GENERAL METHODOLOGY: The synthesis of 1,2-HOPO-6-carboxylic acid was carried out with reference to the method reported by Xu [34] with appropriate adaptations. As shown in Scheme 1, the synthesis process consists of two major consecutive steps. The specific operation was as follows: first, a 6-hydroxypyridine carboxylic acid solution was prepared by dissolving 5 g (0.036 mol) of 6-hydroxypyridine carboxylic acid in a mixed solvent of trifluoroacetic acid (30 mL) and glacial acetic acid (10 mL). Meanwhile, acetic anhydride (30 mL) was mixed with 30% H2O2 solution (10 mL) and stirred for 4 hours under ice bath cooling conditions to prepare peracetic acid solution. Subsequently, 6-hydroxypyridine carboxylic acid solution was slowly added to the peracetic acid solution. The reaction mixture was stirred and refluxed at 80 °C for 10 h, during which a white precipitate was observed to be generated. Upon completion of the reaction, excess trifluoroacetic acid was removed by vacuum distillation. The precipitate was filtered, washed with cold methanol and dried to give 1,6-dihydroxypyridinecarboxylic acid as a powdery white solid with a yield of 4.63 g (82% yield) and a melting point of 209-210 °C. Infrared spectra (KBr press): ν/cm-1 3440 (broad peak, O-H stretching vibration); 1734.7 (broad peak, C=O stretching vibration); 1211.1 (medium-strong peak, C-O stretching vibration); 926.4 (broad peak, O-H out-of-plane bending vibration).1H NMR (DMSO-d6): δ7.42 (triple peak, 1H, J=8.8, 7.1 Hz); 6.64 (quadruple peak, 2H, J=7,6.8 Hz); 3.43 (single peak, 1H, O-H). This compound was used as a precursor for the synthesis of 1,2-HOPO-6-carboxylic acid. Next, a basic solution of 1,6-dihydroxypyridinecarboxylic acid was prepared by dissolving 4.63 g (0.029 mol) of 1,6-dihydroxypyridinecarboxylic acid in 135 mL of 20% KOH aqueous solution. The solution was refluxed at 70 °C for 12 hours. Upon completion of the reaction, the product was precipitated with 50 mL of cold concentrated HCl in an ice bath, keeping the temperature below 20 °C throughout. The white precipitate was filtered and washed sequentially with 0.1 M HCl (aqueous solution), cold methanol and cold water. The product was dried under vacuum to give 4.37 g (95% yield) with a melting point of 217-218°C [42]. Infrared spectra (KBr press): ν/cm-1 3437.4 (broad peak, O-H stretching vibration); 1785.9 (broad peak, C=O stretching vibration); 1618.4 (medium-strong peak, C=O stretching vibration); 929.7 (broad peak, C-H out-of-plane bending vibration).1H NMR (DMSO-d6): δ 7.43 (double peaks, 1H, J=7,9 Hz ); 6.71 (double peaks, 1H, J=1.6,9Hz); 6.64 (double peaks, 1H, J=1.6,7Hz). Mass spectrum (ESI negative ion mode): m/z 154.01 (C6H4NO4).
