Synthesis of Streptococcus pneumoniae type 3 neoglycoproteins varying in oligosaccharide chain length, loading and carrier protein

The preparation is described of a range of neoglycoproteins containing synthesised fragments of the capsular polysaccharide of Streptococcus pneumoniae type 3, that is β-D-GlcpA(1 → 4)-β-D-Glcp-(1 → O)-(CH2)3NH2 (1), β-D-Glcp-(1 → 3)-β-D-GlcpA-(1 → 4)β-D-Glcp-(1 → O)-(CH2)3NH2 2, and β-D-GlcpA-(1 → 4)-β-D-Glcp-(1 → 3)-βD-GlcpA-(1 → 4)-β-D-Glcp-(1 → O)(CH2)3NH2 (3). A blockwise approach was developed for the synthesis of the protected carbohydrate chains, in which the carboxylic groups were introduced prior to deprotection by selective oxidation of HO-6 in the presence of HO-4 by using TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy radical). After deprotection, the 3-aminopropyl spacer of the fragments was elongated with diethyl squarate (3,4-diethoxy-3-cyclobutene-1,2-dione) and the elongated oligosaccharides were conjugated to CRM197 (cross-reacting material of diphtheria toxin), KLH (keyhole limpet hemocyanin) or TT (tetanus toxoid). The resulting neoglycoconjugates varied in oligosaccharide chain length, oligosaccharide loading and protein carrier. These well-defined conjugates are ideal probes for evaluating the influence of the different structural parameters in immunological tests.