| Summary: | <p>The chemical modification of proteins has significantly expanded the toolkit for numerous studies in chemical biology. Focusing on sustainability, electrochemistry has rapidly grown in protein modification over the past five years, albeit still in its early development stage. This thesis begins with an overview of electrochemical protein functionalization in Chapter 1, discussing potential opportunities while addressing three persistent challenges: low biocompatibility, limited residue specificity, and the necessity for a deeper understanding of applying electrochemical analytical methods in mechanistic studies.</p>
<p>To address these challenges, Chapter 2 demonstrates electrochemical protein modification on boronoproteins, where a minimal-sized boronoalanine (Bal) is pre- incorporated at a specific position. Leveraging the unique chemical reactivity of the non-canonical residue Bal, direct and site-specific 'on-protein' electrochemical activation was achieved with catechol as the coordinating partner. Furthermore, the effectiveness of diverse electrochemical techniques in mechanistic studies was highlighted through a comprehensive investigation of electrochemical Bal activation using a mimicked Bal-containing peptide.</p>
<p>Chapter 3 showcases a Cys-targeted oxidative protein functionalization strategy employing dihydroxybenzene derivatives across a diverse array of protein substrates. With a pronounced emphasis on its biological applications, the chapter successfully demonstrates electrochemical fluorescent cell surface labelling. Subsequent efforts to fine-tune cell labelling intensity through optimizing both electrode effective area and electrolysis time establish the groundwork for achieving single cell labelling with precise spatial control.</p>
<p>Chapter 4 diverges from oxidative approaches to explore radical-mediated reductive electrochemical protein modification using pre-installed heteroaromatic sulfones. Unnatural amino acids functionalised with distinct heteroaromatic sulfones are synthesized and activated electrochemically, successfully demonstrating their potential as 'on-protein' radical precursors. Despite facing challenges in electrochemically activating the incorporated sulfones on proteins within the electrochemical window of water at present, this chapter significantly contributes to the exploration of radical-mediated electrochemical protein modification, highlighting the versatility of heteroaromatic sulfones as radical precursors in the intricate field of electrochemical protein engineering.</p>
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