Development of water-soluble far-red fluorogenic dyes for enzyme sensing

A series of Nile Blue analogs, 5-amino-9-dialkylamino benzo[a]phenoxazine dyes, , were prepared by condensation of N-alkyl or N-sulfo-propyl 4-arylazo-substituted 3-hydroxyaniline with 4-arylazo-substituted 1-naphthlamines or 8-amino-2-naphthalenesulfonic acid in the presence of perchloric acid. These fluorochromes have excitation and emission maxima near 640 and 680 nm, respectively. The fluorescence intensity in aqueous solution increases as additional sulfonate groups are added to the benzo[a]phenoxazine core. Compound , which has two sulfonate groups, is ten-times brighter than Nile Blue. Fluorogenic substrates containing this hydrophilic far-red dye were synthesized and applied to detect enzymatic activities of two model proteases, trypsin and leucine aminopeptidase. Given their excellent fluorogenic property, these novel far-red dyes should be useful for enzyme sensing in biological assays.