Intramolecular side-chain-to-side-chain crosslinking, also termed “stapling”, is an important technology in the development of bioactive peptide-based therapeutics. Among all of the tools, S-alkylation is one of the most flexible approaches, as a wide range of bis-thiol-reactive linkers are commercially available. More importantly, this efficient and mild modification can be applied on large biomolecules by re-bridging their disulfides. At the same time, the handle introduced by the method provides further opportunities for manipulating the bioactivity of the molecule. The first part of this work describes a new peptide-macrocyclisation strategy with an isobutylene graft. The reaction is mild and proceeds rapidly and efficiently both with linear and cyclic peptide substrates. The resulting isobutylene-grafted peptides possess improved passive membrane permeability, and are stable in human plasma and in the presence of glutathione. This strategy can be applied to bioactive cyclic peptides such as somatostatin. Importantly, we found that structural preorganisation forced by the isobutylene graft leads to a significant improvement in the binding affinity of somatostatin to its receptor. Next, the same approach was optimised for re-bridging the disulfides of monoclonal antibodies. A ‘one-pot’ stapling strategy using isobutylene motifs was able to stabilise the interchain disulfides of antibodies by avoiding Fab exchange and disulfide shuffling. This general method was applied to an anti-HER2 Fab fragment and full-length IgGs under mild and biocompatible conditions. The binding affinity of the antibody was enhanced, relative to its native form, after stapling. The stapled structure maintained its effector functions and behaved similarly to its native form in vivo. The last part of the thesis describes a photoactivation decaging method of isobutylene-caged thiols through a UV-initiated thiol-ene reaction. The method was demonstrated with an isobutylene-caged cysteine, cyclic disulfide-peptide, and thiol-containing drug, all of which were rapidly and efficiently released under mild UV irradiation in the presence of thiol sources and a photoinitiator. Importantly, it was shown that the activity of histone deacetylase inhibitor largazole can be switched off when stapled, but selectively switched on within cancer cells when irradiated with non-phototoxic light. Further optimisation also demonstrated a potential application on isobutylene grafted protein-small molecule conjugates. Overall, the work presented here offers a new tool for producing cyclic bioactive peptides, stabilising therapeutic antibodies and photoactivation of thiol-containing drugs.