose residue.
acetyl-b-
D
-galactopyranosyl)-b- -galactopyranosyl]-a-
D
D
-mannopyranose 7.
The suitably protected trisaccharide 7 was ideally placed
to serve as the central point to access both the
H-phosphonate donor 9 as well as the PG-acceptor 10
(Scheme 2) for rapid and iterative synthesis of the target
branched phosphoglycan 11. For this, compound 7 was
divided into two parts; the first part was converted into
the PG donor by (a) anomeric deacetylation by freshly
prepared saturated dimethylamine in acetonitrile at
)20 ꢁC to provide compound 8 in quantitative yield
followed by (b) phosphitylation using triimidazolyl
phosphine generated in situ from PCl3 and imidazole.
These two high yielding steps led to 2,3,6-tri-O-acetyl-4-
Having secured efficient access to both the trisaccharide
H-phosphonate donor 9 and the trisaccharide acceptor
10, their coupling was carried out using pivaloyl chloride
followed by in situ iodine oxidation to give the fully
protected phosphohexasaccharide 11.23 The presence of
(1 fi 6)-phosphodiester linkages between the PG repeats
was confirmed by 31P–13C coupling (doublets, 5 and
7 Hz in 13C NMR) for C-1 and C-2 of the Man units,
and C-5 and C-6 of the corresponding Gal units; these
13C signals were shifted due to the a- and b-phosphor-
ylation effect. The a-configuration of the mannosyl
phosphate linkage was confirmed by an 1H NMR
analysis that showed characteristic coupling (5.45 ppm,
JH;H ¼ 1:9 and JH;P ¼ 7 Hz) for the corresponding ano-
meric proton. This was further confirmed from the 13C
signals of the mannose C-3 and C-5 positions, corre-
sponding to those reported24;25 for a-mannosyl phos-
phate containing phosphoglycans. The protected
phosphohexasaccharide 11, on global deprotection with
HF–CH3CN and NaOMe–CH3OH conditions, pro-
vided free phosphoglycan 12 (with two branched PG
repeats).
O-[2,4-di-O-acetyl-6-O-TBS-3-O-(2,3,4,6-tetra-O-acetyl-b-
galactopyranosyl)-b- -galactopyranosyl]-a- -mannopyr-
D-
D
D
anosyl H-phosphonate 9, which was isolated as its tri-
ethyl ammonium bicarbonate salt. The 1H and 31P NMR
of 9 clearly showed22 characteristic signals for its ano-
7
a
OAcOTBS
AcO
c
OAcOAc
O
OAc
O
O
O
O
AcO
AcO
Evidently the protected phosphohexasaccharide 11 is
fairly well placed for further iterative extension of the
phosphoglycan chain either towards the upstream non-
reducing the 60-OH end (removal of the TBS group
followed by coupling with H-phosphonate donor 9), or
downstream reducing the 1-OH end (anomeric-deacet-
ylation, phosphitylation and coupling with the trisac-
charide acceptor 10) following the same methods as
described above. Furthermore, this synthetic approach
is eminently suitable for adaptation on the solid-phase
mode for preparation of larger branched phosphogly-
cans of L. major by the application of our recently
described15 cis-allyloxyphosphoryl linker strategy.
OAc
OAc
OH
8
OAcOH
O
OAcOAc
O
OAc
O
AcO
b
O
O
AcO
AcO
OAc
OAc
OAc
10
OAcOTBS
OAcOAc
O
OAc
O
AcO
O
O
O
AcO
AcO
OAc
OAc
O
O P
H
9
O-Et3NH+
d
In conclusion, a versatile synthesis for the rapid
assembly of the branched phosphoglycans, antigenic
virulence factors of L. major, has been designed that
allows the PG chain to grow in either direction. We are
now extending this approach on solid phase for the
preparation of larger phosphoglycans, and their conju-
gation with suitable carrier antigenic proteins such as
KLH to test such neo-glycoconjugates as potential vac-
cines against L. major infection. Full details of the
solution and solid phase synthesis and immunological
results will be reported in due course.
OAcOTBS
OAcOAc
O
OAc
O
AcO
O
O
O
AcO
AcO
OAc
OAc
O
O P O-
O
OAc
Extension
OAcOAc
O
OAc
AcO
O
O
O
O
AcO
AcO
OAc
OAc
OAc
11
12
e
Free phosphoglycan
Scheme 2. (a) Me2NH, CH3CN, )20 ꢁC, 3 h, 95%; (b) PCl3, imidazole,
CH3CN, 0 ꢁC, 2 h, TEAB work-up, 87%; (c) HF–CH3CN, 0 ꢁC, 2 h,
85%; (d) compounds 9 and 10, pivaloyl-Cl, pyridine, rt, 1 h; iodine in
aq py, 0.5 h, TEAB work-up, 79%; (e) 48% HF–CH3CN, 5:95, 0 ꢁC,
2 h; NaOMe–MeOH, 95%.
Supplementary material
Experimental and characterization data for all new
compound 2–5 and 7–12.

2592
D. Ruhela, R. A. Vishwakarma / Tetrahedron Letters 45 (2004) 2589–2592
HRMS (ESMS+): m=z 451.1420 (M+Na)þ (C20H28O10Na
Acknowledgements
requires 451.1580).
