To find new ways to diagnose and cure disease, scientists have turned to post-translational modifications of proteins – the addition of functional groups or other proteins to a protein freshly made from its genetic guidance. Post-translational modifications change the properties of the original proteins, which can greatly expand their structures and functions and regulate cell activity and reaction sites. For example, the addition of one or more units of the protein ubiquitin to other proteins plays an important role in regulating the occurrence, development and metastasis of various types of cancer.

In nature, the cell’s protein modification process has been refined through millions of years of evolution, but only works for natural amino acids. Chemical techniques for achieving certain modifications without limitation face certain challenges. On the one hand, the reaction must be carried out in the water phase, since the protein is often only soluble in water. Second, the chemical reaction conditions must be mild enough to avoid protein inactivation. Third, the chemical reactions must be somewhat selective, as proteins often have many functional groups with different properties. Therefore, the application of site-specific biomolecule modification is more restricted and, at this early stage of development, is mainly restricted to peptides rather than whole proteins.

In 2016, Benjamin Davis of the University of Oxford, UK brought free radicals to the field of protein modification and reported the first general method of creating a new carbon-carbon bond at the dehydroalanine site of a protein to add both natural and unnatural ones Side chains (Figure 1).1 Dehydroalanine is a highly active free radical receptor that contains polarized double bonds. It also has considerable synthetic flexibility and can be easily installed in peptide and protein sites that need to be modified to participate in reaction with the side chain. This differs from previous methods used to introduce post-translational modifications to proteins that formed new bonds with heteroatoms in the side chain through processes such as phosphorylation, glycosylation, and disulfide bond formation. Even bioorthogonal chemistry can only form carbon-heteroatom bonds, not carbon-carbon bonds.

A picture with Figure 1

Since then, scientists have developed free radical reactions that are capable of modifying entire proteins. This was a difficult task because proteins contain many functional groups with redox activity, so the reaction conditions must be mild enough to selectively react with the target free radical precursor instead of the protein side chain.

In 2020, Davis and Veronique Gouverneur, also from the University of Oxford, led teams working together on a new method of visible-light-induced protein functionalization.2 They used catechol borate (BACED) derivatives and pyridine difluoroalkyl sulfone (PySOOF) as two complementary free radical precursors to functionalize proteins with dehydroalanine in the water phase (Figure 2). BACEDs are generated in situ as reducing free radical precursors to RH. to produce2C · which can then form a link between the protein and the post-translational modification. PySOOF produces RF as an oxidizing free radical2C under the action of Fe (ii) to get a modified link that is a CF. contains2 Etiquette. This provides a powerful method for studying protein activity through 19thF-NMR.

A picture with Figure 2

The two free radical precursors can be easily obtained and modified by controlling the oxidation – reduction properties of the reaction. A high rate of conversion can be achieved and damage to the protein can be reduced, as well as adding functional groups to the protein under neutral redox conditions is convenient. This strategy works with more than 50 types of residue and side chain modifications.

The modified proteins have good biological activity. In addition, the use of catechol borates to add active sites for carbon-halogen bonds to the target protein enables crosslinking with neighboring proteins. Complex protein inhibition can be achieved by modifying the crosslinking of residues of the active site, with an effect similar to that of new targeted low molecular weight covalent inhibitors.

Post-translational modification of proteins is a hot research area these days, and new methods and technologies are constantly being developed. Although they are not yet perfect, they still have tremendous potential for use. The site-specific nature of the modifications could help in the future to develop protein agents and covalent inhibitors that form a link between modern organic chemistry and the life sciences.


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