|
Synthesis and biosynthesis of glycosyl
phosphatidyl inositol (GPI) cell surface molecules of Leishmania parasite |
|
Principal Investigator : Ram A Vishwakarma Project Associates Ph D Students Collaborator The project aims at chemical synthesis and
biosynthesis of cell surface glycosyl-phosphatidyl-inositol (GPI) cell surface
molecules, lipophosphoglycan (LPG) and glycosyl inositol-phospholipids (GIPLs)
of Leishmania donovani parasite. The GPIs are expressed abundantly on the
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 drug design. The main objectives of this project
include i) synthesis of GPI molecules, LPG structural domains (such as PI,
GPI, core, PG repeat and terminating cap), GIPLs and their analogues, ii)
synthesis of isotope-labeled precursors and their biosynthetic incorporation
in parasite to elucidate the steps involved in the LPG/GIPLs assembly, iii)
design and synthesis of mechanism based inhibitors of GPI pathway and iv) role
of parasitic GPIs and their synthetic analogues in PKC and PI-3-kinase
mediated signaling in the macrophages. The first distinct step in GPI biosynthesis is the
generation of N-acetylglucosaminyl-phosphatidylinositol (GlcNAc-PI) from
UDP-GlcNAc and specific PI substrate (different PI pools are used for GPI
anchor, LPG and GIPLs pathway in the parasite) catalyzed by the GPI-N-GlcNAc-transferase
(GPI-GnT), the GlcNAc-PI is then N-deacylated to form Glucosaminyl
phosphatidylinositol (GlcN-PI). This has been proposed that the synthesis of
GlcN-PI occurs on the cytoplasmic side of ER and then these intermediate GlcN-PI
and/or GlcN-acyl-PI translocate to the luminal side (mediated by a putative
“flippase”) where first three mannosyl residues are transferred from Dol-P-Man.
In order to establish microsomal enzyme system to
study substrate-specificity, stereochemical requirement and inhibition of
these enzymes (GPI-GnT, N-de-acylase and flippase), radiolabeled natural D
isomer of short-chain PI and its unnatural L analogue were synthesized using
phosphoramidite chemistry. Key intermediate D-2, 3, 4, 5, 6- penta- O- benzyl-
myo- inositol-1- O-benzyl- diisopropylphosphoramidite was prepared in a
multi-step sequence from racemic myo-inositol. The early steps of this
synthesis included, (a) preparation of bis-1, 2 :4, 5 -isopropylidene-myo-inositol
(cyclohexanone, PTSA) from racemic myo-inositol, (b) selective
4,5-de-acetalation by PTSA, EtOH, (c) benzylation and 1,2-deacetation by BnBr,
NaH followed by PTSA/Ethanol, (d) dibutyltin-oxide mediated regioselective
C1-OH allylation and C2-OH benzylation, (e) C1-OH de-allylation with Pd/C
conditions, (f) preparation of D and L camphanates (with L-camphanic-acid
chloride) and resolution of enantiomers to obtain 2, 3, 4, 5,
6-penta-O-benzyl-myo-D-inositol. This chiral intermediate was activated with
benzyl-NNNN,-tetra-isopropylphosphoramidite using
di-isopropylammonium-tetrazolite catalyst to get
2,3,4,5,6-penta-O-benzyl-myo-D-inositol-1-(O-benzyl-N,N-di-isopropylphosphoramidite.
This key intermediate was coupled (tetrazole as catalyst) with optically pure
radioactive [3-3H]-1,2-isopropylidene-sn-glycerol (prepared separately by
Swern oxidation and NaB3H4 reduction, 100 mCi, 500 mCi/mmole), followed by
oxidation (MCPBA, -40°C) to provide
[3H]-benzyl-1,2-isopropylidene-sn-glycerol-2,3,4,5,6-penta-O-benzyl-myo-D-inositol-phosphate.
