BODIPY-Equipped Benzo-Crown-Ethers as Fluorescent Sensors for pH Independent Detection of Sodium and Potassium Ions

Graphical Abstract

Fluoroionophores: In this work, we introduce a highly Na⁺ selective and Na⁺ sensitive fluorescent probe, which show a similar Na⁺ induced fluorescence enhancement over a broad pH value range from 3 to 10. Moreover, this probe is a universal fluorescent indicator for the analysis of Na⁺ levels in biological settings.

Abstract

Herein, we report on fluorescent probes which enable the selective quantification of Na⁺ and K⁺ in water by fluorescence enhancement (FE) independent of the pH value. These fluorescent probes, so called fluoroionophores, consist of benzo-crown-ether derivatives as ionophores, a benzo-15-crown-5 (cf. 5) or a benzo-18-crown-6 (cf. 6), respectively, to selectively bind either Na⁺ or K⁺ and a 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY) fluorophore. The fluoroionophore 5 shows a FE factor of 7.3 in the presence of 1000 mM Na⁺ and 6 of 4.7 upon addition of 250 mM K⁺ in an acidic environment. The dissociation constants (K_d) for 5+Na⁺ is 276 mM and for 6+K⁺ is 18 mM enabling the fluorometric determination of biological relevant Na⁺ or K⁺ levels. The fluorescence intensities of 5 and 6 were not impacted over a broad range of proton concentrations (pH stable from 3.5 to 9.5) and by the presence of other biologically relevant cations.

Introduction

The alkali metal ions Na⁺ and K⁺ play a central role in numerous biological processes such as transmission of stimuli in nerve cells, the excitation of muscles on the motor endplate, the adjustment of the cellular membrane potential, regulation of cell volume and formation of the cell internal pressure as well as the stabilization of the 3D structure of proteins.^1a-1d Maintaining Na⁺ and K⁺ ion homeostasis is essential for all living organisms and aberrant pathophysiological dysregulation has severe and instantaneous consequences. For example, low Na⁺ blood serum level (5.5 mM–6.0 mM) or moderate (6.1 mM–6.9 mM) causes hyponatremia and triggers low blood pressure, nausea, fatigue or cardiac arrhythmia.^1c High blood serum K⁺ levels (>5.5 mM) causes hyperkalemia. In animal cells, the Na⁺ concentration range differs in the intra- (5 mM–30 mM) and extracellular (100 mM–150 mM) space.^1d In both compartments, K⁺ is the competitive cation and shows an extra- (1 mM–10 mM) and intracellular (100 mM–150 mM) concentration gradient contrary to Na⁺.^1d These high concentration differences make it very challenging to develop a Na⁺ or K⁺ selective tool for the determination of low Na⁺ concentrations in the presence of high K⁺ concentrations and vice versa.² The standard determination methods for Na⁺ and K⁺ in the laboratory are for instance atomic absorption spectrometry (AAS) and inductively coupled plasma optical emission spectrometry (ICP-OES). These determination setups for Na⁺ and K⁺ are space-consuming, time intensive and expensive. Furthermore, these methods cannot be used in real time in vivo and do not allow any statements about the spatial and temporal distribution of Na⁺ or K⁺ within a living cell. Due to the wide biological and medical impact of Na⁺ and K⁺, a precise, fast, and inexpensive analysis of these ions is of great importance. Fluorescence spectroscopy is a simple, sensitive, and precise method that has already been used successfully in living cells.³ Further, Na⁺ and K⁺ are not able to fluoresce themselves and must be made accessible to fluorescence spectroscopy by using Na⁺ or K⁺ binding molecular fluorescent tools so called fluoroionophores, which consisting of a fluorescence signaling unit (fluorophore) and a highly cation selective binding moiety (ionophore). In addition, the K_d value of the cation-fluoroionophore-complex must fit in the concentration range of the corresponding analyte to be determined. A variety of highly selective fluoroionophores for the fluorometric detection of Na^+[4a–l] and K^+[5a–p] in aqueous solutions have been reported. These Na⁺- and K⁺-probes often show a high selectivity, a good sensitivity, a moderate water solubility and suitable K_d values to measure extra- or intracellular ion levels along with an extraordinary fluorescence performance. However, most of the reported Na⁺- or K⁺-probes are based on aza-crown ethers or aza-cryptands as ionophores. A key role for the fluorescence determination of Na⁺ and K⁺ plays the nitrogen atom of the ionophore, which acts as an electron donor. The ion pair of the nitrogen donor is an electron communicator between the ionophore and the fluorophore, which modulates the fluorescent properties of the probe. Upon Na⁺ or K⁺ binding a reduction of the donor ability of the nitrogen causes a change of the fluorescence signal. While the use of nitrogen donor-based mechanism allows the design to tailor-made fluorescent probes with a high selectivity and with extraordinary fluorescence sensing properties, but it makes the fluorescent probe highly sensitive to interfering protonation under varying physiological pH-values. Due to this pH dependency, it is very difficult to accurately determine the Na⁺ or K⁺ concentration, in particular in an acidic environment since an elevated H⁺ concentration will interfere with the nitrogen donor metal interaction and will lead to an erroneous interpretation of the fluorescence signal change. Several efforts were made to circumvent the protonation of the nitrogen donor at physiological relevant pH values by designing more pH-tolerant nitrogen donors.⁶ However, in cells acidic organelles such as late endosomes or early lysosomes the pH varies from 4 to 5,^7c which thus precludes a precise determination of luminal Na⁺ and K⁺ levels in endolysosomes by the use of the common and pH-sensitive aza-ionophore based fluorescent probes. Notwithstanding there is a significant demand to determine the Na⁺ and K⁺ concentration in these acidic organelles to allow the investigation of cellular processes such as endocytosis² and get a deeper understanding of the influence of Na⁺ and K⁺ during intracellular transport and digestion.^{2, 8} For example, Na⁺ is required for the function of some lysosomal transporters^7a and K⁺ regulates the lysosomal membrane potential.^7b A preliminary analysis of luminal Na⁺ and K⁺ by using an indirect null-point titration method revealed for K⁺ a concentration of around 60 mM and for Na⁺ of around 20 mM in these organelles.^9a Further, Wang et al. estimated for luminal Na⁺ concentrations a 100-fold higher value than for K⁺ by using ultracentrifugation and mass spectrometry.^9b Overall, the so far used detection methods show large deviations for the determined luminal Na⁺ and K⁺ levels and intrinsically do not allow the analysis of the direct spatial and temporal distribution of Na⁺ or K⁺ concentrations within these cellular organelles. However, selective fluorescence sensors for alkali metal ion detection, which allow a pH independent determination of biological relevant Na^+[4g,10a] or K^+[10b] levels were rarely described. Therefore, we wanted to design a fluorescence sensor with the potential to exactly measure selective endosomal or lysosomal Na⁺ and K⁺ levels by fluorescence enhancement over a large pH range.

