Conformationally Constrained Phosphonate Backbone Linkages Enhance CpG Oligonucleotide Activation of Toll-Like Receptor 9.

The effect of flexible versus conformationally constrained phosphonate internucleotide linkages on CpG-mediated TLR9 activation was investigated. Sixteen analogues of the CpG oligonucleotide ODN 2006, a well-known TLR9 agonist, uniformly modified at all CpG motifs, were synthesized and evaluated in a human TLR9 (hTLR9) reporter cell line and peripheral blood mononuclear cells. Analogues bearing conformationally constrained 2',3'-cytidine phosphonate modifications (ODN1-ODN10) generally exhibited enhanced activity relative to ODN 2006, with ODN1 (incorporating modification I) displaying ∼30-fold increased potency. Analogues with flexible phosphonate linkages (ODN11, ODN13, ODN15, ODN16) showed comparable activity to that of the parent ODN, whereas additional sugar modification abolished activity (ODN12, ODN14). A hexamer (ODN-B) incorporating the most potent modification I, when combined with CpG DNA, enhanced hTLR9 activation, consistent with interaction at the auxiliary 5'-xCx site. This study evaluates phosphonate internucleotide linkages in CpG oligonucleotides and demonstrates that a conformationally constrained phosphonate backbone in ODN 2006 analogues enhances hTLR9 activation, offering a potential strategy for the development of potent immunostimulatory oligonucleotides.