Fluorene-Based Metal-Ion Sensing Probe with High Sensitivity to Zn²⁺ and Efficient Two-photon Absorption

Abstract

The photophysical, photochemical, two-photon absorption (2PA), and metal ion sensing properties of a new fluorene derivative (E)-1-(7-(4-(benzo[d]thiazol-2-yl)styryl)-9,9-bis(2-(2-ethoxyethoxy)ethyl)-9H-fluoren-2-yl)-3-(2-(9,10,16,17,18,19,21,22,23,24-decahydro-6H dibenzo[h,s][1,4,7,11,14,17]trioxatriazacycloicosin-20(7H)-yl)ethyl)thiourea (1) were investigated in organic and aqueous media. High sensitivity and selectivity of 1 to Zn²⁺ in THF and a water/ACN mixture were shown by both absorption and fluorescence titration. The observed complexation processes corresponded to 1:1 stoichiometry with the range of binding constants ~ (2–3)·10⁵ M⁻¹. The degenerate 2PA spectra of 1 and 1:Zn²⁺ complex were obtained in the 640–900 nm spectral range, with the maximum values of two-photon action cross section for ligand:metal complex ~ (90–130) GM, using a standard two-photon induced fluorescence methodology under femtosecond excitation. The nature of the 2PA bands was analyzed by quantum chemical methods and a specific dependence on metal ion binding processes was shown. Ratiometric fluorescence detection (420/650 nm) provided a good dynamic range (10⁻⁴ to 10⁻⁶ M) for detecting Zn²⁺, which, along with the good photostability and 2PA properties of probe 1. makes it a good candidate in two-photon fluorescence microscopy imaging and sensing of Zn ions.

1. Introduction

Two-photon absorbing (2PA) organic molecules with specific chemical affinity and linear photophysical properties have found utility in two-photon fluorescence microscopy (2PFM) as chemosensors for detecting metal ions in different organic and aqueous media.^1–4 Well-known advantages of 2PA processes, such as a higher excitation selectivity, deeper penetration, and photochemical stability of chemosensors under IR irradiation,^5–7 facilitate measurements of metal ion concentrations and their volumetric distribution in the medium.^8–10 To date, these investigations are of great interest for biophysical and biomedical applications,^{4, 11–14} along with the development of new environmental sensing 2PA materials.^{7, 9, 15} The investigations of 2PA compounds for metal ion sensing are quite limited however,^{2, 5} in contrast to a large variety of organic chromophores that are known to be effective one-photon sensors.^16–19 The latter have been employed for 2PFM, though they are not optimized for two-photon excitation and result in less than satisfactory 2PA sensitivity.

The combination of efficient molecular nonlinear absorption properties with a high selectivity to metal ions is a challenging task in the development of new types of 2PA chemosensors. As an example, two aminostilbene derivatives were developed from the well-known one-photon sensor for Ca²⁺, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA),^{20, 21} with increased 2PA cross sections due to introduction of a 1,2-diphenylethene-based structural unit.² High Ca²⁺ affinity together with efficient two-photon action cross sections δ_2PA ·Φ ~100 GM (δ_2PA and Φ are the 2PA cross section and fluorescence quantum yield, respectively) make such probes suitable in quantitative determination of Ca²⁺ in biological samples. New 2PA sensors for Pb²⁺ and Cd²⁺ based on phenylethynyl phosphine oxide derivatives with several fluorescent arms were recently reported.⁵ These compounds are of interest as the first efficient 2PA sensors for Cd²⁺ and possess potential for the detection of toxic heavy metals in the environment. The effects of Mg²⁺ binding on linear spectroscopic and 2PA properties of mono- and bis-(aza-15-crown-5-ether) substituted molecules were described and the possibility of their efficient practical use in 2PFM was shown.⁹

Among other metal ions, Zn²⁺ plays a vital role in the numerous physiological processes,^{16, 22} and the development of new 2PA organic molecules for selective detection of Zn²⁺ is extremely important for the investigation of biological systems by 2PFM methods. High selectivity and sensitivity for Zn²⁺ were reported for a novel 7-substituted, quinoline-based fluorescent probe (7-MOQ) in aqueous solution by the two-photon induced fluorescence method.¹ The observed fluorescence enhancement was explained by blocking the photoinduced electron transfer pathway²³ in 7-MOQ due to binding with Zn²⁺. A branched chromophore, tris[p-(4-pyridylethynyl)phenyl]amine with pyridine terminal groups, was investigated for its ability to sense a small amount of zinc ions via a two-photon induced fluorescence enhancement mechanism.²⁴ Increase in the acceptor strength and a large change in dipole moment were suggested to contribute to the enhancement of the 2PA cross section upon Zn²⁺ addition. Other types of 2PA organic compounds with specific mechanisms of zinc ions sensing have also been reported.^{7, 8, 25, 26}

