A new approach to construct full-length glycosylphosphatidylinositols of parasitic protozoa and [4-deoxy-Man-III]-GPI analogues

Article abstract of DOI:10.1039/b414119a

A new [2 + 2 + 2] approach to construct GPI molecules through the efficient synthesis of glucosamine-inositol and tetramannose intermediates led to a total synthesis of a GPI-anchor of Trypanosoma cruzi, and also afforded a key intermediate for the synthe

Full text of DOI:10.1039/b414119a

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COMMUNICATION
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A new approach to construct full-length glycosylphosphatidylinositols of
parasitic protozoa and [4-deoxy-Man-III]-GPI analogues{
Asif Ali,a D. Channe Gowdab and Ram A. Vishwakarma*a
Received (in Cambridge, UK) 17th September 2004, Accepted 13th October 2004
First published as an Advance Article on the web 2nd December 2004
DOI: 10.1039/b414119a
chiral auxiliary on the way to GPIs, making a number of early
A new [2 + 2 + 2] approach to construct GPI molecules through
steps redundant. To test this proposition, the racemic 1-O-PMB-
2,3,4,5-tetra-O-benzyl-myo-inositol 76 was glycosylated with a
2-azido glycosyl donor 64b to get pseudodisaccharide 15
(Scheme 1), which on deacylation (16) and benzylidenation, gave
4,6-cyclic acetal protected disaccharide. To our surprise, this
quantitative reaction led to a clean separation (Rf difference of 0.1)
of two enantiomeric disaccharides 17 and 18 by a simple silica
column (for a comparison of their NMR spectra, see Fig. S1 of
ESI{). The next two steps, benzylation at 3-OH and regioselective
opening of benzylidene acetal by NaCNBH3, provided the key
building block 5 and the undesired isomer 19. The spectral and
[a]D data of 17 and 5 were identical to that reported6 for the
compounds prepared by an alternative route. The method also
worked with 2-O-allyl-1-O-PMB-3,4,5-tri-O-benzyl- and 1-O-allyl-
2,3,4,5-tetra-O-benzyl inositols showing its generality. Since good
separation was obtained for 17/18 and their benzylated pairs,
benzylidene-protected donors can also be used. A resolution at
disaccharide level was tried7 earlier but not used due to a problem
in separation.
the efficient synthesis of glucosamine-inositol and tetramannose
intermediates led to a total synthesis of a GPI-anchor of
Trypanosoma cruzi, and also afforded a key intermediate for the
synthesis of valuable [4-deoxy-Man-III]-GPI analogues.
Since the discovery1 of the glycosylphosphatidylinositol (GPI)
anchor as a novel and alternative mode of membrane-association
of cell-surface proteins, the biology of these complex glycolipids
has remained in focus.2 Subsequently, several GPI-anchors and
protein-free GPIs have been isolated across the eukaryotic species
including humans. Surprisingly, the GPIs are produced in high
abundance by protozoan parasites (Trypanosoma, Leishmania and
Malaria) compared to that in higher organisms, and are essential
virulence factors that allow these parasites to infect, proliferate and
subvert the host immune system. Marked differences in the
structure and biosynthesis of GPIs from the parasites and human
cells have been identified2 providing valuable targets3 for drug and
vaccine design. Even among the parasites, various species express
GPIs with subtle structural differences that manifest in remarkable
and, at times, opposing biological functions in the host.
The multi-gram scale synthesis of 5 encouraged us to apply this
new method to the total synthesis of structurally and biologically
challenging GPIs. For this, we decided to construct a GPI anchor
of the Trypanosoma cruzi IG7 antigen8a (Fig. 1) because (a)
synthesis of the GPIs from this parasite has not been reported and
(b) purified materials from T. cruzi have shown8b extraordinary
pro-inflammatory activities (pM), comparable to those of bacterial
Their structural complexity and biological function have
inspired widespread chemical interest and a number of synthetic
approaches towards GPIs (Thy-1, yeast, T. brucei, sperm CD-52,
Leishmania and P. falciparum) have been reported.4 However,
despite the concerted efforts of several leading groups, the total
synthesis of a full-length GPI-anchor remains a daunting task,
which is further complicated by the presence of (a) structural and
functional differences among the species and (b) significant micro-
heterogeneity in their lipid and glycan domains. Arguably, the
most demanding aspect of GPI synthesis has been to access
suitably protected glucosamine-inositol intermediates requiring an
optically pure protected D-myo-inositol acceptor and a 2-azido-2-
deoxyglucosyl donor. This has mainly been done4 either by the
painstaking resolution of bis-cyclo hexylidene-myo-inositols using
costly camphanate auxiliaries/enzymes or through a multi-step
synthesis from D-glucose by the Ferrier reaction. In our ongoing
investigations5 into the chemical biology of the GPI molecules,
such an intermediate was required for fluorescent GPI analogues.
Instead of following the reported methods based on the a priori
resolution of myo-inositol, we reasoned, based on structural
modeling, that if sufficient strain is built through a cyclic protective
group, the azidoglycosyl unit itself could function as an efficient
{ Electronic supplementary information (ESI) available: experimental and
characterisation data for key compounds. See http://www.rsc.org/supp-
data/cc/b4/b414119a/
Scheme 1 (a) TMSOTf, CH2Cl2, 0 uC; (b) NaOMe, MeOH; (c)
PhCH(OCH3)2, CH3CN; (d) BnBr, NaH, DMF; HCl–Et2O, NaCNBH3.
*ram@nii.res.in
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Chem. Commun., 2005, 519–521 | 519

