RESUMO
Aryl azide-mediated photo cross-linking has been widely used to obtain structural features in biological systems, even though the reactive species generated upon photolysis in aqueous solution have not been well characterized. We have established a mechanistic framework for the formation of adducts between photoactivated 5-azido-2-nitrobenzoyl reagents and protein functional groups. Photolysis of the aryl azide tethered to biotin via an amide linkage yields a cross-link with streptavidin. The ability of the pre-irradiated reagent to form a similar cross-link indicates that it is the long-lived reactive intermediate that contributes to the cross-link formation. The reactive intermediate forms an adduct with tryptophan. The sequence of the labeled peptide is found to be GlyTrp(*)ThrValAlaTrp(*)LysAsn, corresponding to residues 74-81 of the streptavidin sequence, where Trp(*) designates the modified Trp-75 and Trp-79. A peak at m/z 1455.1 corresponding to the calculated [M(peptide)+aryl nitrene+2O](+) molecular ion value has been observed for the labeled peptide. Product structure identification experiments support the assignment that the long-lived reactive intermediate is a p-nitro-N-arylhydroxylamine, which undergoes a number of transformations in aqueous solution leading to nitroso derivatives. A plausible mechanism of the interaction between tryptophan and nitroso compound is discussed.
Assuntos
Azidas/química , Reagentes de Ligações Cruzadas/química , Compostos Nitrosos/química , Estreptavidina/química , Marcadores de Afinidade/química , Sequência de Aminoácidos , Biotina/química , Fotólise , Estreptavidina/metabolismo , Triptofano/químicaRESUMO
We show that iron(II)-phthalocyanines are able to catalyze guanosine oxidation by molecular oxygen in the presence of reducing agents such as ascorbic acid and 2-mercaptoethanol. The products of 5'-monophosphate-2'-deoxyguanosine (dGMP) oxidation were directly analyzed using the HPLC-ESI/MS method. The main oxidation products were 5'-phospho-2'-deoxy-8-oxo-7,8-dihydroguanine and the 1,N2-glyoxal adduct of the 5'-monophosphate-2'-deoxyguanosine.
Assuntos
Nucleotídeos de Desoxiguanina/química , Indóis/química , Ferro/química , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Antivirais/farmacologia , Ácido Ascórbico/química , Catálise , Química Farmacêutica/métodos , Cromatografia Líquida de Alta Pressão/métodos , Desenho de Fármacos , Humanos , Radical Hidroxila , Isoindóis , Mercaptoetanol/química , Modelos Químicos , Oxigênio/química , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
Design of chemically modified oligonucleotides for regulation of gene expression has attracted considerable attention over the last decades. One actively pursued approach involves antisense or antigene constructs carrying reactive groups, many of these based on transition metal complexes. The complexes of Co(II) with phthalocyanines are extremely good catalysts of oxidation of organic compounds with molecular oxygen and hydrogen peroxide. In this study, we have investigated the kinetics and thermodynamics of sequence-specific modification of DNA with deoxyribooligonucleotide linked to Co(II)-tetracarboxyphthalocyanine (PtcCo(II)) in the presence of H(2)O(2).
