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Title: Rational design of inhibitors of porphobilinogen deaminase
Author: Ahmed, R. A. A.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2002
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The thesis describes the synthesis of new analogues (53-57) of PBG and their inhibition of and mechanistic studies on the enzyme PBG deaminase from E. coli. The analogues with an additional alkyl group, 53 and 54, were successfully obtained in good yields in 10 steps. The caged compound 97 was also obtained but the final step of hydrogenolysis of the benzyl protecting groups also caused reduction of the nitro group on the 2-nitrobenzyl substituent. Testing of analogues 53 and 54 with the enzyme (PBGD), however, showed no inhibition. A new route for the synthesis of the conformationally restricted analogue 6, 11-ethanoPBG 56 was developed in this work and one stereoisomer of 56 was obtained (the cis-isomer). When this analogue was tested as an inhibitor it also showed no inhibition. This was thought to be due to the stereochemistry of the compound since modelling studies had predicted that the trans-isomer would bind tightly to the enzyme not the cis-isomer. Therefore, the 11-hydroxy-6,11-ethanoPBG analogue 57 was prepared and tested as an inhibitor with the enzyme. The result was only a modest effect on the enzyme's kinetics. 6-Methyl-PBG 55 which has the extra methyl group on the acetate side chain of PBG, was prepared to test the importance of this chain. When the compound was tested as an inhibitor of PBG deaminase it showed strong inhibition and a K1 value of about 3mM was determined. This is the strongest inhibitor ever obtained for the enzyme. When this analogue was tested as a substrate for the enzyme it appeared by UV/visible spectroscopy to form a novel porphyrin, although due to the small quantity, it was not possible to isolate this porphyrin. In addition, it was possible to isolate the covalent enzyme/analogue complexes by the use of the FPLC technique. It was also possible to confirm the formation of complexes of the enzyme with one and two molecules of the substrate analogue bound for the first time by the use of the LC-mass spectrometry. This work also describes attempts to crystallise some of the enzyme/analogue intermediates, which were isolated by the FPLC. These attempts were successful and single crystals were obtained. These crystals diffract well and currently further work is going on to solve their structure. DesaminoPBG 26 has been previously prepared and gave a K1of 75 mM with PBG deaminase from human erythrocytes. In this project it has been tested with the PBG deaminase from E. coli and shown to be a less powerful inhibitor. A new approach for preparing 2,3-disubstituted pyrroles was developed and the 2-methyl-3-pentylpyrrole 59 was obtained in 70% yield. This compound is used as a building block in the biosynthesis of the red pigment prodigiosin 168, one of a class of naturally occurring polypyrroles which exhibit antimicrobial and cytotoxic properties.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available