Deacetylation of uridyldiphospho-3-O-(R-hydroxydecanoyl)-N-acetylglucosamine by LpxC is the first committed step in the Pseudomonas aeruginosa biosynthetic pathway to lipid A; homologous enzymes are found widely among Gram-negative bacteria. As an essential enzyme for which no inhibitors have yet been reported, the P. aeruginosa LpxC represents a highly attractive target for a novel antibacterial drug. We synthesized several focused small-molecule libraries, each composed of a variable aromatic ring, one of four heterocyclic/spacer moieties, and a hydroxamic acid and evaluated the LpxC inhibition of these compounds against purified P. aeruginosa enzyme. To ensure that the in vitro assay would be as physiologically relevant as possible, we synthesized a tritiated form of the specific P. aeruginosa glycolipid substrate and measured directly the enzymatically released acetate. Several of our novel compounds, predominantly those having fluorinated substituents on the aromatic ring and an oxazoline as the heterocyclic moiety, demonstrated in vitro IC(50) values less than 1 microM. We now report the synthesis and in vitro evaluation of these P. aeruginosa LpxC inhibitors.

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Treatment of DNA with nitrous acid results in the formation of DNA-DNA cross-links. Two cross-link lesions have previously been isolated and their structures assigned based on spectroscopic data. The major lesion has been proposed to consist of two deoxyguanosine (dG) nucleosides sharing a common N2 atom (1), while the structure of the minor lesion has been proposed to consist of a common nitrogen atom linking C2 of a dG nucleoside to C6 of deoxyadenosine (2). The chemical synthesis of 1 and 2, utilizing a palladium-catalyzed coupling, is described herein. It is demonstrated that the spectroscopic properties of synthetic 1 are identical to that of lesion 1 obtained from nitrous acid cross-linked DNA, thus providing a proof of its structure. Comparison of the limited spectroscopic data available for lesion 2 originating from nitrous acid cross-linked DNA to synthetic 2 supports its structural assignment. The synthetic approach used for synthesis of 1 and 2 is shown to be a general method for the preparation of a variety of N2-substituted dG nucleosides in good yields.

An important component of protein-DNA recognition is the charge neutralization of DNA backbone phosphates and subsequent protein-induced DNA bending. Replacement of phosphates by neutral methylphosphonates has previously been shown to be a model for protein-induced bending. In addition to bending, the neutralization process may change the inherent flexibility of the DNA-a feature never before tested. We have developed a method to measure the differential flexibility of duplex DNA when methylphosphonate substitutions are made and find that the local flexibility is increased up to 40%. These results imply that backbone-neutralization-dependent DNA flexibility augments DNAbinding motifs in protein-DNA recognition processes.electrostatics ͉ DNA bending ͉ methylphosphonate ͉ phantom proteins T wenty years ago, Mirzabekov and Rich suggested that neutralization of the phosphate backbone of duplex DNA by cationic amino acids contributed to the bending and flexibility of DNA bound to histones (1). Recently this idea has been tested by using model systems in which specific phosphates on DNA have been neutralized (2-4) by replacing phosphate groups with methylphosphonates (MPs). It was shown that strategically placed runs or patches of MPs induced bends of 3-4°per base pair in DNA. The magnitude of bends induced in DNA upon binding positively charged mutants of GCN4, a bZIP transcription factor, compared favorably with the bends generated in the unbound DNA by substituting equal numbers of MPs for phosphates adjacent to the basic region in GCN4 (5). These and other examples (5-7) illustrate the significant contribution of chargeneutralization-induced DNA bending to the binding energetics of protein recognition processes.Increased DNA flexibility could contribute to the energetics of protein-DNA binding by allowing the DNA to accommodate a greater degree of bending and a wider range of structural conformations at the same energy. Transient electric birefringence studies of meroduplexes (in which one side of the duplex DNA has no sugar-phosphate backbone and no charge) found no bends between base pairs (6°per base pair is the lower detection limit of the experiment) but found a suggestion of increased flexibility (8). Theoretical estimates indicate that the electrostatic contribution to the static bending ranges from negligible (9-11) to about a 5°bend per base pair (12). Theoretical estimates of the electrostatic contribution to the dynamic bending also range from increased flexibility (1, 2) through a negligible effect on flexibility (13, 14) to reduced flexibility (15), heightening the importance of experimentally testing whether MP-modified DNA actually alters the internal flexibility of duplex DNA. This report demonstrates that MP substitution does increase the flexibility and that the increased flexural root-mean-square (rms) bending is comparable to the mean bend induced by the MP substitution. The entropic and electrostatic contributions attributable to MP substitution are also investigated.Site-specific duplex DNA f...