Horseradish peroxidase oxidation of 2,3-diaminopyridine and related compounds

The substrate 2,3 diaminopyridine and several structurally similar compounds were oxidized enzymatically and chemically. The resulting products were analyzed. The enzymatic oxidation was conducted using horseradish peroxidase as the enzyme and hydrogen peroxide was used as the co-oxidizing agent. The chemical oxidation was conducted using ferric chloride as the oxidizing agent. The ultraviolet and fluorescent characteristics of the starting materials and products were different. Because the starting material is non-fluorescent and the products are fluorescent, the materials are good substrates for the ELISA assay. The oxidations of o-phenylenediamine, 4-methoxy-o-phenylendiamine, 2,3-diaminotoluene, 3,4-diaminotoluene, and 2,3-diaminopyridine were studied. The enzymatic and chemical oxidations of 4-methoxy-o-phenylenediamine produced 8-methoxy-o-phenylenediamine as the major product. This was confirmed by mass spectra, nuclear magnetic resonance spectra, infrared, and ultraviolet spectra comparisons. The enzymatic oxidation of 2,3-diaminotoluene produced either 2,3-diamino-1,8-dimethylphenazine, or 2,3-diamino-1,5-dimethylphenazine, or a mixture of both compounds. In addition, the enzymatic oxidation of 3,4-diaminotoluene produced a product larger than the phenazine product predicted. The enzymatic oxidation of 2,3-diaminopyridine produced a mixture of compounds. The reaction is pH dependent. As the pH and reaction time are altered different compounds are formed. The majority are large, conjugated molecules of varying sizes. The mass spectrum data suggests that azo compounds are also formed. UV studies were conducted to determine the effect of time, pH, substrate concentration, enzyme concentration, and hydrogen peroxide concentration on the reaction. The compound 5-aminoindazole was also studied by UV to determine the absorbance of product over time. The investigation of the substrates and subsequent oxidation products of the reactions studied in the research project has led to a better understanding of their chemical properties. The new fluorescent products that were found may be useful in enzyme immunosorbant assays.