Disulfide bonds are widely present in protein structures and play a crucial role in stabilizing protein structures. Disulfide bonds are generally formed through oxidation of the thiol groups of two Cys in the sequence. There are many methods for forming disulfide bonds: air oxidation, DMSO oxidation, hydrogen peroxide oxidation, etc. The synthesis process of disulfide bonds can be monitored through Ellman detection and HPLC detection methods.
If the peptide only contains one pair of Cys, the formation of disulfide bonds is simple. Peptides are synthesized in solid or liquid phases and then oxidized in a solution at pH 8-9.
When two or more pairs of disulfide bonds need to be formed, the synthesis process is relatively complex. Although the formation of disulfide bonds is usually completed in the final stage of the synthesis scheme, sometimes the introduction of pre formed Disulfide is beneficial to the connection or extension of peptide chains. The mercapto Protecting group commonly used include trt, Acm, Mmt, tBu, Bzl, Mob, Tmob and other groups.
Selection of Disulfide Bond Reaction Conditions
The disulfide bond is the S-S Covalent bond formed by the oxidation of the sulfhydryl group (- SH) of two different sites Cys in the protein or polypeptide molecule. The disulfide bond formed between amino acids at different positions on a peptide chain can fold the peptide chain into a specific spatial structure. Polypeptide molecules usually have large molecular weight and complex spatial structure. The formation of disulfide bond in the structure requires that the two cysteine molecules are close in spatial distance. In addition, the thiol group in the reduced state of the peptide structure has active chemical properties and is prone to other side reactions. Additionally, other side chains on the peptide chain may undergo a series of modifications. Therefore, the oxidant and oxidation conditions selected for peptide chain modification are key factors in the reaction, and the reaction mechanism is also relatively complex, which may be either a free radical reaction or an ion reaction. There are multiple options for reaction conditions, such as mild oxidation processes such as air oxidation and DMSO oxidation, as well as intense reaction conditions such as H2O2 and mercury salts.
Air oxidation method:
The formation of disulfide bond by air oxidation is the most classic method in Peptide synthesis. Generally, the peptide with sulfhydryl group in reduced state is dissolved in water and reacted for more than 24 hours under near neutral or weakly alkaline conditions (pH 6.5-10). To reduce the possibility of disulfide bond formation between molecules, this method usually needs to be carried out under low concentration conditions.
Iodine oxidation method:
Dissolve the peptide in 25% methanol aqueous solution or 30% acetic acid aqueous solution, add 10-15mol/L iodine dropwise for oxidation, and react for 15-40 minutes. When the peptide chain contains Tyr, Trp, Met and His residues that are sensitive to iodine, the oxidation conditions should be controlled more accurately. After oxidation, vitamin C or Sodium thiosulfate should be added immediately to remove the excess iodine.
When there are two or more pairs of disulfide bonds in a sequence that need to be looped, there are usually two situations:
Natural random ring formation:
Cys in the sequence randomly form rings, similar to the ring formation conditions of a pair of disulfide bonds
Fixed point looping:
Fixed point cyclization refers to the formation of disulfide bonds by Cys in the sequence according to design requirements, and the reaction process is relatively complex. Before Solid-phase synthesis of polypeptides, it is necessary to design the sequence and method route for the formation of several pairs of disulfide bonds in advance, select different side chain mercapto Protecting group, and take advantage of their differences in nature to form two or more pairs of disulfide bonds step by step. The mercapto Protecting group commonly used include trt, Acm, Mmt, tBu, Bzl, Mob, Tmob and other groups.
