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)‐β‐D‐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. Ideal probes made of well‐defined conjugates have been synthesised (see for example figure) using di‐, tri‐ and tetrasaccharide fragments, related to the capsular polysaccharide of Streptococcus pneumoniae type 3, and different protein carriers. These conjugates were then used for the evaluation of the influence of the different structural parameters in immunological tests.