Antigen 85C-mediated acyl-transfer between synthetic acyl donors and fragments of the arabinan.

Antigen 85 (ag85) is a complex of acyltransferases (ag85A-C) known to play a role in the mycolation of the D-arabino-D-galactan (AG) component of the mycobacterial cell wall. In order to better understand the chemistry and substrate specificity of ag85, a trehalose monomycolate mimic p-nitrophenyl 6-O-octanoyl-beta-D-glucopyranoside (1) containing an octanoyl moiety in lieu of a mycolyl moiety was synthesized as an acyl donor. Arabinofuranoside acceptors, methyl alpha-D-arabinofuranoside (2), methyl beta-D-arabinofuranoside (3), and methyl 2-O-beta-D-arabinofuranosyl-alpha-D-arabinofuranoside (9) were synthesized to mimic the terminal saccharides found on the AG. The acyl transfer reaction between acyl donor 1 and acceptors 2, 3, and 9 in the presence of ag85C from Mycobacterium tuberculosis (M. tuberculosis) resulted in the formation of esters, methyl 2, 5-di-O-octanoyl-alpha-D-arabinofuranoside (10), methyl 5-O-octanoyl-beta-D-arabinofuranoside (11), and methyl 2-O-(5-O-octanoyl-beta-D-arabinofuranosyl)-5-O-octanoyl-alpha-D-arabinofuranoside (12) in 2 h, 2 h and 8 h, respectively. The initial velocities of the reactions were determined with a newly developed assay for acyltransferases. As expected, the regioselectivity corresponds to mycolylation patterns found at the terminus of the AG in M. tuberculosis. The study shows that D-arabinose-based derivatives are capable of acting as substrates for ag85C-mediated acyl-transfer and the acyl glycoside 1 can be used in lieu of TMM extracted from bacteria to study ag85-mediated acyl-transfer and inhibition leading to the better understanding of the ag85 protein class.