This journal is c The Royal Society of Chemistry 2011 Chem. Commun., 2011, 47, 8781–8783 8781 Cite this: Chem. Commun., 2011, 47, 8781–8783 Bromopyridazinedione-mediated protein and peptide bioconjugationw Vijay Chudasama, a Mark E. B. Smith, a Felix F. Schumacher, a Danai Papaioannou, b Gabriel Waksman, b James R. Baker a and Stephen Caddick* a Received 12th May 2011, Accepted 20th June 2011 DOI: 10.1039/c1cc12807h Bromopyridazinedione-mediated bioconjugation to a cysteine containing protein and a disulfide containing peptide is described. The conjugates are cleavable in an excess of thiol, including cytoplasmically-relevant concentrations of glutathione, and show a high level of hydrolytic stability. The constructs have the potential for four points of chemical attachment. 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, physio- logical 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 1–3 and a variety of fluorescent, 4–6 radiolabelled 7 and affinity tags 8–10 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 bio- logy methods. 11–12 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 con- structs 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. 13 Using bromomaleimides we have been able to demonstrate the modular construction of complex bioconjugates, without requirement for reagent pre- activation, 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 monobromo- and dibromo- 1,2-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 bromomaleimide- linked bioconjugates. 13d Our initial studies focused on the functionalisation of a single cysteine mutant (L111C) of the SH2 domain of the Grb2 adapter protein 3, 13a 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 ratio- nalised by the acidic nature of its hydrazide protons. At pH 8, PD-4 will predominantly exist in an unreactive, anionic form. 14 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 knowl- edge, 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 diethyl- hydrazine followed by sequential dibromination/elimination steps (Scheme 2). Fig. 1 (Di)bromo-1,2-dihydro-pyridazine-3,6-diones. a Department of Chemistry, University College London, 20 Gordon Street, London, WC1H 0AJ, UK. E-mail: s.caddick@ucl.ac.uk; Tel: +44 (0)20 3108 5071 b Institute of Structural and Molecular Biology at UCL/Birkbeck, Malet Street, London, WC1E 7HX, UK w Electronic supplementary information (ESI) available: Full experi- mental details and characterisation. See DOI: 10.1039/c1cc12807h ChemComm Dynamic Article Links www.rsc.org/chemcomm COMMUNICATION Open Access Article. Published on 07 July 2011. Downloaded on 8/13/2022 12:34:27 PM. View Article Online / Journal Homepage / Table of Contents for this issue