Doctor of Philosophy
Department of Chemistry
Manthey, Michael Kurt, Mechanism and structural elucidation of protein modification 3-hydroxyanthranilic acid, Doctor of Philosophy thesis, Department of Chemistry, University of Wollongong, 1990. http://ro.uow.edu.au/theses/1162
In order to model the reaction of proteins towards 3-hydroxyanthranilic acid 1, a structural, synthetic and mechanistic study of the autoxidative chemistry of 1 was undertaken, the results of which are presented in this thesis.
The autoxidation of 1 under physiological conditions yielded three primary autoxidation products. The major autoxidation product was found to be the p-benzoquinone 30. Two minor components, cinnabarinic acid 7. and the novel benzocoumarin 31, were also isolated and characterised.
Cinnabarinic acid was found to be unstable to hydrogen peroxide generated during the autoxidation process, rapidly decomposing into a variety of compounds including 1 and the unstable secondary autoxidation product 37.
On the basis of oxygen labelling, electrochemical and pulse radiolysis studies, and the role of reactive oxygen species in the autoxidation process, an inter-related mechanism for the formation of 7, 30, and 31 was proposed.
Extensive modelling studies employing amino acids and amino acid analogues suggested that lysine and tyrosine would be the protein residues most likely to undergo covalent interaction with 1 under autoxidising conditions. The adducts isolated from the autoxidative reaction of 1 with lysine and tyrosine were characterised and identified as the lysyl/p-quinone adduct 54. and the benzocoumarin 69. respectively. An independent and efficient synthesis of 69. and its methyl and ethyl analogues 66. and 67 respectively, was carried out and served as structural proof for these adducts.
The reactivity pattern of 1 towards homo and co-polymers of amino acids conformed with the model studies; lysyl and tyrosyl residues being found to undergo covalent interaction with 1 under autoxidising conditions.
Extending the autoxidative reaction to bovine serum albumin (BSA) which served as a representative protein, an analogous reactivity pattern was exhibited, with the protein undergoing a gradual progression in pigmentation with increasing time of modification. Based on the spectral characteristics of the modified protein, interaction was proposed to occur primarily via the formation of amino/p-quinoid adducts. Tyrosyl residues of BSA were also found to undergo covalent interaction with 1, as demonstrated by the isolation of 69. in acid hydrolysates of the modified protein.
A preliminary study of the silk sclerotization process was undertaken on the moth species Hyalophera gloveri and Samia cynthia. The nuclear attachment of 1 to silk proteins was demonstrated by the isolation of 69. in acid hydrolysates of sclerotized silks. Based on the autoxidative modelling studies undertaken, a mechanism was proposed for the involvement of 1 in the oxidative silk sclerotization process.