Development and mechanistic investigation of an 1,2 aminothiol-based bioorthogonal reaction

Wu, Chenyang 2025. Development and mechanistic investigation of an 1,2 aminothiol-based bioorthogonal reaction. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Site-specific modification serves as a powerful strategy for studying the structure and function of peptides and proteins. While diverse bioorthogonal reactions, such as alkyne–azide cycloaddition and Diels–Alder cycloaddition, have been developed for studying peptides in their native environments, significant limitations persist. Many labelling systems suffer from slow kinetics or poor biocompatibility, rendering them unsuitable for live-cell labelling. To address these limitations, we recently reported a novel bioorthogonal reaction where 1,2-aminothiols react rapidly, specifically, and efficiently with 2-[(alkylthio)(aryl)methylene]malononitrile (TAMM) under biocompatible conditions (Figure 1). 1 In this thesis, I present a detailed mechanistic investigation of this TAMM reaction and an analogous dithioester reaction, focusing on kinetics, substituent effects, thermodynamics, and the structure of intermediates. Chapter 1 reviews the literature concerning protein labelling methodologies, classical bioorthogonal reactions, and reactions involving 1,2-aminothiols. Chapter 2 establishes the kinetics of the TAMM reaction, which is a two-step process between 1,2-aminothiols and TAMM reagents. Kinetic analysis revealed that the first step of the TAMM reaction is second order, with the observed rate constants exhibiting significant pH dependence. By comparing the observed rate constants at different pH values (kobs = 5.31 M−1 s −1 at pH 7.4 vs. kobs = 0.25 M−1 s −1 at pH 6.0), I determined the pKa of the thiol moiety in the model peptide to be 8.13. Additionally, I examined the influence of various para-substituents on the TAMM aryl ring on reaction rates. I also demonstrated that the pKa of the TAMM leaving group correlates with enhanced reaction rates. By combining this insight with aryl ring substituent optimisation, I iv achieved remarkably high second-order rate constants. Collectively, these findings elucidate some fundamental aspects of the TAMM reaction. To deepen the mechanistic understanding, Chapter 3 further probes the structural and mechanistic aspects of the TAMM reaction. First, I successfully characterised the key intermediates through techniques such as isolation, NMR, MS, and reactivity studies with thiol-capturing agents, providing conclusive evidence for the proposed structures of the intermediates and refining the mechanistic picture. Furthermore, I determined the activation parameters (Ea, ΔG ‡ , ΔH ‡ , ΔS ‡ ) for the initial thiolate-exchange step and subsequent malononitrile-elimination reaction step, which, along with the intermediate characterisation, elucidated the detailed stepwise mechanism of the TAMM reaction. Extending the investigation to a related system, Chapter 4 explores the analogous dithioester reaction (1,2-aminothiol + dithioester). Similar to the investigation of the TAMM reaction, my research on dithioester reaction focuses on elucidating the role of leaving groups in determining reaction rates. I also examined how the polarization of C=E double bonds affects their kinetics, where E represents S for dithioesters, O for thioesters, and C(CN)2 for TAMM. Given the structural similarities between dithioesters and TAMM compounds, a similar stepwise mechanism involving a transient thiol intermediate is proposed and substantiated through kinetic analysis. Furthermore, I analysed the aqueous stability of dithioester reagents to assess their potential for precise protein modification. In conclusion, this systematic mechanistic investigation offers a foundational framework for the rational design of next-generation TAMM and dithioester reagents. Additionally, it establishes both reactions as robust, versatile tools for bioorthogonal labelling. These advancements are particularly promising for real-time studies in live cells and complex biological environments.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Schools > Chemistry
Date of First Compliant Deposit:	30 January 2026
Last Modified:	30 Jan 2026 11:37
URI:	https://orca.cardiff.ac.uk/id/eprint/184280

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