Kerry B. Reimer

Small-scale solid-phase O-glycopeptide synthesis of linear and cyclized hexapeptides from blood-clotting factor IX containing O-(α-D-Xyl-1→3-α-D-Xyl-1→3-β-D-Glc)-L-ser

Glycopeptide sequences corresponding to residues 51–56 of the EGF-like domains of human and bovine blood-clotting factor IX have been synthesized using the Fmoc/Dhbt strategy. Building blocks consisting of Fmoc-Ser(R)-OPfp, where R=β-D-Glc or α-D-XyI-(1–3)-α-D-XyI-(1–3)-α-D-Glc, have been synthesized. The building blocks were prepared by treatment of the unprotected glycosylated serine compounds with N-(fluoren-9-ylmethoxycarbonyl)succinimide in 1,4-dioxane, followed by treatment with pentafluorophenol and dicyclohexylcarbodiimide in tetrahydrofuran. The glycosylated building blocks were then used in a solid-phase peptide synthesis to give the corresponding glycopeptides. Cyclic glycopeptides were prepared from the acetamidomethylprotected linear glycopeptides by treatment with thalliunl(III) trifluoroacetate in trifluoroacetic acid. The cyclic glycopeptides were fully characterized by NMR spectroscopy and mass spectrometry.

Immunochemical characterization of polyclonal and monoclonal Streptococcus group A antibodies by chemically defined glycoconjugates and synthetic oligosaccharides.

Synthetic oligosaccharides of increasing complexity that represent different epitopes of the Streptococcus Group A cell-wall polysaccharide were used as haptens and glycoconjugates of bovine serum albumin (BSA) and horse hemoglobin (HHb) to characterize polyclonal and monoclonal antibodies. Rabbits were immunized with the BSA glycoconjugates of a linear trisaccharide, branched trisaccharide, and branched pentasaccharide. The binding specificities of the polyclonal antisera were determined by a series of inhibition ELISA studies in which disaccharide through pentasaccharide haptens were used as inhibitors of antibody-glycoconjugate binding. Monoclonal antibodies derived from mice immunized with a killed bacterial vaccine were selected for their binding to native polysaccharide antigen coupled to BSA and the BSA glycoconjugates of the di- and linear tri-saccharides. Polyclonal antibodies were moderately specific for the oligosaccharide epitope of the immunizing glycoconjugate and only those antibodies raised to the branched pentasaccharide antigen showed cross-reaction with the bacterial antigen. The behaviour of selected monoclonal antibodies parallels the binding profile of polyclonal antibodies in that the two highest-titre antibodies were directed toward an epitope displayed by the branched pentasaccharide.

Synthesis and n.m.r. analysis of branched trisaccharide and pentasaccharide haptens of the β-hemolytic streptococci group A and the preparation of synthetic antigens☆

The synthesis of branched trisaccharide and pentasaccharide portions of the cell-wall polysaccharide of the beta-hemolytic Streptococci Group A is described. The key dissaccharide acceptors, allyl or 8-(methoxycarbonyl)ocytol 3-O-(3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-4-O -benzyl - alpha-L-rhamnopyranoside, in conjunction with a selectively blocked alpha-L-rhamnopyranosyl chloride under Koenigs-Knorr conditions, afforded the branched trisaccharides in 81 and 62% yield, respectively. Analogously, glycosylation of the 8-(methoxycarbonyl)octyl disaccharide with a protected beta-D-GlcpNAc-(1----3)-alpha-L-Rhap-(1----3)-alpha-L-Rhap chloride gave the pentasaccharide in 43% yield. The key disaccharide acceptors were obtained, in turn, from the allyl or 8-(methoxycarbonyl)octyl rhamnoside acceptors and 3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl chloride under Koenigs-Knorr conditions. The latter glycosyl donor has not been described previously. Removal of the protecting groups afforded the trisaccharide haptens as their 1-propyl and 8-(methoxycarbonyl)octyl glycosides and the pentasaccharide as its 8-(methoxycarbonyl)octyl glycoside. The compounds have been subjected to detailed analysis by two-dimensional n.m.r. methods. Preparation of the synthetic antigens followed coupling of the 8-(methoxycarbonyl)octyl glycosides to bovine serum albumin via the acyl azide intermediates.