In this paper, a new fluorene derivative, (E)-1-(7-(4-(benzo[d]thiazol-2-yl)styryl)-9,9-bis(2-(2-ethoxyethoxy)ethyl)-9H-fluoren-2-yl)-3-(2-(9,10,16,17,18,19,21,22,23,24-decahydro-6H dibenzo[h,s][1,4,7,11,14,17]trioxatriazacycloicosin-20(7H)-yl)ethyl)thiourea (1), that exhibited high selectivity to Zn²⁺ and efficient 2PA properties was investigated. The ligand architecture is based on a macrocycle receptor that has shown preferential binding to zinc,²⁷ connected to a fluorene backbone via a thiourea moiety. An analytical description of the complexation processes and quantum chemical calculations of the changes in the electronic parameters of 1 upon metal ion binding were performed. A comprehensive analysis of the linear absorption, fluorescence, and lifetime properties of 1 and its complexes with Zn²⁺ in organic solvents and aqueous medium are presented along with photochemical stability measurements. The 2PA spectra of 1 were obtained by the two-photon induced fluorescence (2PF) method²⁸ using 1 kHz femtosecond excitation. The effects of metal ion binding on 2PA efficiency of 1, sensitive ratiometric Zn ion sensing parameters, and its potential applications in 2PFM are presented.

2. Experimental Section

2.1 Materials and Synthetic Procedures

1,7-Bis(2-formylphenyl)-1,4,7-trioxaheptane (A), oxaaza macrocycle ligand (B), and 2-(4-(2-(9,9-bis(2-(2-ethoxyethoxy)ethyl)-2-isothiocyanato-fluoren-7-yl)vinyl)phenyl)benzothiazole (C) were prepared as described previously.^{27, 29, 30} All reagents and solvents were used as received from commercial suppliers unless otherwise noted. ¹H and ¹³C NMR spectroscopic measurements were performed using a Varian 500 NMR spectrometer at 500 or 125 MHz, respectively, with tetramethysilane (TMS) as internal reference; ¹H (referenced to TMS at δ = 0.0 ppm) and ¹³C (referenced to CDCl₃ at δ = 77.0 ppm). Chemical shifts of ¹H and ¹³C spectra were interpreted with the support of CS ChemDraw Ultra version 5.0. High-resolution mass spectrometry (HR-MS) analysis was performed in the Department of Chemistry, University of Florida, Gainesville, FL.

Synthesis of (E)-1-(7-(4-(benzo[d]thiazol-2-yl)styryl)-9,9-bis(2-(2-ethoxyethoxy)ethyl)-9H-fluoren-2-yl)-3-(2-(9,10,16,17,18,19,21,22,23,24-decahydro-6H-dibenzo[h,s][1,4,7,11,14,17]trioxatriazacycloicosin-20(7H)-yl)ethyl)thiourea (1)

Oxaaza macrocycle ligand B (0.13 g, 0.31 mmol) was dissolved in anhydrous DMF (3 mL) under N₂, followed by slow addition of fluorene isothiocyanate C (0.18 g, 0.26 mmol). The clear solution was stirred at room temperature, gradually changing to a fluorescent yellow color. After 14 h, the starting material was completely consumed as determined by TLC (silica gel, EtOAc/MeOH 9:1). DMF was removed by vacuum distillation, yielding brown oil. This was then diluted with four volumes of ether to precipitate the product. The solid was collected by filtration, redissolved in a minimum volume of DMSO, and then reprecipitated by addition of ether. The solid was collected and dried to afford 0.25 g of yellow solid (85% yield, m. p. 93–94 °C). ¹H NMR (500 MHz, CDCl₃) δ/ppm 8.08, (q, J= 8.5, 3H), 7.92 (d, J= 7.5, 1H), 7.68–7.23 (m, 20H), 4.23 (bs, 6H), 3.90 (bs, 4H), 3.41 (m, 16H), 3.02 (m, 10H), 2.42 (s, 8H), and 1.25 (t, J= 6.5 Hz, 6H). ¹³C NMR (125 MHz, CDCl₃): δ/ppm 180.7, 167.8, 157.2, 154.3, 149.7, 149.4, 140.4, 140.3, 135.8, 135.1, 132.5, 128.0, 127.9, 127.1, 126.9, 126.6, 126.3, 125.4, 125.0, 123.2, 123.1, 121.7, 121.1, 120.9, 120.1, 119.8, 112.2, 70.2, 69.7, 69.5, 67.2, 66.6, 66.5, 51.1, 41.2, 39.7, 15.2, 15.1. HR-MS-ESI theoretical m/z [M+H]⁺ = 1119.54 and [M+Na]⁺ = 1141.52, found [M+H]⁺ = 1119.54 and [M+Na]⁺ = 1141.52.

