Structure-Activity Studies of Nitroreductase-Responsive Near-Infrared Heptamethine Cyanine Fluorescent Probes

Two new classes of near-infrared molecular probes were prepared and shown to exhibit "turn on" fluorescence when cleaved by the nitroreductase enzyme, a well-known biomarker of cell hypoxia. The fluorescent probes are heptamethine cyanine dyes with a central 4'-carboxylic ester group on the heptamethine chain that is converted by a self-immolative fragmentation mechanism to a 4'-caboxylate group that greatly enhances the fluorescence brightness. Each compound was prepared by ring opening of a Zincke salt. The chemical structures have either terminal benzoindolinenes or propargyloxy auxochromes, which provide favorable red-shifted absorption/emission wavelengths and a hyperchromic effect that enhances the photon output when excited by 808 nm light. A fluorescent probe with terminal propargyloxy-indolenines exhibited less self-aggregation and was rapidly activated by nitroreductase with large "turn on" fluorescence; thus, it is the preferred choice for translation towards in vivo applications.