Disulfide Bond Modification

Disulfide bonds are integral components in the structure of many peptides and proteins. We can find these covalent bonds in almost all extracellular peptides and protein molecules. These bonds are important in maintaining the structural integrity and stability of peptides and proteins. If the peptide contains cysteine, disulfide bonds can form between the sulfhydryl groups of the cysteine side chains. However, since disulfide bonds are formed randomly, this is a challenge for peptides containing multiple cysteine residues. After long-term and in-depth research in the field of peptides, Alfa Chemistry has accumulated a lot of valuable experience. We have successfully delivered to customers a large number of peptides containing one or more pairs of disulfide bonds.

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Alfa Chemistry has mature multiple pairs of disulfide bond cyclization technology, which can construct disulfide bonds between designated cysteines. The disulfide bond modifications we specialize in include:

• Formation of two pairs of disulfide bonds within the molecule
• Formation of two pairs of disulfide bonds between molecules
• Formation of three pairs of disulfide bonds within the molecule
• Formation of three pairs of disulfide bonds between molecules
• Formation of two pairs of disulfide bonds between different peptide sequences

Methods for Forming A Pair of Disulfide Bonds

Peptides usually have large molecular weights and complex spatial structures. The formation of a disulfide bond in the structure requires two cysteines to be close in spatial distance. In addition, the reduced sulfhydryl group in the peptide structure is chemically active and prone to other side reactions, and other side chains on the peptide chain may also undergo a series of modifications. Therefore, the oxidant and oxidation conditions selected for modification of the peptide chain are key factors in the reaction. Commonly used methods are:

• Air oxidation method. The formation of disulfide bonds by air oxidation is the most classic method in peptide synthesis. This method usually involves dissolving the peptide with the sulfhydryl group in the reduced state in water, and reacting for more than 24 hours under nearly neutral or weakly alkaline conditions (PH value 6.5-10). In order to reduce the possibility of disulfide bond formation between molecules, this method usually needs to be carried out at low concentration conditions.
• Iodine oxidation method. The iodine oxidation method is to dissolve the peptide in 25% methanol aqueous solution or 30% acetic acid aqueous solution, add 10-15 mol/L iodine drop by drop for oxidation, and react for 15-40 minutes. When the peptide chain contains tyrosine (Tyr), tryptophan (Trp), methionine (Met) and histidine (His) residues that are sensitive to iodine, the oxidation conditions need to be controlled more accurately. After oxidation, vitamin C or sodium thiosulfate is added immediately to remove excess iodine.

Methods for Forming Two or More Pairs of Disulfide Bonds

When two or more pairs of disulfide bonds need to be formed in the peptide sequence, there are usually two situations:

• Natural random ring formation. Natural random ring formation refers to random ring formation between cysteines in the sequence, which is similar to the ring formation conditions of a pair of disulfide bonds.
• Fixed-point ring formation. Fixed-point ring formation means that the cysteines in the sequence form disulfide bonds according to the design requirements, and the reaction process is relatively complicated. In response to this situation, before solid-phase synthesis of peptides, it is necessary to design the sequence and method route for the formation of several pairs of disulfide bonds in advance, and select different side chain sulfhydryl protecting groups. Then, the differences in properties of different protecting groups are used to oxidize step by step to form two or more pairs of disulfide bonds. Commonly used sulfhydryl protecting groups include trityl (trt), acetamidomethyl (Acm), tert-butyl (tBu), p-methoxybenzyl (Mob), p-methylbenzyl (Meb) and other groups.

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