The maleimide motif is widely used for the selective chemical modification of cysteine residues in proteins. Despite widespread utilization, there are some potential limitations, including the irreversible nature of the reaction and, hence, the modification and the number of attachment positions. We conceived of a new class of maleimide which would address some of these limitations and provide new opportunities for protein modification. We report herein the use of mono- and dibromomaleimides for reversible cysteine modification and illustrate this on the SH2 domain of the Grb2 adaptor protein (L111C). After initial modification of a protein with a bromo- or dibromomaleimide, it is possible to add an equivalent of a second thiol to give further bioconjugation, demonstrating that bromomaleimides offer opportunities for up to three points of attachment. The resultant protein−maleimide products can be cleaved to regenerate the unmodified protein by addition of a phosphine or a large excess of a thiol. Furthermore, dibromomaleimide can insert into a disulfide bond, forming a maleimide bridge, and this is illustrated on the peptide hormone somatostatin. Fluorescein-labeled dibromomaleimide is synthesized and inserted into the disulfide to construct a fluorescent somatostatin analogue. These results highlight the significant potential for this new class of reagents in protein modification.

Controlling maleimide hydrolysis allows the modular construction of bromomaleimide-mediated bioconjugates which are either stable or cleavable in an aqueous, thiol-mediated reducing environment.

Bromomaleimides are versatile scaffolds that allow facile conjugation of thiolated biomolecules. Here we demonstrate that bromomaleimide‐linked GFP–rhodamine FRET pairs cleave in the cytoplasm of mammalian cells. We believe that bromomaleimide scaffolds provide a potential core structure for prodrugs designed to release bioactive cargo following cell internalisation.

A major obstacle to the efficient production of antibody conjugates for therapy and diagnosis is the non-ideal performance of commonly used chemical methods for the attachment of effector-molecules to the antibody of interest. Here we demonstrate that this limitation can be simply addressed using 3,4-substituted maleimides to bridge and thus functionalize disulfide bonds to generate homogeneous antibody conjugates. This one-step conjugation reaction is fast, site-specific, quantitative and generates products with full binding activity, good plasma stability and the desired functional properties. Furthermore, the rigid nature of this modification by disulfide bridging enables the successful detection of antigen with a spin labeled antibody fragment by continuous-wave electron paramagnetic resonance (cw-EPR), which we report here for the first time. Antigen detection is concentration dependent, observable in human blood and allows the discrimination of fragments with different binding affinity. We envisage broad potential for antibody based in-solution diagnostic methods by EPR or ‘spinostics'.