Convergent synthesis of higher-order oligosaccharides corresponding to the cell-wall polysaccharide of the β-hemolytic streptococci group A. a branched hexasaccharide hapten

A convergent synthesis of a hexasaccharide corresponding to the cell-wall polysaccharide of the beta-hemolytic Streptococci Group A is described. The strategy relies on the preparation of a key branched trisaccharide unit alpha-L-Rhap-(1----2)-[beta-D-GlcpNAc-(1----3)]-alpha-L-Rhap which functions both as a glycosyl acceptor and donor. The hexasaccharide is obtained after only three glycosylation reactions. This fully functionalized unit can serve, in turn, as a glycosyl acceptor or donor for the synthesis of higher-order structures. Deprotection gives a hexasaccharide for use as a hapten in immunochemical studies. The characterization of all compounds by high resolution 1H- and 13C-n.m.r. spectroscopy is also described.

Oligosaccharides corresponding to biological repeating units of Shigella flexneri variant Y polysaccharide: part 3. Synthesis and 2D-nuclear magnetic resonance analysis of a heptasaccharide hapten

The block synthesis of a heptasaccharide portion of the biological repeating unit, [2)-α-L-Rhap-(1→2)-α-L-Rhap-(1→3)-α-L-Rhap-(1→3)-β-D-GlcpNAc-(1], of the Shigella flexneri variant Y polysaccharide is described. The synthetic strategy relies on the use of the key trisaccharide intermediate α-L-Rhap-(1→2)-α-L-Rhap-(1→3)-α-L-Rhap, both as a glycosyl acceptor and as a donor. Thus, the trisaccharide bromide in conjunction with the β-D-GlcpNPhth-(1→2)-α-L-Rhap-(1→2)-α-L-Rhap-(1→3)-α-L-Rhap unit under Helferich conditions yielded the blocked heptasaccharide in 86% yield. The latter unit was obtained, in turn, from the key trisaccharide intermediate functioning as an acceptor molecule. Attempts at coupling the tetrasaccharide donor, α-L-Rhap-(1→2)-α-L-Rhap-(1→3)-α-L-Rhap-(1→3)-β-D-GlcpNPhth, with the trisaccharide acceptor α-L-Rhap-(1→2)-α-L-Rhap-(1→3)-α-L-Rhap, to give the heptasaccharide under a variety of conditions were unsuccessful. The blocked derivatives were synthesized as their allyl glycosides. Removal of the blocking groups, hydrogenation of the allyl group, and N-acetylation yielded the heptasaccharide hapten, α-L-Rhap-(1→2)-α-L-Rhap-(1→3)-α-L-Rhap-(1→3)-β-D-GlcpNAc-(1→2)-α-L-Rhap-(1→2)-α-L-Rhap-(1→3)-α-L-Rhap, as its propyl glycoside, for use in inhibition studies with complementary monoclonal antibodies, and in NMR and X-ray studies. The detailed NMR analysis of the protected and deprotected heptasaccharides by use of two-dimensional NMR techniques is also described.

Synthesis of oligosaccharides corresponding to the antigenic determinants of the β-haemolytic Streptococci Group A. Part 1. Overall strategy and synthesis of a linear trisaccharide

The overall strategy for the synthesis of higher-order oligosaccharides corresponding to the repeating unit of the cell-wall polysaccharide of the β-haemolytic Streptococci Group A is described. The trisaccharide, β-D-GlcpNAc-(1→3)-α-L-Rhap-(1→3)-α-L-Rhap has been synthesized by a series of Konigs-Knorr reactions. The selectively protected rhamnose derivative, allyl 2-O-benzoyl-4-O-benzyl-α-L-rhamnopyranoside, reacted with 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl bromide to give the blocked disaccharide. Deallylation, followed by treatment with N,N-dimethyl(chloromethylene)ammonium chloride then gave the corresponding disaccharide chloride. In conjunction with the same rhamnose monosaccharide unit or 8-methoxycarbonyloctyl 2,4-di-O-benzoyl-α-L-rhamnopyranoside, the synthesis of the blocked trisaccharide, as its allyl glycoside or its 8-methoxycarbonyloctyl glycoside, respectively, was accomplished. Transesterification, followed by hydrazinolysis, selective N-acetylation, and hydrogenolysis afforded the pure trisaccharide, as its propyl glycoside or 8-methoxycarbonyloctyl glycoside, for use as a hapten in binding studies and n.m.r. studies or for use in the preparation of glycoconjugates, respectively. Similar treatment of the blocked disaccharide afforded the hapten, β-D-GlcpNAc-(1→3)-α-LRhap, as its propyl glycoside.