Rhodamine spirolactams (RSLs) have recently emerged as popular fluorescent pH probes due to their fluorescence turn-on capability and ease of functionalization at the spirolactam nitrogen. Design of RSLs is often driven by biological targeting or compatibility concerns, rather than the pH sensitivity of the probe, and the relationship between RSL structure and pK is not well understood. To elucidate the relationship between pK values and the properties of substituents attached to the spirolactam nitrogen, a series of 19 aniline-derived RSLs is presented. RSLs derived from di-ortho-substituted anilines exhibit pK tunability across the moderately acidic region (ca. pH 4-6). Evaluation of pK data using the Fujita-Nishioka model for ortho substituent effects reveals that both steric and electronic substituent properties influence RSL pH responsiveness, with pK values increasing as substituent size and electron withdrawing character increase. These trends are attributed to changes in the RSL structure induced by large substituents, and to electronic influences on the protonated spirocyclic reaction intermediate. To demonstrate the practical applicability of these probes in completely aqueous environments, RSL-doped conjugated polymer nanoparticles that exhibit a ratiometric fluorescence response to changing pH levels are presented.

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