Design, synthesis and immunological evaluation of novel monosaccharide TLR4 agonists

Monophosphoryl lipid A (MPLA), a Toll-like receptor 4 (TLR4) agonist, has been utilized as vaccine adjuvant in several approved human vaccines. However, the complex disaccharide structure of MPLA presents significant challenges for chemical synthesis. To address this limitation, we designed a series of glucosamine-based alkyl 4-phosphate (GAP) compounds. These are monosaccharide lipid A analogues, synthesized by replacing the reducing glucosamine disaccharide of MPLA with an N-diaminoalkyl glycoside unit. Three novel TLR4 agonists, GAP210, GAP212 and GAP214, were successfully synthesized by modulating the length of the secondary acyl chains at the C-2 and C-3 positions of the non-reducing sugar, and were subsequently evaluated as vaccine adjuvants. The results revealed that the length of the secondary acyl chains significantly influenced immune activation. Notably, GAP214-adjuvanted group exhibited 2.5-fold higher IgG level than the positive TLR4 agonist control after three immunizations. These findings identify the GAP analogues as effective TLR4 agonists and promising candidates for vaccine adjuvant development.