Fluorescent chemodosimeter based on NHC complex for selective recognition of cyanide ions in aqueous medium

Abstract

Novel N-heterocyclic carbene based mono and di-nuclear silver probes having the anthracene chromophore act as chemodosimeters for selective and sensitive detection of cyanide ions in the aqueous medium and signal the event through visible enhancement in fluorescence intensity.

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Cyanide ions are extremely toxic and lethal to mammals due to their high affinity towards the heme unit¹ as well as the active site of cytochrome a3.² Such interactions cause decrease in oxygen concentration in the tissue, especially of the central nervous (CNS) and cardiovascular (CVS) tissues, thereby resulting in hypoxia.³ Of the various sources, hydrogen cyanide gas and the crystalline metal cyanides are the main cause of the cyanide poisoning and their maximum contaminant levels in drinking water is limited to 200 ppb.⁴ The hazardous impact of the cyanide ions on both physiological and environmental conditions, has led to an intensive research efforts for the design and development of efficient probes for their detection.

A variety of probes based on optical and electrochemical techniques have been reported for the detection of biologically relevant anions.^5–7 Of which, the optical probes, especially based on fluorescence have gained great importance because of their easy, fast and sensitive detection. Among the various fluorescent probes for the cyanide ions, the reaction based probes have attracted much attention due to their high selectivity towards the analyte.^8–10 Most of these fluorescent probes are based on the reactions such as complexation/decomplexation,^11a nucleophilic addition,^11b and benzyl cyanide reaction.^11c Especially, the probes involving the displacement approach utilize the affinity of the CN⁻ ions towards various metal ions to form stable [M(CN)_x]ⁿ⁻ complexes.¹² Herein, we report two novel N-heterocyclic carbene (NHC) based mono- and dinuclear silver complexes as probes for the selective and sensitive detection of the cyanide ions in aqueous medium. Uniquely, these probes act as chemodosimeters and recognize the cyanide ions through the visible enhancement in the fluorescence intensity.

Synthesis of the NHC complexes 2 and 4 was achieved in ca. 70% and 69% respectively through refluxing their corresponding imidazolium precursors 1 and 3 with silver oxide (Scheme 1). In this reaction, Ag₂O not only generates carbene intermediate through deprotonation of the imidazolium moiety but also acts as the metal center which generally adopts a linear coordination in the di-nuclear complexes.¹³ The products, thus obtained were unambiguously characterized by spectral and analytical evidence; ¹H NMR, ¹³C NMR, MALDI-TOF MS and elemental analysis. The formation of the metal carbon (Ag–C) bond in the complexes 2 and 4 can be easily evidenced through the ¹H and ¹³C NMR analysis. The ¹H NMR spectra of the precursors 1 and 3 in CD₃CN showed the peaks corresponding to the proton (H₂) attached to carbon-2 of the imidazolium moiety as singlets at δ 8.79 and 8.13 ppm respectively, which disappeared upon complexation with Ag⁺ ions.

Scheme 1 Synthesis of NHC probes 2 and 4.

We have recorded proton NMR of the complexes in D₂O, wherein the singlets at δ 8.82 and 8.14 ppm, corresponding to carbene proton of 1 and 3, respectively were disappeared upon complexation with Ag⁺ ions. Similarly, the singlets at δ 7.37 and 7.46 ppm for 1 and δ 7.32 and 7.35 ppm for 3, corresponding to the imidazolium protons showed downfield shift upon metal ion complexation. Similarly, in the ¹³C NMR spectra, we observed a downfield shift for the carbene carbons from δ 135.7 and 135.5 ppm to δ 179.5 and 179.9 ppm when 1 and 3 were converted to the NHC complexes 2 and 4, respectively (Fig. S1–S4, ESI†). The binding stoichiometry of the complexes was determined from the MALDI-TOF MS analysis. In the case of the complex 2, we got a 1 : 2 (Ag : ligand) ratio (Fig. S5, ESI†), while a 1 : 1 ratio of (Ag : ligand) was observed for the complex 4. For a better understanding of the stability of the complexes under low pH conditions, we have checked the ¹H NMR spectra of both the complexes after the addition of trifluoroaceticacid (TFA). The spectra showed negligible changes, indicating thereby that these complexes are quite stable even at low pH conditions in the aqueous medium (Fig. S6 and S7, ESI†).

The complex 2 and its precursor 1 (Scheme 1) showed absorption maximum at 394 nm while their fluorescence spectra exhibited peaks in the range 380–500 nm having maximum at 416 nm in the aqueous medium. The fluorescence quantum yields of complex 2 and its imidazolium precursor 1 were determined and these values are found to be ca. 0.37 ± 0.01 and 0.70 ± 0.01, respectively (λ_ex = 365 nm). Further the radiative and non-radiative decay constants for the systems have been calculated from the fluorescence quantum yield and lifetimes. The complex 2 exhibited a radiative decay constant of 0.053 (k_r) and non-radiative decay constant of 0.090 (k_nr) whereas the corresponding imidazolium precursor 1 showed 0.087 (k_r) and 0.037 (k_nr) respectively, which supports the changes in the fluorescence quantum yields observed for these systems.

