Enzyme substrate comprising a functional dye and associated technology and methods

摘要

Enzyme substrates and associated technology of the present invention are provided. An enzyme substrate of the invention may comprise a biologically functional fluorescent dye and an enzyme-specific substrate moiety attached in such a way that the functionality of the functional dye is diminished. An enzymatic reaction may cleave at least a portion of the substrate moiety from the enzyme substrate to provide a more functional product dye. This product dye may be nonfluorescent or weakly fluorescent, in general, and relatively fluorescent, in a particular condition, such as when bound to a partner biological molecule or an assembly of partner biological molecules. An enzyme substrate of the present invention may thus be useful in fluorescence detection, and/or in any of a variety of useful applications, such as the detection of enzymatic activity in a cell-free system or in a living cell, the screening of drugs, or the diagnosis of disease.

主权项

1. A substrate selected from a compound having a structural formula, DYE-(B)m, and a pro-enzyme substrate thereof, wherein DYE, when independent of DYE-(B)m, comprises a dye selected from a biologically functional fluorescent dye and a fluorogenic dye, the dye being capable of binding to a partner biomolecule, partner biomolecules, or an assembly of partner biomolecules; wherein m is selected from 1, 2, 3, 4 and 5; and wherein each of at least one B, independently, comprises an enzyme substrate moiety that is capable of enzymatic transformation comprising cleavage of a bond involving the dye and at least one B, cleavage of a bond within at least one B, or formation of a bond involving at least one B; and
wherein the dye is a phenanthrodium dye having a structural formula,wherein:
B comprises a peptide or an amino acid; at least one ligand of ligands R1 and R2 comprises B, where the C-terminal carbonyl of B is capable of forming an enzymatically cleavable peptide bond with an amino group of the dye; if any one ligand of ligands R1 and R2 does not comprise B, said any one ligand comprises H; ligand R3 comprises a substituted or an unsubstituted C1 to C10 alkyl; ψ comprises a cation selected from H+, Na+, K+, NH4+, N,N,N-triethylammonium, and N,N-diisopropylethylammonium, or an anion selected from a halide, a sulfate, a phosphate, a perchlorate, a hexafluorophosphate, and a tetrafluoroborate; and d is a number of ψ sufficient to render overall charge of the substrate neutral.