Substituent effect on fluorophores instead of ionophores: its implication in highly selective fluorescent probes for Zn²⁺ over Cd²⁺

Abstract

Three fluorescent molecules with different substituent modification on 5-position showing different extents of fluorescent response to Zn²⁺ and distinct discriminating effect of R_Zn/R_Cd have been readily synthesized. The addition of a dithiol group induces the discriminating effect of R_Zn/R_Cd to be obviously improved from twice to 32 times.

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As the second most abundant transition metal ion in the human body, Zn²⁺ plays an important role in biological processes including gene transcription, regulation of metalloenzymes, DNA synthesis, neural signal transmission and apoptosis, as well as catalysis of proteins.¹ The disorder of Zn²⁺ has been associated with a number of pathological processes, such as Alzheimer's disease, epilepsy, Parkinson's disease, ischemic stroke and infantile diarrhea.² The accurate measurement of Zn²⁺ in physiological media has been considered as an essential factor and addressed target in these diseases. Among the methods developed for Zn²⁺ sensing, fluorescent probes have attracted considerable attention due to their simple operation, high sensitivity and real time detection.³ At present, most of fluorescent probes for Zn²⁺ utilize di-2-picolylamine (DPA) as recognition component with photo-induced electron transfer (PET) sensing mechanism.⁴ The quenched fluorophores by PET effect would recover after Zn²⁺ coordinating with DPA. The selectivity of these probes mainly relies on the binding capability of ionophores (DPA) to Zn²⁺ ions.⁵ Due to the similar binding characters of Cd²⁺ to DPA, most of them suffered from poor selectivity to Zn²⁺. To resolve this issue, more efforts have been devoted into the exploration of DPA derivatives (Scheme 1).⁶ Lippard's group made great efforts to alter the Zn²⁺-binding group by replacing one of the pyridines to specifically detect Zn²⁺.⁷ Peng et al. for the first time reported the ethylene substituted DPA which is linked to BODIPY to discriminate Zn²⁺ from Cd²⁺.⁸ Recently, Jiang and Yoon's groups developed a method of introducing additional binding unit (an amide group) to a DPA-type receptor for distinguishing Zn²⁺ and Cd²⁺.⁹ Up to now, all of Zn²⁺ probes are focused on the optimization of ionophore to sufficiently enlarge the difference of coordination affinity for Zn²⁺ and Cd²⁺. However, this strategy encountered difficulty in labor-intense synthetic process for recognition unit and the guide of probe design for other ions. In addition, introduction of more binding sites usually trend to strongly bind more kinds of metal ions, reducing the sensing selectivity.¹⁰ Alternatively, as a concept-of-principle method, alternation of substitution on fluorophores instead of ionophores for endowing simple sensing subunit the different binding affinity to differentiate Zn²⁺ from Cd²⁺ would be hopeful. At present, most of reported Zn²⁺ fluorescent probes are mainly based on PET or intramolecular charge transfer (ICT) mechanisms which are disadvantageous in the design of highly selective and versatile probes through substituent effect on fluorophores because of the susceptibility of electron transfer to substitution. In contrast, fluorescent probes based on excited-state intramolecular proton transfer (ESIPT) mechanism can address this problem.¹¹ ESIPT has recently received considerable attention due to its unique photophysical properties.¹² Different from other organic chromophores, ESIPT molecules exhibit dual emissions from both the excited enol and keto tautomers. Fluorescent sensing of metal ions could realize by prohibiting ESIPT through the coordination of metal ion with ESIPT centers, resulting in detectable spectral change. 2-(2′-Hydroxyphenyl)benzoxazole (HBO), a typical ESIPT molecule, has been reported for cations and anions sensing.¹² Although the effect of substituent on the fluorescent properties of enol and keto tautomers has been investigated,¹³ none of them showed that the alternation of substituent on fluorophores is feasible to increase the selectivity for metal ions sensing. In addition, it is desirable for Zn²⁺ fluorescent probes to provide near infrared (NIR) fluorescent signal (700–900 nm) to avoid photodamage, scattering light and strong interference derived from short wavelength emission in biological media.³ For ESIPT molecules, large Stokes shift (ca. 150–200 nm) enables their fluorescent wavelength to reach in NIR region.¹⁴ Thus, they are ideal candidates for NIR fluorescent probes. Herein, we present a simple fluorescent ESIPT molecule (H-3) for Zn²⁺ sensing with high sensitivity and selectivity (Scheme 2). Moreover, the Zn²⁺ distinguishing capability from Cd²⁺ can be readily tuned through simply substituent modification on 5-position of ESIPT molecules instead of sensing unit (Scheme 3). To the best of our knowledge, this is the first demonstration that introduction of substituent to fluorescent signal moiety rather than direct sensing unit enhances sensitivity and selectivity, which sets up a promising strategy for the rational design of fluorescent probes.

