Site-selective functionalisation of peptides and proteins via free-radical-induced dechalcogenation

The development of chemistry that enables researchers to decorate proteins at defined positions with a range of functional molecular 'tags' has the potential to generate significant scientific impact. The application of such techniques to the modification of protein-based 'biologic' drugs, a class that currently dominates the pharmaceutical market, facilitates the development of more effective treatments for disease. Within the context of fundamental science, the preparation of proteins that carry naturally occurring modifications provides tools to interrogate biological pathways and enable a greater understanding of cellular biochemistry. As a result, bioconjugate techniques are used routinely in academic and industrial labs across the world. This proposal describes the development of a novel and versatile approach to protein functionalisation that presents advances over current methods.

Due to the characteristic chemical properties of proteins, synthetic methods that facilitate modification of these biological molecules must operate under a specific set of conditions. Successful bioconjugation reactions proceed in water at ambient temperature, afford high yield at low concentrations and demonstrate selectivity for one of the 21 naturally occurring (canonical) amino acid residues. Reactions that follow this template enable modification of a target protein without degradation of the amino acid sequence or the formation of undesired by-products. With these aspects in mind, we have developed a novel, efficient and selective reaction that can be tuned to target two natural amino acids; cysteine and selenocysteine. The method is rapid, quantitative and sustainable, proceeding under conditions ideal for protein chemistry. The protocol requires no precious metal catalyst or costly/unstable reagents and is operationally simple, allowing application across scientific disciplines to maximise impact within the chemical and biological sciences.

This New Investigator Award will enable us to establish a multidisciplinary team focused on the development and application of our new synthetic platform. It is envisaged that a broad range of functionality could be installed into a protein of interest using this approach; the inclusion of contrast agents for medical imaging, drug-conjugates for therapy and reactive tags or natural protein modifications to facilitate the investigation of complex biological processes will be evaluated. Proteins modified with chemical groups that will enable further investigation of protein degradation and tools to target, image and treat highly invasive forms of cancer will also be delivered within the remit of this project. Furthermore, this collaboration will enable us to establish a base of expertise, technology, and training in protein bioconjugation within the School of Chemistry, University of Nottingham.

The development of new, versatile chemistry that can be applied to install a range of functional groups into a protein, to suit many specific applications, would be a powerful addition to the methodology currently available to researchers.

Site-selective protein functionalisation is a vital area of research within the chemical and biological sciences. This project will deliver a novel method to enable the introduction of a library of functionality into a peptide or protein sequence, presenting advances on the current methodology and resulting in positive academic, industrial and societal impact.

The biopharmaceutical market has grown rapidly over the past two decades, in 2017 seven of the top ten best-selling drugs were proteins; the sector is now valued at over $200 bn. As the healthcare industry moves towards a reliance on these 'biologic' therapeutics, the development of synthetic tools to enable functionalisation of protein sequences becomes increasingly relevant. This proposal details novel and versatile bioconjugation chemistry that can be utilised to modify peptides and proteins, produced via biological, semi-synthetic and fully synthetic approaches, with an extensive range of moieties. Residue-specific chemistry allows researchers and clinicians to exploit the remarkable affinity and specificity inherent to native polypeptides to develop enhanced therapeutic and diagnostic tools. Such synthetic advances also enable a further understanding of the role proteins play in disease, which ultimately provides scientists with the tools to improve treatments.

Selective modification at cysteine is presently the most utilised approach towards protein modification; among several advances, our platform delivers a carbon-carbon bond forming reaction at this residue to facilitate rapid, quantitative modification without reliance on a precious metal catalyst and with no impact on native stereochemistry of the polypeptide backbone. The protocol is operationally simple and can, therefore, be employed by researchers without substantive training in the chemical sciences. The possibility of offering a range of novel commercially available bioconjugation 'tags' to be utilised by the biological chemistry/molecular biology community would increase the utility and impact of our technology. Furthermore, the development of a novel one-pot ligation-functionalisation strategy, selective in the presence of the canonical amino acid residues, including cysteine, would allow chemoselective functionalisation of protein sequences without interfering with the structure and activity of the peptide or protein, an advance that is rarely achievable using the reactions currently favoured by the community.

The development of chemistry to facilitate the installation of desirable groups into a polypeptide sequence of interest will impact those fields that rely on the use or understanding of proteins including; proteomics, chemical biology, medical imaging and drug discovery. A focus on the installation of PTMs, cytotoxic groups and contrast agents will yield synthetic advances to allow further development of basic science, drug conjugates and imaging probes, respectively. By broadening the scope and repertoire of bioconjugate chemistry, this project will facilitate access to clinically relevant polypeptides engineered to enhance and exploit the biological properties of these native biomolecules. In this context, the development of the described versatile chemistry may ultimately further reduce the barriers that exist between the lab and the clinic. Such advances will directly contribute to an improvement in the quality of healthcare in the UK and thus decrease the patient burden on the healthcare system.