The NHC complex 4 and its imidazolium precursor 3 also exhibited absorption maximum at 394 nm and characteristic emission peaks in the range 380–500 nm, but showed relatively quenched fluorescence yields of Φ_F = 0.34 ± 0.01 and 0.54 ± 0.03 respectively in the aqueous medium. The complex 4 exhibited a radiative decay constant of 0.042 (k_r) and non-radiative decay constant of 0.082 (k_nr) and the precursor 3 (k_r = 0.066, k_nr = 0.056) also behave similarly with respect to their emission profiles.

As the NHC complexes 2 and 4 exhibited favourable fluorescence quantum yields, good solubility in the aqueous medium and possesses Ag⁺ ions, we evaluated their potential as probes for anions. Addition of tetrabutylammonium cyanide (TBACN) to an aqueous solution of the NHC complex 2 (5 μM), we observed negligible changes in its absorption properties (Fig. 1A). However, in the emission spectrum (λ_ex = 365 nm), we observed a gradual increase in the fluorescence intensity at 416 nm and reached saturation upon addition of 20 μM of CN⁻ ions (Fig. 1B). Similar changes were observed in the case of the probe 4 with the addition of CN⁻ ions under identical conditions (Fig. S8, ESI†).

Fig. 1 Changes in the (A) absorption and (B) fluorescence spectra of the probe 2 (5 μM) with the addition of CN⁻ ions in the aqueous medium. [CN⁻] (a) 0, and (e) 20 μM. λ_ex 365 nm, respectively.

In order to understand the effect of pH on the reaction, we have carried out the pH dependent absorption and fluorescence studies of the complexes 2 and 4 upon titration with cyanide ions. These studies were carried out in phosphate buffer by varying pH between 2 and 12. The absorption spectra of both the complexes 2 and 4 showed negligible changes upon the addition of cyanide ions, whereas the emission spectra showed ca. 2–3 fold enhancement in the fluorescence intensity (Fig. S9, ESI†). The emission spectrum of the complexes showed similar behaviour in the pH range 2–12 as observed earlier in the aqueous medium, which clearly indicates that pH has negligible effect on the interaction of the complexes 2 and 4 with the cyanide ions.

To understand the nature of interactions, we have monitored the changes in the ¹H NMR spectra of the silver complexes 2 and 4 with the addition of tetrabutylammonium cyanide solution. As shown in Fig. 2, upon addition of CN⁻ in CD₃CN at 25 °C, we observed the appearance of a peak corresponding to the carbene proton (H₂) at 8.7 ppm. Similar changes were observed in the case of the complex 4 with ca. 4 equivalents of CN⁻ ions (Fig. S10, ESI†). The authenticity of the reaction was confirmed by isolating the product, after the addition of the excess CN⁻ ions to the complexes 2 and 4. The ¹H NMR spectra of the isolated products were found to be essentially identical to those of the precursors 1 and 3. These results clearly indicate that the imidazolium precursors 1 and 3 were regenerated due to the decomplexation of the complexes 2 and 4, respectively, upon reaction with the cyanide ions. The observation of enhanced fluorescence intensity upon addition of CN⁻ ions furthermore confirms the decomplexation of the silver complexes 2 and 4 in the presence of the strong nucleophilic cyanide ions.

Fig. 2 ¹H NMR spectra of (A) the probe 2 alone and (B–E) with the addition of different concentrations of tetrabutylammonium cyanide solution in CD₃CN.

The interaction between the silver NHC complexes 2 and 4 with the cyanide ions was subsequently analyzed through picosecond time-resolved fluorescence analysis. The complex 2 exhibited a mono-exponential decay with an excited state lifetime of 7.1 ± 0.05 ns in the aqueous medium. Upon addition of saturated amount of tetrabutylammonium cyanide solution (20 μM) to this complex resulted in a new species with an increase in the excited state lifetime of 8.07 ± 0.01 ns. In order to check the nature of the new species that was formed after the reaction, we have recorded the lifetime of the corresponding imidazolium precursor 1. The precursor 1 showed a mono-exponential decay with an excited state lifetime of 8.08 ± 0.01 ns, which is identical to the species obtained after the addition of the cyanide ions to the complex 2 (Fig. 3A). Similar experiments were carried out in the case of the complex 4. Interestingly, the complex 4 showed a mono-exponential decay with an excited state lifetime of 8.11 ± 0.03 ns. After addition of the cyanide ions, the fluorescence lifetime of the complex was changed to 8.19 ± 0.03 ns and which correspond to the imidazolium precursor 3 (8.21 ± 0.01 ns) (Fig. 3B). These results confirm the formation of the imidazolium precursors 1 and 3 after the decomplexation of the NHC complexes 2 and 4, respectively upon interaction with the cyanide ions.