Scheme 1 Modification on sensing unit of Zn²⁺ fluorescent probes based on ESIPT mechanism, where the red circles show Zn²⁺ binding and recognition sites.

Scheme 2 Modification on fluorophores of Zn²⁺ fluorescent probes based on ESIPT mechanism.

Scheme 3 Tactics for Zn²⁺ fluorescent sensors with distinguishing capability to Cd²⁺.

The detailed synthetic procedure was described in the ESI.† Briefly, H-2 was first readily synthesized by condensation reaction between 2,4-diformylphenol and 2-aminophenol. Subsequently, H-3 could be prepared from the reaction of H-2 with 1,3-ethanedithiol in the presence of I₂. H-1 as referenced compound was synthesized according to the literature procedure.¹⁵ All these compounds were fully characterized by ¹H NMR, ¹³C NMR and HRMS.

For comparison, spectra of these compounds and their response to metal ions have been studied in identical conditions (aqueous ethanol solution (1 : 1, v/v, pH 7.2)). The wavelength of maximal emission intensity is at 480 nm in the absence of Zn²⁺, which is assigned to keto emission of ESIPT. After adding of Zn²⁺, ESIPT was inhibited as a consequence of the Zn²⁺ coordination with OH groups. Accordingly, the wavelength of maximal emission intensity shift to 420 nm (enol emission).¹² Upon addition of Zn²⁺, the fluorescence intensity of these compounds showed large enhancement to different extents (λ_ex = 380 nm). When 80 equivalents of Zn²⁺ were added to the solution of these compounds, about 88-, 31- and 376-fold fluorescence enhancement at 420 nm was observed for H-1, H-2 and H-3 respectively (Fig. 1). More interestingly, a new peak in near infrared region (ca. 850 nm) appeared along with fluorescence intensity gradual increasing (Fig. S4, S10 and S18†). The excited spectra of emission at 420 and 850 nm are identical (Fig. S25†), suggesting that the output at 420 and 850 nm come from the same species (H-3 + Zn²⁺). Accordingly, about 169-, 43- and 219-fold fluorescence enhancement at 850 nm was also observed with more than 450 nm Stokes shift. To the best of our knowledge, this is the first sample that Zn²⁺ ions trigger simple molecules to emit near infrared fluorescence with such large Stokes shift. Compared to H-1 and H-2, dithiol group substituted H-3 induces greater intensity increasing (F₄₂₀/F₀ and F₈₅₀/F₀, where F₄₂₀ and F₈₅₀ stand for the intensity intensities at 420 and 850 nm in the presence of different amount of Zn²⁺, and F₀ stand for the intensity intensities at corresponding wavelength in the absence of Zn²⁺) in the presence of identical amounts of Zn²⁺, suggesting that H-3 shows higher sensitivity to Zn²⁺ at two given wavelengths. The quantum yield of H-3 and the Zn + H-3 complexes are 0.030 and 0.164, respectively. In order to further study the discriminating capacity to competitive ions, the fluorescence responses of these compounds to Cd²⁺ were also investigated. As shown in Fig. S1 and S2,† H-1 and H-2 showed gradual fluorescence intensity enhancement at short and long wavelength with increasing of Cd²⁺ amounts. The discriminating effect of R_Zn/R_Cd (R = F₄₂₀/F₀ or R = F₈₅₀/F₀) is small (2–6 times) for H-1 and H-2, implying that H-1 and H-2 showed nearly same fluorescent response to Zn²⁺ and Cd²⁺ and they can hardly discriminate Zn²⁺ from Cd²⁺. In sharp contrast, H-3 shows no obvious fluorescence intensity change upon addition of Cd²⁺, and the discriminating effect of R_Zn/R_Cd reached 32 and 12 times at 420 and 850 nm respectively (Fig. 2), indicating that H-3 can effectively discriminate Zn²⁺ from Cd²⁺. Moreover, the fluorescence spectra of H-3 with increasing concentration of Zn²⁺ in the presence of 10 equivalents of Cd²⁺ still shows obvious fluorescence enhancement, indicating that H-3 can selectively detect Zn²⁺ without the interference of Cd²⁺. H-3 also shows same response of fluorescence enhancement to Zn²⁺ in EtOH–HEPES buffer (pH 7.4, 1 : 1, v/v) (Fig. S26†). Compared to the reported method of modification on binding subunit, facile substituent change on fluorophore segment can greatly improve the discriminating effect between Zn²⁺ and Cd²⁺.