2.2. Linear photophysical and photochemical measurements

The molecular structure of the new fluorene-based macrocyclic chemosensor 1 is illustrated in Figure 1. All linear photophysical parameters were measured in spectroscopic grade toluene, tetrahydrofuran (THF), dichloromethane (DCM), dimethylsulfoxide (DMSO), acetonitrile (ACN), methanol (MeOH), and in an aqueous mixture of water/ACN (1:1). One-photon electronic absorption (1PA) spectra of 1 were determined with an Agilent 8453 UV–visible spectrophotometer for molecular concentrations 10⁻³ M ≤ C ≤ 10⁻⁶ M, using 0.1, 1, and 10 mm path lengths quartz cuvettes. The steady-state fluorescence and excitation anisotropy spectra were measured with a PTI QuantaMaster spectrofluorimeter in 10 mm spectrofluorometric quartz cuvettes for low concentration solutions C ≤ 10⁻⁶ M. All fluorescence spectra were corrected for the spectral responsivity of the PTI detection system. The excitation anisotropy spectra were obtained in L- format configuration geometry³¹ with the extraction of pure solvent emission and scattered light. The fundamental anisotropy values, r₀, were determined in viscous solution, polyTHF (pTHF), at room temperature, where the rotational correlation time, θ≫ τ (τ is the molecular fluorescence lifetime), and the experimentally observed anisotropy was r= r₀/(1+θ/τ) ≈ r₀.³¹ The values of fluorescence quantum yields, Φ, were obtained in low concentration solutions by a standard method relative to 9,10 diphenylanthracene in cyclohexane (Φ ≈0.95) at room temperature.³¹ Fluorescence lifetimes of 1 were determined with a time-correlated single photon counting system PicoHarp 300 under 76 MHz femtosecond laser excitation (MIRA 900, Coherent) with the instrument response function (IRF) ≈80 ps (FWHM). Linear polarization of the laser beam was oriented by the magic angle, while the concentration in 10 mm quartz cells was C ≤ 2·10⁻⁶ M. The quantum yields of the photochemical decomposition of 1 under one-photon excitation, Φ_1PA, were obtained by a previously described absorption method,³² using 405 nm cw laser diode irradiation with average power ≈50 mW.

Figure 1.

Synthesis of fluorene-based chemosensor 1.

2.3. Two-photon measurements

The degenerate 2PA spectra of 1 and metal-ligand complexes were measured in THF and an aqueous mixture of water/ACN (1:1) over a broad spectral region by the relative 2PF method.²⁸ Rhodamine B in methanol, whose comprehensive characterization has been reported,^{33, 34} was used as a standard. Two-photon induced fluorescence spectra were obtained with a PTI QuantaMaster spectrofluorimeter coupled with a femtosecond Clark-MXR CPA-2010 laser pumped optical parametric generator/amplifier (TOPAS). This laser system generates ≈140 fs pulses (FWHM) at a 1 kHz repetition rate. The tuning range of 600–940 nm and pulse energies E_p ≤ 0.15 μJ were used. Fluorescence measurements were performed in 10 mm fluorometric quartz cuvettes with dye concentrations ~(1–2)·10⁻⁵ M. The values of 2PA cross sections, δ_2PA, were determined by the expression:²⁸

$${\delta }_{2PA}^S={\delta }_{2PA}^R·\frac{{\langle F(t)\rangle }_S·C_R·{\varphi }_R·{\mathrm{\Phi }}_R·{\langle P(t)\rangle }_R^2}{{\langle F(t)\rangle }_R·C_S·{\varphi }_S·{\mathrm{\Phi }}_S·{\langle P(t)\rangle }_S^2},$$

where 〈F(t)〉 C, ϕ, Φ,, and 〈P(t)〉 are the average integrated fluorescence intensity, molecular concentration, geometric factor, fluorescence quantum yield, and excitation power, respectively. Subscripts S and R correspond to the sample and reference. The quadratic dependence of 2PF intensity on the excitation power was verified for each excitation wavelength, λ_ex. No spectral dependence Φ = f(λ_ex) was observed for 1 in all solvents utilized in 2PA measurements.

3. Analytical model and quantum chemical calculations

3.1. Complexation analysis

The formation of the 1:1 ligand:metal complex in solution can be described by the equation:³⁵

$$[DM]=K·[D]·[M],$$ (1)

where [DM] is the equilibrium concentration of complex, [D] and [M] are the concentration of ligand (dye) molecules and free metal ions, respectively, and K is the binding constant. Taking into account that [D] = D − [DM] and [M] = M − [DM] (D and M are the total concentrations of ligand and metal ions, respectively), eq. (1) can be rewritten as:

$$[DM]=K·(D-[DM])·(M-[DM]).$$ (2)

