Efficient approach to synthesis of two-chain asymmetric cysteine analogs of receptor-binding region of transforming growth factor-α

The putative receptor-binding region of human transforming growth factor-α (TGF α) has been shown to be contributed by two fragments: an A-chain (residue 12-18) and a 17-residue carboxyl fragment (residue 34-50) that includes a disulfide-containing C-loop (residue 34-43). An approach to the synthesis of two-chain analogs containing an intermolecular disulfide linked A-chain and the 17-residue carboxyl fragment (C-fragment) possessing receptor-binding activity is described. The synthesis was achieved by the solid-phase method using the Boc-benzyl protecting group strategy. The single Cys of the A-chain was activated as a mixed disulfide with 2-thiopyridine to form the intermolecular disulfide bond with Cys41 or Cys46 of the C-fragment on the resin support. Prior to this reaction, the acetamido (Acm) protecting group of Cys41 or Cys46 was removed by Hg(OAc)₂ on the resin support. The peptide and side chain protecting groups including the S-methylbenzyl moiety of the Cys34 and Cys43 were concomitantly cleaved by high HF. The intramolecular disulfide with two unprotected Cys was formed in the presence of an intermolecular disulfide. This intramolecular disulfide bond formation was usually not feasible under the traditionally-held scheme at basic pH since disulfide interchange would occur faster than intramolecular oxidation. To prevent the disulfide interchange, a new method was devised. The intramolecular disulfide bond oxidation was mediated by dimethylsulfoxide at an acidic pH, at which the disulfide interchange reaction was suppressed. The desired product was obtained with a 60-70%, yield. In contrast, the conventional scheme of using I₂ to form the intermolecular disulfide between the Cys(Acm) of the A-chain and C-fragment with the preformed intramolecular disulfide bond in solution phase did not result in any product. The purified two-chain analogs were found to be unstable and rearranged to the homo-dimers. This reaction was greatly accelerated in I₂, which explained the difficulty associated with the conventional scheme. When assayed against A431 and NRK clone 49F cells, both the A-chain and the C-fragment did not exhibit any biological activity independently, but the two-chain analogs showed low receptor-binding activity with an IC₅₀ at 0.3 mM level. Unexpectedly, dimeric C-fragment, which resulted from the rearrangement reaction, also showed receptor-binding activity. Our results demonstrate that the two-chain analogs exhibit low but distinct biological activity and provide evidence that the putative TGFα receptor binding region may be discontinuous. In addition, we also provide an efficient approach to further explore the two-chain receptor-binding analogs of TGFα.