This on deacetation (PTSA, MeOH), esterification (DCC/DMAP/Butyric-acid), and
final hydrogenolysis (Pd-C, H2, 50 psi) provided desired
[3H-1,2-di-O-acyl-sn-glyceryl-1-myo-D-inositol-phosphate (water soluble
short-chain PI). Similar approach was followed for preparation of unnatural
L-analogue. These materials are being used for establishment of microsomal
enzyme preparation of Leishmania parasite. This synthetic approach is also being extended for the
synthesis of PI-3-phosphate (PIP), PI-3,4-bisphosphate (PIP2) and
PI-3,4,5-triphosphate (PIP3) which are key substrates and effector molecules
involved in PI-3 kinase/akt signaling. Our efforts on devising a convergent synthetic
methodology towards parasitic GPI molecules have continued; this included
preparation of suitable inositol, lipid and glycan intermediates and bringing
them together to assemble required GPIs. The myo-inositol intermediate
1-O-PMB-2,3,4,5-tetra-O-benzyl-myo-D-inositol was prepared following two
independent approaches. The first route used optical-resolution approach
similar to the one described above for PI synthesis. The second and more
flexible route mediated by Ferrier reaction utilized D-glucose as starting
material. D-glucose was converted in four straightforward steps (glycosidation,
6-O-tritylation, benzylation, and detritylation) to
2,3,4-tribenzyl-aD-methyl-glucoside. This material was oxidized under Swern
condition (oxalyl chloride, DMSO, -78°C), converted (Ac2O, K2CO3) to the
corresponding 5-enol-acetate, which on Ferrier rearrangement (mercuric
acetate) provided D-enantiomer of 1-O-acetyl-3,4,5-tri-O-benzyl-6-inosose. The
stereospecific reduction of this inosose intermediate with sodium-acetoxyborohydride
followed by deacetylation gave 3,4,5-tri-O-benzyl-myo-D-inositol. Now the
important task was to obtain selectivity among three vicinal hydroxyl groups
and to block axial C2-OH in presence of equatorial C6-OH and C1-OH, and this
was feasible (although in lower yields) by initial C-1/C-2-dibutyltin
complexation followed by p-methoxybenzylation at C-1 and benzylation at C-1
position. The glycan intermediate
3,4,6-tri-O-benzyl-2-deoxy-2-azido-D-glucopyranosyl fluoride was prepared from
tri-O-acetylglucal by azidonitration chemistry. The glycerolipid intermediate
1, 2- isopropylidene- sn-glycerol- 3- (O-benzyl-N,N-di-isopropyl)-phosphoramidite
was prepared as described before. The inositol, glycan and lipid intermediate
are now being assembled. The most striking feature of LPG structure is the
variable phosphoglycan (PG) domain composed of [6-Gal(b1,4)Man(1a-PO4) repeats
linked together by phosphodiester groups. The PG repeats are signature motif
of phosphoglycan family of molecules expressed both in the promastigote (lipid
linked phosphoglycan such as LPG) and amastigote (protein linked phosphoglycan
PPG) phase of the parasite. The biosynthesis of PG repeats occurs inside the
Golgi (after the pre-assembled GPI core is translocated from ER to Golgi) and
involve a set of putative initiating and elongating Man-1a-PO4-transferases (iMPT
and eMPT respectively). These MPTs are unique to Leishmania parasite and are
capable of transferring intact Man-a-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 and trafficking of PG repeats and involvement of 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 and inhibitors of elongating-MPTs and GDP-Man
transporter activities. This year we have synthesized radio labeled
lipid-linked PG substrate, and also set up in vitro LPG biosynthetic and
GDP-Man-transporter assays using L.donovani Golgi vesicles prepared by sucrose
gradient ultra-centrifugation. We have also been able to devise a chemical
route for re-iterative synthesis of PG repeats of varying sizes, and
availability of these materials would enable us to study their solution
conformation by NMR and to use them for identifying MPT recognition elements.
The main feature of this glycal chemistry based phosphoglycan synthesis is the
application of single intermediate to generate higher oligomer of the PG
repeats through H-phospphonate chhemistry, which allowed PG chain to grow
either on C6’-OH or C1-OH ends. We have also established a synthetic route
for preparation of GDP-Man analogues. This involves preparation of TEA salt of
mannose-1-aphosphate (or deoxy/fluoro counterparts) and its coupling with
dicyclohexylammonium salt of GMP-morpholidate in presence of tetrazole
catalyst under argon. This synthetic route is being adopted for preparation of
deoxy- and fluoro- analogues of GDP-Man for Golgi transport inhibition
experiments. 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 this domain must involve at least three
interesting activities; (a) UDP-Galp-mutase to transform UDP-Galactopyranose
to UDP-Galactofuranose, (b) UDP-Galf transporter, and (c) UDP- Galf
transferase. This year we have 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. Publications Original peer-reviewed articles 1. Upreti M, Ruhela D and Vishwakarma RA (2000) Synthesis of the tetrasaccharide cap domain of the antigenic cell surface lipophosphoglycan of Leishmania donovani parasite. Tetrahedron 56:6577-6585. 2. *Sahai P, Chawala M and Vishwakarma RA (2000) 13C labeling and electrospray mass spectrometry reveal de novo biosynthesis route for inositol in Leishmania parasite. J Chem Soc, Perkin Trans 1, 1283-1290 (*in press last year, since published). 3. Nakra P, Manivel V, Vishwakarma RA and Rao KVS (2000) B cell responses to a primary response is thermodynamically regulated. J Immunol 164:5615-5625. 4. 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 (in press). Patents 1. Jain DC, Bhakuni RS, Saxena S, Kumar S and Vishwakarma RA (2000) New synthetic method for preparation of antimalarial arteethers from dihydroartemisinin, US patent application # 09/535513 filed on 24 Mar 2000. 2. Balakrishnan A and Vishwakarma RA (2000) Lignan compounds with anti-proliferative properties, Indian patent application # PCT/IN00/00063 filed on 16 Jun 2000. |