pH-Responsive N^C-Cyclometalated Iridium(III) Complexes: Synthesis, Photophysical Properties, Computational Results, and Bioimaging Application

Abstract

Herein we report four [Ir(N^C)₂(L^L)]ⁿ⁺, n = 0,1 complexes (1-4) containing cyclometallated N^C ligand (N^CH = 1-phenyl-2-(4-(pyridin-2-yl)phenyl)-1H-phenanthro[9,10-d]imidazole) and various bidentate L^L ligands (picolinic acid (1), 2,2'-bipyridine (2), [2,2'-bipyridine]-4,4'-dicarboxylic acid (3), and sodium 4,4',4″,4‴-(1,2-phenylenebis(phosphanetriyl))tetrabenzenesulfonate (4). The N^CH ligand precursor and iridium complexes 1-4 were synthesized in good yield and characterized using chemical analysis, ESI mass spectrometry, and NMR spectroscopy. The solid-state structure of 2 was also determined by XRD analysis. The complexes display moderate to strong phosphorescence in the 550-670 nm range with the quantum yields up to 30% and lifetimes of the excited state up to 60 µs in deoxygenated solution. Emission properties of 1-4 and N^CH are strongly pH-dependent to give considerable variations in excitation and emission profiles accompanied by changes in emission efficiency and dynamics of the excited state. Density functional theory (DFT) and time-dependent density functional theory (TD DFT) calculations made it possible to assign the nature of emissive excited states in both deprotonated and protonated forms of these molecules. The complexes 3 and 4 internalize into living CHO-K1 cells, localize in cytoplasmic vesicles, primarily in lysosomes and acidified endosomes, and demonstrate relatively low toxicity, showing more than 80% cells viability up to the concentration of 10 µM after 24 h incubation. Phosphorescence lifetime imaging microscopy (PLIM) experiments in these cells display lifetime distribution, the conversion of which into pH values using calibration curves gives the magnitudes of this parameter compatible with the physiologically relevant interval of the cell compartments pH.

Keywords: orthometalated iridium(III) complexes; pH-dependent luminescence; phosphorescence lifetime imaging.

Scheme 1

Synthesis of N^CH.

Scheme 2

Synthesis of complexes 1–4.

Figure 1

Perspective view of 2 in the solid state showing thermal ellipsoids at the 40% probability level. Hydrogen atoms are omitted for clarity.

Figure 2

Spectral changes of N^CH in CH₂Cl₂ upon titration with TFA: (A,C) absorption spectra, (B,D) emission spectra λ_ex = 365 nm; (E) excitation and emission spectra of the representative solutions at different stages of the N^CH protonation chosen from the panels B and D (i—N^CH, ii—N^CH + TFA 10:1, iii—N^CH + TFA 1:1, iv—N^CH + TFA excess); and (F) CIE 1931 chromaticity diagram with an embedded photograph of the N^CH solution in CH₂Cl₂ at various TFA concentrations, λ_ex = 365 nm.

Figure 3

Normalized excitation (dashed line) and emission (solid line) spectra of 1–4 (A–D respectively). Emission spectra were recorded under excitation at 360 nm, excitation spectra were recorded at emission band maxima, dichloromethane, 1 × 10⁻⁵ M. A weak emission at 410 nm is generated by solvent (water) Raman resonance signal (*).

Figure 4

¹H NMR spectra of N^CH upon addition of TFA. Bottom: N^CH emission spectra recorded in NMR tubes, λ_ex = 365 nm.

Figure 5

The decrease (blue) and increase (red) of electron density in the S₁→S₀ electronic transitions for N^CH, {N^CH+H⁺(Py)} containing protonated pyridine group, {N^CH+2H⁺} containing protonated pyridine and imidazole groups; calculated and experimental emission wavelengths are given in the bottom rows.

Figure 6

The decrease (blue) and increase (red) in electron density for S₀→S₁ and T₁→S₀ electronic transitions for complexes 1–4, and calculated emission wavelengths. Note that {3−2H⁺} is the anion formed by the dissociation of two protons from the ligand carboxylic functions.

Figure 7

Dependence of emission intensity (A,D), lifetime of the excited state (B,E), and normalized emission decay curves (C,F) of 3 (A–C, λ_ex = 355 nm, λ_em = 600 nm) and 4 (D–F, λ_ex = 355 nm, λ_em = 560 nm) upon pH variations in aerated aqueous buffer solution (pH 12.5, 8.0, 7.0, 6.0, 2.5—phosphate buffer solution, pH 4.75, 4.5, and 3.5—citrate buffer solution, pH 9.0 and 9.6—borate buffer solution, for 3 buffer/acetone 9/1; v/v), 293K. Lifetime (LT) of the probes were calculated from bi- (for 4) and tri- (for 3) exponential decay fit as intensity-weighted average lifetime (τ_av, see Equation (1)).

Figure 8

Left: MTT assay of CHO-K1 cells after incubation with complexes 1–4 for 24 h at different concentrations. Cell viability of control cells (without probe) was taken for 1. The data are shown as mean ± standard deviation. N = 6 repetitions for CHO-K1 cells. Right: Confocal microscopy of CHO-K1 cells incubated with complexes 1–3 (5 µM, 24 h) and 4 (25 µM, 24 h). Green, red, and DIC (differential interference contrast) channels are merged. Scale bar 20 µm.

Figure 9

Co-staining of CHO-K1 cells with complexes 3 (5 µM, 24 h) and 4 (25 µM, 24 h) and Lysotracker Deep Red (50 nM, 30 min). Pearson’s (P) and Mander’s (M1) coefficients are given in the merged picture.

Figure 10

Confocal and PLIM-images of CHO-K1 cells incubated with complex 3 (Top, 5 µM, 24 h) and 4 (Bottom, 25 µM, 24 h). Left: confocal image and merged confocal image and DIC. Central: PLIM image. Right: lifetime distribution for the PLIM image. Excitation 405 nm, 37 °C and 5% CO₂, normoxia.