IMPROVED SYNTHETIC METHODS OF FIREFLY LUCIFERIN DERIVATIVES FOR USE IN BIOLUMINESCENT ANALYSIS OF HYDROLYTIC ENZYMES - CARBOXYLIC ESTERASE AND ALKALINE-PHOSPHATASE

Improved synthetic methods of firefly luciferin derivatives, such as 6-O-alkanoylluciferins and 6-O-phosphonoluciferin, are described. These derivatives proved to be useful as substrates for the bioluminescent assay of hydrolytic enzymes such as carboxylic esterase and alkaline phosphatase. The synthetic methods previously reported gave fairly low yields because of direct derivatization of unstable luciferin itself and are difficult to be applied to large-scale synthesis because of unavoidable HPLC purification. We first modified the hydroxyl group of 2-cyano-6-hydroxybenzothiazole and then condensed the resulting stable products with D-cysteine to give the appropriate derivatives in good yields without HPLC purification. 6-O-Phosphonoluciferin was unstable as free acid form but stable as its sodium salt.