Near-infrared fluorescent probe for imaging nitroxyl in living cells and zebrafish model
Graphical abstract
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.
References (51)
- et al.
A comparison of the chemistry associated with the biological signaling and actions of nitroxyl (HNO) and nitric oxide (NO)
J Inorg Biochem
(2013) - et al.
Mechanisms of inhibition of aldehyde dehydrogenase by nitroxyl, the active metabolite of the alcohol deterrent agent cyanamide
Biochem Pharmacol
(1998) - et al.
The pharmacology of nitroxyl (HNO) and its therapeutic potential: not just the janus face of NO
Pharmacol Therapeut
(2007) - et al.
Nitroxyl (HNO): the Cinderella of the nitric oxide story
Trends Pharmacol Sci
(2008) - et al.
Nitroxyl, a new generation of positive inotropic agent for heart failure
Engineering
(2015) - et al.
Detection of nitroxyl (HNO) by membrane inlet mass spectrometry
Free Radical Biol Med
(2011) - et al.
Reduced glutathione-resisting 19F NMR sensors for detecting HNO
Bioorg Med Chem
(2012) - et al.
Recent advances in the chemical biology of nitroxyl (HNO) detection and generation
Nitric Oxide
(2016) - et al.
Fluorescent probes for the detection of nitroxyl (HNO)
Free Radical Biol Med
(2018) - et al.
The chemistry and biology of nitroxyl (HNO): 10 - fluorescent probes for HNO detection
(2017)
Visualization of methylglyoxal in living cells and diabetic mice model with a 1,8-naphthalimide-based two-photon fluorescent probe
Chem Sci
Novel fluorescent ESIPT probe based on flavone for nitroxyl in aqueous solution and serum
Sensor Actuator B Chem
Single probe giving different signals towards reactive oxygen species and nitroxyl
Dyes Pigments
A mitochondria-targetable near-infrared fluorescent probe for imaging nitroxyl (HNO) in living cells
Dyes Pigments
A highly sensitive and reductant-resistant fluorescent chemodosimeter for the rapid detection of nitroxyl
Sensor Actuator B Chem
The chemistry and biology of nitroxyl (HNO): 11 - phosphine-based HNO detection
Localization of nitric oxide synthase indicating a neural role for nitric oxide
Nature
Nitric oxide as an inhibitory non-adrenergic non-cholinergic neurotransmitter
Nature
Role of nitric oxide in cardiovascular disease: focus on the endothelium
Clin Chem
Nitric oxide and the immune response
Nat Immunol
Reaction between S-nitrosothiols and Thiols: generation of nitroxyl (HNO) and subsequent chemistry
Biochemistry
The chemistry and biology of nitroxyl (HNO): a chemically unique species with novel and important biological activity
Chembiochem
Discussing endogenous NO•/HNO interconversion aided by phenolic drugs and vitamins
Inorg Chem
A fast and selective near-infrared fluorescent sensor for multicolor imaging of biological nitroxyl (HNO)
J Am Chem Soc
Time-resolved electrochemical quantification of azanone (HNO) at low nanomolar level
Anal Chem
Cited by (31)
Small molecule fluorescent probes for the detection of reactive nitrogen species in biological systems
2023, Coordination Chemistry ReviewsVisualizing biothiols in vivo using a dual-channel sensitive fluorescent probe
2023, Dyes and PigmentsA near-infrared self-assembled micellar nanoprobe for highly effective detection of cysteine in vitro and in vivo
2023, Sensors and Actuators B: ChemicalAn activatable near-infrared fluorescent probe for tracking nitroxyl in vitro and in vivo
2023, Dyes and Pigments
- 1
These authors contributed equally to this work.