The solution of eq. (2) gives:³⁶

$$[DM]=\frac{\alpha (D,M)}{2}=\frac{\mathrm{\Delta }A(\lambda )}{L·\mathrm{\Delta }\epsilon (\lambda )},$$ (3)

where $\alpha (D,M)=\left[\frac{1}{K}+D+M-\sqrt{{\left(\frac{1}{K}+D+M\right)}^2-4·D·M}\right]$, ΔA(λ) is the change in absorbance of the solution at a particular wavelength, λ, L is the path length of the cuvette, and Δ(λ) = ε_D(λ) − ε_DM(λ) is the difference in the extinction coefficients between pure ligand ε_D(λ) and ligand:metal complex ε_DM(λ) at the same λ. The value of binding constant K was obtained from the fitting of the experimental dependence ΔA = f(M) by eq. (3). In the case of low ligand absorption sensitivity on metal ion binding processes, changes in the corresponding fluorescence spectrum can be utilized for the determination of K. In this case, the equilibrium 1:1 ligand:metal complex concentration can be expressed as:

$$[DM]=M+\frac{F-F_0}{K·(F-F^{max})},$$ (4)

where, F₀ and F^max are the relative fluorescence intensity of the solution at a particular wavelength, λ, for M = 0 and M≫D, respectively. From eqs. (3) and (4) one can find:

$$\frac{F}{F_0}=\frac{1+(F^{max}/F_0)·K·[\alpha (D,M)/2]}{1+K·[\alpha (D,M)/2]}.$$ (5)

In the case of fluorescence measurements, the value of binding constant K can be determined from the fitting of the experimental dependence $\frac{F}{F_0}=f(M)$ by eq. (5). It should be mentioned that this complexation analysis is not restricted by the frequently used assumption [M] ≈ M^{9, 35, 36} and includes a full range of metal concentration.

3.2. Quantum chemical calculations

The electronic properties of free ligand and 1:1 ligand:metal complex were analyzed by quantum chemical calculations using Gaussian 2009, Rev. A2 suite of programs.³⁷ To save computer time, aliphatic side chains in the calculated molecules were replaced with methyl groups. This is a reasonable approach since the substituents in the 9-position of the fluorene ring are not in conjugation with the aromatic system and exhibit no substantial effect on the electronic distribution of the chromophore system.³⁸ The ground-state geometries of 1 and 1:Zn²⁺ were optimized using DFT with the B3LYP exchange-correlation functional and the standard 6-31G* basis set. Initial (before optimization) structures of 1 and 1:Zn²⁺ were constructed using X-ray crystal data for a dibenzosubstituted oxaaza macrocycle reported previously.²⁷ Because the experiment was carried out in a polar solvent (THF) and aqueous solution (water/ACN), the THF parameters were used with the Polarizable Continuum model (PCM) for geometry optimization and simulation of excitations. The permanent and state-to-state transition dipoles were obtained using a posteriori Tamm-Dancoff approximation (ATDA),³⁹ implemented in a locally modified version of the Gaussian 2009 code. The values of 1PA and 2PA cross sections were calculated at the TD-B3LYP/6-31G* level of theory and the Sum-Over-States expressions⁴⁰ with the empirical damping parameter Γ = 0.1eV.

4. Results and discussion

4.1. Synthesis

Fluorene-based chemosensor probe 1 (Figure 1) is comprised of a fluorenyl π-electron bridge with an unsymmetrical chemical structure. Figure 1 illustrates the strategy used to incorporate an oxaaza macrocyclic ligand containing an amine terminal pendant arm to the fluorenyl 2-position via an addition reaction. The synthesis of the oxaaza macrocyclic ligand B was derived from cyclocondensation of O¹,O⁷-bis(2-formylphenyl)-1,4,7-trioxaheptane and diethylenetriamine in methanol, followed by an in situ reduction using NaBH₄, as described in Ref 37. Details of the synthesis and characterization of fluorene isothiocyanate was previously reported.³⁰ Conjugation reaction between the oxaaza macrocyclic ligand and the isothiocyanate fluorene reactive probe was carried out in DMF under N₂ at room temperature. Full characterization, including ¹H and ¹³C NMR, and HR-MS analysis, confirmed the molecular structure of 1. In the ¹³C NMR spectrum, the thiocarbonyl carbon signal was observed at 180.7 ppm. Additionally, FT-IR analysis revealed the disappearance of the strong −NCS stretch at 2111 cm⁻¹ present in fluorene isothiocyanate C precursor.

