Application of MPEG soluble polymer support in the synthesis of oligo- phosphosaccharide fragments from the Leishmania lipophosphoglycan

Article abstract of DOI:10.1016/S0040-4039(00)00153-2

A polymer (MPEG) supported synthesis of the phosphorylated tetra- and hexa-saccharide fragments of the lipophosphoglycan from Leishmania has been developed using mono- and di-saccharide H-phosphonates for construction of the phosphodiester bridges. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)00153-2

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 2449–2452
Application of MPEG soluble polymer support in the synthesis of
oligo-phosphosaccharide fragments from the Leishmania
lipophosphoglycan
Andrew J. Ross, Irina A. Ivanova, Adrian P. Higson and Andrei V. Nikolaev ∗
Department of Chemistry, University of Dundee, Dundee DD1 4HN, UK
Received 26 November 1999; accepted 18 January 2000
Abstract
A polymer (MPEG) supported synthesis of the phosphorylated tetra- and hexa-saccharide fragments of the
lipophosphoglycan from Leishmania has been developed using mono- and di-saccharide H-phosphonates for
construction of the phosphodiester bridges. © 2000 Elsevier Science Ltd. All rights reserved.
Keywords: carbohydrates; polymer support; phosphorous acid and derivatives; phosphoric acids and derivatives.
One of the major molecules forming the glycocalyx of the Leishmania parasite is the lipophosphogly-
can (LPG), which is produced by the infectious promastigote stage of all species of the parasite. The LPG
has been shown to be essential for parasite infectivity and survival1 thus making the enzymes responsible
for its biosynthesis of great interest. It contains a polymeric phosphoglycan region consisting of (1→6)-
linked β-D-galactosyl-(1→4)-α-D-mannosyl phosphate repeating units, which has been shown2 to be
assembled in vitro by the sequential action of the Leishmania α-D-mannopyranosylphosphate transferase
(MPT) and β-D-galactopyranosyl transferase (GT). We have recently described chemical syntheses of
oligo-phosphosaccharide substrates3 and substrate analogues4 for characterization of the MPT. Here we
report the chemical synthesis of the tetraglycosyl diphosphate 1 and hexaglycosyl triphosphate 2, which
are fragments of the LPG containing an α-D-Manp phosphate unit at the non-reducing end and are
designed to be acceptor substrates for the GT in the Leishmania. Both compounds contain a dec-9-enyl
aglycone moiety to assist biochemical assays.
We have demonstrated previously that oligo-phosphosaccharides composed of glycosyl phosphate re-
peating units can be prepared using H-phosphonate condensation protocols and stepwise,3,5 blockwise,6
or polycondensation7 chain elongation strategies. However, no polymer-supported synthesis of an
oligo(glycosyl phosphate) has been reported so far. Compounds 1 and 2 were prepared in a stepwise
∗
Corresponding author.
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)00153-2
tetl 16433

2450
manner using the H-phosphonates 33 and 48 for the chain elongation, the β-D-galactoside derivative 9
(Scheme 1) as a DMT-protected hydroxyl acceptor and monomethyl polyethylene glycol (MPEG, M
5000, loading capacity 0.2 mmol/g) as a polymer support. The advantage of MPEG is that it allows for
both solid-phase and solution-phase techniques.9 MPEG-bound products can be precipitated from ether
or crystallized from cold ethanol greatly simplifying their purification. They are, however, soluble in
most other organic solvents; this helps to avoid the pseudo-high dilution problems (at the condensation
step) associated with solid-phase approaches.
Scheme 1. Reagents: (i) NaOMe, MeOH; (ii) TrCl, DMAP, pyridine; (iii) Me2C(OMe)2, TsOH·H2O, MeCN; (iv) BzCl,
pyridine; (v) 80% AcOH, 70°C; (vi) (EtO)3CPh, TsOH·H2O, MeCN; (vii) 80% AcOH, rt; (viii) DMTCl, pyridine; (ix) succinic
anhydride, DMAP, pyridine; (x) (a) MPEG, MSNT, 1-methylimidazole, DCM; (b) Ac2O, pyridine
A succinic diester linker9 was used for binding the first β-D-galactoside unit 9 to the polymer.
It allowed, in combination with benzoic esters (in 3, 4 and 9) as permanent O-protecting groups,
for one-step total deprotection to produce the final products 1 and 2. The 3-O-succinoyl derivative
9 was chosen over its 2-O- or 4-O-succinoyl isomers to avoid closeness of the linker to either the
hydrophobic dec-9-enyl aglycone (that could impede its effective coupling to MPEG) or 6-HO-group
(that could hinder effective formation of the (1→6)-phosphodiester linkage). Compound 9 was prepared
(Scheme 1) starting from acetobromogalactose and dec-9-en-1-ol, which reacted in the presence of
Hg(CN)2/HgBr2 to give the galactoside 510 (60%). Consecutive deacetylation, 6-O-tritylation, treatment
with 2,2-dimethoxypropane and conventional benzoylation produced the 2-benzoate 610 (86%). It was
converted to the 2,4-dibenzoate 810 (45%) by successive acid hydrolysis (→710), reaction with triethyl
orthobenzoate to form the corresponding 3,4- and 4,6-orthoesters and their acidic opening, which gives,
preferentially,11 the axial 4-benzoate. Treatment of 8, first with DMTCl in pyridine and then with succinic
anhydride and DMAP resulted in the 6-O-DMT-3-O-succinoyl-β-D-galactoside 910 in 78% yield.
The derivatization of MPEG began by coupling with 9 in the presence of 1-(2-mesitylenesulfonyl)-3-
nitro-1,2,4-triazole (MSNT) and 1-methylimidazole followed by precipitation of the product 10 from
ether. The extent of loading 9 on the support was found to be 0.182 mmol/g (91%), as determined

