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Synthesis and biosynthesis of glycosyl-phosphatidyl-inositol (GPI) cell surface molecules of Leishmania parasite |
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Principal Investigator : Ram A Vishwakarma Project
Associates/Assistants
PhD
Students
Collaborators The
project aims at chemical synthesis and biosynthesis of the
glycosyl-phosphatidyl-inositol (GPI) cell surface molecules (lipophosphoglycan,
proteophosphoglycans, and free GPIs) of Leishmania parasite, and design
and synthesis of the structural and functional mimics and inhibitors of GPI
pathway. The GPIs are expressed abundantly on the cell surface of Leishmania,
Trypanosoma and Plasmodium species, and have been implicated in
the infectivity, transmission and intracellular survival of the parasites. The
parasitic GPIs have distinct structural features, compared to mammalian
protein GPI anchors, and are interesting targets for chemical synthesis,
biosynthesis and molecular design. The
main objectives of the program include, (i) design and synthesis of GPI
anchor, LPG structural domains (PI, GPI, conserved glycan-core, variable
phosphoglycan repeats and terminating cap), and GPI analogues, (ii) synthesis
of labeled biosynthetic precursors and their incorporation in the parasite to
elucidate the steps involved in the LPG/GPI assembly, (iii) design and
synthesis of structural and functional mimics, and mechanism based inhibitors
of GPI biosynthetic pathway, and (iv) role of parasitic GPIs and their
synthetic analogues in PKC and PI-3-kinase mediated events in macrophages. The
intriguing structure of the LPG consists of four distinct domains: alkyl-lyso-GPI
anchor, conserved glycan core with internal galactofuranose residue, variable
phosphoglycan repeats and neutral mannose cap. The unique feature of LPG is
the variable phosphoglycan domain made of phosphodisaccharide [6Galp-b1,4-Manpa1-phosphate]n repeats linked through phosphodiester linkages
between anomeric-OH of Man of one repeat and 6-OH of Gal of adjoining repeat.
Several biochemical and biophysical experiments to probe the function,
biosynthesis and conformation of Leishmania LPG/PPG, require efficient
chemical synthesis of the phosphoglycans and their structural and functional
mimics. Since the phosphoglycans are labile (due to presence of anomeric
phosphodiester linkages), their synthesis remains fairly challenging. We have
been working towards this objective for last few years, and now we have
succeeded in designing a new, iterative and efficient route for the synthesis
of Leishmania phosphoglycans. Significantly this approach does not
involve any glycosidation step, and the PG chain can be extended either
towards non-reducing (6¢-OH) end or reducing (1-OH) end in
high yielding iterative steps. Important features of our synthesis are: glycal
mediated gluco®manno transformation and
regioselective 6¢-protection of lactal to a key
protected Galb1,4Man building block, extension of
the PG repeats in either direction by selective deprotection at the
non-reducing 6¢-position or at reducing 1-position
and a-phosphitylation, followed by iterative PG coupling cycles. The
starting material lactal is prepared in
straightforward three steps (acetylation, bromination, reductive elimination
and deacetylation) from lactose, high yield obtained in reductive elimination
step by Zn/Vitamin-B12
reagent. Next task was to selectively block 6-position of Gal residue of
lactal and this was achieved, after considerable efforts, by dibutyltinoxide
mediated silylation exclusively to 6¢-O-TBS-lactal. It
should be mentioned here that under similar conditions most of other
protection groups (benzyl, PMB and allyl) led to C3¢-OH protected
lactals. The next step involved stereoselective gluco®manno
transformation of 6¢-O-TBS-lactal (mCPBA) to the
manno product, which on acetylation gave a key intermediate, 1,2,3,6-tetra-O-acetyl-4-O-(2,3,4,-tri-O-acetyl-6-O-TBS-b-D-galacto
pyranosyl)-a-D-mannopyranose, which served as a central point to both the donor
as well as acceptor for iterative assembly of the PG repeats. This
