The synthesis of a light activated proximity labelling technique for the study of protein interactions

Pearson, Charlotte (2025) The synthesis of a light activated proximity labelling technique for the study of protein interactions. Masters by Research thesis (MSc), Manchester Metropolitan University.

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Abstract

Understanding the spatial positioning and bimolecular interactions of proteins within the cell is an important area of biological research. The presence and abundance of proteins has been widely studied but there is less information known on the occurrence of protein�protein interactions within cells. Proximity labelling methods have been developed to study these interactions, this is a set of techniques that involves the labelling and identification of nearby biological macromolecules to a protein of interest. First-generation methods for studying protein proximity in a cellular environment involved the genetic tagging of proteins of interest with mutated forms of bacterial ligases to label nearby proteins. More recently second-generation techniques have utilised light activated photocatalysts to trigger the generation of reactive species which can label proteins within close proximity without requiring genetic tagging. Both generations have been successful at labelling proteins, however there are limitations associated with each technique. This project synthesised the first proof-of-concept chemical labelling probe based on a nitrobenzyl photocage. The compound consisted of an alkyne handle as the specific chemical labeller, a light activated photocage and an electrophilically activated chemical core. The bound photocage inactivates the labelling compound, once the photocage is activated by light of a specific wavelength, it is cleaved off and the active labelling compound is released. Two proof-of-concept compounds, 27 and 34 were synthesised containing a UV-active nitrobenzyl photocage. Compound 48 was then synthesised as a blue-light-activated photocage based on a functionalised coumarin derivative, exhibiting a red shifted absorption maximum compared to compound 27. Proof-of-concept compound 27 was tested for electrophile formation and quinone methide activity using light irradiation at 365 nm followed by LC-MS analysis. Compound 27 showed successful photocage removal after ten minutes when irradiated with 365 nm light and quinone methide reactivity was successfully demonstrated by reaction with a ten-fold excess of biological nucleophile cysteine.