Syed, Aisha Jabeen; (2019) The rational design, synthesis and properties of conformationally-locked infra-luciferin analogues. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The opening chapter of this thesis gives a brief introduction to the history of bioluminescence, how the factors influencing bioluminescent emission were initially discovered and later scientifically quantified. Following that, there is greater focus on firefly bioluminescence and its two vital components – the luciferase enzyme and the substrate D-luciferin. The importance of bioluminescence in biotechnology is discussed in the form of bioluminescence imaging, and the characteristics of an ideal bioluminescent reporter probe are examined. A comprehensive review of the literature is presented which includes the synthesis of D-luciferin and its analogues, their bioluminescent properties and their applications. The introductory chapter closes with a discussion of the limitations in the currently available suite of bioluminescent reporters, and following this, the aim of this project to explore if conformationally restricted infra-luciferin analogues give brighter bioluminescent emission than infra-luciferin. The results and discussion start with the design of and synthesis of a conformationally restricted infra-luciferin analogue based on the pyridobenzimidazole motif, and its flexible counterpart analogue based on the benzimidazole motif. The highlight of this work was that the synthesis of pyridobenzimidazole infra-luciferin was achieved in only 2 steps, in a protecting group free manner. The bioluminescence results of the two analogues showed that the conformationally restricted one was brighter than its flexible counter-part, but both were dimmer than infra-luciferin. Following these results, a conformationally restricted infra-luciferin analogue was designed based on the conformation of infra-luciferin in the active site of the luciferase enzyme inferred from the x-ray co-crystal structure of a stable analogue of infra-luciferin with the luciferase enzyme. A rotationally restricted infra-luciferin molecule based on a dibenzothiophene motif and its flexible counterpart based on a benzothiophene motif were designed to mimic the conformation of infra-luciferin in the luciferase active site. The synthesis and bioluminescence properties of these molecules are discussed. The highlight of this work was that both the new analogues had brighter bioluminescent emission than infra-luciferin. Suggestions for future work are given based on the results obtained from these two sets of analogues. The final results and discussion chapter focuses on work done towards other infra-luciferin analogues including an acetylene linked infra-luciferin structure and luciferin molecules with more than one electron-donating group. The experimental section presents detailed preparative methods and analytical data for all compounds.

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