To measure alkali metal ion concentrations in a highly selective and pH independent fashion by fluorescence spectroscopy, fluoroionophores are required, that show a near pH independent fluorescence signal change. As ionophores, we selected therefore nitrogen-free benzo-crown ether derivatives. These well-known ionophores can be tailored to bind alkali metal ions selectively. In water the benzo-15-crown-5 shows an affinity to Na^+[11a] and the benzo-18-crown-6 is K⁺ selective.^11b Thus, our selected fluoroionophores 4, 5 and 6 consist of the ionophore elements, a benzo-12-crown-4 in 4, a benzo-15-crown-5 in 5, and a benzo-18-crown-6 in 6, respectively (cf. Scheme 1). While benzo-crown-ethers are electron poorer donors than phenyl-aza-crown ethers avoiding the aforementioned pH dependent fluorescence enhancement they show in general a much lower metal ion induced fluorescence enhancement by blocking a photoinduced electron transfer (PET).¹² According to Rehm-Weller equation, which allows estimation of the thermodynamic driving force for a possible PET process in a fluorescent probe¹³ the usage of benzo-crown-ether as ionophores, requires a very electron poor fluorophore. Therefore, we chose as a strongly electron-deficient fluorophore the 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY) moiety in 4, 5 and 6 due to its high stability against chemical and physical influences, extraordinary spectroscopic properties such as a high quantum yield, an emission maxima higher than 500 nm and a high photostability as well as a small spectroscopic influence to changing solvent polarities and pH value fluctuations.¹⁴ This assumption is based on previously studied BODIPY equipped fluorescent probes which were already used to detect Na^+[4e,f,15] and K^{+[5a,c,d,e,l,16a,b]} in neutral aqueous or slightly acidic solutions.

