Solid-Phase Synthesis of s-Tetrazines: Method Development and Applications in Chemical Biology

Abstract

Since the emergence of bioorthogonal chemistry in the 20th century, s-tetrazines stand out from the bioorthogonal toolbox due to their superior reaction kinetics over azide/alkyne chemistries, and high chemical selectivities. Specifically, s-tetrazines selectively undergo inverse electron-demand Diels-Alder reactions with dienophiles within complex biological systems, which has allowed for their use in various biological scenarios such as in sensing, imaging and drug delivery. However, despite the growing applications of tetrazine bioorthogonal chemistry, their development has been hampered due to a lack of practical approaches that facilitate the emergence of new applications. In this thesis, an efficient and high-yielding solid-phase route to s-tetrazines was successfully developed without the need for metal catalysts or harsh reaction conditions. This method provides a versatile route to the synthesis, under mild conditions, of unsymmetrical aryl and alkyl tetrazines, as well as more reactive monosubstituted tetrazines, with possible applications in bioorthogonal chemistry. To explore the bioorthogonal activation of tetrazines and of mycobacteria labelling, a small library of fluorescent probes was initially developed based on the mycobacterial membrane-associated disruption peptide (MAD1), with evaluation of both enantiomers of the MAD1 peptides, decorated with four different fluorophores (ranging from green to far-red), including solvato-fluorogenic dyes. Fluorescent labelling on M. smegmatis, showed robust and stable labelling. Two of the D-probes (5-CF-D-MAD1 and NBD-D-MAD1) successfully labelled Mtb. A norbornene-labelled MAD1 peptide was then synthesised, which was used to bind M. smegmatis, and trigger the activation of the quenched fluorophore (BODIPY-Tz). This process facilitated a 61-fold amplification of the fluorescent signal upon bioorthogonal activation, allowing efficient free-wash labelling.