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 synt hesised fragments of the capsular polysaccharide of Streptococcus pneumonia e type 3, that is beta -D-GlcpA- (1 --> 4)-beta -Glcp-(1 --> O)-(CH2)(3)NH2 (1), beta -Glcp-(1 --> 3)-beta -GlcpA-(1 --> 4)-beta -D-Glcp-(1 --> O)-(CH 2)(3)NH2 (2), and beta -D-GlcpA-(1 --> 4)-beta -D-Glcp-(1 --> 3)-beta -D-Gl cpA-(1 --> 4)-beta -D-Glcp-(1 --> O)-(CH2)(3)NH2 (3). A blockwise approach was developed for the synthesis of the protected carbohydrate chains, in wh ich the carboxylic groups were introduced prior to deprotection by selectiv e oxidation of HO-6 in the presence of HO-4 by using TEMPO (2,2,6,6-tetrame thyl-1-piperidinyloxy radical). After deprotection, the 3-aminopropyl space r of the fragments was elongated with diethyl squarate (3,4-diethoxy-3-cycl obutene- 1,2-dione) and the elongated oligosaccharides were conjugated to C RM197 (cross-reacting material of diphtheria toxin), KLH (keyhole limpet he mocyanin) or TT (tetanus toxoid). The resulting neoglycoconjugates varied i n oligosaccharide chain length, oligosaccharide loading and protein carrier . These well-defined conjugates are ideal probes for evaluating the influen ce of the different structural parameters in immunological tests.