4.2. Linear photophysical properties

Probe 1 is an unsymmetrical fluorene-based molecule containing a dibenzosubstituted oxaaza macrocycle, a metal ion binding construct.^{27, 41} The conjugation length of 1 was increased via the addition of a polarizable π-system (styryl) between the fluorenyl moiety and the benzothiazole acceptor group. In the 7-position, thiourea NH moiety acts as electron-donating group. The steady-state absorption spectra of 1 were nearly independent of solvent properties (Figure 2a, curves 1–5), with small changes (≤ 10 %) in the corresponding maxima extinction coefficients ε^max (Table 1). In contrast, the steady-state fluorescence spectra (Fig. 2a, curves 1′-5′) exhibited a strong solvatochromic behavior associated with an increase in the stationary dipole moment of 1 upon electronic excitation, typical for unsymmetrical fluorene derivatives.^{42, 43} The values of the Stokes shifts increased up to 157 nm (in DMSO), while no strict correspondence to the Lippert equation³¹ was observed. Excitation anisotropy spectra of 1 (Figure 2b, curves 1, 2) revealed the nature of the main 1PA band. A nearly constant value of anisotropy r(λ) over the 340–430 nm spectral range corresponded to one electronic transition S₀ → S₁ (S₀ and S₁ are the ground and first excited electronic states of 1, respectively). In viscous pTHF, r ≈ r₀ and relatively high values of anisotropy in the main absorption band were observed, r₀ ≈ 0.35, corresponding to a small angle (< 20°) between absorption (S₀ → S₁) and emission (S₁ → S₀) transition dipole moments. This allows one to estimate that the transition dipoles S₀ → Sn (Sn, n = 0, 1, 2, 3, ….) have mutual orientations.³¹ The values of fluorescence quantum yields were sufficiently high (Φ > 0.5), and exhibited a weak and complicated dependence on solvent properties with no correlation with solvent polarity Δf (see Table 1).

Figure 2.

(a) Normalized linear absorption (1–5) and fluorescence (1′-5′) spectra of 1 in toluene (1′), THF (2′), DCM (3′) ACN (4′) and DMSO (5′). (b) Excitation anisotropy spectra of 1 in THF (1) and pTHF (2) (observed wavelengths correspond to the fluorescence maxima) and normalized linear absorption spectrum in THF (3).

Table 1.

Linear photophysical and photochemical parameters of 1 in organic solvents of different polarity Δf ^*: absorption ${\lambda }_{\mathit{abs}}^{max}$ and fluorescence ${\lambda }_{fl}^{max}$ maxima, Stokes shifts, maximum extinction coefficients ε^max, fluorescence quantum yields Φ and lifetimes τ, quantum yields of photochemical decomposition under one-photon irradiation Φ_1PA.

N/N	Toluene	THF	DCM	DMSO	ACN
Δf	0.0135	0.209	0.217	0.263	0.305
${\lambda }_{\mathit{abs}}^{max}$, nm	391 ± 1	391 ± 1	386 ± 1	391 ± 1	382 ± 1
${\lambda }_{fl}^{max}$, nm	473 ± 1	496 ± 1	504 ± 1	548 ± 1	517 ± 1
Stokes shift, cm−1	4430 ± 100	5410 ± 100	6070 ± 100	7330 ± 100	6840 ± 100
εmax ·10−3, M−1·cm−1	-	61 ± 3	59 ± 3	58 ± 3	56 ± 3
Φ	0.72 ± 0.04	0.83 ± 0.05	0.94 ± 0.05	0.54 ± 0.03	0.78 ± 0.04
τ, ns**	1.08 ± 0.08	1.44 ± 0.08	1.45 ± 0.08	1.59 ± 0.08	1.63 ± 0.08
Φ1PA ·104	-	1.6 ± 0.4	2.0 ± 0.4	1.0 ± 0.3	6.3 ± 2

^*orientation polarizability Δ f = (ε − 1)/(2ε +1) − (n² − 1)/(2n² +1) (ε and n are the dielectric constant and refraction index of the medium, respectively).³¹

^**All lifetime measurements correspond to goodness-of-fit parameters χ² ≥ 0.99.

Fluorescence lifetimes corresponded to a single-exponential decay process with the observed values in the range of 1.0 < τ < 1.7 ns, gradually increasing with Δf. The unique characteristics of the linear photophysical parameters of 1 in a number of organic solvents allow one to assume noticeable changes in its equilibrium molecular geometry and electronic structure in the S₁ state.

The photostability of 1 under one-photon excitation in the main long wavelength absorption band was determined, resulting in the photochemical quantum yield 10⁻⁴ ≤ Φ_1PA ≤ 7·10⁻⁴ (see Table 1) that noticeably depended on the nature of the medium. The highest level of photostability was observed for 1 in DMSO (Φ_1PA ≈ 10⁻⁴) and substantially decreased in ACN (Φ_1PA ≈ 6.3·10⁻⁴). A comparative analysis of the obtained and known values of Φ_1PA^{44, 45} indicated that photostability of the new fluorene-based macrocyclic probe 1 is reasonably high and suitable for practical applications.

