N5-METHYLASPARAGINE AND ASPARAGINE AS NUCLEOPHILES IN PEPTIDES - MAIN-CHAIN VS SIDE-CHAIN AMIDE CLEAVAGE

The chemistry of peptides containing N5-methylasparagine (NMA) was inv estigated by incubating the synthetic peptides Ile-Ala-Pro-Gly-Gly-Asn -Gly-Tyr and Ile-Ala-Pro-Gly-Gly-NMA-Gly-Tyr at 60-degrees-C in 0.1 M NaPO4, pH 7.4, to assay for peptide deamidation. The Asn-Gly octapepti de deamidates to Ile-Ala-Pro-Gly-Gly-isoAsp/Asp-Gly-Tyr with a half-li fe of 2.17 h and activation energy of 18.6 kcal/mol. The NMA-Gly octap eptide partitions between main-chain cleavage and side-chain deamidati on in 2.7:1 ratio. Analysis of products diagnostic for each of these N MA peptide reactions yields indistinguishable activation energies for each pathway: 22.6 kcal/mol. The half-life for NMA side-chain deamidat ion is 98 h, commensurate with a 2.5 kcal/mol difference in activation free energies for deamidation at Asn and NMA sites. These results ind icate that methylation provides a substantial (45-fold) stabilization against intramolecular C-N cleavage. The identical activation energy f or the alternative pathways of NMA peptide reactivity suggests the dif ferences in the rates may be due to the preexponential portion of the rate equation reflecting small differences in DELTAS(double dagger). M olecular mechanics studies were performed to account for these pattern s. The computational studies disclose 3-fold more conformers in the Bo ltzmann population for the tetrahedral intermediate leading toward mai n-chain cleavage. This result supports the hypothesis that the 2.7-fol d difference in NMA peptide partitioning rates is attributable to diff erences in DELTAS(double dagger).