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80 uC; 1M HCl–acetone, 1 : 9, 60 uC). The per-acetylation of
the resultant triol, selective removal of the anomeric acetyl
(Me2NH, MeCN, 220 uC) followed by Schmidt activation
(CCl3CN, DBU) provided the desired mannobiose 9. It needs to
be mentioned that the two acetyls at positions 4- and 6-OH were
deliberately placed keeping in view our future target, the [4-deoxy-
Man-III]-GPI analogue. Lower mannobiose 10 was prepared by
the glycosylation (TESTf, NIS) of allyl-2,3,4-tri-O-benzyl-a-D-
mannopyranoside (14, from D-Man in 4 steps) with 3,4,6,-tri-
O-benzyl-b-D-man-1,2-pent-4-enylorthobenzoate 134c followed by
the removal of benzoyl from the 2-position.
Having both mannobiose donor 9 and acceptor 10 in hand,
further glycosylation, which required considerable optimization,
provided a fully protected tetramannose in an acceptable 65%
yield. This, after anomeric allyl-removal (palladium chloride,
NaOAc, AcOH–water, rt) and activation (CCl3CN, DBU),
afforded the desired tetramannose donor 8. To our satisfaction,
the next critical step of the [4 + 2] glycosylation of glucosamine-
inositol 5 with the above tetramannose 8 went smoothly
(TMSOTf, DCM, 0 uC, 70%) to provide a pseudohexasaccharide
3a as the central point for both the GPI-anchor as well as the
deoxy-GPI analogues. For the synthesis of the GPI-anchor, two
acetyls were first removed and the primary 6-OH of the diol 3b
was silylated (TBDPSCl, imidazole) to get 3c, followed by
benzylation of the 4-OH (BnBr, NaH) and TBDPS removal
(TBAF, THF) to obtain the pseudohexasaccharide acceptor 3d
ready for phosphorylation with ethanolamine. A part of the
diol 3b (with free 4- and 6-OH of the Man-III) was used
for 4-deoxygenation (CSCl2,Bu3SnH, AIBN) by Barton’s
cyclic-thiocarbonate method for further synthesis of [4-deoxy-
Man-III]-GPI probes. The coupling of 3d with NHCbz-ethanola-
mine-phosphoramidite4c (4) was carried out with 1H-tetrazole
followed by mCPBA oxidation. Now the PMB group from the
1-position of the myo-inositol residue was removed (CAN,
MeCN–DCM–H2O) and the product was phospholipidated with
1-O-alkyl18 0-2-O-acyl18 0-sn-glycero-H-phosphonate (2)4a by
Fig. 1 Proposed backbone structure of the GPI anchor of T. cruzi.
LPS (lipopolysaccharide). The issues regarding the structural
features responsible for such unusual activity can only be resolved
through synthesis. Herein, we present a new strategy for the
synthesis of parasitic GPIs (Scheme 2, retrosynthetic analysis). The
synthetic design also accommodates a feature that allows access to
valuable [4-deoxy-Man-III]-GPI analogues to address a funda-
mental biological question: why proteins are transferred only to
the 6-OH of a Man-III residue9 and what happens if the
conformation of this residue is disturbed by 4-deoxygenation.
After the efficient access to glucosamine-inositol intermediate 5,
we designed a new and convergent [2 + 2] approach for the
construction of the tetramannose building block 8. This was
