Peptide Synthesis - Regioselective Disulfide Formation

Regioselective Disulfide Formation

The formation of multiple native disulfides remains one of the primary challenges of native peptide synthesis by solid-phase methods. Random chain combination typically results in several products with nonnative disulfide bonds. Stepwise formation of disulfide bonds is typically the preferred method, and performed with thiol protecting groups (PGs). Different thiol PGs provide multiple dimensions of orthogonal protection. These orthogonally-protected cysteines are incorporated during the solid-phase synthesis of the peptide. Successive removal of these PGs to allow for selective exposure of free thiol groups, leads to disulfide formation in a stepwise manner. The order of removal of these PGs must be considered so that only one group is removed at a time. Using this method, Kiso et al. reported the first total synthesis of insulin by this method in 1993.

The thiol PGs must possess multiple characteristics. First, the PG must be reversible with conditions that do not affect the unprotected side chains. Second, the protecting group must be able to withstand the conditions of solid-phase synthesis. Third, the configuration of the removal of the thiol protecting group must be such that it leaves intact other thiol PGs, if orthogonal protection is desired. That is, the removal of PG A should not affect PG B. Some of the thiol PGs commonly used include the acetamidomethyl (Acm), tert-butyl (But), 3-nitro-2-pyridine sulfenyl (NPYS), 2-pyridine-sulfenyl (Pyr), and triphenylmethyl (Trt) groups. Importantly, the NPYS group can replace the Acm PG to yield an activated thiol.

In the stepwise formation of disulfides to synthesize insulin by Kiso et al., the authors synthesize the A-chain with following protection: CysA6(But); CysA7(Acm); CysA11(But). Thus, CysA20 is unprotected. Synthesis of the B-chain is performed with the following protection: CysB7(Acm) CysB19(Pyr). The first disulfide bond, CysA20-CysB19, was formed by mixing the two chains in 8 M urea, pH 8 (RT) for 50 min. The second disulfide bond, CysA7-CysB7, was formed by treatment with iodine in aqueous acetic acid to remove the Acm groups. The third disulfide, the intramolecular CysA6-CysA11, was formed by the removal of the But groups by methyltrichlorosilane with diphenyl sulfoxide in TFA. Importantly, formation of the first disulfide in 8 M urea, pH 8 does not affect the other PGs, namely Acm and But groups. Likewise, formation of the second disulfide bond with iodine in aqueous acetic acid does not affect the But groups.

Important to the discussion of disulfide bond formation is the order in which disulfides are formed. From a logical standpoint, the order in which the thiol groups are exposed to form disulfides should be of little consequence, since the other cysteines are protected. Practically, however, the order in which disulfides are formed can have a significant effect on yields. This may be because the formation of the CysA20-CysB19 disulfide may place the thiol group of CysB7 in close proximity with both CysA6 and CysA7, leading to multiple disulfide products. This is one manifestation of the reality that solid-phase peptide synthesis is as much art as it is science.