2451
spectrophotometrically12 at 498 nm after quantitative release of the DMT-cation under acidic conditions.
A capping step (Ac2O/pyridine) was used to block any residual MPEG hydroxyl groups.
A chain elongation cycle for the preparation of the protected oligomers 13 and 14 (Scheme 2) is listed
in Table 1. It started by cleavage of the DMT-ether 10 with 1% TFA in DCM followed by neutralization
and precipitation of the product from ether. The first elongation step was performed via pivaloyl chloride
mediated coupling of the disaccharide H-phosphonate 3 followed by oxidation with iodine to provide
the MPEG-bound trisaccharide 11. It should be noted that a standard work-up procedure is required
after the oxidation (see Table 1 and Refs 3–8) to allow the complete precipitation of the product from
ethanol. The use of ethanol (not ether) provided the isolation of the pure polymer-bound material (11)
free of any H-phosphonate contaminants. For the preparation of compound 1, the above set of procedures
and the monosaccharide H-phosphonate 4 were used to end the chain extension (11→13). Subsequent
deprotection (0.1 M methanolic NaOMe) afforded the oligo-phosphosaccharide 1, which was isolated by
anion exchange chromatography13 in 40% yield based on the galactoside 9 (corresponding to an average
89% yield per step).
Scheme 2. Reagents: (i) TFA, DCM; (ii) pivaloyl chloride, pyridine; (iii) I2, pyridine–water; (iv) NaOMe, MeOH
Table 1
Protocol for the chain elongation cycle
The preparation of the hexaglycosyl triphosphate 2 was done in a similar fashion, but was accom-
plished using an optimized protocol for chain elongation: (1) a double amount of the H-phosphonates

