Encapsulation within a coordination cage modulates the reactivity of redox-active dyes

Abstract

Confining molecules within well-defined nanosized spaces can profoundly alter their physicochemical characteristics. For example, the controlled aggregation of chromophores into discrete oligomers has been shown to tune their optical properties whereas encapsulation of reactive species within molecular hosts can increase their stability. The resazurin/resorufin pair has been widely used for detecting redox processes in biological settings; yet, how tight confinement affects the properties of these two dyes remains to be explored. Here, we show that a flexible Pd^II₆L₄ coordination cage can efficiently encapsulate both resorufin and resazurin in the form of dimers, dramatically modulating their optical properties. Furthermore, binding within the cage significantly decreases the reduction rate of resazurin to resorufin, and the rate of the subsequent reduction of resorufin to dihydroresorufin. During our studies, we also found that upon dilution, the Pd^II₆L₄ cage disassembles to afford Pd^II₂L₂ species, which lacks the ability to form inclusion complexes - a process that can be reversed upon the addition of the strongly binding resorufin/resazurin guests. We expect that the herein disclosed ability of a water-soluble cage to reversibly modulate the optical and chemical properties of a molecular redox probe will expand the versatility of synthetic fluorescent probes in biologically relevant environments.

Fig. 1. Encapsulation of resorufin within coordination cage 1.

a Structural formulas of coordination cage 1 (counterions = 12 NO₃⁻), resorufin 2, and a cartoon representation of the inclusion complex 2₂⊂1. The two types of acidic imidazole protons in 1 are denoted by black and white circles. b ¹H NMR spectrum of 2 (600 MHz, D₂O, 320 K). c Partial ¹H NMR spectrum of 2₂⊂1 (600 MHz, D₂O, 320 K) (for full-range ¹H and ¹³C spectra, see Supplementary Figs. 1 and 2). d Partial ¹H NMR spectrum of 1 (600 MHz, D₂O, 320 K). e Crystal structure of 2₂⊂1 co-crystallized with 1 equiv of unencapsulated 2; i.e., (2₂⊂1)⋅2. f Crystal structure of 2₂⊂1 co-crystallized with 4 equiv of unencapsulated 2; i.e., (2₂⊂1)⋅2₄. Nitrate counterions and water molecules were omitted for clarity.

Fig. 2. Spectroscopic characterization of the resorufin complex.

a Bottom to top: ¹H NMR spectra of 2 in the presence of increasing amounts of cage 1 (400 MHz, D₂O, 298 K). b A series of UV–Vis absorption spectra accompanying the titration of 2 with cage 1. No changes in the >400 nm region are visible beyond ~0.5 equiv of 1, indicating a 2:1 stoichiometry of the complex. c Decrease in the ratio of absorbance at 571 nm (Abs_571nm) (high for 2; low for 2₂⊂1) to absorbance at 542 nm (Abs_542nm) (high for 2₂⊂1; low for 2) as a function of the amount of 1 added. d A series of fluorescence spectra (λ_exc = 530 nm) accompanying the titration of 2 with cage 1. e Decrease of emission at 587 nm as a function of the amount of 1 added. f Cartoon representation of fluorescence quenching due to inclusion complex formation during the titration of 2 with cage 1.

Fig. 3. Reversible disassembly and guest-templated reassembly of cage 1.

a Bottom to top: ¹H NMR spectra of cage 1 in the presence of increasing amounts of guest 2 (deprotonated with excess TMEDA) (400 MHz, D₂O, 298 K). b UV-Vis absorption spectra of cage 1 in the presence of increasing amounts of 2. c Changes in the ratio of Abs_571nm (high for 2; low for 2₂⊂1) to Abs_542nm (high for 2₂⊂1; low for 2) as a function of the amount of 2 added. The sharp increase of Abs_571nm/Abs_542nm at ~1.4 equiv 2 indicates that 2 added at that point is not complexed. d A series of fluorescence spectra (λ_exc = 530 nm) accompanying the titration of cage 1 with 2. e Following the emission at 584 nm (due to uncomplexed 2) as a function of the amount of 2 added. The sharp increase of emission at ~1.2 equiv 2 indicates that the added 2 is not complexed. f Cartoon representation of fluorescence fluctuations during the titration of a dilute solution of cage 1 with 2.

