Protein Modification, Bioconjugation, and Disulfide Bridging Using Bromomaleimides

Abstract

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.

Selective chemical protein modification

“…Notably, some derivatives react with N-nucleophiles 18 . However, commercial availability and ease of use and synthesis of maleimide derivatives 13,19 have led to widespread use (for example, vaccine candidates 20 or modified enzymes 21 ) 13 . Their use results in a reaction typically considered irreversible, yet it has been suggested that this can be reversed by competitive thiols 22 .…”

“…Thus, potential degradation may be an important consideration, particularly when instability may give rise to an unwanted mixture of products. Interestingly, use of bromomaleimides has opened up the possibility of reversible conjugation, allowing modulation of activity and in vivo monitoring, while also allowing the bridging and stabilisation of native disulfides 19,24 . The rare 21st amino acid, selenocysteine, can also be engineered into proteins and used to react with maleimides; greater Se nucleophilicity can allow conjugation selectivity over cysteine residues 25 .…”

Synthesis and antibacterial evaluation of some teicoplanin pseudoaglycon derivatives containing alkyl- and arylthiosubstituted maleimides

“…Applying various approaches including squaric acid conjugation method, azide-alkyne cycloaddition reaction or three-component isoindole formation, we have prepared a large set of new derivatives exhibiting high antibacterial [10][11][12][13] and, in some cases, robust antiinfluenza virus activity. [14][15][16][17] Recently, Caddick, Baker and coworkers [18][19][20][21] reported on applications of 3,4-dibromomaleimides for site-specific protein modification and bioconjugation. The method is based on addition-elimination reaction of thiols to the bromomaleimides leading to regeneration of the double bond resulting in thiomaleimide products (Scheme 1).…”

Reversible surface functionalisation of emulsion-templated porous polymers using dithiophenol maleimide functional macromolecules

“…17 Recent studies have shown that dibromomaleimides (DBMs) are able to conjugate to cysteine residues 18 and to re-bridge peptides containing disulfide bonds following in situ reduction. 19 Dithiophenol maleimides (DTMs) have also been demonstrated as highly efficient and rapid re-bridging agents for the disulfide containing peptides. 20 Reaction with thiols proceeds via an addition, elimination mechanism resulting in the retention of the maleimide functionality, which lends itself to reversibility or further modification.…”