1 PERKIN DOI: 10.1039/b002701o J. Chem. Soc., Perkin Trans. 1, 2000, 2023–2036 2023 This journal is © The Royal Society of Chemistry 2000 Synthesis and in vitro enzyme activity of peptide derivatives of bacterial cell wall biosynthesis inhibitors Russell J. Cox,* Helen Jenkins, James A. Schouten, Rosie A. Stentiford and Katrina J. Wareing School of Chemistry, University of Bristol, Cantock’s Close, Clifton, Bristol, UK BS8 1TS Received (in Cambridge, UK) 5th April 2000, Accepted 3rd May 2000 Published on the Web 9th June 2000 The enzyme diaminopimelate aminotransferase (DAP-AT) is a good potential target for the design of novel antibacterial agents. We have synthesised a series of peptide hydrazines based on the structure of the natural substrate of DAP-AT. These compounds show varied inhibition properties in vitro vs. DAP-AT from E. coli as well as moderate antimicrobial activity vs. E. coli. Examination of the kinetics of inhibition reveals that hydrazine, as well as the substituted hydrazino-peptides, shows two-phase slow-binding inhibition. Possible mechanisms for inhibition are discussed. Introduction Interest in novel antimicrobial compounds has increased recently as the problem of antibiotic-resistant pathogens has become more prevalent. 1 Resistance to almost all commercially available antibacterial drugs has been observed in both ‘wild type’ and laboratory strains of disease-causing bacteria. Worry- ingly, resistance is building up in bacteria which can cause major human epidemics, such as Mycobacterium tuberculosis, the causative agent of TB. 2 Resistance has emerged for a num- ber of reasons. Many antimicrobial drugs are, or are closely related to, natural products. Many of these compounds are produced through fermentation of strains of bacteria and fungi. In order that these antibiotic producing organisms do not kill themselves they utilise a variety of mechanisms to ameliorate the action of the antibiotics. These resistance mech- anisms are genetically encoded and under appropriate condi- tions resistance genes can propagate through the environment. The spread of resistance mechanisms often negates treatment by entire classes of antimicrobial compounds. Under these cir- cumstances the development of novel classes of antimicrobial compounds is required. We have been studying specific enzymes involved in bacterial cell wall biosynthesis as potential targets for new classes of antimicrobial compounds. In particular the biosynthesis of -lysine 1 in bacteria (Scheme 1) has interested us because of the central role of -lysine and its precursors, meso- and -diaminopimelic acid (DAP, 2), as key cross-linking elements in the strength-bearing peptidoglycan layer of the prokaryote cell wall. 3–5 The biosynthesis of the peptidoglycan structure is the target for successful antimicrobial drug classes including the penicillins and other β-lactams and the vancomycins and other glycopeptides. 6 Of course, -lysine itself is also crucial to bacterial growth and development because of its requirement for protein synthesis. The biosynthesis of -lysine, however, does not appear to be a target for existing naturally occurring compounds and resistance mechanisms may be absent. An additional attractive feature of this pathway is that it is absent from mammals (where -lysine is obtained solely through the diet) and specific enzyme inhibitors could avoid mammalian side effects. We have developed a series of compounds designed to inhibit a key enzyme in the bacterial -lysine biosynthetic pathway. The hydrazines 3 and 4 are very potent, slow-binding inhibitors of the enzyme -N-succinyldiaminopimelate aminotransferase (DAP-AT) from E. coli (Scheme 1). 7,8 The most potent of them, 3, possesses a K I * of 22 nM and is an extremely effective in vitro inhibitor of -lysine biosynthesis. Other related compounds, where the N-succinyl group has been replaced by, for example, N-Cbz (e.g. 4), are also potent inhibitors of DAP-AT. On com- plex growth media (which contain -lysine and DAP isomers) 3 shows very little activity vs. E. coli, but on minimal growth Scheme 1 Later steps during the biosynthesis of -lysine by E. coli.