TFA Cleavage Strategyfor Mitigation of <i>S</i>‑<i>t</i>ButylatedCys-Peptide Formation in Solid-PhasePeptide Synthesis

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-<i>tert-</i>butylated Cys residues results from the reaction of the liberated Cys thiol with the <i>t</i>Bu cations coming from the <i>t</i>Bu-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 <i>m</i>-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(<i>t</i>Bu) and/or Thr(<i>t</i>Bu), the reduction of the TFA content led to the incomplete removal of the <i>t</i>Bu 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.