intermediate was divided into two parts; the first part was transformed to the
PG donor by anomeric deacylation (Me2NH
at –20 0C) followed by
phosphitylation (tri-imidazolyl phosphine) to provide 2,3,6-tri-O-acetyl-4-O-[2,3,4-tri-O-acetyl-6-O-(TBS)-b-D-galactopyranosyl]-a-D-mannopyranosyl
H-phosphonate. The second part was converted to hepta-acetyl PG-acceptor by
removing TBS from 6-position of Gal residue. The coupling of PG-H-phosphonate
donor with PG-acceptor (pivaloyl chloride) followed by in-situ iodine
oxidation afforded protected phosphotetrasaccharide; which on full
deprotection provided free phosphoglycan (with two PG repeats). Now
the protected phosphotetrasaccharide was well placed for further extension of
phosphoglycan chain either towards upstream direction (non-reducing 6¢-end)
or downstream direction (reducing 1-OH end). For upstream extension of PG
domain, the TBS from 6¢-position of Gal at the non-reducing
end was removed, and resulting phosphotetrasaccharide was coupled with PG-H-phosphonate
donor, to provide phosphohexasaccharide in high yield. The global deprotection
led to free phosphoglycan with three PG repeats. For downstream extension of
the PG domain, the central intermediate was deacylated (Me2NH at –20 0C) at the reducing-end anomeric position and
converted to H-phosphonate as described above. This H-phosphonate was then
coupled with hepta-O-acetyl-disaccharide to provide the phosphohexasaccharide
ready for deprotection or further extension to higher oligomers. The
presence of (1®6)-phosphodiester linkages between the
PG repeats was confirmed by 31P-13C NMR coupling for C-1 and C-2 of the mannose
units, and the C-5 and C-6 of the corresponding Gal units; these 13C signals were shifted due to a-
and b- phosphorylation effect. The a-configuration of
mannosyl phosphate fragments was confirmed by 1H,
13C and 31P NMR analysis. It is obvious from above that
the phosphoglycan chain can further be extended to the desired length in
either direction, and instead of 2+4 (disaccharide +tetrasaccharide) coupling,
4+4 coupling can also be carried out for rapid access to desired PGs. We are
now extending this solution synthesis on the solid phase (Merrifield resin) so
that the synthesis can be automated for preparation of the PGs of required
length and repeat number. For this purpose, a new solid phase linker has been
designed to facilitate iterative PG assembly and final deblocking of PGs from
resin without affecting labile phosphodiester linkages. We have also got
access to an important intermediate for synthesis of L. major PG
repeats, which are more complex than that of L. donovani and L.
mexicana. The
biosynthesis of PG repeats occurs inside Golgi (after the pre-assembled GPI
core is translocated from ER to Golgi) and involves a set of initiating and
elongating Man-1a-PO4-transferases
(MPTs). The MPTs are unique to Leishmania and are capable of
transferring intact Man-1a-phosphate (and not
just the Man) from the GDP-Man nucleotide sugar donor. Interestingly a unique
GDP-Man transporter (GMP antiporter) has recently been identified in Leishmania
Golgi vesicles. The biosynthetic assembly of PG repeats, unique MPTs and
GDP-Man-transporter are interesting target for synthesis, conformation and
inhibitor design. Our interest in LPG biosynthesis is to design the
substrates/inhibitors of MPTs and GDP-Man transporter activities. Last year we
reported synthesis of radiolabelled lipid-linked PG substrate and also set up
in-vitro LPG biosynthetic and GDP-Man-transporter assays using L. donovani
Golgi vesicles. This year we have made further progress and established
synthetic routes for novel b-lactam analogues of
phosphoglycan repeat, monofluoro-, difluoro and C-analogues of GDP-Man. These
synthetic analogues will soon be evaluated for inhibition of MPTs and GDP-Man
transport. This year efforts have also been initiated towards synthesis of
hybrid LPGs and multivalent glycodendrimers based on the phosphoglycan motif. Another
distinct feature of LPG structure is the presence of a galactofuranose (Galf) residue right in the middle of the conserved
glycan core, which makes it an attractive point to understand and intervene.
