A highly sensitive and responsive fluorescent probe based on 6-azide-chroman dye for detection and imaging of hydrogen sulfide in cells
Graphical abstract
We synthesized a novel fluorescent probe (AC-N3) for H2S by introducing azide into 6-position of chroman dye and utilized it to detect the change of H2S level in cells.
Introduction
Hydrogen sulfide (H2S), as a colorless gas, is produced by a variety of cells in the body and by the sulfate reducing bacteria in the lower gastrointestinal (GI) tract [1]. Emerged as an important gasotransmitter and signaling molecule, H2S regulates many physiological functions in GI tract, cardiovascular, immune, endocrine, and nervous systems [2], and also plays an important role in diabetes and insulin regulation [3], [4], [5], [6]. The exposure of high level can lead to diseases such as Down syndrome, diabetes and Alzheimer's disease [7], [8]. The enzymatic machineries for endogenous production of H2S are pyridoxal-5′-phosphate (PLP)-dependent enzymes such as cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST) [9], [10], [11], [12], [13]. The CSE is mainly expressed in the kidney, liver, ileum, thoracic aorta, uterus, portal vein, placenta and pancreas [11], [14], [15], [16], and the CBS is highly expressed in the brain, kidney, liver, uterus, ileum and placenta [17], [18]. There is a great difference in the concentration of hydrogen sulfide ranging from nanomolar to micromolar in tissues [19]. In mouse liver and brain, H2S production is at nanomolar range, whereas H2S level in human whole blood can reach as high as 35–80 μM [20], [21]. The safety window of H2S between physiological level and toxicological level is narrow with less than twofold in the brain tissues [22]. Thus, it is necessary to develop a sensitive assay for measurement of H2S and elucidation of its role in physiology and pathology [23].
Over the past few decades, H2S has been quantitatively measured by the methods of electrochemical analysis, gas chromatography, colorimetry and fluorescence assays [24], [25], [26], [27], [28]. Among them, the fluorescence-based assay received a widespread attention because of its high sensitivity, convenience, rapid implementation, noninvasive monitoring capability and simplicity in fluorescent imaging of living cells and tissues [29], [30], [31], [32], [33]. These fluorescent probes for detection of H2S are mainly based on several reactions including reduction and addition of azide, nitro groups or chalcone on a fluorophore such as fluorescein, naphthalimide, rhodamine, cyanine and coumarin [34], [35]. However, these probes are limited for their use in measurement of unpretentious change of H2S level both in vitro and in vivo because of their high detection limits. Chromones (4H-chromen-4-ones, 4H-1-benzopyran-4-ones, or benzo-γ-pyrones) bearing diversity bioactivities such as antifungal, antimicrobial and antimycobacterial [36], are widely distributed in organisms, implying an excellent biocompatibility which enhances membrane permeability and improves bioavailability. This excellent biocompatibility can be beneficial for chromone-based fluorescent probes suitable for applications in vivo. However, chromone was rarely used in fluorescent probe design (Scheme 1). The possible reasones are (1) chromone precursor has no fluorescence emission, and (2) its derivatives having strong fluorescence require complicated synthesis process, which limites it used as fluorophor in fluorescent probe. Therefore, exploring the relationship between chromone structure and fluorescence emission will enrich its application in probe design. Several chromone analogues have been employed as fluorescent probes by mainly introducing conjugate groups for enhancing the fluorescent emission [37], [38]. Especially, the introduction of a conjugated group at the 2-position of chromone can produce a large red shift and quantum yield [39], and the push-pull electron of conjugate groups can regulate the fluorescent emmision of chromone analogues, indicating 2-position is a sensitive site for changing the fluorescent properties of chromone [40]. In light of conjugated effect, we reasoned that 6-position (para-position) of chromone may be a trigger of fluorescent properties. To test this hypothesis, first we introduced electron-withdrawing nitryl at 6-position of chromone to obtain 6-nitrochomeone derivative (6-NC) that shows little fluorescence like chromone. Next, we reduce the nitro group to an electron-donating amino group to obtain 6-nitrochomeone derivative (6-NHC). Fortunately, 6-NHC displays strong fluorescence at 500 nm upon excitation with 360 nm. This conversion of push-pull electron group can trigger the change of fluorescence, which provides the basis for the development of oxidation-reduction probes based on chromone.
In this study, we synthesized a novel fluorescent probe (AC-N3) for detection of H2S by introducing azide into 6-position of chroman dye and evaluated the properties of the dye (Scheme 2). Azide is a strong electron-withdrawing group like nitryl and is highly sensitive to hydrogen sulfide. The free probe AC-N3 shows little fluorescence. When AC-N3 encountered with H2S, AC-N3 was converted into AC-NH2 showing strong fluorescence at 500 nm upon excitation at 360 nm. Moreover, AC-N3 exhibits the excellent properties of high selectivity, outstanding sensitivity, little cytotoxicity and large stokes-shift (140 nm).
Section snippets
Materials and apparatus
Unless otherwise stated, all reagents were purchased from Macklin Company and used without further purification. Twice-distilled water was used throughout all experiments. 1H NMR (500 MHz) and 13C NMR (125 MHz) spectra were acquired on a Bruker Avance-500 spectrometer, with CDCl3 or d6-DMSOused as a solvent and tetramethylsilane (TMS) as an internal standard. High-resolution mass spectrometry (HRMS) involved a Q-TOF6510 spectrograph (Agilent). The absorbance of MTT was measured by microplate
The pH- and time-dependent fluorescence intensity of AC-N3 probe
The pH dependence and response time are essential factors for practical use of a fluorescent probe in vivo. To evaluate the pH effect on AC-N3, fluorescence emission of free AC-N3 and AC-N3 with H2S was measured at the different range of pH. Stock solutions of H2S were prepared through adding sodium sulfide solid into aqueous solution [41]. As shown in Fig. 1A, little fluorescence of free AC-N3 was observed at 500 nm upon excitation at 360 nm in the range of pH 4–10, indicating that AC-N3
Conclusions
In this paper, a simple fluorescent probe AC-N3 for detection of H2S was designed through introducing azide into 6-position of chroman dye. The probe AC-N3 exhibits a better selectivity with undetectable interference from analytes, high sensitivity and little cytotoxicity. The probe AC-N3 can detect the change of H2S level in live cells, and its novel structure can provide a basis for further design of other fluorescent probes based on chroman dye.
Acknowledgements
The project is supported by the research grants to K.W. Wang from National Natural Science Foundation of China (NSFC81573410), and from Ministry of Science and Technology of China (2013CB531302).
Conflicts of interest
There are no conflicts to declare.
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