Design and Synthesis of a Fluorescent Probe Based on Copper Complex for Selective Detection of Hydrogen Sulfide

Abstract

A novel fluorescence probe NA-LCX was rationally designed and synthesized for the sequential recognition of Cu²⁺ and H₂S by the combination of hydroxyl-naphthalene and diformylphenol groups. The response properties of NA-LCX for Cu²⁺ ions and H₂S with “on-off-on” manner were investigated by fluorescence emission spectra. A highly selective and sensitive response of complex NA-LCX-Cu²⁺ for H₂S over other competing amino acids was observed with a limit of detection at 2.79 μM. The stoichiometry of NA-LCX toward Cu²⁺ ions was determined to be 1 : 1 by the UV-Vis absorption spectrum, and the coordination configuration was calculated by density functional theory (DFT) calculations. Moreover, probe NA-LCX was applied successfully for the recognition of Cu²⁺ ions and H₂S in living cells.

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. Introduction

Hydrogen sulfide (H₂S), the simplest biomercapto compound, is not only a rotten egg smelling gas pollutant but also the third gasotransmitter and cellular signaling molecule after CO and NO [1, 2]. The endogenous H₂S could regulate vascular smooth muscle tension and cardiac contractile function, anti-inflammatory and antioxidative stress, neurotransmitter transmission, and insulin signaling inhibition, which plays an important role in the physiological and pathological processes of the cardiovascular, nervous, immune, and digestive systems [3–7]. The concentrations of H₂S in the normal metabolism often maintain dynamic equilibrium, while abnormal changes of the H₂S level could induce serious health problems, such as heart diseases [8, 9], chronic obstructive pulmonary diseases [10, 11], cirrhosis [12, 13], and Alzheimer [14, 15]. Hence, it is crucial to exploit a highly sensitive and selective method for the detection of hydrogen sulfide in living systems.

Many conventional methods for H₂S detection have been developed, including colorimetric method [16, 17], electrochemical analysis [18, 19], liquid chromatography mass spectrometry [20, 21], and fluorescence analysis [22–24]. Among them, fluorescence analysis is more desirable due to its simple operation, high sensitivity, wide dynamic range, high fluorescence quantum yield, good biocompatibility, noninvasiveness, and ability of in situ real-time detection in living systems [25]. In recent years, many fluorescent probes for H₂S detection have been reported on account of different types of strategies such as reduction reactions [26, 27], nucleophilic addition reactions [28, 29], dinitrophenyl ether/sulfonyl ester cleavage [30, 31], and metal sulfide precipitation reaction [32–40]. However, there are some limitations to those reaction methods as well as the products obtained via those reactions. For example, those reactions are insensitive, complex, and time-consuming; moreover, fluorescent probes prepared via those reactions are sometimes not biocompatible and sometimes unstable in the presence of biological thiols (glutathione, cysteine, etc.) [31]. The strategy by using a metal displacement approach is in high demand for its fast response and high sensitivity and selectivity. Sulfide is known to react with copper ion to form very stable CuS with a very low solubility product constant $K_{\text{sp}}=6.3\times {10}^{-36}$ (for cyanide, $K_{\text{sp}}=3.2\times {10}^{-20}$). Thus, the utilization of the higher affinity of Cu²⁺ towards sulfide for designing a specific Cu²⁺ sensor to sequentially identify H₂S has received considerable attention because it can effectively eliminate the interference of other analytes in the system.

Naphthalene derivatives with an electron donor-π-acceptor (D-π-A) structure have been widely used due to good optical properties, such as high fluorescence quantum yield, good biocompatibility, and light stability. Herein, we synthesized a new fluorescent probe NA-LCX based on hydroxyl-naphthalene and diformylphenol which have excellent coordination ability to metal ions. The probe showed an obvious “on-off” fluorescence quenching response toward Cu²⁺, and the NA-LCX-Cu²⁺ complex showed an “off-on” fluorescence enhancement response toward H₂S in a DMSO/HEPES (3 : 2 $v/v$, $\text{pH}=7.4$). The photophysical capabilities of probe NA-LCX for Cu²⁺ and NA-LCX-Cu²⁺ for H₂S were studied in details from fluorescence spectroscopy, absorption spectroscopy, and fluorescence images in vivo.

2. Experimental Section

2.1. Reagents and Materials

2,6-Diformyl-4-methylphenol was purchased from Shanghai TCI Chemical Industry Development Co. Ltd. 3-Hydroxy-2-naphthoyl hydrazide was purchased from Sinopharm Chemical Reagent Co. Ltd. All the other chemicals and reagents were commercially available and were analytical grade. All solvents were purified by standard procedures. Aqueous solutions ($2.0\times {10}^{-2}\text{M}$) of perchlorates of various metal ions (Al³⁺, K⁺, Na⁺, Mg²⁺, Ca²⁺, Cr³⁺, Mn²⁺, Fe²⁺, Fe³⁺, Co²⁺, Ni²⁺, Zn²⁺, Cd²⁺, Hg²⁺, Ag⁺, Pb²⁺, and Cu²⁺) and various amino acids (Asn, Glu, Cys, Phe, Pro, Gln, Arg, Trp, Asp, Tyr, Ile, Thr, His, Gly, Met, Leu, Ala, Val, Ser, Lys, Cys, GSH, Hcy, and NAC) were prepared before use.

2.2. Apparatus and Instruments

The following are the apparatus and instruments used in the study: UV-Vis spectrophotometer (UV-2600, Shimadzu Corporation), fluorescence spectrophotometer (FS5, Edinburgh, UK), nuclear magnetic resonance spectrometer (NMR) (Ascend™ 400, Bruker Co., USA), precision pH meter (PHS-3E, Zhengzhou Tailai Instruments Co., Ltd.), and inverted fluorescence microscope (Leica DMI8, Leica Microsystems, Germany).