Fig. 1 The relative fluorescence intensity changes at 420 (a) and 850 nm (b) of these three compounds (10 μM) in aqueous solution upon addition of Zn²⁺ (λ_ex = 350 nm).

Fig. 2 The relative fluorescence intensity changes at 420 (a) and 850 nm (b) of H-3 (10 μM) in aqueous solution upon addition of Zn²⁺ and Cd²⁺ (λ_ex = 350 nm).

To further exclude the possible assistant binding of dithiol group on H-3 to Zn²⁺, NMR titration experiments of H-3 to Zn²⁺ and Cd²⁺ ions in identical conditions were performed. H-3 showed almost identical NMR spectra change upon addition of Zn²⁺ or Cd²⁺ (Fig. S22 and S23†). This result confirms that the great discriminating effect of H-3 to Zn²⁺ and Cd²⁺ is ascribed to intrinsic characteristic but not additional binding sites.

The fluorescent intensities of H-3 solution at 437 and 850 nm were linearly proportional to the amounts of Zn²⁺, demonstrating that H-3 is applicable to dual-output fluorescent detection of Zn²⁺ (Fig. S13†). To check the selectivity, the response of H-3 to other cations was investigated. It can be seen that only Zn²⁺ promotes significant fluorescence intensity enhancement, whereas other metal ions cause no detectable spectra change except that Cu²⁺ induces fluorescence quenching under the identical conditions (Fig. 3). To explore the possible utility of H-3 as fluorescent sensor for Zn²⁺, competitive experiments were carried out in the presence of 30 equivalents of Zn²⁺ and 30 equivalents of various other cations. Although a number of transition metals exert a clear quenching effect on the probe, these free cations would have little influence in vivo since they exist at very low concentrations.¹⁶ The fluorescence intensity of H-3 at 437 and 850 nm were not influenced in the presence of other competing cations, indicating that H-3 has specific selectivity for Zn²⁺.

Fig. 3 Emission intensity of H-3 (5 μM) at 420 (a) and 850 nm (b) in EtOH–H₂O (1 : 1, v/v) in the presence of different metal ions (150 μM) with the excitation at 350 nm (blank bar). Black bars represent the intensity with subsequent addition of Zn²⁺ ions (150 μM).

