Lysine biosynthesis: synthesis of enzyme inhibitors and substrates

Campbell, Robert Alexander (1998) Lysine biosynthesis: synthesis of enzyme inhibitors and substrates. PhD thesis, University of Glasgow.

Abstract

Two distinct biosynthetic pathways to the essential amino acid L-lysine (A) are found in nature. The -aminoadipate pathway operates in fungi and yeasts. The diaminopimelate (DAP) pathway occurs in bacteria and higher plants. Our studies were concerned with the DAP pathway and particularly with the first two steps of this pathway. These steps involve condensation of L-aspartate--semialdehyde (ASA) (B) with pyruvate (C) to form L-2,3-dihydrodipicolinic acid (DHDPS) (D) and subsequent reduction to L-2,3,4,5-tetrahydrodipicolinic acid (THDPA) (E). The first step is catalysed by the enzyme dihydrodipicolinate synthase (DHDPS). The second step is catalysed by the enzyme dihydrodipicolinate reductase (DHDPR) and utilises NADPH as a co-factor. (Fig 6269A)

Our primary objective was the inhibition of this biosynthetic pathway. Inhibitors of this pathway have potential as antibacterial or herbicidal agents. A number of substrate analogues of the DHDPS and DHDPR enzymes were prepared and tested as inhibitors of these enzymes. In previous studies by our group, heterocyclic compound (F) showed promising activity. In our work, a number of analogues of compound (F) were prepared. Inhibition studies with these compounds constituted a valuable insight into the characteristics of these enzymes. The level of inhibition with these compounds and for a range of other substrate analogues indicate high substrate selectivity for the enzymes. (Fig 6269B)

The condensation catalysed by DHDPS is mechanistically interesting. In chemical terms, C-C bond formation is commonly a high energy process involving highly reactive compounds such as organometallic agents or strong bases. In such cases the strategy must be directed towards protecting other functionality or introducing it at a later stage. Clearly, achieving this task under mild conditions with the high regio- and stereoselectivity associated with enzymic catalysis could be of tremendous advantage.

Once again, however, we were restricted by the high substrate specificity of the DHDPS enzyme. In earlier studies by our group, Dr J.E. McKendrick found evidence of substrate activity for 2- and 3-methyl substituted ASA, (G) and (H), respectively. In our work, an improved preparation of these compounds was developed and the subsequent biotransformations with DHDPS were examined both qualitatively and quantitatively. Interestingly, compound (G) was shown to display a greater substrate activity than ASA. The preparation of ASA methyl ester (I) was achieved. This compound also displayed a moderate level of substrate activity and was the only compound found to show substrate activity for a DHDPS/DHDPR coupled substrate assay. (Fig 6269C)

A significant proportion of our effort was concentrated on the investigation of glutamate--semialdehyde (GSA) (J), the higher homologue of ASA, as a substrate of DHDPS. Problems were encountered with earlier studies in this area because of cyclisation of GSA, even in protected forms. To counter this problem two novel strategies were considered. The first involved in situ enzymic deprotection of the N-acetyl derivative of GSA. Although this was successful for N-Acetyl-ASA, problems with cyclisation were once again experienced for the GSA equivalent. The second strategy utilises the reversible nature of enzymic catalysis. The synthesis of the proposed 7-membered heterocyclic product (K) of DHDPS catalysed condensation of GSA and pyruvate is not a trivial task. Some progress was made and further direction detailed. Further validation for this study was demonstrated on preparation of 5-hydroxyproline (L). Compound (L) is a cyclic equivalent of GSA and was found to display clear evidence of substrate activity. (Fig 6269D)

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: Q Science > QD Chemistry
• Colleges/Schools: College of Science and Engineering > School of Chemistry
• Supervisor's Name: Robins, Prof. D.J.
• Date of Award: 1998
• Depositing User: Angi Shields
• Unique ID: glathesis:1998-3981
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 12 Feb 2013 12:38
• Last Modified: 12 Feb 2013 12:38
• URI: https://theses.gla.ac.uk/id/eprint/3981