Recent Advances in Site-Specific Modification of Peptides and Proteins

Proteins and peptides are the core functional media of life activities, and natural post-translational modifications in vivo can precisely regulate the vitality, localization and stability of proteins. Human completion of peptide and protein site non-specific modification, that is, the introduction of fluorescent groups, drug molecules, sugar chains or isotope labels in the preset individual amino acid sites, is a key technology in the field of chemical biology and protein drug research and development.

The review of "Recent Advances in Site-Specific Modifications of Peptides and Proteins" systematically combs the latest research results from this perspective, and divides the existing technologies into four categories: chemical modification based on amino acid microenvironment gap, logo-mediated modification, adjacent drive decoration and enzymatic modification. The analysis of various technical principles, advantages and limitations and biomedical applications provides a clear framework for the subsequent development of this field. Traditional protein modification methods mostly rely on the inherent chemical reactivity of amino acid side chains, such as the nucleophilic reaction of lysine amino group and cysteine thiol group. However, there are generally multiple similar residues in the protein sequence, reflecting the lack of site discrimination ability. The product is a mixture of various decorative isomers, which cannot meet the precise requirements of basic research and biomedicine. The core goal of site-specific modification technology is to break the limitation of "the same amino acid has the same reactivity", and only complete covalent coupling at the target site without destroying the overall folding structure of the protein.

Based on the modification strategy of the difference of the chemical environment of the protein part, the difference of the microenvironment brought by the protein folding is used to distinguish the amino acid residues on the protein with the same chemical properties but different spatial environment. After protein folding, some lysine residues are buried in the hydrophobic core and some are exposed to the solvent; the charge and hydrogen bond microenvironment around the exposed residues will change the pKa value of the amino group, thus achieving single-site selective reflection. The biggest advantage of this strategy is that there is no need to carry out genetic modification of the protein, directly to the natural protein to carry out post-modification, easy to operate. However, the universality of this method is limited, and the selectivity is highly dependent on the three-dimensional structure of the protein itself. When the microenvironment of the target site is different from other residues, it is difficult to obtain high purity products. Tag-and-modify is one of the most widely used strategies.

According to genetic engineering, this method inserts a short peptide identifier or a non-natural amino acid at the target position of the protein, and the label can specifically identify the corresponding chemical probe, and an efficient and orthogonal biological coupling reaction occurs. The common system consists of a tetracysteine identifier with a double arsenic stain, unnatural amino acid-mediated changes in click chemistry. The site of the technology is accurate, highly selective, not affected by the three-dimensional structure of the protein, widely used in living cells, protein fluorescent labeling, protein probe construction. The disadvantage is that the protein must be genetically edited, the introduction of additional amino acid sequences, may affect the natural folding and biological function of the target protein, in vitro natural protein, clinical protein drug transformation scenarios have limitations. Adjacent drive chemical modification, with the help of intermolecular affinity effect, will reflect the probe gathered around the target amino acid, with the help of part of the high concentration to complete site selective identification. The typical representative is the affinity-guided catalytic system: after the small molecule ligand binds to the specific selection point of the protein, the catalytic group is partially activated at the binding site, and only decorates the adjacent amino acids. This strategy does not need to insert a long peptide tag, and only relies on ligand-protein interaction to achieve the site identification of natural proteins, which is suitable for the origin decoration of targeted enzymes and receptor proteins. However, this method relies on the design of high affinity ligands, which is difficult to be fully promoted for proteins lacking non-specific small molecule ligands, and is prone to non-specific background identification. Enzymatic modification strategy uses biological enzymes to identify non-specific natural substrates and catalyze covalent modification of target sites. For example, transglutaminase, Sortase, halogenase, etc., can identify specific short peptide sequences and precisely catalyze the attachment of glycosyl, lipid, fluorescent groups or drug molecules to polypeptides and proteins. Enzymatic reaction standard is soft, can be carried out in solution, physiological temperature, good biocompatibility, less side effects, in the antibody drug conjugate (ADC) preparation industry has great potential.

The short board of enzymatic decoration lies in the limitation of the substrate sequence of the enzyme itself. Most enzymes only identify specific short peptide motifs and must store the identification sites in the protein sequence. Some commercial enzymes are expensive and difficult to produce in large quantities. Non-specific decoration of peptides and protein sites opens the key channel from protein chemical transformation to life system analysis and biological drug development.