To further gain the insight into the recognition mechanism and the merits of H-3, scanning electron microscope (SEM) images of H-3 before and after addition of Zn²⁺ or Cd²⁺ were investigated by solution drop-casting. As shown in Fig. 4, different morphological characters were obtained. H-3 formed needle crystal with length of 3 micron. The absorption spectra of H-3 with increasing concentration showed the reduced ratio of A₂₉₆/A₃₂₃ (A₂₉₆ and A₃₂₃ stand for the absorption intensity at 296 and 323 nm respectively), indicating that H-3 easily trends to form aggregation due to intermolecular hydrogen bonds (Fig. S27†). Upon addition of Zn²⁺, morphology transforms to dendritic structure. In contrast, upon addition of Cd²⁺ rod-like crystals with size of 1 × 4 micron were clearly formed. The different morphological structures triggered by Zn²⁺ and Cd²⁺ are presumed to the high selectivity and sensitivity.¹⁷ Stoichiometry of 1 : 1 with Zn²⁺ or Cd²⁺ from Job's plot can be readily achieved (Fig. S15†). Associate constant between H-3 and Zn²⁺ was estimated as 5.85 × 10⁴ M⁻¹ with detecting limit of 6.11 × 10⁻⁸ M. As shown in Fig. S16,† the fluorescent intensities at 437 and 850 nm in the absence and presence of Zn²⁺ are pH independent in the range of 6–8, demonstrating that H-3 can detect Zn²⁺ in biological system.

Fig. 4 SEM images of H-3 in the presence of Zn²⁺ or Cd²⁺, where (A) and (B) without addition of metal ions, (C) and (D) after addition of Zn²⁺, (E) and (F) after addition of Cd²⁺.

It should be noted that K⁺ triggers 2-fold signal enhancement for Zn²⁺ detection, which is presumed to the synergistic effect because K⁺ cannot induce any signal change of H-3 in the absence of Zn²⁺. This may be due to the building blocks of H-3 + Zn²⁺ fitting well for the accommodation of K⁺. On the other hand, K⁺ can still interact with another lone pair of electrons on the oxygen atom of OH groups after it has coordinated with one lone pair of electrons to Zn²⁺, which can increase molecular rigidity to reduce the nonradiative decay of the excited state and lead to the fluorescence enhancement.¹⁸ It is well known that K⁺ with suitable atomic size can stabilize the G-quadruplexes derived from G-rich DNA sequences through coordination with eight oxygen atoms of carbonyl groups which exist on two planes of tetrad.¹⁹ Based on these, a plausible binding mode between Zn²⁺ and H-3 was presented (Scheme 4), where the driving force for layer stacking is supposed to π–π interaction or coordination by K⁺. The stacking mode is consistent with the observed photographs of SEM.

Scheme 4 The plausible binding mode between Zn²⁺ and H-3 in the presence of K⁺.

In summary, facile HBO-based fluorescent molecules through different substituent modification on 5-position showing different extents of fluorescent response to Zn²⁺ and distinct discriminating effect of R_Zn/R_Cd have been readily synthesized. The addition of a dithiol group results in that the discriminating effect of R_Zn/R_Cd was obviously improved from twice to 32 times. Instead of sophisticated tuning of recognition units, simple substitution varied on fluorophore subunit provides a new strategy for design of Zn²⁺ fluorescent sensors. Although some drawbacks for Zn²⁺ sensing, for example, too be short excited wavelength and low binding constant to apply for confocal microscopy analysis in vivo, need to be further optimized, as a concept-of-principle method, variation of substitution on fluorophores instead of ionophores to differentiate similar interferents would be hopeful. Endeavors to further increase the sensitivity of sensing and expand the analytes based on this strategy are in process in our laboratory.

This work was supported by the National Natural Science Foundation of China (Grant no. 21206137) and the Scientific Research Foundation of Northwest A&F University (Z111021103 and Z111021107).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Details of experimental operation, images and additional spectra. See DOI: 10.1039/c3ra46468g

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