TFA Cleavage Strategy for Mitigation of S-tButylated Cys-Peptide Formation in Solid-Phase Peptide Synthesis

Abstract

Cysteine (Cys) is the most versatile amino acid-forming part of a peptide chain but at the same time the most complex. Its presence is associated with a large number of side reactions. In particular, the formation of S-tert-butylated Cys residues results from the reaction of the liberated Cys thiol with the tBu cations coming from the tBu-based protecting groups. Here, we have studied this side reaction using different scavengers such as alkyl and aryl thiols (DTT, 1,4-BDMT), thioethers (DMS, thioanisole), and sulfur-free compounds such as m-cresol, anisole, PPh₃ and TCEP in addition to TIS and H₂O. Three of these scavengers (DTT, 1,4-BDMT, PPh₃) are disulfide-reducing agents. Furthermore, the study also considered the cleavage duration and the TFA content in the cleavage mixtures. In peptides containing Ser(tBu) and/or Thr(tBu), the reduction of the TFA content led to the incomplete removal of the tBu protecting group. After this feasibility study, it can be concluded that the combined use of thioanisole and DMS in slightly higher quantity than TIS and H₂O in the presence of 1% DTT is beneficial. Furthermore, optimal results are obtained if the cleavage is carried out in two steps: initial treatment of the peptide with TFA/TIS/H₂O/thioanisole/DMS/1% DTT (70:5:5:10:10) for 30 min followed by TFA addition up to an 80% proportion and continued treatment for 150 min.