2452
3 and 4 (threefold molar excess instead of 1.5 molar) was used in coupling reactions, and (2) at the
end of each cycle, the residual 6-HO-groups of D-galactose were blocked by repeating the condensation
and oxidation steps (see Table 1). This enhanced the efficiency of each elongation cycle (determined, as
above, spectrophotometrically at 498 nm for the preparation of 11 and 12; Scheme 2) to 97%. After the
final chain extension (→14) and total deprotection (0.05 M methanolic NaOMe, 3 h), the oligomer 2 was
isolated13 in 57% yield (corresponding to an average 95% yield per step starting from 9). The structures
of compounds 1 and 2 were confirmed by NMR14 and electrospray-MS data.15
To summarize, the first polymer-supported synthesis of fragments of a natural phosphoglycan compos-
ed of glycosyl phosphate units has been performed using the H-phosphonate methodology. A biochemical
evaluation of compounds 1 and 2 will be published elsewhere in due course.
Acknowledgements
This work was supported by the Wellcome Trust both International (for I.A.I) and Project (for A.P.H.)
Grants. One of us (A.J.R.) thanks the BBSRC for the award of a studentship. The research of A.V.N was
supported by an International Research Scholar’s award from the Howard Hughes Medical Institute.
References
1. McConville, M. J.; Ferguson, M. A. J. Biochem. J. 1993, 294, 305.
2. Carver, M. A.; Turco, S. J. J. Biol. Chem. 1991, 266, 10974.
3. Nikolaev, A. V.; Rutherford, T. J.; Ferguson, M. A. J.; Brimacombe, J. S. J. Chem. Soc., Perkin Trans. 1 1995, 1977; Higson,
A. P.; Tsvetkov, Y. E.; Ferguson, M. A. J.; Nikolaev, A. V. J. Chem. Soc., Perkin Trans. 1 1998, 2587; Higson, A. P.; Tsvetkov,
Y. E.; Ferguson, M. A. J.; Nikolaev, A. V. Tetrahedron Lett. 1999, 40, 9281.
4. Ivanova, I. A.; Ross, A. J.; Ferguson, M. A. J.; Nikolaev, A. V. J. Chem. Soc., Perkin Trans. 1 1999, 1743; Ross, A. J.;
Higson, A. P.; Ferguson, M. A. J.; Nikolaev, A. V. Tetrahedron Lett. 1999, 40, 6695.
5. Nikolaev, A. V.; Ivanova, I. A.; Shibaev, V. N. Carbohydr. Res. 1993, 242, 91.
6. Nikolaev, A. V.; Rutherford, T. J.; Ferguson, M. A. J.; Brimacombe, J. S. J. Chem. Soc., Perkin Trans. 1 1996, 1559.
7. Nikolaev, A. V.; Chudek, J. A.; Ferguson, M. A. J. Carbohydr. Res. 1995, 272, 179.
8. Nikolaev, A. V.; Ivanova, I. A.; Shibaev, V. N.; Kochetkov, N. K. Carbohydr. Res. 1990, 204, 65.
9. MPEG was used for polymer-supported syntheses of peptides (Bayer, E.; Mutter, M. Nature 1972, 237, 512),
oligonucleotides (Bonora, G. M.; Scremin, C. L.; Colonna, F. P.; Garbesi, A. Nucleic Acids Res. 1990, 18, 3155),
ribosylribitol phosphate oligomers (Kandil, A. A.; Chan, N.; Chong, P.; Klein, M. Synlett 1992, 555) and oligosaccharides
(Krepinsky, J. J. In Modern Methods in Carbohydrate Synthesis; Khan, S. H.; O’Neill, R. A., Eds.; Harwood Academic
Publishers: Amsterdam, 1996; pp. 194–224).
10. NMR data and elemental analysis for compound 5, 6, 7, 8 and 9 are consistent with the structures.
11. Lemieux, R. U.; Driguez, H. J. Am. Chem. Soc. 1975, 97, 4069.
12. Gait, M. J.; Mathes, H. W. D.; Singh, M.; Sproat, B. S.; Titmas, R. C. Nucleic Acids Res. 1982, 10, 6243.
13. Compounds 1 and 2 were isolated on a column of Fractogel TSK DEAE-650 (S), HCO3−-form, eluted with a linear gradient
(0→0.3 M) of NH4HCO3 in 40% aq. propan-2-ol. [α]D values (in degrees, at 24°C in water): for 1 +23.5 (c 1); for 2 +19 (c
0.3).
14. 31P NMR data (121 MHz, D2O): for 1 δP −2.02; for 2 δP −1.97. Selected 13C NMR data (75 MHz, D2O): for 1 δC 61.32
(C-6, Man), 61.92 (C-6, Man0), 65.20 (JC,P 3.4, C-6, Gal), 65.55 (JC,P 3.4, C-6, Gal0), 70.90 (JC,P 8.0, C-2, Man), 71.45
(JC,P 8.0, C-2, Man0), 74.44 (JC,P 8.1, C-5, Gal), 74.80 (JC,P 8.1, C-5, Gal0), 96.93 (JC,P 4.9, C-1, Man), 97.17 (JC,P 4.9, C-1,
Man0), 103.82 (C-1, Gal), 104.39 (C-1, Gal0); for 2 δC 61.30 (2C, C-6, Man and Man0), 61.90 (C-6, Man00), 65.20 (JC,P 3.7,
C-6, Gal), 65.47 (2C, br, C-6, Gal0 and Gal00), 70.94 (2C, JC,P 8.1, C-2, Man and Man0), 71.48 (JC,P 7.1, C-2, Man00), 74.45
(JC,P 8.1, C-5, Gal), 74.77 (2C, JC,P 8.1, C-5, Gal0 and Gal00), 96.88 (2C, JC,P 4.0, C-1, Man and Man0), 97.18 (JC,P 5.2, C-1,
Man00), 103.79 (C-1, Gal), 104.36 (2C, C-1, Gal0 and Gal00).
15. The main signals in the ES(−) mass spectra corresponded to the pseudo-molecular ions for the diphosphate 1 (m/z 480.90
[M−2 H]2−) and triphosphate 2 (m/z 454.95 [M−3 H]3−).