Streptococcus pneumoniae (SP) is a key pathogen that induces various infectious diseases such as pneumonia, otitis media, and meningitis. As a core immunogen, its capsular polysaccharide serves as the key target for the research and development of pneumococcal vaccines. The chemical synthesis of structurally well-defined and homogeneous oligosaccharides plays a pivotal role in elucidating the structure-activity relationship underlying the immunological activity of polysaccharides and in the development of novel glycoconjugate vaccines. This study achieved the efficient chemical synthesis of two trisaccharide intermediates of the capsular polysaccharide from Streptococcus pneumoniae serotype 9N. Starting from readily available D-mannose and D-glucose, a reducing-end to non-reducing-end synthetic strategy was adopted. By optimizing key reaction parameters including protecting group screening and solvent effect regulation, two types of 1,2-cis glycosidic linkages were efficiently constructed. Specifically, the yield of the 1,2-cis-α-glycosidic bond reached 90%, whereas that of the 1,2-cis-β-glycosidic bond was tripled, with its stereoselectivity significantly improved. Furthermore, the issue of low nucleophilicity at the C4 position of amino sugars was resolved by the introduction of electron-donating protecting groups at the hydroxy groups adjacent to the receptor glycosylation sites. Eventually, two target trisaccharide intermediates were efficiently synthesized in 11 and 10 total steps, respectively, laying a solid foundation for the preparation of the pentasaccharide repeating unit and longer-chain oligosaccharides of the Streptococcus pneumoniae serotype 9N capsular polysaccharide.

Pneumococcal vaccine plays a key role in preventing diseases such as pneumonia and meningitis caused by Streptococcus pneumoniae(S.pneumoniae).Capsular polysaccharide,C-polysaccharide and phosphorus content are important indicators for quality control of polysaccharide antigens in vaccine development and production.In this study,a quantitative 1H NMR and 31P NMR method based on a single internal standard hexamethylphosphoramide(HMPA)was developed to achieve simultaneous determination of capsular polysaccharide,C-polysaccharide and phosphorus content in 6A,6B,18C,19A,19F and 23F S.pneumoniae polysaccharide antigens.Using the internal reference comparison method,the effect of solubility of polysaccharide on quantitative 1H NMR determination was investigated.It was found that the determination results of quantitative 1H NMR were affected by the viscosity and concentration of polysaccharide solution.It was found that high viscosity polysaccharides at 3-15 mg/mL and low viscosity polysaccharides at 5-25 mg/mL were the optimal detection solution concentration range.This"one internal standard three quantitative"NMR method has good precision,specificity and accuracy,and provides a valuable new strategy for the quality control of pneumococcal vaccine.