1,2,4,5-Tetrazine mediated decaging of biologically relevant molecules
Many cancer drugs are inherently cytotoxic, leading to severe toxicity towards healthy tissues and organs. One effective strategy to circumvent these side effects is to mask a cytotoxic drug with a “caging group” which, once at the target tissues, releases the masked drug upon an appropriate trigger. In this study, 1,2,4,5-tetrazines which undergo bioorthogonal inverse electron demand Diels-Alder cycloadditions, were used as “chemical triggers” to unmask a “caged cargo”. The vinyl carbamate bond was successfully used as a caging group to ‘cage coumarin’ and was decaged with trigger ‘dimethyl 1,2,4,5-tetrazine-3,6-dicarboxylate’. However the trigger underwent hydrolysis under physiological conditions. Various 1,2,4,5- tetrazines were synthesized and hydrolytic stable 1,2,4,5- tetrazines were evaluated for decaging of caged coumarin but with no success. In depth stability of 1,2,4,5-tetrazines under physiological conditions was investigated before exploration alternative caging groups. The results obtained showed basic and physiological conditions had a deteriorating impact on the stability of 1,2,4,5-tetrazine with rapid degradation (t1/2 5– 50 min) in culture medium. The half-lives were significantly increased in acidic pH compared to basic pH (with up to a 187-fold increase from pH 9 to pH 5). Exploration of ketene-N,S-acetals, enamides, thioether-norbornene, enolethernorbornene and vinyl ether as caging groups was conducted. Only vinyl ethers proved to be a success with caging of two fluorophores i.e. fluorescein and resorufin. Two 1,2,4,5-tetrazines were effective in the decaging of the vinyl ether caged fluorophores in water and PBS. Bis-vinyl fluorescein was successfully decaged in HeLa cells showing the potential of vinyl ethers as a caging groups. In future, the vinyl ether would be used to cage a cytotoxic drug and its activation by 1,2,4,5-tetrazine functionalised with tumor targeting peptide or antibodies would result in localised and selective drug release.