Interest in linear peptides and peptidomimetics experienced rapid growth within the pharmaceutical industry due to their ability to disrupt protein-protein interactions; and their ability to modulate protein function by allosteric regulation and competitive binding. However, many linear peptides that show therapeutic potential cannot be readily developed into bioavailable pharmaceuticals due to their poor lipophilicity and protease stability; and their unpredictable secondary structures. Macrocyclic peptides show increased lipophilicity and protease stability; and their constrained secondary structures often lead to improved target binding affinity and selectivity. Consequently, there has been a resurgence of interest within the pharmaceutical industry towards peptide based therapeutics and increased research towards novel peptide macrocyclisation strategies. Progress towards a novel solid-phase peptide macrocyclisation strategy based on the inverse electron demand Diels-Alder reaction of 1,2,4,5-tetrazines is reported in this thesis. The solid-phase oxidation activation peptide macrocyclisation strategy uses the in situ oxidation of a dihydro-1,2,4,5-tetrazine to trigger the inverse electron demand Diels-Alder reaction of a 1,2,4,5,-tetrazine and therefore peptide macrocyclisation. Also, the solid-phase oxidation activation peptide macrocyclisation strategy enables selective late stage incorporation of the dihydro-1,2,4,5-tetrazine moiety onto the peptide backbone in the presence of a dienophile. A successful proof of concept in situ oxidation and inverse electron demand Diels-Alder reaction of a dihydro-1,2,4,5- tetrazine was achieved. However, the poor stability of a strained dienophile towards an oxidant was also highlighted. To overcome this problem a library of alternative oxidants and a library of unstrained dienophiles were screened to successfully optimise the proof of concept in situ oxidation and inverse electron demand Diels-Alder reaction of a dihydro-1,2,4,5-tetrazine. The optimised in situ oxidation and inverse electron demand Diels-Alder reaction of a dihydro-1,2,4,5-tetrazine was successfully transferred onto solid-phase using resin-bound dienophiles. However, attempts to synthesise resin-bound dihydro-1,2,4,5-tetrazine derivatised amino acids were unsuccessful. Therefore, an alternative dihydro-1,2,4,5-tetrazine was selected for future development of the solid-phase oxidation activation peptide macrocyclisation strategy.

In the course of attempting to prepare 3,6-bis(hydroxy-methyl)-s-tetrazine via the reduction of 3,6-bis(carboxy)-s-tetrazine with lithium aluminum hydride, it became apparent that the tetrazine ring was cleaved.

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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.

The need for chemoselective bond formation within complex biological systems has driven much research in chemical biology and chemical medicine and has allowed control over the structure and biological properties of a range of chemical entities. Reactions that are highly biocompatible, selective and occur at low concentration are classified as being bioorthogonal. Although bioorthogonal reactions have been successfully applied to bioconjugation and imaging in living systems, only a few examples exist of bioorthogonal reactions being utilised for the activation of prodrugs. The tetrazine mediated inverse electron demand Diels-Alder reaction is characterized by excellent reaction rates and high biocompatibility in both in vitro and in vivo applications. To date, this chemistry has found only limited application in prodrug activation or drug release strategies. Herein, a series of tetrazine-trigger systems are reported in which an active drug is liberated from its inactive form upon triggering with tetrazine. It is shown that the release of encapsulated and conjugated drugs from polymeric nanoparticles can be triggered by tetrazines providing an on-demand release within biological systems. In a totally new approach that fully complies with the principle of bioorthogonality by avoiding the generation of any by-products, tetrazine was utilised as a prodrug scaffold leading to symbiotic and traceless dyadic prodrug activation. The simultaneous formation of two active drugs (here the anticancer drug camptothecin and a known micro RNA inhibitor) was confirmed and validated within a biological environment. The use of tetrazines as a trigger to activate or release an active drug will open new directions in the field of chemical biology/medicine.