2.3. Synthesis of the Probe NA-LCX

3-Hydroxy-2-naphthohydrazide (0.40 g, 2.0 mmol) and 2,6-diformyl-4-methylphenol (0.164 g, 1.0 mmol) were dissolved in 30 mL of ethanol, respectively. Then, the solution was mixed dropwise and refluxed for 6 hours. The obtained mixture was filtered, washed, and vacuum dried to afford ligand NA-LCX (Scheme 1). ¹H NMR (400 MHz, d⁶-DMSO), δ 8.76 (s, 2H), 8.48 (s, 2H), 7.94 (d,$J=8.2\text{Hz}$, 2H), 7.78 (d,$J=8.2\text{Hz}$, 2H), 7.61 (s, 2H), 7.53 (t,$J=7.6\text{Hz}$, 2H), 7.37 (dd,$J=14.7$, 7.1 Hz, 4H), 2.36 (s, 3H). ¹³C NMR (101 MHz, d⁶-DMSO), δ 164.23, 155.37, 154.54, 147.20, 136.36, 130.95, 130.72, 129.15, 128.76, 127.24, 126.35, 124.31, 120.70, 120.33, and 111.02, 20.41. MS: calculated [(M+Na)⁺] 555.1644; found 555.1638.

[figure omitted; refer to PDF]

To further explore whether probe NA-LCX-Cu²⁺ could be used as a highly selective H₂S sensor, the fluorescence response of probe NA-LCX-Cu²⁺ (20 μM) to different amino acids was tested. As described and shown in Figure 4, only the addition of H₂S instantly caused an obvious fluorescence enhancement. The fluorescence intensity of probe NA-LCX-Cu²⁺ remains unchanged in the presence of 10 equiv. of different mercapto-amino acids such as glutathione, cysteine, N-acetyl-L-cysteine, homocysteine, and non-mercapto-amino acids. And other reactive sulfur species (S₂O₃^2-, SO₄^2-, and HSO₃^-) also did not cause obvious fluorescence changes. The competitive experiments further showed significant fluorescent enhancement without being interfered by other amino acids and reactive sulfur species, which further indicated the good selectivity of the probe NA-LCX-Cu²⁺ for H₂S detection.

[figure omitted; refer to PDF]

3.4. DFT Calculation

To gain further insight into the nature of coordination configuration and optical response of sensor NA-LCX toward Cu²⁺, the different coordination structures of NA-LCX-Cu²⁺ were examined by density functional theory calculation. All calculations were performed by Gaussian 09 program. The geometries were optimized at the B3LYP/6-31G(d)/SDD level, and the interaction energies were calculated based on the single point energies obtained at the B3LYP/6-31+G(d)/SDD level. As shown in Figure S9, it was obvious that the interaction energy of structure C was higher than structures A and B, which verified the experimental results and presumed the complexation mode of probe NA-LCX with Cu²⁺.

3.5. Effect of pH on the Performance of Probe NA-LCX and Complex NA-LCX-Cu²⁺

To investigate the effect of pH value, fluorescence intensity of probe NA-LCX, complex NA-LCX-Cu²⁺, and complex NA-LCX-Cu²⁺ in the presence of S^2- was investigated in a wide range of pH values. No significant changes in fluorescence intensity were found at lower pH ($\text{pH}\le 6$) (Figure S10). However, It could be observed that significant fluorescence changes in ligand NA-LCX and NA-LCX-Cu²⁺-Na₂S at $\text{pH}>6$, indicating potential of probe NA-LCX-Cu²⁺ to detect H₂S in physiological environments.

3.6. Cell Imaging Experiments

Inspired by the excellent selectivity at physiological pH levels, the cell imaging application of sensor NA-LCX for detection of Cu²⁺ and H₂S was further investigated. Prior to the cell imaging experiment, the MTT cell toxicity assay for probe NA-LCX-Cu²⁺ was performed in human liver cancer cells (HepG-2) shown in Figure S11, and no significant cytotoxicity was found in the range of 0~10 μM, even after incubating for 24 h. As shown in Figure 5, significant intracellular green fluorescence in the HepG-2 cells was observed in the presence of probe NA-LCX when excited with blue light (Figure 5(a)), indicating that the sensor NA-LCX was well permeable. However, the complex NA-LCX-Cu²⁺ was added to the wells, and the green fluorescence in HepG-2 cells was quenched to a large degree, as expected (Figure 5(b)). Upon subsequent addition of 2 and 5 equiv. of Na₂S solution, obvious fluorescence recovery was observed (Figures 5(c) and 5(d)). The fluorescence imaging results suggested the potential of probe NA-LCX-Cu²⁺ for in vivo detection of H₂S.

[figures omitted; refer to PDF]

4. Conclusion

In this study, a novel two-armed naphthalene derivative probe NA-LCX was synthesized and its spectral performance for sequential recognition of Cu²⁺ and H₂S was studied. The probe NA-LCX showed an obvious “on-off-on” fluorescence response toward Cu²⁺ and H₂S. The probe NA-LCX showed a 1 : 1 binding stoichiometry to Cu²⁺ with a complexation constant of $2.6\times {10}^4{\text{M}}^{-1}$. Fluorescence study indicated sensitivity and selectivity of the probe NA-LCX-Cu²⁺ for H₂S detection without interference from other amino acids. The detection limit for H₂S was calculated to be 2.79 μM. The cell imaging results further showed the potential of the probe for Cu²⁺ and H₂S detection in living cells.

Authors’ Contributions

Guanglan Mao and Chenxi Liu contributed equally to this work.

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