Modification of bacterial lipopolysaccharide (LPS) by addition of 4-Amino-4-deoxy-L-arabinose (Ara4N) to lipid A and Kdo/Ko inner core units has been implicated as a major cause of antibiotic resistance in Gram-negative bacteria due to the masking of anionic charges.[1-3] 4-Amino-4-deoxy-L-arabinose transferases (ArnT) for which functional and structural information is still scarce, utilize α-Ara4N undecaprenylphosphate as donor substrate. In order to study the enzyme mechanism and explore substrate inhibition, a series of both anomeric forms of Ara4N-phosphodiester linked derivatives have been synthesized. First, TIPDS-protected hemiacetal were converted into a separable mixture of the corresponding αand βH-phosphonates, followed by formation of the phosphodiester derivatives in fair yields upon PivCl mediated activation and oxidation. Cleavage of the silyl ether and conversion of the 4-azido into the 4-amino group through a Staudinger reduction gave the target compounds containing linear alkyl and isoprenyl lipid extensions (Fig. 1A). Preliminary activity assays using a crude membrane preparation of ArnT and Kdo2-lipid A (KLA) as acceptor and α-L-Ara4N-P-neryl 4αas donor resulted in a robust in vitro conversion of 40% of the Kdo2-lipid A precursor to mono-substituted Kdo2-lipid A containing a single L-Ara4N unit (Fig. 1B).
Financial support from Austrian Science Fund (FWF, grant P28826-N28).
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