*4.8. Purification of 3-(2,4-Dihydroxyphenyl)-propionic Acid Bioconversion Product*

The supernatant containing the 3-(2,4-dihydroxyphenyl)-propionic acid oxidation product was incubated with 1.2 M ammonium chloride at room temperature overnight [39,50]. Reaction mixture was concentrated to ~100 mL volume and adjusted to pH 1 with concentrated HCl. The remains of substrate were extracted by five consecutive changes of 25 mL of ethyl acetate, and then aqueous fraction was purified using a reverse phase C18 column, equilibrated with water. Column was washed with at least 100 mL of water and then eluted with linear gradient of 0–60% methanol solution at a flow rate of 2 mL/min. Aqueous fractions were combined and evaporated (40 ◦C). Brownish crystals were dissolved in 0.1% formic acid solution and again loaded onto a reverse phase C18 column, previously equilibrated with 0.1% formic acid solution. Column was washed with at least 30 ml of 0.1% formic acid solution and then eluted with 60% methanol solution. Picolinic acid derivative was eluted with 0.1% formic acid solution. Fractions containing the product were collected, combined, and evaporated (40 ◦C). Picolinic acid yield from 145 mg of 3-(2,4-dihydroxyphenyl)-propionic acid fermentation was 34 mg, 24% of the theoretical yield. The product had traces of formic acid impurities, which aided the dissolution of the analyte in D2O for NMR analysis.
