The shikimate pathway : enzymes and antimicrobials
(6S)-6-Fluoroshikimic acid 1 was developed as a potential antimicrobial which was converted to 6-fluorochorismate 2 and hence targeted the post-chorismate pathway to p-aminobenzoic acid 2 and folic acid (fig. 1). Although successful against microbial challenge, resistance developed rapidly. This resistance was found to be due to mutation at the shiA locus which codes for a transporter required for the entry of shikimic acid into bacterial cells. (Fig. 11006A) In the initial part of our research, we aimed to synthesise five peptide-based 'smugglins' 4-8 which would target oligopeptide permease and hence provide a route for the entry of (6S)-6-fluoroshikimic acid into bacterial cells (fig. 2). (Fig. 11006B) Attempts to synthesise the required (6S)-6-fluoroshikimic acid 1 by biotransformation from erythrose-4-phosphate and fluoro-phosphoenolpyruvate were not successful. However, a published route from shikimic acid 9 eventually provided sufficient material for the synthesis of the test compounds (fig. 3). The details of these syntheses are described in Chapter 2. (Fig. 11006C) The smugglins were synthesised using standard Fmoc-based solid-phase peptide synthesis using DIC/HOBt to catalyse peptide coupling and BOP/ET3N to catalyse the coupling of (6S)-6-fluoroshikimic acid to the peptide moiety. Testing of the phenylalanine and leucine-based smugglins 5 and 6 (fig. 2) for antibacterial activity against wild-type Escherichia coli has so far been unsuccessful. Details of these syntheses and investigations are described in Chapter 3. As a secondary aim to route to pure 3-dehydroquinic acid (DHQ) 10, required for the kinetic analysis of 3-dehydroquinate dehydratase (DHQase), was investigated. Initial attempts using acetal protecting groups developed to be selective for diequatorial diols were unsuccessful. However, modification of a published route to halogenated derivatives of dehydroquinic acid gave DHQ in good yield on a gram scale (fig. 4). (Fig. 11006D) This pure DHQ was then used for the kinetic investigation of the type II DHQase from Helicobacter pylori and a mutant type II DHQase: Bacillis subtilis F28Y.