prepared from two protected mannobiosides, the activated donor
9 and acceptor 10. The donor 9 was prepared by the coupling of
allyl 3-O-benzyl-4,6-O-benzylidene-a-D-mannoside (12, prepared
in 4 steps from D-Man) with 2,3,4,6-tetra-O-benzyl-a-D-mannosyl
trichloroacetimidate (11, made from D-Man). The glycosylation
(TMSOTf, DCM, 220 uC) went smoothly and the product
was taken to the next stage: simultaneous removal of
anomeric allyl and 4,6-benzylidine groups (KOtBu, DMSO,
:
:
pivaloyl chloride/I2-oxidation, to provide a fully-protected GPI-
anchor. The final step involved global deprotection and azide-
reduction by hydrogenolysis (Pd(OH)2, DCM–MeOH–H2O, H2)
to the target GPI anchor.
Scheme 2 Retrosynthetic analysis showing key building blocks and intermediates.
520 | Chem. Commun., 2005, 519–521
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In conclusion, we designed a new and efficient approach for the
synthesis of GPIs, and showed its utility by accomplishing a total
synthesis of a full-length GPI of T. cruzi. This synthesis represents
the first step in our efforts aiming at related GPIs (e.g. with
unsaturated fatty-acids at sn-2-glycerol and aminoethyl phospho-
nate on a GlcN residue) to probe their biology. The synthesis also
afforded key intermediates for valuable [4-deoxy-Man-III]-GPI
analogues and the GPIs of the malaria parasite, which will be
reported along with the relevant biological studies.
The work was supported by DST, India (SR/S5/OC-11/2002)
and NIAID, NIH-USA (RX4220-105 of ROI A141139-05).
5 (a) P. Sahai and R. A Vishwakarma, J. Chem. Soc., Perkin Trans. 1,
1997, 1845; (b) P. Sahai, M. Chawla and R. A. Vishwakarma, J. Chem.
Soc., Perkin Trans. 1, 2000, 1283; (c) D. Ruhela and
R. A. Vishwakarma, Chem. Commun., 2001, 2024; (d) D. Ruhela and
R. A. Vishwakarma, J. Org. Chem., 2003, 68, 4446; (e) M. Chawla
and R. A. Vishwakarma, J. Lipid Res., 2003, 44, 594.
Asif Ali,a D. Channe Gowdab and Ram A. Vishwakarma*a
aBio-organic Chemistry Laboratory, National Institute of Immunology,
JNU Complex, New Delhi, India. E-mail: ram@nii.res.in;
Fax: +91-11-26162125; Tel: +91-11-26717178
bDepartment of Biochemistry and Molecular Biology, Pennsylvania
State University College of Medicine, Hershey, PA 17033, USA
6 K. Pekari, D. Tailler, R. Weingart and R. R. Schmidt, J. Org. Chem.,
2001, 66, 7432.
7 M. A. Jardine, H. S. C. Spies, C. M. Nkambule, D. W. Gammon and
J. Steenkamp Daniel, Bioorg. Med. Chem., 2002, 10, 875.
Notes and references
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M. A. J. Ferguson, R. A. Dwek, T. W. Rademacher, R. Anand and
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2 For reviews on GPIs: (a) M. J. McConville and A. K. Menon, Mol.
Membr. Biol., 2000, 17, 1; (b) M. McConville and M. A. J. Ferguson,
Biochem. J., 1993, 294, 305.
9 K. Nagamune, K. Ohishi, H. Ashida, Y. Hong, J. Hino, K. Kanagawa,
N. Inoue, Y. Maeda and T. Kinoshita, Proc. Natl. Acad. Sci. USA,
2003, 100, 10682 and references cited therein.
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