Next, we designed a series of two complementary cellular experiments to determine whether the excellent in vitro performance noted above translates within a more complex cellular environment where many esterase isoforms are present. Moreover, we aimed to determine whether the Ph i Pr ester will have a similar effect on other molecules with diverse chemical structures beyond BL660. First, we prepared BL660-Me, a methyl ester derivative of BL660 (Figure ). Next, we cultured 4T1 murine breast cancer cells, stably expressing the luciferase enzyme (4T1-Luc). The cells were then treated with a DMSO vehicle control, BL660-Me or compound 4 (Figure and). We postulate that the BL signal will be lower if the optimized ester is indeed more stable in live cells since less of the luciferase substrate will be liberated and made available to generate light. Analysis of the imaging results revealed that the total luminescence count for BL660-Me-treated 4T1 cells (94.1 ± 5.9 × 10 3 ) were 55-fold and 31-fold more luminescent relative to the vehicle (1.7 ± 0.8 × 10 3 ) and compound 4 (3.1 ± 1.2 × 10 3 ) counterparts, respectively (Figure ). These results indicate the Ph i Pr ester is stable to the collection of intracellular esterases present in 4T1 cells. Next, we modified the two carboxylates in methotrexate (a potent anticancer and immunosuppressive drug) to afford the methyl (MTX-Me2) or Ph i Pr (MTX-(Ph i Pr)2) ester variants (Figure ). Structure-activity relationship analysis has shown that carboxylate masking decreases target engagement, leading to a significant reduction of cytotoxicity (IC50). We hypothesized if the Ph i Pr esters installed onto methotrexate remain intact, cells treated with MTX-(Ph i Pr)2 will remain viable. To test this, A549 cells were cultured and treated with MTX-Me2 or MTX-(Ph i Pr)2 at various concentrations for 24 hours before they were subjected to MTT analyses. Our results revealed the viability was ~63% and 100% at the highest concentration (10 µM) of MTX-Me2 and MTX-(Ph i Pr)2 tested, respectively (Figure and). This experiment demonstrates that the hydrolysis of the esters on MTX-(Ph i Pr)2 is sufficiently slow that the buildup of MTX was inconsequential to the health of the cells. Conversion of Ph i Pr Ester into a Versatile Self-Immolative Linker While we envision nearly any electron-deficient trigger can be installed at the para (or ortho) position of the Ph i Pr ester to yield an ABS probe for NIR BL imaging, we strategically selected to use the nitro group to develop BL660-NTR. In addition to its common use in probe design for other modalities, this biomarker is a feature of gram positive and negative bacteria and thus, can be leveraged for sensing bacterial infections. Moreover, NTR can also be employed to detect tumor hypoxia owing to overexpression in many types of solid tumors. Once our probe engages its target, the aryl nitro moiety can be reduced to afford the electron-rich hydroxylamine or amino products, which initiates self-immolation to release BL660. In the presence of luciferase, this substrate is then enzymatically converted to a dioxetanone, which decomposes to generate oxy-BL660 in an excited state. Relaxation to the ground state is accompanied by the emission of a photon at 660 nm (Scheme 2). The synthesis of BL660-NTR began with subjecting 4-nitrobromobenzyme to phenyllithium to initiate a lithium-halogen exchange reaction. The resulting 4-nitrophenyllihtium 5 intermediate was used directly without purification to attack 2-methyl propanaldehyde to give benzyl alcohol 6 in 25% yield. Then utilizing the acyl fluoride chemistry we optimized above, BL660-NTR was obtained in 10% yield (Fig- ). With BL660-NTR in hand, we performed an in vitro assay to demonstrate that the BL output depends on both NTR and luciferase activity (Figure and). When each component is present, we observed a notable increase in the total luminescence count. However, when NTR (column 2) was absent, the signal was significantly attenuated. Likewise, when luciferase (column 3) or BL660-NTR (column 4) was excluded from the reaction, the signal was minimal and comparable to control wells containing only media and a DMSO vehicle. Next, we subjected BL660-NTR to various biologically relevant analytes that may contribute to off-target probe activation in vivo. For instance, transition metals like Cu(II) can facilitate ester hydrolysis via Lewis acid-mediated ester activation. Additionally, various reactive oxygen and nitrogen that can interact with the ester bond were also tested. Lastly, we also examined reactive sulfur species, which are known to reduce aryl nitro groups. Under no circumstance did we observe activation greater than 10% compared to the NTR positive control (Figure ). Additional experimental details including NMR and MS characterization and dose dependent NTR studies (Figure ), can be found in the supporting information document. Prior to testing BL660-NTR in vivo, we assessed potential cytotoxicity by treating A549 cells with BL660-NTR across a concentration range of 0 to 20 µM for up to 3 hours. No loss of cell viability was observed (Figure ). Next, we performed a cell imaging study to measure hypoxia within a living system. Specifically, A549 cells (not expressing Luc) were cultured in an atmosphere consisting of 1% or 20% oxygen for 2 days to mimic hypoxic or normoxic conditions, respectively. Because aryl nitro reduction mediated by mammalian NTRs can be inhibited by molecular oxygen, we expect there to be greater probe activation under oxygendeficient conditions. After incubating with BL660-NTR for 60 minutes, the cells were washed with fresh PBS, detached, pelleted, and lysed via sonication. The lysates were then transferred to 24-well plates, treated with luciferase and imaged immediately using the IVIS imaging system. As shown in Figure , wells containing lysates obtained from the cells incubated under hypoxic conditions (5.0 ± 0.1 × 10 5 total luminescent counts) were significantly brighter than the corresponding normoxic lysates (1.1 ± 0.03 × 10 5 total luminescent counts) (Figure and). This represents a significant 4.5-fold difference in BL intensity.