Tuning the pK_a of Fluorescent Rhodamine pH Probes through Substituent Effects

Abstract

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.

Two water‐soluble fluorescence probes based on 5(6)‐carboxyl rhodamine for Cu2+ imaging in living cells

“…This makes the spirolactam of T1 more easy to open than that of T2 . [ 40,41 ] In a similar manner, and for the same reason, the quantum yield of T1 was much larger than that of T2 .…”

Rhodamine-Functionalized Mechanochromic and Mechanofluorescent Hydrogels with Enhanced Mechanoresponsive Sensitivity

“…In contrast, when the pH was 1.0, ultrahigh emission intensity centered at 550 nm emerged in the corresponding fluorescence spectrum. It can be learned that the amounts of Rh activated by acid when pH was 1.0 was much more than others’, and the pH needed to trigger the obvious acidichromic property of Rh in hydrogel was much lower than reported before [50,51,52]. This was possibly because the diffusion of H + into the microspheres was difficult; only when the pH was 1.0 was the concentration gradient of H + outside and inside the microspheres high enough, and certain amounts of H + diffused into the microspheres.…”

“…More importantly, the special microspheres within the hydrophilic hydrogels were not completely free of water [47,48,49], and water was beneficial to the mechanical activation of Rh mechanophores [20]. In addition, since Rh spirolactam derivatives exist commonly in a colorless and nonfluorescent form in neutral or alkaline solvents, and they can structurally equilibrate to a colored and fluorescent ring-opened form upon an acidic pH [50,51,52], Rh-based hydrogels may also respond to acid and alkali.…”

Tuning the Photophysical Properties of Spirolactam Rhodamine Photoswitches

“…They proposed a four‐state mechanism to explain the acid‐dependent photoswitching behaviour, and used a mathematical model to extract kinetic parameters. The acid‐dependent properties of N ‐phenyl spirolactam rhodamine and its derivatives have been explored independently by Scott and Harbron, and these properties make molecules of this type useful as fluorometric pH probes [32–35] . The above studies have been aided by advanced synthetic methodology for the synthesis of a wide variety of spirolactam rhodamine derivatives [10,24,32–35] …”