Multiple disulfide bonds form rings

Disulfide bonds are widely present in protein structures and play a very important role in stabilizing protein structures. Disulfide bonds are generally formed by oxidation of the thiol groups of two Cys in the sequence. There are many methods to form disulfide bonds, such as air oxidation, DMSO oxidation, hydrogen peroxide oxidation, etc. The synthesis process of disulfide bonds can be monitored for its reaction progress using Ellman detection and HPLC detection methods.  

If the polypeptide contains only one pair of Cys, the formation of disulfide bonds is simple. Peptides are synthesized in solid or liquid phase and then oxidized in a solution with pH 8-9.

When it is necessary to form 2 or more pairs of disulfide bonds, 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 compounds is beneficial for linking or extending peptide chains. The commonly used thiol protecting groups include various functional groups such as trt, Acm, Mmt, tBu, Bzl, Mob, Tmob, etc. We have listed two routes for the formation of multiple disulfide bonds on peptides synthesized using 2-Cl resin and Rink resin as carriers:

Selection of reaction conditions for disulfide bond

Disulfide bond is an S-S covalent bond formed by the oxidation of thiol groups (- SH) at two different positions of Cys in protein or peptide molecules. The disulfide bonds formed between amino acids at different positions on a peptide chain can fold the peptide chain into specific spatial structures. Peptide molecules typically have a large molecular weight and complex spatial structure, and the formation of disulfide bonds in the structure requires two cysteine residues to be close in spatial distance. In addition, the thiol groups in the reduced state of peptide structures are 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 peptide chain modification are key factors in the reaction, and the reaction mechanism is also relatively complex, which can be either a free radical reaction or an ionic reaction.

There are various reaction conditions to choose from, such as mild oxidation processes like air oxidation and DMSO oxidation, as well as intense reaction conditions like H2O2, I2, and mercury salts.

Air oxidation method:

The formation of disulfide bonds through air oxidation is the most classic method in peptide synthesis. Typically, peptides with thiol groups in a reduced state are dissolved in water and reacted for over 24 hours under near neutral or weakly alkaline conditions (pH 6.5-10). In order 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 a 25% methanol aqueous solution or a 30% acetic acid aqueous solution, add 10-15 mol/L iodine dropwise for oxidation, and react for 15-40 minutes. When the peptide chain contains residues of Tyr, Trp, Met, and His that are sensitive to iodine, the oxidation conditions need to be controlled more accurately. After oxidation, vitamin C or sodium thiosulfate should be added immediately to remove excess iodine.

Two or more pairs of disulfide bond cyclization strategies

When there are two or more pairs of disulfide bonds in a sequence that need to form a ring, there are usually two situations:

Natural random looping:

Cys in the sequence randomly form loops, similar to the conditions for a pair of disulfide bonds to form loops;

Fixed point ring formation:

Fixed point cyclization refers to the formation of disulfide bonds between Cys in a sequence according to design requirements, and the reaction process is relatively complex. Before solid-phase synthesis of peptides, it is necessary to design several sequences and routes for the formation of disulfide bonds, select different side chain thiol protecting groups, and utilize their property differences to form two or more pairs of disulfide bonds through stepwise oxidation.

The commonly used thiol protecting groups include various functional groups such as trt, Acm, Mmt, tBu, Bzl, Mob, Tmob, etc.