Scheme 1

Studied cation-responsive fluorescent probes 4, 5^19b and 6^19c as well as reference compounds 1,^17a 2,^17b 3^17c and 7.^19d

Herein, we synthesized the known benzo-crown-ether substituted BODIPY dyes 5 and 6 to study their so far unknown fluorescence behavior towards Na⁺ and K⁺ over a broad pH range from 3.5 to 9.5 in aqueous solutions. As benzo-crown-ether free references compounds 1,3,5,7-tetramethyl-4,4-difluoroboradiazaindacene (1), 1,3,5,7-tetramethyl-8-phenyl-4,4-difluoroboradiazaindacene (2) and 1,3,5,7-tetramethyl-8-(3,4-dimethoxyphenyl)-4,4-difluoroboradiazaindacene (3) were used to compare their photophysical properties with 4, 5 and 6 as well as the phenyl-aza-15-crown-5 equipped fluoroionophore 7 to illustrate the pH dependency of phenyl-aza-crown ether substituted BODIPY fluorescent probes.

Results and Discussion

Synthesis and characterization of fluorescent probes 1–7

The known reference compounds 1,^17a 2^17b and 3^17c were prepared according to procedures in the literature. Moreover, the novel benzo-12-crown-4 substituted fluoroionophore 4 was synthesized¹⁸ in a four-step one-pot reaction procedure by following a synthetic approach from Wang et al..^19a Further, the known crown-ether equipped fluoroionophores 5,^19b 6^19c and 7^19d were also synthesized in moderate yields, 39 %, 37 % and 39 %, respectively, by using this one-pot procedure.¹⁸ Overall, benefits of this synthetic approach are an easier and faster preparation and purification of 5, 6 and 7 compared to the chosen synthetic routes by Ozlem et al.,^19b by Shao et al.^19c and by Bozdemir et al.^19d for 5, 6 and 7, respectively. The synthesized fluorescent probes 1–7 were characterized by ¹H- and ¹³C-NMR spectroscopy as well as electrospray ionization mass spectrometry.¹⁸ Further, the molecular structure of 3 and 5 were confirmed by X-ray analysis (cf. Figure 1).¹⁸

Figure 1

Molecular structures of 3 and 5 with their typical torsion angles around the BODIPY core and the phenyl ring as well as a CPK presentation of the H-atoms of the methyl groups in position 2 and 7 in light blue and the C-atoms of the phenyl ring in light orange.

Spectroscopic properties of 3–6 in the presence of varying pH values and alkali metal ions in H₂O/DMSO mixtures

To study the fluorescence behavior of 3, 4, 5 and 6 towards different pH-values and alkali metal ions in aqueous solutions, we found that the fluorescent probes 3, 4, 5 and 6 (c=10⁻⁶ M) show good solubility in H₂O/DMSO mixtures (v/v, 999/1). First, we checked the pH influence on the fluorescence signal intensity of the crown ether free reference compound 3 compared to the benzo-crown ether based fluorescent probes 4, 5 and 6 in H₂O/DMSO mixtures (v/v, 999/1) as well as of the aza-crown ether equipped BODIPY dye 7 in H₂O/DMSO mixtures (v/v, 99/1). Figures S5a, 2 and S6c show for 3, 5 and 6 in the pH value range from 3.5 to 9.0 only very small fluorescence intensity changes while 7 shows a significant FE in the pH range from 3 to 5, caused by protonation of the anilino nitrogen donor (cf. Figure 2). This blocks the fluorescence quenching process in 7 and triggers a FE.²⁰ In contrast, the very stable fluorescence signal over the pH ranges from 3.5 to 9.5 of 4, 5 and 6 qualified them as potential candidates for fluorescence titration experiments with alkali metal ions at different pH values.

Figure 2

Fluorescence intensities of 5 (red), 5+150 mM NaCl (green) and 7 (black) at different pH values at 507 nm (c_dye=10⁻⁶ M, λ_ex=475 nm).

