Bridging disulfides with halomaleimides. (360G-Wellcome-086714_Z_08_Z)

The repertoire and functionality of proteins in mammal cells is greatly enhanced by post-translational modifications. These include the addition of polymers and small groups to the protein, the formation and change of the orientation of single bonds and the modification of amino acids. The possibility to artificially mimic the cells' ability to modify proteins yields great opportunities to gain a deeper insight into function and regulation but would also lead to the creation of new applications with a diagnostic and clinical background. One of the main obstacles yet to overcome in the synthetic modification of proteins is the poor selectivity exhibited by the methods that are available at present. In order to sustain the ability of a protein to bind its interaction partner or to accomplish its designated role it is vital that any artificially modification interferes as little as possible with its structure and its function. This can only be achieved if the applied chemistry allows the attachment of one or more functional molecules at one or more well-defined sites with high selectivity. Cysteine is widely considered the easiest amino-acid to selectively modify, due to its high nucleophilicity. However cysteines present in proteins often have a catalytic function or are inaccessible due to their incorporation in disulfide bonds. Free cysteines can be generated by subjecting proteins containing disulfides to mild reducing conditions, but solvent exposed disulfide bridges often fulfil the role of a stabilising feature of the structure and their breakage often leads to denaturation. One possible approach to preserve the protein structure would be the reconnection of the two cysteines after the reaction. We are proposing to develop a new class of reagents, the dihalomaleimides (e.g. dibromomaleimide), for this purpose (Scheme 1). Each cysteine from the disulfide can displace one of the bromines on the maleimide to afford the bridged product. By varying the R group we can, in a highly controlled manner, incorporate a range of functional moieties onto the protein.