Fig. 4. Characterization of “half-cage” 3.

a Structural formula of 3. b Crystal structure of 3. Nitrate counterions and water molecules were omitted for clarity. c Top: Partial ¹H NMR spectrum of 3 (600 MHz, D₂O, 300 K) (for full-range ¹H and ¹³C spectra, see Supplementary Figs. 20–22). Bottom: Partial ¹H NMR spectrum of 1 (600 MHz, D₂O, 300 K). d Cartoon representation of the extraction of axial Pd²⁺ centers from cage 1 using TMEDA, followed by reassembly of cage 1 from 3 using [Pd(TMEDA)]²⁺.

Fig. 5. Encapsulation of resazurin within coordination cage 1.

a Top: Structural formula and ¹H NMR spectrum of resazurin 4 (600 MHz, D₂O, 330 K). Center: Partial ¹H NMR spectrum of 4₂⊂1 (600 MHz, D₂O, 330 K) (for full-range ¹H and ¹³C spectra, see Supplementary Figs. 31 and 32). Bottom: Partial ¹H NMR spectrum of 1 (600 MHz, D₂O, 330 K). b A series of UV–Vis absorption spectra accompanying the titration of 4 with a concentrated solution of cage 1. No changes in the >400 nm region are visible beyond 0.5 equiv of 1, indicating a 2:1 stoichiometry of the complex. c Decrease of absorbance at 601 nm (high for 4; low for 4₂⊂1) as a function of the amount of 1 added. d Crystal structure of 4₂⊂1 co-crystallized with 1 equiv of unencapsulated 4; i.e., (4₂⊂1)⋅4. e Crystal structure of 4₂⊂1. Nitrate counterions and water molecules were omitted for clarity.

Fig. 6. Modulating the reactivity of resazurin and resorufin by encapsulation within cage 1.

a Reduction of resazurin 4 to resorufin 2 and the subsequent reduction of 2 to dihydroresorufin 5. Inset: A transmission electron micrograph of 21 nm citrate-capped Au NPs. b Representative UV–Vis absorption spectra of 4 before (blue) and after exposure to green light in the presence of TMEDA and Au NPs for 12 s (red; mostly 2) and for 151 s (gray; mostly 5). Note that due to the rapid oxidation of 5 back to 2, it was very challenging to eliminate the residual absorption at ~550 nm due to 2. Note also that Au NPs are used in such a small amount that their contribution to UV–Vis spectra is not visible. c Concentrations of 2 and 4 (extracted from UV–Vis spectra) at various reaction times (markers) and fits to first-order kinetics (lines). d Cartoon representation of the sequential reduction of 4₂⊂1 into 2₂⊂1 and then into 5₂⊂1 and the corresponding photographs of the reaction mixtures. The pink color at the water/air interface for 5₂⊂1 is due to the rapid oxidation to 4₂⊂1. e Representative UV–Vis absorption spectra of 4₂⊂1 before (blue) and after exposure to green light in the presence of TMEDA and Au NPs for 3.5 min (red; mostly 2₂⊂1) and for 64 min (gray; mostly 5₂⊂1). f Concentrations of encapsulated 2 and 4 (extracted from UV–Vis spectra) at various times of the reaction (markers) and fits to first-order kinetics (lines). g Photograph of a flower painted using a combination of 4 and 4₂⊂1 dyes (4.5 and 2.25 mm, respectively). The paper was soaked with TMEDA (18 mm) and Au NPs (0.11 nm). h Effect of green light irradiation on the flower’s color.