To investigate the fluorescence behavior of 3, 4, 5 and 6 towards alkali metal ions in acidic aqueous solutions (pH=4.7), we dissolved the fluorescent probes 3, 4, 5 and 6 (c=10⁻⁶ M) in acetate buffered H₂O/DMSO mixtures (v/v, 999/1, acetic acid/acetate buffer, c=10 mM, pH=4.7). The fluorescence spectra of 3, 4, 5 and 6 showed very similar emissions centered at 506 nm or 507 nm, respectively (cf. Table 1). The quantum yields ϕ_F of fluorescent probes 3, 5 and 6 are also very similar to each other, but the ϕ_F value of 4 is obviously higher (cf. Table 1). The low ϕ_F values of 3, 4, 5 and 6 are caused by a PET process.^21a-21e For a more detailed study of the fluorescence quenching PET process in 3, 4, 5 and 6 see the section spectroscopic studies in organic solvents below.

Table 1. Photophysical properties of 3, 4, 5 and 6 in H₂O/DMSO mixtures (v/v 999/1) at pH 4.7 (10 mM acetic acid/acetate buffer).

	λabs [nm]	λf [nm]	ϕf^[a]	FEF^[b]	Kd^[c] [mM]
3 4 4+100 mM Na^+ 4+100 mM K^+ 4+100 mM Li^+ 5 5+1000 mM Na^+ 5+1000 mM K^+	498 498 498 498 498 498 498 498	506 507 507 507 507 506 507 507	0.011 0.072 0.073 0.072 0.072 0.008 0.056 0.023	– – 1.0 1.0 1.0 – 7.3 2.8	– – -^[d] -^[d] -^[d] – 276 342
6 6+250 mM Na^+ 6+250 mM K^+	498 498 498	507 507 508	0.008 0.013 0.035	– 1.8 4.7	– 65 18

• [a] Fluorescence quantum yield, (±15) %. [b] Fluorescence enhancement factor, [FEF=I/I₀], (±0.2). [c] Dissociation constants K_d. [d] K_d values for the corresponding cation complexes could not be determined, caused by low cation induced intensity changes.

In a next step, we performed titration experiments with 3, 4, 5 and 6 in the presence of increasing alkali metal ion concentrations from 1 mM to 1000 mM NaCl, KCl or LiCl in acetic acid/acetate buffered H₂O/DMSO mixtures (v/v, 999/1, acetic acid/acetate buffer, c=10 mM, pH=4.7). Figure 3 shows the fluorescence titration curves of 4+Li⁺, of 5+Na⁺ and 6+K⁺ at a pH value of 4.7. Notably, the fluorescence signal of 4 is not influenced by increasing Li⁺, Na⁺ or K⁺ concentrations (cf. Table 1, Figures 3 and S5c). On the other hand, the fluoroionophore 5 showed a moderate FEF of 3.4 in the presence of 200 mM NaCl and a smaller K⁺ induced FEF of 1.6 in the presence of 200 mM KCl (cf. Figure 4 and Figure S6b). Further, the fluoroionophore 6 exhibits a higher K⁺ induced FEF of 4.7 (6+200 mM KCl) and a lower Na⁺ induced FEF of 1.7 (6+200 mM NaCl, cf. Figure S7b). The ϕ_F values of 5 and 6 were also increased in the presence of Na⁺ or K⁺, respectively (cf. Table 1).

Figure 3

Fluorescence intensity changes of 4 (blue), 5 (black) and 6 (red) in the presence of Li⁺, Na⁺ or K⁺ in H₂O/DMSO (999/1 v/v) mixtures with 10 mM acetic acid/acetate buffer (pH=4.7) at 507 nm (c=10⁻⁶ M, λ_ex=475 nm).

Figure 4

Fluorescence spectra of 5 (c=10⁻⁶ M, λ_ex=475 nm) in the presence of different NaCl concentrations in H₂O/DMSO (999/1 v/v) mixtures with 10 mM acetic acid/acetate buffer (pH=4.7).

