A fluorescent probe with low-background for visualization of nitroxyl in living cells and zebrafish
Introduction
Due to the critical roles of reactive oxygen species (ROS) and reactive nitrogen species (RNS) played in the human body, the development of fluorescent small-molecule probes for the real-time detection and imaging of ROS/RNS has gained great interest in recent years [[1], [2], [3], [4], [5]]. As one of the reduced congeners of nitric oxide (NO), nitroxyl (HNO, also known as nitrosyl hydride) has been recently identified as an elusive RNS and a pharmacological agent [[6], [7], [8], [9]]. HNO is known to be generated through different ways including oxidation of N-hydroxyl-l-arginine, nitric oxide synthase, reduction of NO by cytochrome c, and reaction of S-nitrosothiols with thiols under physiological conditions [10,11]. HNO has been assumed to be a potent cytotoxic agent that reacts with cellular thiols in aldehyde dehydrogenase, resulting in the inhibition of the enzyme's activity [12,13]. Apart from its cytotoxic effect, HNO also displays unique pharmacology properties, such as migrating the myocardial ischemia-reperfusion injury and preventing heart failure [14]. Because of the important physiological roles of HNO, developing reliable detection methods capable of rapid and specific recognition of HNO in living systems is of great importance for understanding the biological profile of HNO. Nevertheless, HNO is highly unstable under physiological conditions, which make the detection of HNO is an immense challenge.
Until now, various analytical methods including electrochemical analysis, high-performance liquid chromatography and mass spectrometry have been developed for the detection of HNO [[15], [16], [17]]. However, these methodologies are not acceptable for the spatiotemporal detection of HNO in living systems due to their destructive and time-consuming disadvantages. In this case, the fluorescent probe has attracted extensive attention because of its noninvasive detection, ultra-sensitivity, and real-time sensing and imaging ability [18]. So far, various fluorescent probes have been constructed to discriminate HNO from the other biological species, based on the reaction between HNO and Cu(II) complexes [[19], [20], [21], [22]], thiols [23], or organic phosphines [[24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49]]. These studies immensely advanced the biological sensing and imaging of fluorescent probes towards HNO. However, some of them still suffer from disadvantages such as poor sensitivity, slow reaction rate, and interference from other ROS. Therefore, fluorescent HNO probes with high sensitivity and selectivity, fast response capability still need to be addressed.
The “covalent-assembly” approach is usually employed for the construction of fluorescent probes with a strong fluorescence “off-on” response [50]. Taking the advantages of high contrast response caused high sensitivity, fluorescent probes based on the “covalent-assembly” approach have been rationally designed to detect chlorodiethylphosphate [50], peroxynitrite [51], fluoride anions [52], and mercury ions [53]. In this work, we present a new fluorescent probe, BT, which is constructed through the “covalent-assembly” strategy for HNO sensing and imaging. The “covalent-assembly” strategy allows the probe to not only display a “turn-on” response to HNO with low background, but also display a low detection limit of 9 nM. Moreover, the probe responds rapidly, sensitively and selectively towards HNO over the other biological species including ROS, RNS, amino acids, cations, and anions, which make it holds great potential for practical applications in living systems. As a proof-of-concept, the probe has been successfully employed for visualizing the HNO in living HeLa cells and zebrafish larvae.
Section snippets
Synthesis of compound 1
To a dichloromethane solution (20 mL) containing 4-(dimethylamino)salicylaldehyde (640 mg, 3.3 mmol) and 2-(diphenylphosphino)benzoic acid (2.0 g, 6.6 mmol), was added 4-dimethylaminopyridine (DMAP) (800 mg, 6.6 mmol) and 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCl) (640 mg, 3.3 mmol) in one portion. The mixture was stirred at room temperature for 6 h. Then the organic layer was washed with saturated Na2CO3 solution, water, and brine, respectively. The organic layer was
Design and synthesis
The synthesis of BT is depicted in Scheme 1. The HNO reaction unit, diphenylphosphinobenzoate moiety, was introduced to 4-diethylaminosalicylaldehyde in the presence of EDCl and DMAP to give compound 1 in a 60% yield. After reacting with benzothiazole-2-acetonitrile, BT was obtained in a 20% yield. Due to the strong electron-donating ability of diphenylphosphinobenzoate moiety, we envisioned that the fluorescence of the probe could be efficiently quenched via the photo-induced electron transfer
Conclusion
In summary, we have developed a new fluorescent probe BT for HNO sensing and imaging. As the precursor of a π-extended iminocoumarin, the probe showed very weak fluorescence and produced iminocoumarin with intense fluorescence through a cascade reaction mediated by HNO. The probe showed rapidly and high selectively response to HNO over the other biological species including ROS, RNS, amino acids, cations, and anions under physiological condition, enabling it to sense and image HNO in living
Credit author statement
Changli Zhang: Conceptualization, Writing - review & editing, Project administration, Supervision. Fengyun He: Conceptualization, Methodology, Investigation, Writing - original draft, preparation. Fang Huang: Investigation, Methodology, Data curation, Validation. Jian Xu: Visualization, Investigation. Yaojuan Hu: Visualization, Investigation.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgment
This work is financially supported by the Key University Science Research Project of Jiangsu Province (17KJA150004) and the Scientific Research Projects of Nanjing Xiaozhuang College (2018NXY49).
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2023, Dyes and PigmentsCitation Excerpt :In contrast, fluorescent probes have been widely exploited as a powerful tool for tracking bio-targets due to their advantages of ultra-sensitivity, non-invasion, and high spatiotemporal resolution [12–24]. Up to now, several fluorescent probes for HNO have been developed based on the reduction of Cu(II) to Cu(I) [25–28], reaction with thiols [29], nitroxide to hydroxylamine [30] or organic phosphines derivative [31–37]. Despite their potential for practical applications [38–44], a majority of fluorescent probes still suffer from drawbacks of short emission wavelength, poor water-solubility, and suffer from interference [30,31,39].
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