The published sequence of the human genome revealed encoding for only one tenth of the total proteome. The role of protein post-translational modification has therefore become of intense interest in understanding complex, physiological processes. Combining synthetic organic chemistry with molecular biology has enabled the construction of a range of proteins and peptides as probes to aid in this venture. This has allowed the introduction of synthetic post-translational modifications and a variety of fluorescent, radiolabelled and affinity tags into a variety of protein and peptide structures. The selective modification of cysteine remains a popular method for achieving protein modification. This is due to its low natural abundance, high nucleophilicity and the ease with which cysteine can be introduced at desired locations in protein sequences using modern molecular biology methods. In our laboratory, we have recently become interested in the construction of protein bioconjugates that are cleavable in a reducing environment. We believe that such an approach could deliver reversible affinity or fluorescent labels, or constructs cleavable under cytoplasmic conditions with potential as prodrugs. Maleimides are known to react rapidly and selectively, but irreversibly, with thiols. However, we have recently demonstrated an approach to reversible cysteine bioconjugation using bromomaleimides, where retention of the maleimide double bond allows cleavage of the constructs via conjugate addition reaction. Using bromomaleimides we have been able to demonstrate the modular construction of complex bioconjugates, without requirement for reagent preactivation, that have three points of chemical attachment. Following on from this success, we are interested to determine whether the ability to construct reversible constructs is something that is restricted to maleimides or whether other cyclic or acyclic haloeneamide systems will afford alternative opportunities. We report herein the use of both monobromoand dibromo1,2-dihydro-pyridazine-3,6-diones (MBPDs 1 and DBPDs 2) (Fig. 1) to assemble cleavable bioconjugates. The bioconjugates generated demonstrate exceptional hydrolytic stability with the potential for four points of chemical attachment. We have previously shown that hydrolytic stability is crucial in preserving the thiol-cleavable property of bromomaleimidelinked bioconjugates. Our initial studies focused on the functionalisation of a single cysteine mutant (L111C) of the SH2 domain of the Grb2 adapter protein 3, a protein that does not otherwise contain any cysteine residues, with pyridazinedione (PD) 4 (100 mol eq., 37 1C, 16 h). No reaction was seen to occur (Scheme 1). The absence of reactivity observed with PD-4 may be rationalised by the acidic nature of its hydrazide protons. At pH 8, PD-4 will predominantly exist in an unreactive, anionic form. However, treatment of protein 3 with PD-5 (100 mol eq., 37 1C, 16 h) yielded quantitative conversion to the desired conjugate 6 (Scheme 1). As this protein sequence contains eight lysine residues, the reaction with PD-5 demonstrates remarkable selectivity for cysteine as was evidenced following an absence of reaction with Ellman’s reagent. To our knowledge, this is the first reported example of a pyridazinedione being used to modify cysteine. Encouraged by the successful functionalisation of protein 3 with PD-5, we sought to synthesise and evaluate MBPD-7 and DBPD-8 as reagents for the reversible bioconjugation of proteins. Synthesis of both 7 and 8 was achieved in high yield by the initial condensation of maleic anhydride with diethylhydrazine followed by sequential dibromination/elimination steps (Scheme 2).