In a further step, we carried out titration experiments with 5 and 6 in the presence of increasing NaCl and KCl concentrations (from 1 mM to 1000 mM) in neutral aqueous solutions (Tris buffered H₂O/DMSO mixtures (v/v, 999/1, Tris buffer, c=10 mM, pH=7.2)). The fluorescence titration curves of 5+Na⁺ and of 6+K⁺ at a pH value of 7.2 are shown in the Supporting Information (cf. Figures S6e and S7e). The course of the curves at pH=4.7 and 7.2 were very similar to each other. Furthermore, the fluoroionophores 5 and 6 show in the presence of 150 mM NaCl or 150 mM KCl in a pH range from 3.5 to 9.5 almost constant FEFs of 3±0.1 and 5±0.25, respectively (cf. Figure 2 for 5+Na⁺ and Figure S7c for 6+K⁺). This illustrates the applicability of fluorescent probes 5 and 6 to measure biological relevant Na⁺ or K⁺ levels over a wide pH range.

The dissociation constants (K_d) of 5 and 6 in the presence of Na⁺ and K⁺ were calculated from fluorescence intensity changes at a pH value of 4.7.¹⁸ For 5+Na⁺ and K⁺, we obtained K_d values of 276 mM and 342 mM and for 6+Na⁺ and K⁺ values of 65 mM and 18 mM, respectively (cf. Table 1). As expected, the benzo-15-crown-5 substituted fluoroionophore 5 shows a higher affinity to Na⁺ and the benzo-18-crown-6 substituted fluoroionophore 6 forms a more stable complex with K⁺ than with Na⁺. Therefore, we conclude from the K_d values that 5 has an appropriate Na⁺/K⁺ selectivity to measure extracellular Na⁺ levels in the presence of extracellular K⁺ concentrations and 6 has a higher K⁺ affinity to determine physiological relevant K⁺ levels from 1 mM to 50 mM in the presence of low Na⁺ levels.

A better comprehension of the Na⁺- and K⁺-selectivity of 5 and 6 is by comparing the FEF at physiological extracellular Na⁺ (100 mM–150 mM) or extracellular K⁺ (1 mM–10 mM) concentration levels. Here the fluorescence of 5 increased by a factor of 2.8 in the presence of 140 mM NaCl and only enhanced by a factor of 1.05 in the presence of 10 mM KCl. For 6+140 mM NaCl and for 6+10 mM KCl, we found FE factors of 1.7 and 2.3, respectively. These FE differences show that the fluorescence signal of 5 is only slightly influenced by extracellular K⁺ levels while the fluorescence signal of 6 is detrimentally perturbed by high extracellular Na⁺ levels. Thus, fluorescent probe 5 shows a good extracellular Na⁺/K⁺ selectivity based on fluorescence intensity changes.

To further verify the Na⁺ selectivity of 5 and the K⁺ affinity of 6 towards other important biological inorganic cations such as NH₄⁺, Mg²⁺, Ca²⁺ and Zn²⁺, we measured the fluorescence intensities in the presence of these ions (cf. Figure 5 for 5 and Figure S7d for 6). Overall, these cations show only a very minor influence on the fluorescence behavior of 5 and 6. In the case of other more biologically relevant cations such as Sr²⁺, Ba²⁺ and Pb²⁺ we noted that the fluorescence of 6 is however significantly enhanced (cf. Figure S7d), which was similarly observed for a series of benzo-18-crown-6 based fluorescent probes in previous studies.^{22a, 22b} However, after the addition of Na⁺ to 5 in the presence of K⁺, NH₄⁺, Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺, Li⁺, Rb⁺, Cs⁺, Al³⁺, Pb²⁺, Cr³⁺, Mn²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Ag⁺, Zn²⁺, Cd²⁺ or Hg²⁺, 5 shows a FEF, which is comparable to the FEF induced only by 150 mM Na⁺ (cf. Figure 5). Thus, 5 is a suitable fluorescent tool to selectively detect and quantify extracellular Na⁺ or K⁺ levels in the presence of other biologically relevant cations and the most toxic heavy metal ions.

Figure 5

Fluorescence intensities changes of 5 (c=10⁻⁶ M, λ_ex=475 nm) in H₂O/DMSO mixtures (v/v 999/1) at 507 nm with 10 mM acetic acid/acetate buffer (pH=4.7) in the presence of 5 mM for K⁺ and NH₄⁺, 2 mM for Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺, Li⁺, Rb⁺, Cs⁺ and 0.1 mM for Al³⁺, Pb²⁺, Cr³⁺, Mn²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Ag⁺, Zn²⁺, Cd²⁺, Hg²⁺ (orange bars) as well as subsequent addition of 150 mM of Na⁺ (blue bars).