4.3. Sensing properties

The investigation of metal ion sensing behavior of 1 was performed in THF and water/ACN (1:1) solutions. The addition of Zn²⁺ ions to THF solution of 1 resulted in visible changes in the absorption and fluorescence spectra (Figure 3a, b). The increase of total ion concentration [Zn²⁺] from 2·10⁻⁶ to 2.4·10⁻⁴ M in the 2.5·10⁻⁵ M THF solution of 1 led to a hypsochromic shift in the absorption spectrum to 381 nm and ≈10 % increase in extinction coefficient (see Figure 3a and Table 2). Well defined isobestic points are indicative of the presence of only two absorbing species. Under the highest metal ion concentration, [Zn²⁺] ≈2.4·10⁻⁴ M, nearly all ligand molecules were in the complexed form and no changes in absorption were observed for additional increase in [Zn²⁺].

Figure 3.

(a) Absorption spectra of 1 in THF (D ≈ 2.5·10⁻⁵ M) for different values of total ion concentration [Zn²⁺]. (b) Fluorescence spectra of 1 in THF (D ≈ 2·10⁻⁶ M) for different values of [Zn²⁺] (λ_ex = 370 nm). The arrows indicate the decrease in [Zn²⁺]. (c) Change in absorbance at 360 nm as a function of [Zn²⁺] for 1 in THF (D ≈ 2.5·10⁻⁵ M). The solid line corresponds to the best fit by eq. (3). (d) Dependence F_s = f ([Zn²⁺]) for 1 in THF (D ≈ 2·10⁻⁶ M). The absolute accuracy of each experimental point is ±10%.

Table 2.

Linear photophysical and photochemical parameters of 1 and 1:Zn²⁺ complexes in organic (THF) and aqueous (water/ACN) medium: absorption ${\lambda }_{\mathit{abs}}^{max}$ and fluorescence ${\lambda }_{fl}^{max}$ maxima, Stokes shifts, maximum extinction coefficients ε^max, fluorescence quantum yields Φ, lifetimes τ and quantum yields of photochemical decomposition under one-photon irradiation Φ_1PA.

N/N	THF + Zn2+ (M ≈ 2.4·10−4M)	Water/ACN (1:1)	Water/ACN (1:1) + Zn2+ (M ≈ 2.4·10−4M)
${\lambda }_{\mathit{abs}}^{max}$, nm	381 ± 1	380± 1	379± 1
${\lambda }_{fl}^{max}$, nm	462 ± 1	534 ± 1	498 ± 1
Stokes shift, cm−1	4600 ± 100	7590 ± 100	6300 ± 100
εmax ·10−3, M−1·cm−1	72 ± 5	49 ± 3	52 ± 3
Φ	0.73 ± 0.05	0.46 ± 0.03	0.51 ± 0.03
τ, ns*	1.18 ± 0.08	1.41 ± 0.08	1.27 ± 0.08
Φ1PA ·104	3.0 ± 0.8	5.1 ± 2	6.0 ± 2

^*All lifetime measurements correspond to goodness-of-fit parameters χ² ≥ 0.99.

The dependence of the change in absorbance ΔA at 360 nm as a function of [Zn²⁺] (Figure 3c) can be reasonably fit by eq. (3), indicative of 1:1 ligand:metal complex formation (i.e. a single- step complexation equilibrium). The value of binding constant K ≈ (3 ± 0.3) ·10⁵ M⁻¹ corresponded to the best fit of experimental dependence ΔA = f (M). The fluorescence spectrum of 1 in THF (D ≈ 2·10⁻⁶ M) exhibited a hypsochromic shift up to 30 nm with an increase in intensity upon addition of Zn²⁺ in concentrations up to 2.4·10⁻⁵ M (Figure 3b). A sharp isobestic point and a single-exponential fluorescence decay process for the highest Zn²⁺ concentration (see Table 2) were consistent with 1:1 ligand:metal complex formation and nearly full conversion of ligand molecules into the complexed form. The analysis of the ratio of fluorescence intensities from the solution with ligand:metal mixture at 420 and 650 nm, F_s, revealed good selectivity to Zn²⁺ ions with a relatively large dynamic range (see Figure 3d), spanning 1 × 10⁻⁴ to 1 × 10⁻⁶ M, useful for ratiometric measurements of biologically-relevant concentrations.^{6, 35} It should be mentioned that fluorene- based probe 1 did not exhibit comparable selectivity to Mg²⁺, Ca²⁺, and K⁺ ions under the same experimental conditions.