18. Characterization data for 3:1H NMR (acetone-d6) d 0.07
(s, 6H), 0.89 (s, 9H), 3.49–3.58 (m, 2H), 3.60–3.68 (m, 3H),
3.72–3.87 (m, 11H), 4.07 (d, 1H), 4.23 (d, 1H), 4.46 (d,
J ¼ 7:8 Hz, 1H), 4.57–4.69 (m, 3H), 6.27 (d, J ¼ 6:2 Hz,
This work was generously supported by a research grant
from the Department of Science and Technology (Govt.
of India). The Department of Biotechnology (Govt. of
India) is acknowledged for providing core infrastructure
grant to the Institute.
1H), 6.87 (d, J ¼ 8:4 Hz, 2H), 7.33 (d, J ¼ 8:4 Hz, 2H); 13
C
NMR (acetone-d6) d )6.32 to )6.23, 17.94, 25.36, 54.51,
61.21, 62.45, 65.32, 68.16, 70.30, 70.83, 75.55, 77.12, 81.07,
81.50, 103.04, 104.57, 113.38, 129.18, 143.08; HRMS
(ESMS+): m=z 565.2430 (M+Na)þ (C26H42O10SiNa re-
quires 565.2445).
References and notes
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(s, 9H), 1.97–2.11 (m, 18H), 3.42–3.88 (m, 4H), 4.08–4.13
(m, 2H), 4.28–4.43 (m, 3H), 4.56–4.63 (m, 2H), 4.94–5.01
(m, 3H), 5.19 (br d, 1H), 5.32 (dd, J ¼ 4:8, 8.1 Hz, 1H),
5.55 (br d, 1H), 5.98 (d, J ¼ 1:8 Hz, 1H), 6.83 (d,
J ¼ 8:4 Hz, 2H), 7.21 (d, J ¼ 8:4 Hz, 2H); 13C NMR
(CDCl3) d )5.77, 18.07, 20.46–20.73, 25.65, 55.15, 60.41,
68.24, 69.39, 70.77, 70.87, 71.01, 73.49, 73.74, 76.38, 90.28,
101.29, 113.70, 129.34–130.01, 159.26–170.39; HRMS
(ESMS+): m=z 851.3015 (M+Na)þ (C38H56O18SiNa re-
quires 851.3134).
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HRMS
(ESMS+):
m=z
1061.3512
(M+Na)þ
(C44H66O26SiNa requires 1061.3509).
22. Selected data for 9:1H NMR (CDCl3) d 0.05 (s, 6H), 0.86
(s, 9H), 1.30 (t, 9H, TEA ion), 2.04–2.14 (m, 27H,
9 · OAc), 3.10 (q, 6H, TEA ion), 3.50 (m, 4H), 4.08–4.15
(m, 6H), 4.25–4.40 (m, 3H), 4.56–4.63 (m, 4H), 4.93–5.20
(m, 4H), 5.20 (m, 2H), 5.32 (dd, J ¼ 4:7, 8.1 Hz, 1H), 5.48
(dd, J ¼ 1:9, 8.0 Hz, 1H), 6.90 (d, JH;P ¼ 642 Hz, 1H); 13C
NMR d )5.70, 13.90, 20.45–20.80 (9 · OCOCH3), 60.38,
60.89, 61.17, 66.60, 67.43, 68.03, 68.35, 69.15, 70.30, 70.97,
72.36, 73.08, 73.67, 77.11, 92.54, 101.2, 101.5, 168.0–
169.23 (9 · OCOCH3); 31P NMR: d ¼ 0:13; HRMS
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1059.3142).
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(s, 9H), 1.30 (t, 9H), 1.96–2.15 (m, 57H), 3.00 (q, 6H),3.50
(m, 6H), 3.87 (m, 2H), 3.94 (m, 2H), 4.07–4.14 (m, 2H),
4.35 (m, 1H), 4.39 (m, 4H), 4.40 (m, 1H), 4.48 (m, 1H),
4.52 (m, 1H), 4.94 (d, J ¼ 7:7 Hz, 3H), 5.28 (m, 5H), 5.43
(m, 1H), 5.45 (dd, JH;H ¼ 1:9 and JH;P ¼ 7:0 Hz, 1H), 5.46
(m, 1H), 6.01 (d, J ¼ 2:7 Hz, 1H); 13C NMR: )5.75, 17.95,
25.57, 8.5, 45.50 (TEA ion), 20.48–20.79 (19 peaks), 60.06,
60.42 (d, JC;P ¼ 8 Hz), 62.22, 62.63, 66.55, 67.46, 68.27,
68.64, 69.37, 69.66, 69.84, 70.14, 70.75, 70.88, 71.20, 73.31,
73.76, 74.24, 77.15, 78.95, 90.41, 91.69, 101.08, 101.29,
168; 31P NMR: d )2.90 (dt, JP;H ¼ 7.5, 10 Hz); HRMS
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requires 1981.5732).
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17. Characterization data for 2: H NMR (acetone-d6) d 3.53
(dd, J ¼ 3:3, 9.6 Hz, 1H), 3.71–3.79 (m, 10H), 3.80–4.00
(m, 6H), 4.15 (d, 1H), 4.32 (d, 1H), 4.54 (d, J ¼ 7:8 Hz,
1H), 4.63–4.77 (m, 3H), 6.28 (d, J ¼ 6:2 Hz, 1H), 6.86 (d,
J ¼ 8:4 Hz, 2H), 7.33 (d, J ¼ 8:4 Hz, 2H); 13C NMR
(acetone-d6) d 54.36, 61.25, 65.80, 68.24, 70.35, 70.72,
75.52, 77.20, 80.91, 102.96, 104.39, 113.42, 129.26, 143.23;
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