Cation sensing mechanism of fluorescent probes 5 and 6 in organic solvents

To get a better understanding of the fluorescence sensing mechanism of Na⁺ and K⁺, we measured and analyzed the UV/Vis absorption spectra, the fluorescence emission spectra and the fluorescence quantum yields ϕ_f of the fluorescent probes 4, 5 and 6 as well as of the reference compounds 1, 2 and 3 in organic solvents. The solubility of the fluorescent dyes 1–6 is appropriate in common polar and non-polar organic solvents. At first, we measured the UV/Vis-absorptions spectra of 1, 2, 3, 4, 5 and 6 in CH₃CN (cf. Figure S1). The UV-Vis absorption spectra of 1, 2, 3, 4, 5 and 6 in the range from 400 nm to 550 nm are very similar to each other. We found the most intense absorption band for 1, 2, 3, 4, 5 and 6 with a local maximum (λ_max) at about 500 nm with a shoulder at about 475 nm. This intense absorption can be assigned to the S₀→S₁ transition, where the absorption at about 500 nm is the vibronic 0–0 state and the shoulder at about 475 nm is the vibronic 0–1 state.^{23a, 23b} This long-wavelength absorption is more relevant for the mechanistic study than the other more energetic π-π* transitions in the UV-Vis absorption spectra of 1, 2, 3, 4, 5 and 6. The molar extinction coefficients ϵ_λ at λ_max for 1, 2, 3, 4, 5 and 6 are also very similar to each other (cf. Table S1) and in the range of structurally related BODIPY dyes.^{23a, 23b} These data suggest that the phenylic substituent in meso-position of the BODIPY's in 2, 3, 4, 5 and 6 does not essential extend the π-electron system of the BODIPY chromophores. In 2, 3, 4, 5 and 6 the phenyl derivative is not fully conjugated with the BODIPY core, caused by the steric hindrance of the bulky methyl groups in position 1 and 7, which prevent free rotation of the phenyl derivative.¹⁴ As observed in the X-ray structure of 3 and 5 the phenyl ring is almost orthogonal to the planar BODIPY core (cf. Figure 1). Thus, in 3, 4, 5 and 6 the BODIPY fluorophore is virtual decoupled from the benzo-crown-ether ionophore as already found for other BODIPY based fluoroionophores.^{20, 23c}

We recorded fluorescence emission spectra of 1, 2, 3, 4, 5 and 6 (λ_exc=475 nm, c=10⁻⁶ M) under the same conditions in CH₃CN. As a result, the fluorescence emission maxima λ_f of 1, 2, 3, 4, 5 and 6 were nearly the same for each fluorescent dye (for example: 506 nm for 1 and 506 nm for 5, cf. Table S1), but they differ in their fluorescence intensities. Further, we determined the ϕ_f values of 1, 2, 3, 4, 5 and 6 in different organic solvents by varying the polarity (cf. Table S2). The ϕ_f of the BODIPY derivative 1 in polar and non-polar solvents has nearly the same high value of about 0.85.¹⁸ The phenyl substituted BODIPY dye 2 shows a lower ϕ_f value than 1 in solution.¹⁸ This reduced ϕ_f value is caused by the rotational freedom of the phenyl substituent in meso position, which enhances a non-radiative deactivation of S₁ in 2.¹⁴ An even lower ϕ_f value of 0.175 in DMSO show the dimethoxybenzene substituted BODIPY dye 3 (cf. Table S2). This additional quenching, compared to 1 and 2, can be explained by a photoinduced electron transfer (PET) from the dimethoxybenzene electron donor to the excited electron acceptor the BODIPY fluorophore.^21a-21e For the 15- and 18-membered benzo-crown ether derivatives 5 (0.105) and 6 (0.120), we determined similar ϕ_f values in DMSO, but for the 12-membered benzo-crown ether derivative 4, we found a higher ϕ_f value of 0.551 in DMSO. The low ϕ_f values of fluorescent probes 4, 5 and 6 compared to 1 in polar solvents (cf. Table S2) could be also caused by a quenching PET process.^21a-21e The ϕ_f value difference of 4 and 5 might be by the fact, that the benzo-15-crown-5 in 5 is more sterically demanding than the benzo-12-crown-4 in 4. Consequently, in solution the bulky benzo-15-crown-5 unit is nearly orthogonal to the BODIPY core, which leads to a strong decoupling of these two π-systems and to an efficient fluorescence quenching of 5. On the other hand, the smaller benzo-12-crown-4 in 4 allows in solution a higher torsional flexibility between the phenyl and the BODIPY unit, which results in a poorer orthogonal orientation of these moieties. Therefore, the benzo-12-crown-4 in 4 has a stronger π-coupling to the BODIPY core via the meso-phenyl ring and a higher ϕ_f value compared to 5 is observed. Further, solvent effects on the ϕ_f value are found for 3, 4, 5 and 6 (cf. Table S2) because their PET processes are accelerated in polar solvents. We favor a PET process for the fluorescence quenching in alkoxy-benzene substituted BODIPYs 3, 4, 5 and 6 from the dimethoxy electron donor to the excited BODIPY electron acceptor due to the following observations. We estimate a negative driving force for the PET process in 3, 5 and 6.^21a-21e In addition to it, we could not detect any red-shifted emission in 3, 4, 5 and 6 from an energetically lower CT state in polar solvents. Moreover, the view of a PET quenching process is also in line with recent mechanistic studies in a series of dimethoxybenzene or phenolate substituted BODIPY dyes^{23a, 24a-24d} and in 18-membered benzo-crown ether substituted BODIPY fluoroionophores.^24e

