The late-stage site-specific functionalisation of peptides and proteins via polyoxometalate-mediated H-atom abstraction

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Problems and importance:The last two decades have witnessed an increase in the regulatory approval of protein-based therapeutic and diagnostic agents. The unparalleled affinity and selectivity with which proteins bind to their target, ensures that such 'biologics' can be employed efficaciously for medical use. Additionally, the application of native proteins to the treatment of disease enables the development of therapeutics which may exhibit fewer side effects and thus reduced toxicity. Within oncology especially, protein-based biologics have stepped to the forefront of cancer diagnosis and treatment. However, despite advances within this field, safer and more efficacious approaches are actively sought. Functionalising clinically relevant protein sequences with imaging agents or cytotoxic compounds further enhances their therapeutic application and has led to significant interest in protein modification techniques. The development of efficient, chemoselective and generally applicable bioconjugation chemistry will enable the production of more advanced biologic agents which may ultimately have a substantial impact in the clinic.Relevance to sustainability:Despite the development of several site-specific protein modification techniques, the most utilised reactions to-date target cysteine residues, which are integral to protein structure and function, or lysine, the abundance of which causes the production of heterogeneously modified isoforms. Alternative techniques that do not make use of native residues may employ non-standard amino acids, a strategy that involves directed evolution of the relevant biologically machinery. Semi-synthetic techniques involving chemically synthesised peptides have also been widely utilised, however, such methods employ toxic reagents and use large quantities of solvent which limits the sustainability of this approach. The development of an efficient and mild site-selective bioconjugation reaction that could be broadly applied to any biologically expressed protein sequence, targeting native residues that are not integral to protein structure and function, would be a highly impactful advance. Proposed solution and methodologyWe propose the development of a strategy for the late-stage site-specific functionalisation of peptides and proteins utilising radical chemistry. Polyoxometalate (POM) clusters have previously been reported to enable H-atom abstraction from unactivated aliphatic tertiary centres, facilitating the fluorination of selected amino acids. We wish to further explore this POM-mediated strategy, expanding the scope of the approach to enable the broader functionalisation of peptides and proteins in a site-selective manner. By combining this H-atom abstraction method with a small library of modified radical traps, we aim to install desired functionality (e.g. radioactive nuclides or cytotoxic compounds) at the site of radical formation on several amino acid residues. Starting with a simple decatungstate cluster, we will explore and utilise the tunable properties of the POM to increase control and site-selectivity. The reaction will be mild, proceeding rapidly in an aqueous solution, requiring only catalytic addition of the POM. Our strategy will be tailored to enable the introduction of a toolbox of functionality which could be applied to any chosen protein. Ultimately, we will seek to employ our technique to functionalise proteins that target receptors expressed by highly invasive triple-negative breast cancer.