N5-Methylasparagine and Asparagine as Nucleophiles in Peptides: Main-Chain vs Side-Chain Amide Cleavage

Article abstract of DOI:10.1021/jo00077a014

The chemistry of peptides containing N⁵-methylasparagine (NMA) was investigated by incubating the synthetic peptides Ile-Ala-Pro-Gly-Asn-Gly-Tyr and Ile-Ala-Pro-Gly-Gly-NMA-Gly-Tyr at 60 deg C in 0.1 M NaPO4, pH 7.4, to assay for peptide deamidation.The Asn-Gly octapeptide deamidates to Ile-Ala-Pro-Gly-Gly-isoAsp/Asp-Gly-Tyr with a half-life of 2.17 h and activation energy of 18.6 kcal/mol.The NMA-Gly octapeptide partitions between main-chain cleavage and side-chain deamidation in 2.7:1 ratio.Analysis of products diagnostic for each of these NMA peptide reactions yields indistinguishable activation energies for each pathway: 22.6 kcal/mol.The half-life for NMA side-chain deamidation is 98 h, commensurate with a 2.5 kcal/mol difference in activation free energies for deamidation at Asn and NMA sites.These results indicate that methylation provides a substantial (45-fold) stabilization against intramolecular C-N cleavage.The identical activation energy for the alternative pathways of NMA peptide reactivity suggests the differences in the rates may be due to the preexponential portion of the rate equation reflecting small differences in ΔS(excit.).Molecular mechanics studies were performed to account for these patterns.The computational studies disclose 3-fold more conformers in the Boltzmann population for the tetrahedral intermediate leading toward main-chain cleavage.This result supports the hypothesis that the 2.7-fold differences in NMA peptide partitioning rates is attributable to differences in ΔS(excit.).