Synthesis and Testing of Inhibitors of Dihydrodipicolinate Synthase

McKendrick, John E (1995) Synthesis and Testing of Inhibitors of Dihydrodipicolinate Synthase. PhD thesis, University of Glasgow.

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There are two distinct biosynthetic pathways to the essential amino acid L-lysine (A). The diaminopimelate pathway to L-lysine occurs in higher plants and bacteria. The second pathway known is the alpha-aminoadipate pathway and is found to operate in fungi and yeasts. This thesis will deal with only the diaminopimelate pathway to L-lysine and in particular with the first step, which involves the condensation of L-aspartic acid beta-semialdehyde (ASA) (B) with pyruvate (C) to form L-dihydrodipicolinate (DHDP) (D). The mechanism of formation of L-DHDP (D) was studied using electrospray mass spectrometry. The synthesis and testing of potential inhibitors of dihydrodipicolinate synthase (DHDPS) was also studied. [diagram] L-ASA is a substrate of the first enzyme of the diaminopimelate pathway to L-lysine. A former co-worker in the group, Dr D. Tudor had developed a route to 1-ASA as the trifluoroacetate salt. This route was low yielding, approximately 14% for four steps, thus a higher yielding route was developed utilising the para-methoxybenzyl (PMB) ester protecting group. This material was not suitable for use in the biochemical assay as an impurity from the deprotection stage was found to absorb strongly at the wavelength used for the our enzyme assay system. An improved procedure for the synthesis of L-ASA was developed increasing the overall yield of the procedure to approximately 48% for the same four steps. The synthesis of L-ASA as its trifluoroacetate salt allowed a number of analogues of 1-ASA to be prepared with some synthetic modification of the original route. The compounds prepared this way were alkylated derivatives of L-ASA. The alpha-methyl ASA (E) was prepared by the reaction of methyl iodide with the anion generated from treating diprotected allylglycine with lithium diisopropylamide (LDA). It proved to be a poor inhibitor but initial studies suggest that it may be a reasonably good substrate for DHDPS. A number of other derivatives were prepared including beta-methyl ASA (F) which again was a poor inhibitor but was found to be a good substrate for DHDPS (beta-methyl ASA is utilised at approximately 20% of the rate that L-ASA is consumed). A number of other derivatives and analogues of L-ASA were prepared. A number of heterocyclic compounds were prepared as analogues of DHDP and were tested for inhibitory effects with DHDPS. These compounds were prepared by a 1,3-dipolar cycloaddition of a nitrile oxide onto an alkene or alkyne. The isoxazolines produced had a general structure (G). These compounds were found to be poor inhibitors of DHDPS with none showing inhibition below 1 mM. The ring opened isoxazolines (H) were prepared as analogues of pyruvate but again they proved to be poor inhibitors of DHDPS. [diagram] An attempt to synthesise glutamic acid gamma-semialdehyde (I) starting from glutamic acid was undertaken. The compound isolated from the series of reactions was found to have cyclised and was stable as the carbinolamine (J). This route was abandoned due to the cyclisation of the product. [diagram] A number of pyridinedicarboxylic acid derivatives and analogues were prepared to test for inhibitory activity. The pyridine-2,6-dicarboxylic acid N-oxide (K) and the pyridine-2,6-dinitrile (L) showed very good inhibitory activity. These compounds were studied in detail to determine the type of inhibition they showed. The two compounds (K) and (L) were found to be non-competitive inhibitors of DHDPS. A number of other saturated and unsaturated analogues of L-DHDP were prepared and tested for inhibitory action. A study of the mechanism of DHDPS was undertaken using electrospray mass spectrometry to detect enzyme bound intermediates. The electrospray mass spectrometer was able to provide evidence for a number of pyruvate analogues bound to the enzyme as Schiff's bases. No evidence for L-ASA bound to DHDPS could be found. Further to these studies was the need to preserve stocks of DHDPS used for inhibitor testing and biotransformations. A study of the immobilisation of DHDPS on Eupergit resins was undertaken to determine the feasibility of this technique as a method of obtaining reusable DHDPS. The studies found that only up to 18% of the initial sample of DHDPS was bound to the beads. This suggests that this may not be a suitable method for the immobilisation of DHDPS, however DHDPS bound to the beads was found to have long term stability.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: D J Robins
Keywords: Organic chemistry, Biochemistry
Date of Award: 1995
Depositing User: Enlighten Team
Unique ID: glathesis:1995-75433
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 20:08
Last Modified: 19 Nov 2019 20:08

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