Because the primary objective of this work is to develop ABS probes for the BL modality via carboxylate-masking, we chose to investigate the potential of benzyl esters. Triggers based on para-substituted benzyl alcohol, are the most ubiquitous self-immolative linkers used in modern probe design. Typically, an electron-deficient analyte-responsive trigger occupies the para-position, and the immolative linker is connected to a reporter through a stable ether, carbamate, or carbonate bond. Upon interaction with the target analyte, the trigger is activated to yield a more electron-rich moiety. Flow of electron density into the aromatic ring facilitates formation of a quinone methide (or aza-quinone methide) intermediate which is coupled to the spontaneous release of the latent reporter. We hypothesized inclusion of additional phenyl or alkyl groups at the benzyl position can impart stability by shielding the ester from water or other nucleophiles. Further, we envisioned installing these onto BL660 would enable monitoring of stability using BL imaging. Our envisioned panel of esters initially consisted of 1 (Ph,H), 2 (Ph2), 3 (PhMe), and later, included optimized ester 4 (Ph, i Pr) shown in Figure . While the synthesis of 1-3 was routine, the esterification of BL660 to afford 4 proved to be challenging owing to the bulky nature of this benzyl alcohol. Conventional reaction conditions to form esters including Fischer, Mitsunobu, Steglich and Yamaguchi esterification failed to give products in appreciable yields beyond trace conversion. Moreover, we observed racemization of the chiral center housing the carboxylate. This is detrimental because stereochemistry at this position is required for luciferase recognition. Similarly, activation to the acyl chloride, followed by addition of the benzyl alcohol gave complex, inseparable mixtures. Our efforts to control the rate of alcohol addition, molarity of the reaction, stoichiometry of the components, and temperature were all unsuccessful. We also found that diazo chemistry was incompatible with our system. Ultimately, we employed cyanuric fluoride at -20 °C to access the less reactive acyl fluoride intermediate, which upon reaction with the corresponding benzyl alcohol yields the desired esters. Of note, no attempts were made to separate the stereoisomers for 3 and 4. All subsequent analyses were performed on the mixtures and reported as 'apparent' results. We began our analysis of stability by incubating BL660 esters 1-3 at 37 °C in PBS (pH 7.4). After 1, 6, and 12 hours, luciferase was added to initiate BL production. The rate of hydrolysis, reported as total luminescence counts per hour, indicates hydrolysis in descending order is: 1 (13.9 ± 1.4 × 10 3 ), 2 (3.3 ± 0.6 × 10 3 ), and 3 (1.0 ± 0.1 × 10 3 ) (Figure and S1). These results show the methyl substituent is over three-fold more effective at blocking hydrolysis than the phenyl group. We speculate hydrolysis of 3 may involve an SN1 mechanism, thus favoring formation of a double benzylic carbocation. On the contrary, when the panel was treated with porcine liver esterase (0.1 units/mL, 37 °C, pH 7.4) for 20, 40, and 60 minutes, we found that 2 was most resistant toward enzymatic degradation. The enzymatic rates, reported as total luminescence counts per minute, in descending order are: 1 (31.0 ± 6.8 × 10 2 ), 3 (9.5 ± 1.2 × 10 2 ), and 2 (0.6 ± 0.1 × 10 2 ) (Figure and). The greater stability imparted by the aromatic ring is not surprising since esterase reactivity can be readily attenuated with bulkier substituents. With this data, we hypothesized the hydrolytic stability of 3 can be retained (or improved) by switching to a larger alkyl group with multiple rotatable bonds (i.e., isopropyl ( i Pr)), while simultaneously suppressing esterase reactivity. To our delight, we observed significant improvements of both properties with this modification. Relative to the parent benzyl ester, 4 is 140.5-fold more resistant to hydrolysis and exhibits 67.8-fold greater esterase stability (Figure and). Following this set of experiments, we subjected 4 to acidic (pH 5) and alkaline (pH 9) conditions to account for the various pH gradients in the body. For these experiments we used the Britton-Robinson buffer system. As reported in total luminescence counts per hour, 4 was most stable at pH 7.4 (8.6 ± 1.3 × 10 2 ), followed by pH 5.0 (25.7 ± 9.6 × 10 2 ), and finally pH 9.0 (52.8 ± 20.7 × 10 2 ) (Figure ). Lastly, we examined the stability of 4 against a panel of enzymes beyond porcine liver esterase that are known to possess estercleaving activity. These include lipase, human carboxyesterase-2, monoacylglycerol lipase, fatty acid amide hydrolase, and aldehyde dehydrogenase 1A1. Remarkably, exposure to high levels of these enzymes over a period of one hour resulted in minimal activation (Figure ).