Ascorbyl-6-O-oleate obtained from lipase-catalyzed synthesis through esterification and transesterification reaction

Ascorbyl-6-O-esters are hydrophobic antioxidants with acyl groups attached to the 6th hydroxyl group of ascorbic acid, potentially exhibiting ionizable surfactant properties. However, commercial ascorbyl-6-O-esters mostly consist of saturated fatty acids, leading to low solubility in a lipophilic medium. In this study, we propose a method of lipase-catalyzed synthesis and purification for ascorbyl-6-O-oleate. The results showed an esterification synthesis (i.e., bonding oleoyl group to ascorbic acid) rate of 19.7% when using acetone as the reaction solvent. Moreover, the transesterification synthesis (i.e., exchanging the acyl group with oleic acid in ascorbyl-6-O-palmitate) rate increased to 73.8% (when substrate molar ratio of ascorbyl-6-O-palmitate:oleic acid, 1:3). Thereafter, the esterification product was purified through liquid-liquid extraction using ethyl acetate (2 vol) and water (1 vol) followed by hexane (3 vol) and acetonitrile (1 vol), and ascorbyl-6-O-oleate showed 94.8 area% in HPLC analysis. When 90% methanol was replaced by acetonitrile, AO was 97.2 area%. Similarly, the transesterification product showed 94.3 area% of ascorbyl-6-O-esters (ascorbyl-6-O-palmitate: ascorbyl-6-O-oleate=8.9:91.1) through sequential re-crystallization followed by liquid-liquid extraction using the same method mentioned above. Finally, in 1H-NMR spectrum, the purified ascorbyl-6-O-oleate from both syntheses revealed peaks corresponding to hydroxyl groups at the 2nd and 3rd carbons (11.10 and 8.41 ppm) while 13C-NMR indicated that oleic acid selectively esterified at the 6th carbon, showing upfield shift of the 5th carbon (65.94 ppm) with downfield shift of the 6th carbon (64.88 ppm). In addition, FT-IR confirmed the ester bond (1733 cm-1) and olefin structure of oleic acid (3006 cm-1), while LC-ESI-MS/MS identified the m/z 439.3 peaks representing ascorbyl-6-O-oleate (molecular weight=440.575 g/mol), showing the identical fragmentation pattern that the standard (ascorbyl-6-O-palmitate). Finally, DSC observed that purified ascorbyl-6-O-oleate showed broad endothermic curves ranging from 23.1 to 46.7°C. These analysis data suggest the successful lipase-catalyzed synthesis and purification process of ascorbyl-6-O-oleate.