A fluorescent probe with low-background for visualization of nitroxyl in living cells and zebrafish

https://doi.org/10.1016/j.dyepig.2020.108889Get rights and content

Highlights

  • A new fluorogenic probe (BT) with low-background for detecting HNO was developed.

  • BT shows specific HNO-induced emission “turn-on”.

  • BT has been utilized to realize the HNO imaging in living HeLa cells and zebrafish larvae.

Abstract

The development of a fluorescence probe with a low-background is essential to improve the sensitivity of the “turn-on” fluorescence response. In this paper, we present a new activatable fluorescent probe with low background for HNO sensing and imaging. The probe exhibits very weak fluorescence in solution and undergoes a cascade reaction mediated by HNO to generate highly fluorescent iminocoumarin. Under physiological conditions, the probe can rapidly and selectively responds to HNO in the presence of other biological-related species including ROS, RNS, amino acids, cations, and anions. Importantly, the probe has been successfully used to visualize HNO in living HeLa cells and zebrafish larvae.

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).

References (56)

  • Z. Li et al.

    A novel dual-channel fluorescent probe for nitroxyl detection and its application in HepG2 cells

    Sens Actuators, B

    (2020)
  • J. Kim et al.

    A boronate-based fluorescent probe for the selective detection of cellular peroxynitrite

    Chem Commun

    (2014)
  • S. Peng et al.

    A two-photon fluorescent probe for hno rapid visualization in endoplasmic reticulum

    Sens Actuators, B

    (2020)
  • X. Chen et al.

    Recent progress in the development of fluorescent, luminescent and colorimetric probes for detection of reactive oxygen and nitrogen species

    Chem Soc Rev

    (2016)
  • L. Wu et al.

    Reaction-based fluorescent probes for the detection and imaging of reactive oxygen, nitrogen, and sulfur species

    Acc Chem Res

    (2019)
  • D. Andina et al.

    Ratiometric fluorescent probes for the detection of reactive oxygen species

    Chem Eur J

    (2017)
  • X. Bai et al.

    Small-molecule-based fluorescent sensors for selective detection of reactive oxygen species in biological systems

    Annu Rev Biochem

    (2019)
  • Q. Sun et al.

    Ultrafast detection of peroxynitrite in Parkinson's disease models using a near-infrared fluorescent probe

    Anal Chem

    (2020)
  • F. Doctorovich et al.

    The chemistry and biology of nitroxyl (HNO)

    (2017)
  • S.A. Suarez et al.

    Nitric oxide is reduced to hno by proton-coupled nucleophilic attack by ascorbate, tyrosine, and other alcohols. A new route to hno in biological media?

    J Am Chem Soc

    (2015)
  • M.A. Sharpe et al.

    Reactions of nitric oxide with mitochondrial cytochrome c: a novel mechanism for the formation of nitroxyl anion and peroxynitrite

    Biochem J

    (1998)
  • S.A. Suarez et al.

    Hno is produced by the reaction of no with thiols

    J Am Chem Soc

    (2017)
  • J.M. Fukuto et al.

    The pharmacological activity of nitroxyl: a potent vasodilator with activity similar to nitric oxide and/or endothelium-derived relaxing factor

    J Pharmacol Exp Therapeut

    (1992)
  • J.L. Favaloro et al.

    The nitroxyl anion (HNO) is a potent dilator of rat coronary vasculature

    Cardiovasc Res

    (2007)
  • S.A. Suarez et al.

    A surface effect allows HNO/NO discrimination by a cobalt porphyrin bound to gold

    Inorg Chem

    (2010)
  • I. Johnson et al.

    The molecular probes hand-book: a guide to fluorescent probes and labeling technologies

    (2010)
  • A.S.M. Islam et al.

    Phenazine-embedded copper(II) complex as a fluorescent probe for the detection of no and hno with a bioimaging application

    ACS Applied Bio Mater

    (2019)
  • D. Maiti et al.

    Dansyl-appended Cu(II)-complex-based nitroxyl (HNO) sensing with living cell imaging application and DFT studies

    Dalton Trans

    (2019)
  • Cited by (10)

    • An iminocoumarin based covalent-assembly red-emitting fluorescent probe for detection of β-galactosidase activity in ovarian cancer cells

      2023, Dyes and Pigments
      Citation Excerpt :

      Therefore, red/NIR-emitting probes for β-Gal with different detection mechanisms are worth developing. Covalent-assembly is another approach for the design of biosensors [26–29]. In comparison with other types of sensing mechanisms, the covalent-assembly mechanism has several outstanding advantages, such as a large Stokes shift, higher sensitivity, and higher contrast response.

    • An activatable near-infrared fluorescent probe for tracking nitroxyl in vitro and in vivo

      2023, Dyes and Pigments
      Citation 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].

    • Recent development in fluorescent probes based on attacking of double bond and masking of functional group

      2022, Chinese Chemical Letters
      Citation Excerpt :

      Several diverse plans have been projected for the detection of HNO in chemical, enzymatic and biological systems. There were four classes of HNO reactive probes: (1) Triphenylphosphine-based probes [48,49], (2) copper(II) complexes based probes [50], (3) nitroxide-based fluorescent probes [51,52] and (4) esters of fluorescent dyes (2-mercapto-2-methylpropionic acid) [53]. Ye’s group reported dual-channel fluorescent probe 21 to trace HNO in living HepG-2 cells [54].

    View all citing articles on Scopus
    View full text