The L-lysine biosynthetic pathway provides a unique and exciting target for the design of
novel antibacterial agents through inhibition of the synthesis of meso—DAP or L—lysine,
crucial components of the bacterial peptidoglycan cell wall. The first two enzymes in this
pathway—dihydrodipicolinate synthase (DHDPS) and dihydrodipicolinate reductase
(DHDPR)—were targeted for inhibition. DHDPS catalyses the first committed step to
L—lysine biosynthesis, the condensation of pyruvate and (S)-aspartate B-semialdehyde to
give DHDP. DHDPR, the next enzyme in the pathway, then reduces DHDP in an NAD(P)H
dependent reaction to give THDP.
A suite of inhibitors were designed and synthesised to act as product- or substrate-based
analogues of DHDPS and DHDPR, respectively. Heterocyclic compounds based on
chelidamic acid or thiazane-3,5-dicarboxylates were synthesised as potential inhibitors of
DHDPS. Stereoselective procedures for the oxidation of thiazane-3,5-dicarboxylates were
developed. It was found that when direct oxidants were used (such as sodium periodate,
peroxides and peracids) the axial sulfur lone-pair reacts preferentially, providing the axial
S—oxide. Oxidation via a two step mechanism using bromine/water gives the epimeric
equatorial S—oxide. Acyclic analogues of the heterocyclic compounds were also synthesised
as potential inhibitors of the intermediate in the DHDPS reaction pathway.
Two methods of synthesising the DHDPS substrate aspartate B-semialdehyde (ASA) were
investigated. The first four step procedure beginning from racemic allylglycine, installed the
aldehyde moiety through an osmium tetroxide/sodium periodate reaction. The ASA
produced by this procedure was of variable yield and purity. The second method of
synthesising ASA was achieved by reduction of a Weinreb amide derivative of aspartic
acid. This procedure gave ASA in excellent yield and purity and was the method of choice
for preparing ASA for use in kinetic studies. Additionally the required enzymes, DHDPS
and DHDPR were purified to homogeneity as judged by SDS-PAGE visualised by
Coomassie brilliant blue staining and specific activity tables.
The inhibitors synthesised were tested for inhibition of DHDPS and DHDPR. The
heterocyclic compounds were not found to be potent inhibitors of DHDPS. Furthermore,
chelidamic acid and its dimethyl ester were shown NOT to be competitive inhibitors of
DHDPS. Unfortunately, none of the analogous acyclic compounds proved to be potent
DHDPS inhibitors either.
Two potent acyclic irreversible inhibitors synthesised en route to other target inhibitors of
DHDPS were discovered, possessing micromolar—millimolar activity. These compounds
provide a new lead in the design of more potent DHDPS inhibitors.
From the DHDPS inhibitory results obtained in this study the mechanism of DHDPS was
revised. It was postulated that the DHDPS-catalysed reaction proceeds via a protonated
aldehyde intermediate that is consistent with the inhibitory data obtained herein.
None of the synthesised inhibitors evaluated against DHDPR were found to be more potent
then the known inhibitor dipicolinic acid. Consistent with the literature results dipicolinic
acid was found to be a competitive inhibitor of DHDPR, however dimethyl chelidamate was
found to be an uncompetitive inhibitor of DHDPR
