Elsevier

Dyes and Pigments

Volume 166, July 2019, Pages 260-265
Dyes and Pigments

Near-infrared fluorescent probe for imaging nitroxyl in living cells and zebrafish model

https://doi.org/10.1016/j.dyepig.2019.03.013Get rights and content

Highlights

  • A near-infrared metal-free fluorescent probe was synthesized.

  • DCM-P exhibits fast response and ultra sensitivity to nitroxyl.

  • DCM-P shows high selectivity and long emission wavelength in detecting of nitroxyl.

  • The probe shows favourable cellular uptaken.

  • DCM-P successfully imaging nitroxyl in different living cells and zebrafish larvae.

Abstract

Nitroxyl (HNO) is one of the important derivatives of nitric oxide (NO). It was intertwined with various biological and pharmacological events as a well-defined active molecule. Developing fluorescent probes for high specific and in situ trapping of HNO in living samples is still challenging. In this project, we constructed a near-infrared (NIR) metal-free fluorescent probe (DCM-P) for monitoring HNO in vitro and in vivo. The novel probe, DCM-P, contains a dicyanomethylene-4H-pyran (DCM) fluorophore as the reporter and a triarylphosphine as the recognition moiety. Upon exposure to HNO, the probe emits fluorescence at the wavelength of 688 nm, which belongs to near-infrared region and endows great beneficial for imaging in vivo. Moreover, DCM-P shows high chemoselective detection and fast response to HNO in the presence of various biological relevant reductants, and is successfully employed to trap nitroxyl in different living cells and zebrafish models. These results suggest that DCM-P is a suitable near-infrared metal-free fluorescent probe for motoring nitroxyl in living samples.

Introduction

Nitric oxide (NO) is an important biological signaling molecule in mammalian system, it plays vital role in many physiological and pathological processes, including neurotransmission regulation [1,2], vasodilation [3], and immune response [4]. Nitroxyl (HNO), a one-electron-reduced and protonated derivative of nitric oxide (NO), displays biological effects which is distinct from that of NO [5]. In living system, HNO as an electrophile can oxdize protein thiols and directly inhibit aldehyde dehydrogenase, cathepsin P and some other thiol-containing enzymes [[6], [7], [8]]; By means of mediating calcium in cardiac tissue, HNO can also serve as a novel therapy for heart failure [9,10]; Even though endogenous generation of HNO has not yet been directly observed in organisms, it still attracted great interest to researchers for its crucial role in living system [[11], [12], [13]]. Therefore, developing efficient analytical methods capable of monitoring biological nitroxyl is of great importance.

To date, several analytical methods including electrochemical analysis, mass spectrometry, NMR, high-performance liquid chromatography, headspace gas chromatography, electron paramagnetic resonance (EPR) spectroscopy and colorimetry [8,[14], [15], [16], [17], [18], [19], [20]], have been developed for the detection of HNO in various samples. Recently, fluorescent probes have attracted extensive attention due to its ultra-sensitivity, non-invasion detection, and excellent spatiotemporal analysis to the targets of interest in vitro and in vivo [[21], [22], [23], [24], [25], [26], [27], [28], [29]], which makes it best done in imaging in living cells. Until now, various fluorescent probes have been developed for the specific recognition of HNO, and they are mainly based on the reduction of Cu (Ⅱ) to Cu (Ⅰ) [12,[30], [31], [32], [33]], nitroxide to hydroxylamine [34], or the reaction between HNO and 2-Mercapto-2-methylpropionic acid [35]. Although these fluorescent probes have some advantages for the detection of HNO, they tend to be disturbed by abundant biological reductants, such as glutathione (GSH) and ascorbate in living systems. To solve this problem, several metal-free fluorescent probes have been developed based on the reaction between HNO and triarylphosphine [[36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46]]. In this procedure, triarylphophines react with HNO and give the corresponding phosphine oxide and aza-ylide. The unstable aza-ylide intermediate will then undergo an intramolecular nucleophilic attack on adjacent carbonyl carbon of an ester bond, leading to the formation of alcohol and amide [17,47]. In this way, several attractive fluorescent probes have been designed which are free from interferences by biological reductants.

However, most of these metal-free fluorescent probes are limited by short emission wavelengths, which probably induce photobleaching, cellular auto fluorescence, and photo-damage to tissues or organelles, making them unsuitable for imaging in vivo. Moreover, quite a lot of reported fluorescent probes have shortcomings associated with long reaction time and undesirable detection limits (Table S1). To address these deficiencies, a near-infrared (>650 nm) metal-free fluorescent probe which could overcome aforementioned disadvantages is urgently needed.

Dicyanomethylene-4H-pyran (DCM) fluorophore has attracted particular interest due to its excellent optical properties, such as controllable emission wavelengths in the NIR region, large stokes shift and high photo-stability [48,49]. On the basis of these facts, we designed and synthesized a metal-free fluorescent probe (DCM-P) for monitoring HNO in vitro and in vivo. The new probe contained a DCM fluorophore as a NIR fluorescence reporter and a triarylphosphine cleavable unit as the HNO-active trigger. In our design, DCM-P was quenched by the opposite electron-withdrawing carbonyl group of the 2-(diphenylphosphine)benzoate moiety. Upon exposure to HNO, an aza-ylide derivative will be produced and followed by an intramolecular nucleophilic attack on carbonyl carbon of the ester, leading to the formation of DCM-O-, which will give a near-infrared fluorescence (Scheme 1) [17,36,47]. Based on this mechanism, we hope our newly designed fluorescent probe (DCM-P) will realize the highly chemoselective detection of HNO in a near-infrared region.

Section snippets

Materials and instruments

Unless otherwise noted, all reagents and solvents were purchased from commercial suppliers in analytical grade and were used as received. The ultrapure water was obtained from a water ultra-purification system. Silica gel (200–300 mesh) was used for flash column chromatography. NMR spectra were recorded on a Bruker AV-600 spectrometer. Mass spectrometry (MS) was performed with an Agilent 1100 Series. UV–vis absorption spectra were acquired on a SHIMADZU UV-1800 instrument. Fluorescence spectra

Design and synthesis of DCM-P

As a typical donor-π-acceptor (D-π-A) configurational fluorophore, DCM shows several advantages, such as high photostability, large Stokes shift and near-infared emission. In particular, DCM-OH as a unique scaffold exhibits a perfect NIR-emitting performance with an easy modifiable hydroxyl site for modulation of electron-donating capability [48,49]. We envisioned that installation of HNO-trigger moiety at the hydroxyl position could quench NIR fluorescence of DCM-OH due to electron-withdrawing

Conclusion

In summary, we have developed a novel near-infrared metal-free fluorescence probe (DCM-P) for recognition of HNO in vitro and in vivo. DCM-P exhibited excellent stability in living samples and realized the highly chemoselective, fast responsive and ultra sensitive detection of HNO in near-infrared region. Moreover, DCM-P showed good cell membrane permeability and was successfully applied for imaging HNO in living cells. In addition, zebrafish experiments further confirmed its ability for

Acknowledgment

This work was supported by the National Natural Science Foundation of China (Grant No. 21702053, 21676075), the Hubei Provincial Department of Education project (Q20171013) and the Hubei Provincial Natural Science Foundation of China (2016CFB126, 2017CFB222) for financial support.

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    These authors contributed equally to this work.

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