Selective Fluorogenic Derivatization with Isotopic Coding of Catechols and 2-Amino Phenols with Benzylamine: A Chemical Basis for the Relative Determination of 3-Hydroxy-tyrosine and 3-Nitro-tyrosine Peptides
2007
Pennington, Justin P | Schöneich, Christian | Stobaugh, John F
The derivatization of catecholamines and catechols, first with diphenylethylenediamine and latter benzylamine, to apparently form the same highly fluorescent product has been described for many years. Based on fluorescence spectral evidence, previous conclusions were that the same product was formed for each analyte class with either reagent. In the present investigation this conclusion has been revisited by isolating and characterizing (X-ray crystal determination, MS, NMR, etc.) the product resulting from reaction of 4-methylcatechol with benzylamine. These investigations definitively establish that catecholamines and catechols undergo this reaction to form related but different 6-substituted 2-phenyl benzoxazole products. Based on a plausible product formation mechanism, it was postulated and shown that 2-aminocresol would participate in this fluorogenic derivatization reaction to form the same product as results from 4-methylcatechol. It appears that this is a general transformation available for analytes bearing the 2-hyroxyl-phenol (catechol) and/or the 2-amino-phenol moieties. Further experimentation confirmed that ²H₅-benzylamine reacts with 4-methylcatechol and 2-aminocresol to form the same product in identical analytical yield as previously noted for ¹H₅-benzylamine, thus establishing a potential chemical basis for the relative quantitation of 3-hyroxy-tyrosine (DOPA) residues and 3-nitro-tyrosine residues (reduction to the corresponding 3-amino-tyrosine residue required) in proteins that have been subjected to oxidative stress. In preliminary experiments relative quantitation was achieved for 4-methylcatechol and the 3-hydroxy-tyrosine containing peptide DOPA-Gly-Gly.
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