1,2,4,5-Tetrazine mediated decaging of biologically relevant molecules
Item statusRestricted Access
Embargo end date31/12/2100
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.