Chemical methods for protein modification are of utmost importance as they allow the mimicking of post-translational modifications that occur in living cells. In addition, they allow exogenous modifications that are the basis for bioconjugation, protein visualisation (fluorescent tagging) and immobilisation (biotin labelling) and many applications in therapeutics. Bromomaleimides are the first of a new class of reagents that could be efficiently used for the highly selective and reversible modification of cysteine and for the bridging of disulfide bonds in proteins. Aiming to prove that these transformations are not restricted to bromomaleimides, the present work presents a library of novel analogues, bearing different leaving groups on the double bond. By controlling the chemistry of this class of compounds we were able to tune properties such as thiol selectivity, reactivity, water-solubility and cross reactivity with reducing agents. The utility of the novel monosubstitued analogues as protein labelling reagents was shown using a single cysteine mutant of protein Grb2 (L111C) as a model system and a kinetic study was designed with the aim to quantify the difference in reactivity of the various selected analogues. The disubstitued analogues were tested as disulfide bridging reagents using Somatostatin, a 14-aminoacid peptide containing a disulfide bridge. Phenoxymaleimides were shown to be less reactive and to display a different reactivity compared to bromomaleimides. They react with reduced disulfides to form a succinimide bridged product. The successful use of phenoxymaleimides in bridging disulfides in peptides and proteins is described. This strategy is fast and efficient and was optimised as to reduce the risk of disulfide scrambling, protein aggregation and loss of activity, which are the common problems associated with disulfide modification. Also, a novel one-pot protocol for the differentially labelling of disulfides in both peptides and proteins was developed. Several dual modified Somatostatin conjugates were prepared using this strategy
