After 75 Years, an Alternative to Edman Degradation: A Mechanistic and Efficiency Study of a Base-Induced Method for N-Terminal Peptide Sequencing

The sequencing of peptides via N-terminal amino acid removal is a classic reaction termed Edman degradation. This method involves repeated treatment of the N-terminal amino group of a peptide with phenyl isothiocyanate (PITC), followed by treatment with trifluoroacetic acid. Spurred by the need for an alternative non-acid-based chemistry for next-generation protein sequencing technologies, we developed a base-induced N-terminal degradation method. Several N-terminal derivatization reagents carrying supernucleophiles were tested. After rounds of iterative designs, compound <b>DR3</b>, with a <i>N</i>-hydroxysuccinimide as a leaving group and hydrazinecarboxamide as the supernucleophile, demonstrated the highest yield for the peptide derivatization step and the most efficient elimination of the N-terminal amino acid in just 1% of a hydroxide salt. The method successfully removed all 20 amino acids at the N-terminus in high yield. The technique demonstrates compatibility with oligonucleic acids, which differs from Edman degradation due to their inherent sensitivity to acidic environments. To demonstrate the practical application of our approach, we sequenced amino acids sequentially from a peptide, effectively determining the sequence of an unknown peptide. Notably, our methodology was successfully applied to mixtures of peptides derived from protein samples, where a significant fraction of the peptides derivatized with <b>DR3</b> underwent elimination of their N-terminal amino acid upon addition of base. Overall, although our method does not outperform Edman degradation in efficiency, it serves as a valuable alternative in cases where base-induced cleavage is advantageous, particularly for preserving acid-sensitive functionalities.