A fluorescent pH probe for an aqueous solution composed of 7-hydroxycoumarin, Schiff base and phenanthro[9,10-d]imidazole moieties (PICO)

Introduction

Intracellular pH (pH_i) regulates various functions of many organelles and plays vital roles in cellular events [1], including proliferation and apoptosis, cell cycle control, cell adhesion and endocytosis [2], [3]. In diverse prokaryotic species and different subcellular compartments of eukaryotic cells, pH_i can vary from highly acidic to basic values [4], [5]. Abnormal pH_i values in organelles are often associated with cellular dysfunction and affect human physiology, which is related to many diseases such as cancer [6] and Alzheimer’s disease [7]. Therefore, monitoring the pH changes inside live cells is crucial to understand the cellular functions and biological and pathological processes. The use of fluorescent pH probes are the most successful approaches due to their nondestructive character, high sensitivity and high specificity [8], [9]. Many excellent pH probes for lysosomes (pH 4.5–5.0) [10], [11], [12], [13], [14] or cytosol (pH 6.8–7.4) [15], [16], [17] have been obtained based on the mechanism of photoinduced electron transfer (PET), internal charge transfer (ICT) and Förster resonance energy transfer (FRET).

The pH sensors based on conjugated Schiff bases have been paid increasing attention due to tautomerism from phenol-imine to ketone-amine forms [18] and their photochromic and thermochromic properties [19]. Among these, Schiff bases containing a hydroxy group are of a special interest [20], [21], [22], [23], [24], [25], [26] because of their characteristic property to form the ionic N⁺-H…O⁻ hydrogen bond [19]. In continuation of our studies on pH fluorescent probes, a new pH sensitive compound 7-hydroxy-4-methyl-8-(((2-(1-phenyl-1H-phenanthro[9,10-d]imidazol-2-yl)phenyl)imino)methyl)-2H-chromen-2-one (PICO) was synthesized as an ICT fluorescent probe which contains o-hydroxycoumarin as an electron donor (D) and phenanthro[9,10-d]imidazole as an electron acceptor (A) mutually conjugated through Schiff base (Scheme 1). Changes in absorption and fluorescence properties of the probe PICO are observed with changes in pH.

Scheme 1

Synthetic route to PICO.

Results and discussion

The straightforward synthesis of PICO is shown in Scheme 1. As can be seen from the proton nuclear magnetic resonance (¹H NMR) spectrum (see Experimental), a single peak at 9.12 ppm is due to the presence of the imino group and the sharp signal at 14.14 ppm can be assigned to the hydroxy group. This signal is located considerably downfield compared to the normal hydroxy proton resonance, which can be a result of the intramolecular hydrogen bond between the phenol group and the adjacent N atoms of the imino group and the imidazole ring.

Figure 1 shows the UV-Vis absorption spectra of PICO at various pH values. Two absorption peaks around 300 nm and 365 nm can be seen. Under strong acidic conditions (pH 2.21), a weak absorption peak is located around 300 nm. With increases in pH, the maximum absorption peak of PICO becomes stronger and is gradually shifted to around 360 nm. Furthermore, the isosbestic point at 335 nm can be assigned to the π−π^* transition of C=N and C=C functions. These results are consistent with the dominance of the enol-imine form under neutral or weakly basic conditions.

Figure 1

Absorption spectra of PICO as a function of pH.

Subsequently, the fluorescence spectra of PICO as a function of pH were investigated (Figure 2) using the isosbestic point excitation wavelength at 335 nm. The emission spectra of PICO are sensitive to pH changes with an ‘off−on’ switching profiles. In strongly acidic solution (pH 2.21), the fluorescence spectrum has a weak maximum emission band at 500 nm with the fluorescence quantum yield Φ of 0.024. When the acidity changes to neutral conditions (pH 7.42), the maximum emission peak of PICO in fluorescence spectrum shows a huge shift from 500 nm to 450 nm. It can be suggested that the 50-nm blue shift is the result of deprotonation of PICO with a concomitant fluorescence enhancement. Under the pH conditions of 12.22, the fluorescence quantum yield Φ is 0.43 with the maximum fluorescence intensity. It can be suggested that the increase of fluorescence intensity is a result of the disruption of the intramolecular hydrogen bond because of the deprotonation of the hydroxy group. In this situation, the oxygen anion is a strong electron donor which also alters the ICT effect and causes the fluorescence emission shift. Meanwhile, the titration curve (inset in Figure 2A) exhibits distinct pH-dependent fluorescence changes in this range, and the maximum fluorescence enhancement factor (F_max/F_min) of PICO is 50-fold (calculated with the fluorescence intensity at 450 nm). The sigmoidal fitting yielded a pKa value of 8.01 and there is a good linearity between emission and pH in this range with R² value of 0.9909 (Figure 2B).

