Stereoisomeric Pam2CS based TLR2 agonists: synthesis, structural modelling and activity as vaccine adjuvantsElectronic supplementary information (ESI) available: Complete details of in silicodocking studies regarding scripts, 1H NMR, 13C NMR, mass spectra of all the intermediates and final compounds, list of interacting residues and surface representation of TLR2/6 heterodimer with the enantiopure final compounds, table to correlate all the available data, including computation, in vitroand in vivodata. See DOI: https://doi.org/10.1039/d1md00372k

Lipopeptides including diacylated Pam2CSK4as well as triacylated Pam3CSK4act as ligands of toll-like receptor (TLR)-2, a promising target for the development of vaccine adjuvants. The highly investigated Pam2CSK4and Pam3CSK4, despite their aqueous solubility have not performed well as vaccine adjuvants which may be attributable to potential denaturation of protein antigens by these cationic surfactant-like lipopeptides. In the present investigation, we synthesized (R), (S) and racemic Pam2CS(OMe) analogs and their N-acetyl derivatives without the tetralysine component to systematically investigate the effect of stereochemistry at the thio-glycerol lipopeptide core of these lipopeptide based TLR2 agonists. The resulting compounds were compared using TLR2 reporter cell-based assays and the ability of the synthesized lipopeptides to stimulate cytokine production (IL-6, IL-10 and TNF-α) by freshly collected human PBMCs and CD40 and CD86 expressions by mouse spleen cells was also investigated. Notably, few synthesized lipopeptides were found to be potent TLR2/6 agonists, inducing cytokine production and upregulating CD40 and CD86 expressions. The TLR2/6 agonistic lipopeptides were further assessed for vaccine adjuvant effects in mice. The results confirmed that the R-stereochemistry at the thio-glycerol lipopeptide core was preferred for maximal TLR2/6 activity, as reflected in Th1 immune deviation, higher antibody levels and enhanced vaccine protection against a lethal influenza challenge.