Preparation of synthetic oligosaccharide derivatives of secondary cell wall polysaccharides from Bacillus stearothermophilus PV72

Recently, the role has been described of two distinct secondary cell wall polymers (SCWP) in the specific anchoring of two different S-layer proteins to the cell walls of Bacillus stearothermophilus PV72 variants p2 or p6 (Egelseer, et al., 1998; Schuster, et al., 1997). In a companion project, the biological characterization and functional application has been undertaken of the little-known, lectin-type protein--carbohydrate interactions between the apposing S-layer proteins and the SCWP (M. Sara and U. B. Sleytr, Crystalline Bacterial Cell Surface Layers (S-Layers), S-Layer Associated Cell Wall Polysaccharides and S-Layer Associated Exoenzymes as Building Blocks for Supramolecular Engineering, Project 12938--MOB). Presently, it is proposed to synthesize oligosaccharide derivatives corresponding to part structures of the p6- and p2-related polysaccharides. Such compounds will supplement and strengthen the efforts of the companion project by serving as construction elements for supramolecular engineering with S-layer proteins, as tools for elucidating the biological function of the polysaccharides, and as model compounds for the study of the underlying protein-- carbohydrate interactions. The SCWP of the p6-variant is a regular polysaccharide containing 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid, N-acetylglucosamine and glucose in a ratio of 2: 1: 1; the structure of the repeating unit has been proposed to be -4)-beta-ManpA2,3(NAc)2 -(1 -3)-alpha-GlcpNAc-(l -4)-beta-ManpA2,3(NAC) 2 -( 1-6)-alpha-Glcp(l- (Messner, et al., 1987; Egelseer, et al., 1998). The more complex, p2-related SCWP is a polysaccharide composed of N-acetylglucosamine and N-acetylmannosamine in a ratio of 2:1 and contains pyruvate ketal groups. The structure of this material is presently being elucidated (Kosma, et al., unpublished). Starting with the p6-related structures, a new model compound corresponding to a beta-ManpA2,3(NAc)2 unit will be prepared during year one of the 3-year project period. At the same time, a practical glycosylation procedure will be developed for the introduction of beta-ManpA2,3(NAC) 2 units into oligosaccharides. In the preferred approach, a new alpha-trichloroacetimidate donor derived from 2-azido-2-deoxy-D-altropyranose will be investigated; after coupling, the plan involves introduction of the second nitrogen function with inversion at C3, and selective oxidation at C-6. In an alternative approach, introduction of the PManpA2,3(NAC) 2 units would be attempted using a glycosyl donor derived from 3-azido-3-deoxy-D-glucose; subsequent to coupling, the second nitrogen function would be introduced as an azido group with inversion at C-2, and the sugar unit selectively oxidized at C- 6. Also during the first year, the suitability will be explored of D-mannopyranose donors as precursors of the alpha- D-glucopyranoside (alpha-Glc) and 2-acetamido-2-deoxy-alpha-D-glucopyranoside (alpha-GlcNAc) units.-- For year 2, the plan is to synthesize disaccharide building blocks and target compounds corresponding to the part structures beta-ManpA2,3(NAc)2 -(l-6)-alpha-Glcp and beta-ManpA2,3(NAc)2 -(1-3)-alpha-GcpNAc. At the same time, model structures will be designed for the GlcNAc/ManNAc-containing, p2-type SCWP, and appropriate monosaccharide precursors explored.-- Finally, for year 3, it is foreseen to assemble tetra- -and octasaccharide target compounds, to produce a set of corresponding neoglycoconjugates, and to prepare some chemically modified oligosaccharide analogs. Regarding the p2-type SCWP, syntheses of a first set of target compounds are envisioned for year 3.-- For the required biochemical and functional studies, the model saccharide derivatives will be made available in the form of glycoconjugates derived from allyl glycosides. Such glycosides can be conjugated to functional molecules by a wide variety of methods described in the literature.