The fluorescence spectrum of 1 in aqueous solution (water/ACN (1:1)) revealed noticeable changes under the addition of Zn²⁺ ions (Figure 4a), as was the case in organic solvent. In contrast, the absorption spectrum of 1 was nearly insensitive to the Zn²⁺ ion bonding in aqueous medium (Figure 5b). In this case, the fluorescence spectra of 1 (D ≈ 1.7·10⁻⁶ M) were obtained for the range of Zn²⁺ concentrations from 1.7·10⁻⁷ M to 2.0·10⁻⁴ M. The change in relative fluorescence intensity $\frac{F}{F_0}$ at 460 nm as a function of [Zn²⁺] is presented in Figure 4b (circles) and can be reasonably fitted by eq. (5) (solid line) with the corresponding binding constant K ≈ (2 ± 0.2)·10⁵ M⁻¹. The experimentally obtained data along with the main photophysical parameters (see Table 2) are consistent with 1:1 ligand:metal complex formation and full transformation of ligand molecules into the complexed form under the highest concentration of Zn²⁺ used.

Figure 4.

(a) Fluorescence spectra of 1 in water/ACN (1:1) solution (D ≈ 1.7·10⁻⁶ M) as a function of [Zn²⁺]. The arrow indicates decreasing [Zn²⁺]. (b) Relative change in fluorescence intensity F / F₀ at 460 nm as a function of [Zn²⁺] for 1 in water/ACN (1:1) solution (D ≈ 1.7·10⁻⁶ M). The solid line corresponds to the best fit by eq. (5). The absolute accuracy of each experimental point is ± 10%.

Figure 5.

Excitation anisotropy (1, 1′) and normalized linear absorption (2, 2′) spectra of 1 (1′, 2′) and 1:Zn²⁺ complexes (1, 2) in THF (a) and water/ACN (1:1) (b).

The values of fluorescence quantum yield Φ and lifetimes τ for 1:Zn²⁺ complex in THF decreased by 15–20 % in comparison with those of the corresponding pure ligand (see Tables 1, 2). In contrast, water/ACN solution of 1 with mixtures of Zn²⁺ exhibited a small increase (by ≈ 10 %) in the fluorescence quantum yield which is not consistent with corresponding observed trend in τ.³¹ Excitation anisotropy spectra of 1 and 1:Zn²⁺ complexes in THF and aqueous solutions (Figure 5a, b) exhibited a close spectral dependence that is indicative of their similar mutual orientations of the transition dipoles S₀ → S₁ and S₀ → Sn (n = 1, 2, 3…). A comprehensive analysis of the main photophysical data revealed a rather complex nature of the influence of Zn²⁺ ion binding processes on the electronic structure of 1. We believe that the mechanism of sensing is based on the coordination of the macrocyclic receptor to the Zn²⁺. This process results in perturbation of the electronic environment of the chromophore by decreasing basicity of the receptor nitrogens.

The photochemical stability of 1 in THF and water/ACN solution exhibited a specific dependence on Zn²⁺ (see Table 2). The photochemical decomposition quantum yield Φ_1PA of the 1:Zn²⁺ complex in THF increased by a factor of ≈2 relative to the pure ligand. In contrast, the photostabilities of 1 and ligand:metal complex in water/ACN (1:1) were nearly the same, with only a small increase in Φ_1PA upon metal ion binding. Thus, probe 1 demonstrated high photostability, suitable for fluorometirc sensing, and particularly important for potential in vivo imaging and sensing applications.

4.4. 2PA properties of 1 and 1:Zn²⁺ complex

The degenerate 2PA spectra of 1 and 1:Zn²⁺ complexes were investigated by the relative 2PF method²⁸ with 1 kHz femtosecond laser system in organic and aqueous media. All nonlinear absorption measurements of 1:Zn²⁺ complexes were performed on solutions with M ≫ D, where only 1:1 bound ligand molecules were present. The 2PA spectra (Figure 6) were characterized by two well-defined short and long wavelength bands, with the maximum at λ_ex/2 = 320–330 nm and 390–400 nm, respectively. The long wavelength 2PA band overlapped quite well with the main 1PA band which is typical for unsymmetrical fluorene derivatives.^46–48 This band was characterized by maximum 2PA cross section δ_2PA ≈130 GM for 1 in THF and decreased by a factor of ≈2 upon Zn²⁺ binding (Figure 6, curves 1, 1′). In contrast, nearly the same values of δ_2PA ≈50 GM were observed for 1 and 1:Zn²⁺ complex in water/ACN solution (Figure 6, curves 2, 2′). Well-defined long wavelength 2PA bands in the spectral range of one-photon allowed transitions are associated with the relatively large changes in the stationary dipole moments of the ligand and ligand:metal complex (|Δμ| ~ 5 – 10 D) under the S₀ → S₁ electronic excitation.^48–50 The values of 2PA cross sections of 1 in the shorter wavelength (higher energy) two-photon allowed band (λ_ex/2 = 320–330 nm) corresponded to ≈240 and ≈140 GM in THF and water/ACN solution, respectively. The addition of Zn²⁺ to the THF solution of 1 resulted in ≈30 % decrease in δ_2PA. In contrast, nearly the reverse effect (but same magnitude of change) was observed for 1 in water/ACN solution, i.e., a higher 2PA cross section was observed for the complex relative to free ligand. It should also be mentioned that the corresponding behavior of the two-photon action cross sections δ_2PA · Φ upon complexation were sufficiently close to the values of δ_2PA due to only a small change in the fluorescence quantum yield upon metal binding. The maximum values of δ_2PA · Φ ≈(90–130) GM for 1:Zn²⁺ complexes are suitable for application in 2PFM techniques.

