Tetrazine-Triggered Bioorthogonal Decaging Reactions for Prodrug Activation

Bioorthogonal decaging reactions have emerged as a promising strategy for the spatially and temporally controlled activation of proteins and drugs. The inverse electron-demand Diels-Alder (IEDDA) reaction between tetrazines and strained alkenes exhibits high reaction rates and selectivity and has been widely applied for the decaging of amine prodrugs. Although amines are common in small molecule drugs, decaging methods for other functional groups are required in order to develop a broadly applicable prodrug activation strategy that can be applied to a wider range of drugs and diseases. This thesis describes the application of the tetrazine-triggered IEDDA reaction to cleave protecting groups from carboxylic acid- and alcohol-containing molecules. The first project relates to the development of both vinyl and trans-cyclooctene (TCO) protecting groups to mask a carboxylic acid-containing anti-inflammatory drug. In each case, the prodrug stability, reaction profile and kinetics of decaging were studied with a range of tetrazines. Whilst vinyl esters suffered from poor stability and release rates, rapid drug release (< 2 minutes) was achieved from a stable TCO prodrug. Importantly, decaging of a non-toxic TCO prodrug in live macrophages was demonstrated, resulting in the reinstation of the anti-inflammatory activity. The second project describes methods to release alcohol-containing molecules via the TCO-tetrazine IEDDA reaction. Work towards three linkers (carbonate, ether and carbamate benzyl ether) is described. The carbamate benzyl ether linker was shown to be both synthetically accessible and stable, which was not the case for the other linkers. Therefore, the tetrazine-triggered decaging of a TCO-carbamate benzyl ether fluorophore was studied in detail. In addition, this reaction was shown to be compatible with living organisms through decaging of a prodrug in the presence of live cells. Overall, the work presented here offers new methods for masking carboxylic acid and alcohol functionality that can be rapidly reinstated through tetrazine-triggered decaging.EPSRC PhD Studentshi