Malik, Samerah (2019) Development of a rapid detection system for the foot and mouth disease virus. (PhD thesis), Kingston University, .
Abstract
The total syntheses of BocAL(Z)QAMC (i) and BocAL(Boc)Q(Trt)AMC (ii) have been accomplished. Synthesis of compound (i) was achieved by initially coupling the amino acid adjacent to the fluorophore, AMC, using selenocarboxylate / azide amidation coupling conditions, followed by the dipeptide coupling on to the third amino acid in the sequence Q using the coupling reagent HATU, under optimised conditions.1 Synthesis of compound (ii) was achieved using automated peptide synthesis of the protected tripeptide sequence and the final coupling to the fluorophore; AMC was achieved using HATU, under optimised conditions. The integrity of compound (i) and its fragment Q-AMC was found to be compromised during purification steps and storage, by spontaneous decomposition resulting in the premature, non-enzyme assisted cleavage of the fluorophore, AMC. This decomposition was minimised by the addition of the bulky trityl protecting group, therefore compound (ii) was used for biochemical testing. Successful deprotection of compound (ii) was accomplished prior to the enzymatic assay, with the target enzyme 3Cpro. Importantly, the proof of concept has been gained through evidence of enzyme assisted breakdown of the detection probe by fluorescence measurements. Also the deprotected version of compound (ii) was found to show selectivity towards the target enzyme FMDV 3Cpro over other enzymes potentially also present in clinical samples. These enzymes include chymotrypsin, thrombin and trypsin in FMDV clinical samples and the carefully selected TEV protease due to its reported cleavage after amino acid Q in peptide sequences.2 However, problems with the stability of the detection probe after deprotection were still apparent, resulting in a very short shelf-life of the probe. Further work is to be done in the stability of the probe but this project has proved 3Cpro recognises and processes shorter peptide fragments, previously reported not to be the case.
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