N-O Bond Fission as the Rate-Determining Step in the Aqueous Conversion of N-Peptidyl-O-(p-nitrobenzoyl)hydroxylamines to p-Nitrobenzoic Acid and Peptidylhydroxamic Acids

Article abstract of DOI:10.1021/jo00286a017

N-Acetyl-, N-alanyl-alanyl-, N-alanyl-prolyl-, and N-Boc-alanyl-prolyl-O-(p-nitrobenzoyl) hydroxylamines, compounds that are mechanism-based irreversible inactivators of some proteolytic enzymes, are degraded in aqueous buffers at neutral pH to p-nitrobenzoic acid and either the corresponding N-acylhydroxamic acid or products of its further degradation such as the diketopiperazine.At neutral pH, the reactants exist as the monoanion, as a result of the acidity of the -CO-NH-O- linkage.The p-nitrobenzoic acid formed in a mixture of 50percent H2(18)O and 50percent H2(16)O contains less than 2percent (18)O, which shows that nucleophilic attack of water at the ester carbonyl is not occuring in the degradation.The decomposition of the N-alanyl-prolyl derivative, labeled with (15)N at the N-O nitrogen, exhibits a kinetic isotope effect k₁₄/k₁₅ = 1.092 +/- 0.056, suggesting that N-O fission is occuring in the rate-determining step of the degradation.Kinetic solvent isotope effects of 1.02-1.15 are inconsistent with an expectation of factors around 2 or greater for spontaneous hydrolysis of the ester linkage.All derivatives have ΔH^* = 24-27 kcal/mol and ΔS^* = +4-7 eu, consistent with unimolecular fission of the substrate N-O to generate p-nitrobenzoate ion and the acyl nitrene.The nitrene must suffer nucleophilic attack at nitrogen very rapidly, producing the hydroxamic acid as the initial product.In the peptide derivatives, further reaction to the cyclized products results.