The biosynthesis of Galf
domain must involve at least three interesting steps; (i) UDP-Galp-mutase to transform UDP-Galp to UDP-Galf
(ii) UDP-Galf
transporter and (iii) UDP- Galf
transferase. Last year we initiated efforts towards synthesis of acceptor
substrates for UDP-Galf
transferase and characterization of UDP-Galp-mutase
and UDP-Galf
transporter activities in the parasite. Towards this objective, two
lipid-linked 1,3-a-linked mannobiose acceptors have been
synthesized. The synthetic acceptors are now being used as exogenous substrate
for UDP- Galf
transferase in Leishmania cell free system. We are also close to
completing synthesis of entire conserved glycan core (hexasaccharide) of LPG
containing galactofuranose residue. Our
efforts on devising a new convergent synthetic methodology towards GPI anchor
and analogues have continued and last year we reported on the preparation of
suitable chiral protected myo-inositol, glycerolipid and glycan intermediates.
This year it has been possible to bring these intermediates together in
correct linkages and stereochemistry to make GPI anchor and its labeled
analogues. This synthesis has allowed us to make hitherto inaccessible a key
proposed biosynthetic GPI intermediate acylated at the C-2 position of
inositol moiety. This synthetic approach will now be exploited for preparation
of specific structural and functional mimics of GPI anchor, such as
multivalent glycodendrimers and hybrid GPIs, to attempt some cytomimetic (memebrane
fusion, nearest-neighbor-recognition and flipping) experiments using synthetic
giant unilamellar vesicles. The GUVs have recently emerged as good model
systems to mimic cell size and membrane dynamics, and synthetic GPI analogues
may help address few functional issues. The
first distinct step in GPI biosynthesis is the generation of GlcNAc-PI from
UDP-GlcNAc and specific PI substrate (different PI pools are used by the
parasite for GPI anchor, LPG and GIPLs biosynthesis) catalyzed by GPI-N-GlcNAc-transferase.
The GlcNAc-PI is then N-deacylated to form GlcN-PI. The biosynthesis of GlcN-PI
occurs on the cytoplasmic side of ER and then the intermediates GlcN-PI and/or
GlcN-acyl-PI are flipped into the lumen where first three Man are transferred
from Dol-P-Man donor. To establish microsomal enzyme system and inhibition of
GPI biosynthesis, last year we reported on the synthesis of both the D and L
enatiomers of water-soluble short-chain phosphatidylinositols and radio active
analogues. These analogues have now been evaluated and early experiments have
shown good inhibition of Plasmodium falciparum by some of these
compounds. In
addition to our primary focus on Leishmania GPIs, we have also
initiated synthetic efforts towards total synthesis of the complex
GPIs/analogues, and their application to study glycobiology of Plasmodium
falciparum (in collaboration with DC Gowda of Penn State University). The
malaria GPIs (also termed as malaria toxins) have recently been identified as
factors contributing to malaria pathology, and are highly immunogenic. Under
the NMILTI program of CSIR, and in collaboration with Dr A Mukhopadhyay and Dr
S K Basu), we have designed and synthesized new Rifampicin drug conjugates
with Poly-G and Fucoidin for scavenger receptor mediated drug delivery of
antitubercular rifampicin to the macrophages. Publications Original
peer-reviewed articles 1.
Ruhela D and Vishwakarma RA (2001) Efficient synthesis of the antigenic
phosphoglycans of Leishmania parasite. Chem Commun (19)
2024-2025. 2.
Khan SR, Deutscher J, Vishwakarma RA, Monedero V and Bhatnagar NB
(2001) The ptsH gene from Bacillus thuringiensis israelensis:
characterization of a novel phosphorylation site on the protein HPr. Eur J
Biochem 268: 521-530. Patents 1.
Balakrishnan A and Vishwakarma RA. A substituted fFurochromenone
derivative exhibiting anti-cancer and anti-proliferative properties. International
patent application No. PCT/IN01/00156 filed on 11 Sep 2001.. |