Fig. 3 Lifetime profiles of (A) the complex 2: (a) alone, (b) after the addition of cyanide ions and (c) the imidazolium precursor 1 and (B) the complex 4: (a) alone, (b) after the addition of cyanide ions and (c) the imidazolium precursor 3, respectively in the aqueous medium.

Scheme 2 shows the proposed mechanism of the decomplexation of the probe 2 by the addition of the cyanide ions. Even though the metal-carbon bond is quite strong, the highly nucleophilic cyanide ions could easily cull the silver ion from the complex through the formation of the highly fluorescent imidazolium precursor 1 and the stable soluble Ag(CN)₂⁻ salt. The plot between the relative changes in the fluorescence intensity versus concentration of cyanide ions for the complex 4 showed a sigmoidal nature, which may be due to the stepwise cleavage of the Ag–C bond in the sensing mechanism.¹⁴ These results thus demonstrate that the strong binding affinity of the silver ions towards the CN⁻ ions make the probes 2 and 4 as efficient chemodosimeters for the recognition of the cyanide ions.

Scheme 2 Possible mechanism for the detection of CN⁻ ions using probe 2.

To investigate the selectivity of the NHC complexes 2 and 4 for the CN⁻ ions, we have studied their interactions with the biologically relevant anions like I⁻, Br⁻, Cl⁻, F⁻, ClO₄⁻, HSO₄⁻, OH⁻, C₆H₅COO⁻, S²⁻, SCN⁻ and N₃⁻ ions. The absorption and emission changes of the probes were monitored by the addition of all these competing anions. We observed negligible changes in the absorption and fluorescence properties of the complexes 2 and 4 even at the addition of ca. 100-fold higher concentrations of these ions than that of the cyanide ions (Fig. 4 and S11†).

Fig. 4 Selectivity plot showing the changes in the fluorescence intensity (I − I₀)/I₀ of the complex 2 with the addition of different anions. λ_ex 365 nm.

To understand the uniqueness of the probes 2 and 4, we have investigated the interactions of the precursors 1 and 3, which lack silver ions with various anions. Both these precursors showed negligible changes in their absorption and fluorescence spectra in the presence of all anions including CN⁻ ions (Fig. S12, ESI†). Furthermore to understand the interaction of the imidazolium precursor 1 with the cyanide ions, we have carried out the ¹H NMR titration experiments in the presence of cyanide ions. The negligible changes in the NMR spectra with the addition of excess cyanide ions, confirms the negligible interaction between the precursor 1 and the cyanide ions. These observations demonstrate that the NHC complexes 2 and 4 show selective interactions with the cyanide ions and the mechanism of the recognition is through displacement of the silver ions from the complexes. To estimate the effect of halides as competing anions, we have investigated the interactions of the both the complexes with the chloride ions by ¹H NMR spectroscopy. Expectedly, we observed negligible changes in which further confirms the selectivity of the cyanide ions over chloride ions.

To determine the sensitivity of the detection, the fluorescence changes of the NHC complexes 2 and 4 were recorded by the addition of various concentrations of the cyanide ions. With the gradual increase in the concentration of the cyanide ions, we observed a regular enhancement in the fluorescence intensity of the probes 2 and 4. Fig. 5A and B show the linear plots of the relative changes in fluorescence intensity vs. concentration of the cyanide ions. The limit of detection of the cyanide ions was found to be 49 (1.89 μM) and 50 ppb (1.94 μM), respectively, by the complexes 2 and 4. Further to determine the unknown concentration of the CN⁻ ions in water, the concentration of the complex 2 was maintained at 5 μM, whilst the CN⁻ ions concentration was varied from 0–20 μM. The fluorescence spectra were monitored at 416 nm and the changes in the fluorescence intensity was plotted against the concentration resulting in a linear relationship (Fig. S14, ESI†). From this plot, we have determined the unknown concentration of the cyanide ions by measuring its fluorescence intensity in the aqueous medium and also in the presence of other anions.

Fig. 5 Calibration curve (I − I₀)/(I_f − I₀) for (A) the probe 2 and (B) the probe 4 as a function of [CN⁻], in μM. λ_ex 365 nm, respectively.

In conclusion, we have developed two novel silver N-heterocyclic carbene based complexes that possess the anthracene chromophore as the optically active unit. These probes exhibited high selectivity towards the cyanide ions when compared to other biologically relevant anions in the aqueous medium. Uniquely these probes act as chemodosimeters for the cyanide ions with a sensitivity as low as ca. 50 ppb (LOD = 50 ppb) and signal the event through the visible enhancement in the fluorescence intensity. Further studies are in progress to extend the strategy of detection to the solid-state and polymer matrix and as well as in natural resources.

Acknowledgements

We acknowledge the financial support from the CSIR-National Institute for Interdisciplinary Science and Technology (no. CSC 0134) Trivandrum. This is contribution no. PPG-346 from CSIR-NIIST, Trivandrum.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: General experimental techniques, synthetic details, Fig. S1–S13 showing the characterization data, spectra of the precursors and the probes under different conditions. See DOI: 10.1039/c4ra09969a

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