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Title: Solid phase synthesis and evaluation of fluorogenic oxazine based protease substrates
Author: Hintner, Eva
ISNI:       0000 0001 3578 9601
Awarding Body: University of Nottingham
Current Institution: University of Nottingham
Date of Award: 2006
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Two to three percent of the genome encode for proteases. These versatile enzymes play important roles in different pathological states and disease processes. Thus, they form an important target for pharmaceutical research. A sensitive tool for the detection and quantification of proteases are fluorogenic substrates, which can be employed in proteolytic assays. Although some substrates are already available for in vitro assays, a wider choice of fluorogenic substrates, which are also applicable for in vivo assays, is still lacking. This thesis is intended as a proof-of-concept for the synthesis and applicability of new fluorogenic cresyl violet-based peptide substrates. These fluorophore-linked peptides Jl?ay find applications in both in vitro and in vivo proteolytic assays. The first part of this thesis describes the investigation and discovery of a novel polymersupported synthetic strategy for CV-based peptides. The presence of two amino groups on CV provides two handles for tethering of CV to a solid support on one side and appending a peptide on the other. To this end, CV was successfully loaded onto pnitrophenyl carbonate Wang resin either as Fmoc-CV or neat CV. Additionally, a novel method for the Fmoc-protection of CV with Fmoc-Cl in NMP has been established. Using the newly-found method, the construction of several CV-based substrates of different peptide sequences was accomplished via the Fmoc SPPS approach. This synthetic approach would also potentially pave the way for the preparation of a wide variety ofpeptide substrates based on oxazine type fluorophores. The second part of the thesis involved probing CV fluorescence properties in aqueous buffers and testing the prepared CV-based peptides in proteolytic fluorescence-based assays. In contrast to free CV, the prepared CV-linked substrates are non-fluorescent at around 625 nm. Subsequent treatment of the CV-appended peptides with selected proteases liberates CV, which is detectable as an increase in fluorescent light. Fluorescence detection ofCV at concentrations around 10 IlM is recommended since CV was found to form a dimer with a different emission maximum at higher concentrations. Hence, CV based substrates are generally suitable for proteolytic assays but warrant further investigation on the suitability ofthe substrates for particular proteases.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available