Technical improvements relevant to peptide bond formation, including the use of new coupling reagents and microwave irradiation, have advanced the peptide chain assembly process. Nevertheless, the synthesis of aggregation-prone peptides, “difficult sequence”, has remained a challenge due to the decrease in coupling efficiency and solubility. The use of aggregation-disrupting units, such as pseudoproline1 and O-acyl isopeptide2, during the stepwise elongation for synthesizing short peptides, including peptide fragments, has facilitated the chain assembly process. However, their application to protein synthesis requiring native chemical ligation (NCL) has been hampered by the following reasons: (1) pseudoproline units revert to the parent Ser or Thr unit upon the acidic deprotection indispensable for the preparation of peptide fragments for NCL; (2) the O-acyl isopeptide suffers O-N acyl transfer in NCL under neutral reaction conditions. Therefore, an NCL-compatible aggregation-disrupting unit has been demanded for facilitating the NCL of aggregation-prone fragments.
In this context, we were interested in selenazolidine (Sez), an acid-resistant pseudoproline cognate that could disrupt the aggregation. As expected, the Sez derivative remained intact under the acidic and neutral reaction conditions required for performing the NCL. More gratifyingly, we found that the Sez unit can be converted to the O-acyl Ser unit by the action of a silver salt under acidic conditions. Consequently, the Sez unit would function as the aggregation-disrupting unit not only as a pseudoproline moiety during NCL coupling but also as O-acyl isopeptide at the following purification step under acidic conditions
Here, we will discuss the performance of the Sez unit in facilitating the NCL-mediated protein synthesis using aggregation-prone and poorly soluble peptide fragments.