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
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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.
KeywordsColumn liquid chromatography DOPA derivatization Catechols Benzylamine 2-Aminophenols 3-Hydoxy-tyrosine 2-Nitro-tyrosine derivatization
This research was supported by NIH grants AG25350 and AG23551, and by a AFPE Fellowship JPP (partial support). The authors would like to thank Dr. David Vander Velde and Sarah Neuenswander (NMR facility) and Dr. Victor Day (X-ray crystallography laboratory) from the University of Kansas Molecular Structures Group.
- 1.Natelson S, Lugovoy JK, Pincus JB (1949) Arch Biochem Biophys 23:157–158Google Scholar
- 2.Weil-Malhebe H, Bone AD (1952) Biochem J 51:311–318Google Scholar
- 4.Armstrong SG, Dean RT (1995) Redox Report 1:291–298Google Scholar
- 8.Davies MJ (2005) Biochim Biophys Acta 1703:93–109Google Scholar
- 12.Ambs S, Merriam WG, Bennett WP, Felley-Bosco E, Ogunfusika MO, Oser SM, Klein S, Shields PG, Billiar TR, Harris CC (1998) Cancer Res 58:334–341Google Scholar
- 21.Marumo K, Waite JH (1986) Biochim Biophys Acta 872:98–103Google Scholar
- 22.Finley KT (1974) The addition and substitution chemistry of quinones. In: Patai S (ed) The chemistry of the quinonoid compounds. Wiley, UK, pp 877–1144Google Scholar