Synthesis of and evaluation of lipid A modification by 4-substituted 4-deoxy arabinose analogs as potential inhibitors of bacterial polymyxin resistance

Abstract

Three sets of novel 4-deoxy-l-arabinose analogs were synthesized and evaluated as potential inhibitors of the bacterial resistance mechanism in which lipid A, on the outer membrane, is modified with 4-amino-4-deoxy-l-arabinose (l-Ara4N). One compound diminished the transfer of l-Ara4N onto lipid A. These results suggest that small molecules might be designed that would effect the same reversal of bacterial resistance observed in genetic knockouts.

Figure 1

Figure 2

Assay of PmrK/ArnT aminoarabinose transferase activity. Membranes from S. typhimurium strain JSG435 were assayed for the transfer of L-Ara4N from endogenous sources to the tetra-acylated lipid A precursor 4’-³²P-lipid IV_A. The protein concentration was 1 mg/ml and the concentration of the inhibitor when present was 1 mM. Assays were carried out as previously described for 0.5 h (Panel A) or for 6 h (Panel B) at 30 C using 5 µM 4’-³²P-lipid IV_A (20,000 cpm/nmol). The reaction products were separated by thin layer chromatography in the solvent chloroform, pyridine, 88% formic acid, water (50:50:16:5, v/v) and visualized by Phosphorimaging. Lipid II_A arises from the addition of L-Ara4N to the starting substrate lipid IV_A. Lipids II_B and IV_B arise from the PagP-dependent addition of palmitate to lipid II_A and lipid IV_A, respectively. Reaction products modified with L-Ara4N are indicated by an asterisk.

Scheme I

a. (i) 1 M HCl/acetone, then NaHCO₃, (ii) 1M HCl, 90 °C, (iii) Li₂CO₃, (iv)Ac₂O, NaOAc, reflux; b. (CH₃)₂NH, CH₃CN, −20 °C; c. (i) 2-chloro-4[H]-benzodioxaphosphorin-4-one, NEt₃, THF-dioxane, 0 °C, (ii) H₂O; d. (i) Dowex 50W × 8, H⁺ form, THF-dioxane, (ii) tBuOOH/decane, cat. I₂, NEt₃; e. (i) tri n-octylalmine/pyridine, (ii) UMP morpholidate, DCCM salt, tetrazole, pyridine

Scheme II

a. (i) n-BuLi, THF, (ii) tetrabenzylpyrophosphate, THF; b. (CH₃)₃P, pyridine, CH₂Cl₂; c. (i) H₂, Pd-C, CH₃OH (ii) NEt₃; d. UMP morpholidate, tetrazole, pyridine

Scheme III

a. (i) ethyl benzyl H-phosphinate, BTSTFA, pyridine (ii) 1:3 CH₃OH-CHCl₃ , Δ; b. (i) H₂, Pd-C, CH₃OH (ii) NEt₃; c. (i) tri-n-octylamine, pyridine, (ii) UMP morpholidate, tetrazole, pyridine