Synthesis and purification of 3-hydroxykynurenine-O-beta-glucoside, a primate lens ultraviolet filter, and its application in a two-step assay for beta-glucosidase activity

3-Hydroxykynurenine-3-O-beta -glucoside (3-HKG) functions in the primate le ns as a filter of 295- 400-nm light, thereby protecting the retina from dam aging UV radiation. Although extensive studies have been conducted to deter mine the functional role of 3-HKG in the primate lens, an efficient method for its synthesis and purification has yet to be developed. Several procedu res have been reported for the synthesis of 3-HKG; however, these procedure s either result in low yields or require numerous sequential reactions and purification steps. In this study, we report a two-step synthesis of 3-HKG with a one-step purification and a two- to eightfold increase in yield over previously reported methods. Additionally, an assay was developed to confi rm the presence of a beta -glycosidic linkage in the purified reaction prod uct and we propose a method by which 3-HKG can be used as a general probe o f B-glucosidase activity. The assay consists of adding glucose oxidase to t he 3-HKG/glucosidase solution and then allowing the hydrogen peroxide, gene rated from the interaction of glucose with glucose oxidase, to oxidize 3-hy droxykynurenine to xanthomattin (XAN) and 4,6-dihydroxyquinolinequinone car boxylic acid (DHQCA). Both XAN and DHQCA absorb strongly between 400 and 50 0 nm and the color change of the solution can be seen by eye. In addition, XAN fluoresces in the visible region with lambda (max) = 527 nm. (C) 2001 E lsevier Science Elsevier Science.