Further support emerges from the investigated absorption and fluorescence intensity changes of 1, 2, 3, 4, 5 and 6 in the presence of increasing NaClO₄ and KPF₆ concentrations in CH₃CN (cf. Figures S3 and S4, Table S1).¹⁸ As a result the fluorescence intensity of 5 an 6 is also enhanced at λ_f(max) by Na⁺ or K⁺ in CH₃CN (cf. Table S1), which is characteristic for PET fluoroionophores.²⁵ On the other hand, 1, 2, 3 and 4 show no fluorescence increase by Na⁺ or K⁺. In addition, the S₀→S₁ transition in the absorption spectra of 5 and 6 peaked at 498 nm is nearly unaffected by Na⁺ or K⁺ in CH₃CN, which is also typical for PET fluoroionophores.²⁵ The coordination of Na⁺ or K⁺ by the benzo-crown ether moiety can be seen from the change in the absorption spectrum of 5 or 6 at around 280 nm (cf. Figure S2).¹⁸ This absorption band peaked at 280 nm is attributed to a π→π^* transition within the benzo-crown ether unit^{26a, 26b} and is enhanced and slightly blue-shifted upon cation complexation.^{18, 26b} Overall, these results indicate that the blocking of a PET quenching process by Na⁺ or K⁺ is responsible for the fluorescence intensity enhancement of 5 and 6.

Conclusion

We have synthesized in a four-step one-pot reaction procedure the benzo-crown ether (ionophore) equipped with BODIPY fluorophores 4, 5 and 6. These fluoroionophores consist of different sized macrocycles for the fluorometric recognition of alkali metal ions. In particular the fluorescent probe 5 shows good Na⁺ selectivity and appropriate Na⁺ sensitivity to determine physiological relevant Na⁺ levels by fluorescence intensity enhancements. Moreover, the fluorescent probe 5 enables the Na⁺ determination independently of physiological pH fluctuations. Hence, 5 is a universal fluorescent indicator for the analysis of Na⁺ concentration levels in biological settings. In contrast to fluorescent probe 4 which shows no fluorometric sensitivity to Li⁺, Na⁺ or K⁺, 6 shows a high affinity to K⁺ over other biological important cations. The fluorescent probe 6 is a capable tool to measure pH independently K⁺ levels in the range from 1 mM to 10 mM by fluorescence enhancement in the presence of low Na⁺ levels. Currently, we are synthesizing Na⁺ selective fluorescent probes with organelle specific targetable groups for the fluorometric detection of endosomal and lysosomal Na⁺ levels.

Acknowledgments

Open Access funding enabled and organized by Projekt DEAL.

Conflict of interest

The authors declare no conflict of interest.

Open Research

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.