Figure 2

(A) Fluorescence spectra of PICO as a function of pH; the inset shows the fluorescence titration of PICO. (B) The sigmoidal fitting plot for fluorescence titration of PICO against pH.

Considering a possible binding of PICO with metal ions and anions, which would cause a possible interference with pH monitoring, its selectivity to pH in the presence of metal ions (Co²⁺, Cr³⁺, Cd²⁺, Fe³⁺, Zn²⁺, Cu²⁺, Hg²⁺, Ni²⁺, Al³⁺, Ba²⁺, Pb²⁺, Na⁺, K⁺, Ca²⁺, Ag⁺, Mg²⁺, 100 μm) and anions (I⁻, Cl⁻, Br⁻, CO₃²⁻, HCO₃⁻, SO₄²⁻, NO₂⁻, AcO⁻, H₂PO₄⁻, HPO₄²⁻, BrO₃⁻, NO₃⁻, SO₂⁻, HSO₃⁻, F⁻, and SCN⁻, 100 μm) were investigated. Figure 3A shows fluorescence intensity changes of PICO in the presence of different metal ions at pH 3.1 and 7.4. The fluorescence intensity of PICO is not affected by the addition of any metal ion under each pH condition. In a similar way, the fluorescence intensity of PICO in the presence of anions is not affected (Figure 3B). These results show excellent selectivity response of PICO in the presence of background metal ions or anions across a wide pH range.

Figure 3

(A) Effect of metal ions (100 μm, Co²⁺, Cr³⁺, Cd²⁺, Fe³⁺, Zn²⁺, Cu²⁺, Hg²⁺, Ni²⁺, Al³⁺, Ba²⁺, Pb²⁺, Na⁺, K⁺, Ca²⁺, Ag⁺, and, Mg²⁺) on the fluorescence intensity of PICO (10 μm) at pH values of 3.1 and 7.4. (B) Effect of anions (100 μm, I⁻, Cl⁻, Br⁻, CO₃²⁻, HCO₃⁻, SO₄²⁻, NO₂⁻, AcO⁻, H₂PO₄⁻, HPO₄²⁻, BrO₃⁻, NO₃⁻, SO₂⁻, HSO₃⁻, F⁻, SCN⁻) on the fluorescence intensity of PICO (10 μm) at pH values of 7.4 and 12.2.

The stability of PICO (10 μm) was tested by measuring the fluorescent response for 3 h under different pH conditions. Figure 4A shows fluorescence at pH 12.2, 7.4 and 2.2 at room temperature. The results show that fluorescence of PICO is stable over time. Reversibility of response is another important parameter to assess the performance of a fluorescence probe. The pH value was modulated back and forth 5 times between 3.1 and 12.2 by the addition of HCl (0.1 m) and NaOH (0.1 m). As shown in Figure 4B, the fluorescence changes are fully reversible in the whole pH range, which means that PICO exhibits a fully reversible response to pH.

Figure 4

(A) The fluorescence intensities of PICO (10 μm) at pH 3.1, 7.4 and 12.2 over a 2-h time course. (B) Reversibility of the fluorescence intensities of PICO between pH 3.1 and 12.2.

It can be suggested that PICO exists in various tautomeric forms as shown in Scheme 2. The enol form 1 is present under neutral conditions and is transformed under basic conditions to the enol form 2 with the oxygen-centered anion. This suggestion is consistent with the ICT effect on the fluorescence change. The enol form 2 with the oxygen anion is the precursor to the stable keto structure. The density functional theory (DFT) calculations for enol and ketone tautomers, obtained at the B3LYP/6-31G* level using the B3LYP/6-31G(d) level of the Guassian 09 program, strongly support this conclusion. The calculated band gaps in water between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of enol and ketone forms are 3.18 eV and 3.04 eV, respectively. Thus, the energy gap between the HOMO and LUMO of the ketone form is only slightly smaller than that of the enol form. The energy parameters also reveal that the ketone form is more stable than the enol form in DMF as well.

Scheme 2

A plausible equilibrium between different forms of PICO.

Experimental

The substrates 2-(1-phenyl-1H-phenanthro[9,10-d]imidazol-2-yl)aniline and 7-hydroxy-4-methyl-2-oxo-2H-chromene-8-carbaldehyde were prepared as previously reported [27].

N,N-Dimethylformamide (DMF) was distilled over calcium hydride. Other commercial solvents were of analytical grade and used without further purification. To maintain constant pH values, the experiments were performed in water/DMF (9:1, v/v) solutions in the presence of ammonia/ammonium chloride buffer (pH>9) and 4-(2-hydroxyethyl)1-piperazineethane sulfonic acid (HEPES) buffer (pH<9.0 or pH 7.4). All pH measurements were made with a Sartorius PB-10 digital pH-meter. Absorption and fluorescence spectra were recorded on a TU–1901 UV-Vis spectrometer and a Perkin-Elmer LS55 fluorescence spectrophotometer, respectively.