An overview of disulfide bonds in chemistry and biology

Chemically, the disulfide bond refers to the R-S-S-R “functional group. Disulfide bonds usually consist of two thiol groups. In biology, disulfide bonds formed between thiol groups in two cysteine residues are an important part of the secondary and tertiary structure of proteins. This bond plays an important role in the three-dimensional structure of the protein molecule.

In mild oxidation processes such as oxidation and DMSO oxidation, intense reaction conditions such as H2O2, I2, and mercury salts can also be used.

Air oxidation method:

The generation of disulfide bonds by oxidation is the most classical method in peptide synthesis, usually by dissolving peptides in the reduced state in water, after reacting in a near-neutral or weakly alkaline environment (PH 6.5-10) for more than 24 hours. To reduce the possibility of intermolecular disulfide bonding, this method usually needs to be performed at low concentration conditions.

An overview of disulfide bonds in chemistry and biology

Iodine oxidation method:

Peptides were dissolved in 25% methanol solution or 30% acetic acid solution and oxidized with 10 to 15mol/L iodine per drop for a reaction of 15 to 40min. “When the peptide chain contains iodine-sensitive Tyr, Trp, and in the process of Met and His residues, the oxidation conditions are controlled more precisely, and after oxidation, vitamin C or sodium thiosulfate is added immediately to remove excess iodine.”

Ring forming strategy of two-to-many disulfide bonds

If the sequence contains two or more pairs of disulfide bonds that need to be looped, there are usually two situations:

Nature is a random loop:

Cys was looped randomly in the sequence, similar to the looping condition of a pair of disulfide bonds.

Fixed point ring:

According to the design requirements, the specified loop, namely Cys in the sequence, produces disulfide bonds, and the reaction process is complex. Before the solid-phase synthesis of peptides, it is necessary to design the sequence and method of producing several groups of disulfide bonds in advance, select different side chain group protection groups, and take advantage of their property differences to gradually oxidize to produce two or more pairs of disulfide bonds. Generally used base protectants are trt, Acm,Mmt,tBu,Bzl,Mob, various groups such as Tmob.

After designing the production sequence of disulfide bonds and the Cys side chain protection strategy, we should take a small amount of resin to cut after ring formation of each disulfide bond pair and perform MS verification of the cleaved device. The MS verification chart will be provided together with the shipment.