Figure 6.

2PA spectra of 1 (1, 2) and 1:Zn²⁺ complex (1′, 2′) in THF (1, 1′) and water/ACN (1:1) (2, 2′). Normalized linear absorption spectrum of 1 in THF (3). Total concentrations of 1 and Zn²⁺ ions in solution were 1.2·10⁻⁵ M and 8·10⁻⁴ M, respectively.

4.5. Quantum chemical calculations for 1 and 1:Zn²⁺ complex

The crystal structure of dibenzosubstituted oxaaza macrocycle²⁷ reveals Zn²⁺ coordinated to the four nitrogens of the ligand. The corresponding optimized molecular geometries for the model compounds of 1 (1a) and 1:Zn²⁺ (2a) are presented in Figure 7. The calculated molecular orbitals (Figure 7) demonstrate weak polarization of the highest occupied molecular orbital upon metal binding, resulting in 5–10% decrease in the absolute values of transition dipoles from 1a to 2a. However their spatial orientations (see values of the relative angle, α, for the main electronic transitions in Table 3) remain close. This result is in good agreement with experimental anisotropy spectra (Figure 5) which are nearly identical for 1 and the 1:Zn²⁺ complex. Calculated energies of the electronic transitions and 2PA cross sections predict a weak dependence on metal ion binding (Table 3) which is also consistent with experimental data. The absolute values of δ_2PA exhibit a relatively large deviation from the corresponding experimental parameters. This may be due to more complicated nature of specific solvation and metal-ligand coordinating processes, a question that is presently under investigation.

Figure 7.

Optimized molecular geometry (upper), and highest occupied molecular orbitals (lower) of model compounds 1a (a) and 2a (b).

Table 3.

Calculated energies, E, oscillator strengths, f, and 2PA cross sections, δ_2PA, of the main electronic transitions for model compounds 1a, 2a and angles of space orientation, α, of the corresponding transition dipoles (relative to S₀ → S₁).

N/N	1a			2a
	S0 → S1	S0 → S4	S0 → S5	S0 → S1	S0 → S2	S0 → S3
E, eV (nm)	2.76 (449)	3.50 (354)	3.62 (343)	2.64 (469)	3.15 (394)	3.47 (357)
f	2.20	0.29	0.08	1.98	0.83	0.03
δ2PA, GM	209	910	510	258	1306	567
α, degree	-	1.6	8.0	-	2.2	10.5

5. Conclusions

Linear photophysical, photochemical, 2PA, and metal ion sensing properties of a new fluorene-based probe 1 containing a dibenzosubstituted oxaaza macrocycle were investigated in organic and aqueous solution as a potential Zn²⁺ sensor for two-photon bioimaging and sensing applications. The steady-state absorption spectra of unsymmetrical 1 were nearly independent of solvent properties, while a strong solvatochromic effect was observed in the fluorescence spectra without dramatic changes in the fluorescence quantum yield. The values of fluorescence lifetimes corresponded to a single-exponential decay and exhibited a weak dependence on solvent polarity. The addition of Zn²⁺ to organic and aqueous solutions of 1 resulted in noticeable changes in absorption and fluorescence spectra, consistent with a 1:1 complexation mechanism with sufficiently high binding constants K ~ (2 – 3)·10⁵ M⁻¹, in contrast to other metal ions, such as Mg²⁺, Ca²⁺, and K⁺. The photochemical stability of 1 and the 1:Zn²⁺ complex was investigated by determining the corresponding photodecomposition quantum yields, Φ_1PA, and were in the range (1 < Φ_1PA < 7)·10⁻⁴. The values of Φ_1PA exhibited a complex dependence on solvent properties and increased slightly upon metal ion binding. The 2PA spectra of 1 and the 1:Zn²⁺ complex were characterized by two well defined absorption bands at ≈650 and 800 nm with maxima cross sections δ_2PA ≈ 140–240 GM and 50–130 GM, respectively. The electronic properties of the 2PA bands of 1 and 1:Zn²⁺ complexes were analyzed based on computational results while a weak dependence of 2PA efficiency on complexation was shown. The high sensitivity and selectivity of the new fluorenyl macrocycle 1 to Zn²⁺ along with excellent ratiometric fluorescence detection parameters, good photochemical stability, and a relatively large two-photon action cross section (90–130 GM) for the 1:Zn²⁺ complex suggests 1 may be potentially useful as a Zn ion sensor in 2PFM bioimaging, a subject of future work.