Synthesis and characterization of luciferin derivatives for use in bioluminescence enhanced enzyme immunoassays. New ultrasensitive detection systems for enzyme immunoassays, I.

Derivatives of luciferin, D-luciferin methyl ester, D-luciferyl-L-phenylalanine, D-luciferyl-L-N alpha-arginine, D-luciferin-O-sulphate and D-luciferin-O-phosphate, were synthesized for use as highly sensitive substrates for enzyme assays. The luciferin derivatives were characterized by ultraviolet and fluorescence spectrophotometry, by amino acid analysis and by fast atom bombardement mass spectrometry. Enzymatic cleavage of the compounds by enzymes leading to the release of D-luciferin was demonstrated. Kinetic constants were determined for the following enzyme/substrate pairs: D-luciferin methyl ester/carboxylic esterase, D-luciferyl-L-phenylalanine/carboxypeptidase A, D-luciferyl-L-N alpha-arginine/carboxypeptidase B, D-luciferin-O-sulphate/arylsulphatase, D-luciferin-O-phosphate/alkaline phosphatase. All compounds proved to be acceptable substrates for the respective enzymes, D-luciferin-O-phosphate being accompanied by an especially high turnover number (kcat = 1010 s-1) with alkaline phosphatase.