Hydrolysis-Resistant Ester-Based Linkers for Development of Activity-Based NIR Bioluminescence Probes

Activity-based sensing (ABS) probes equipped with a NIR bioluminescence readout are promising chemical tools to study cancer biomarkers owing to their high sensitivity and deep tissue compatibility. Despite the demand, there is a dearth of such probes because NIR substrates (e.g., BL660 (a NIR luciferin analog)) are not equipped with an appropriate attachment site for ABS trigger installation. For instance, our attempts to mask the carboxylic acid moiety with standard self-immolative benzyl linkers resulted in significant background signals owing to undesirable ester hydrolysis. In this study, we overcame this longstanding challenge by rationally designing a new hydrolysis-resistant ester-based linker featuring an isopropyl shielding arm. Compared to the parent, the new design is 140.5-fold and 67.8-fold more resistant toward spontaneous and esterase-mediated hydrolysis, respectively. Likewise, we observed minimal cleavage of the ester moiety when incubated with a panel of enzymes possessing ester-hydrolyzing activity. These impressive in vitro results were corroborated through a series of key experiments in live cells. Further, we showcased the utility of this technology by developing the first NIR bioluminescent probe for nitroreductase (NTR) activity and applied it to visualize elevated NTR expression in oxygen deficient lung cancer cells and in a murine model of non-small cell lung cancer. The ability to monitor the activity of this key biomarker in a deep tissue context is critical because it is associated with tumor hypoxia, which in turn is linked to drug resistance and aggressive cancer phenotypes.