In recent years, the research and development of protein and polypeptide drugs have become a hot topic in the field of biomedicine because of their specific action sites and clear curative effects.

Disulfide bonds play an important role in maintaining the spatial structure of polypeptides and proteins and the resulting biological activity. Disulfide bond is the S-S covalent bond formed by oxidation of sulfhydryl group (-SH) at two different sites of Cys in protein or polypeptide molecule. Disulfide bonds formed between amino acids at different points in a peptide chain can fold the peptide chain into a specific spatial structure.

Multi-pair disulfide ring formation is a new technique in the field of protein engineering and synthetic biology, which builds proteins with specific structure and function by ordering multi-pair disulfide bonds.

The development of this technology offers a more flexible approach to designing and manufacturing novel proteins, with important biomedical engineering potential.

At present, multi-pair disulfide bond ring formation technology is in the stage of rapid development. As chemical and biological tools continue to improve, researchers are able to more precisely control the formation and breaking of disulfide bonds, overcoming some of the challenges of synthesis.

Polypeptide molecules usually have large molecular weight and complex spatial structure, and the formation of disulfide bonds in the structure requires two cysteines to be close to each other in space. In addition, the chemical properties of the reduced sulfhydryl group in the polypeptide structure are active and prone to other side reactions, and other side chains on the peptide chain may also undergo a series of modifications, so the oxidizer and oxidation conditions selected for the modification of the peptide chain are important factors in the reaction, and the reaction mechanism is also more complex, which may be a free radical reaction or an ionic reaction.

In the modification of disulfide bonds in polypeptides, the synthesis of a pair of intramolecular or intermolecular disulfide bonds is usually relatively easy, and there are many options for reaction conditions, such as mild oxidation processes such as air oxidation, DMSO oxidation, or intense reaction conditions such as H2O2, I2, and mercury salt. The reaction products are also relatively easy to purify and separate, and obtain higher purity and yield.

The formation of disulfide bonds by air oxidation is the most common method in peptide synthesis, and good results have been obtained in early studies. The use of air oxidation method is generally the thiol group in the reducing state of polypeptides dissolved in water, in near neutral or weakly alkaline conditions (PH 6.5 ~ 10), reaction for more than 24 hours. In order to reduce the possibility of the formation of disulfide bonds between molecules, the method usually needs to be carried out at low concentrations.

Iodine oxidation method is also widely used in peptide synthesis. Generally, the peptide is dissolved in 25% methanol aqueous solution or 30% acetic acid aqueous solution, and 10 ~ 15mol/L of iodine is added to oxidize each drop, and the reaction is 15 ~ 40min. When the peptide chain contains residues of Tyr, Trp, Met and His that are more sensitive to iodine, the oxidation conditions should be controlled more precisely, and vitamin C or sodium thiosulfate should be added immediately after oxidation to remove excess iodine.

When two or more pairs of disulfide bonds need to be formed on a peptide chain, the reaction process becomes relatively complex. Before the synthesis of polypeptide in solid phase, it is necessary to design several pairs of disulfide bond formation sequence and method route in advance, select different side chain sulfhydryl protection groups, and make use of their property differences to form two or more pairs of disulfide bonds by step oxidation. Usually used sulfhydryl protection groups are trt, Acm, Mmt, tBu, Bzl, Mob, Tmob and other groups. We listed the multipara-disulfide bond formation routes on two kinds of polypeptides synthesized with 2-Cl resin and Rink resin as carriers respectively:

* Disulfide ring labeling peptide is a modification method by forming a disulfide ring structure in a polypeptide chain.

This method uses the disulfide bond between two cysteine residues to form a ring structure, which enhances the stability and biological activity of the polypeptide. Disulfide ring-labeled peptides have important applications in antimicrobial peptide research, drug design and protein engineering.

The formation of disulfide bonds has always been a difficult point in peptide synthesis. Motif has quite mature multi-pair disulfide ring formation technology. At present, we can successfully synthesize three - and four-pair disulfide polypeptides with a success rate of more than 85%.

At present, MOTIF has been successfully synthesized as follows: Pyr-GCCSKW*********GPARCC-NH2(Cys3-Cys15 Cys4-Cys21 Cys10-Cys22)  (Figure below)