Development of large Stokes shift, near-infrared fluorescence probe for rapid and bioorthogonal imaging of nitroxyl (HNO) in living cells
Graphical abstract
A near Infrared fluorescent probe with large stokes shift, high sensitivity and fast response is designed for HNO.
Introduction
Nitroxyl (HNO), a member of the reactive nitrogen species (RNS) obtained via one-electron reduction and protonation of nitric oxide (NO), is known as an important signaling molecule in many physiological processes [1], [2]. Earlier studies revealed that HNO had emerged as a potential diagnostic and therapeutic biomarker for different diseases such as cardiovascular disorders [3], [4]. Despite of the advances about the biological roles of HNO, many physiological and pathological functions of HNO in living cell context still remain elusive. Development of fluorescence probes for rapid and sensitive detection of HNO in cellular studies has become a major interest in chemical biology.
Most of reported fluorescent probes for HNO are designed based on the reduction of Cu(II) to Cu(I) [5], [6], [7], or nitroxide to hydroxylamine by HNO [8], [9], [10]. These probes are subjected to the risk of interferences by biological reductants such as ascorbic acid and glutathione (GSH) [11], [12], [13]. More recently, a thiol-based probe which could be oxidized by HNO, triggering the release of fluorophore was developed for HNO detection [14]. However, this design may have issues of stability and complicated synthetic procedures. Recent development has demonstrated that HNO can react highly selectively with phosphines at a high reaction rate constant (up to 9 ×105 M−1 s−1) to generate an aza-ylide, which subsequently undergoes intramolecular cyclization reaction and release a hydroxyl- and amide-containing compound [15], [16]. This Staudinger ligation reaction is bioorthogonal, since phosphines are usually reactively inert toward other endogenous biological substances, which catalyzes the increasing utility of this mechanism for developing of fluorescence probes for HNO imaging. Until recent, a few activatable fluorescent probes were designed by deriving various fluorophores with this HNO-responsive bioorthogonal moiety [17], [18], [19], [20]. To avoid autofluorescence background and minimize photodamage in biological systems, several near infrared fluorescence (NIR) probes were also developed, however, majority of them had short Stokes shifts and sluggish response rates, which may suffer from self-quenching and preclude fast detection and imaging of transient HNO in living cells [21], [22], [23], [24], [25], [26]. Thus, the development of NIR probes with large Stokes shifts for detection and imaging of HNO concentrations in biological systems is still of great interest.
Here we report a NIR fluorescent turn-on probe (DCX-TPP) for rapid and sensitive monitoring of exogenous and endogenous HNO level in living systems. DCX-OH, consisted of a dicyanomethylene-4H-chromene which is conjugated to a xanthene moiety, is chosen as the fluorophore because of its NIR infrared emission and large Stokes shift. DCX-TPP is obtained by masking the phenolic hydroxyl group of DCX-OH with a diphenylphosphinobenzoyl group, a bioorthogonal HNO-recognition moiety. The probe was initially nonfluorescent due to esterification of the hydroxyl group, leading to an interrupted push-pull structure. Nonetheless, HNO could react with phosphine moiety, forming an aza-ylide, which intramolecularly attacked the carbonyl carbon, releasing the initial fluorophore with activated fluorescence signal, and 2-(diphenylphosphoryl) benzamide via intramolecular nucleophilic attack of the carbonyl carbon. DCX-TPP was responsive towards HNO with fast response, high selectivity and high sensitivity in vitro. The probe was also introduced to image exogenous and endogenous HNO activity in living cells with high sensitivity, high specificity and dose-dependent fluorescence signals. We believe our novel probe would provide a useful tool for detection and visualizing HNO in living cells, holding great potential in elucidating its biological functions.
Section snippets
Reagents and apparatus
All reagents were of analytical grade and were purchased from J&K Chemical (Beijing, China). Organic solvents were obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China) and used without further purification. Angeli's salt (Na2N2O3, AS) was purchased from Cayman Chemical. 1H and 13C NMR spectra were recorded on a Bruker DRX-400 spectrometer using CDCl3 and DMSO-d6 as solvent. The chemical shifts (δ) were reported in ppm (relative to TMS as internal standard) and coupling constants
Design rationale and probe synthesis
We aimed to design a NIR fluorescence probe (DCX-TPP) with large Stokes shift to detect HNO in vitro and vivo. DCX-OH, comprised of dicyanomethylene-4H-chromene as the electron-withdrawing group and a hydroxyl xanthene moiety as the electron-donating group, was chosen as the fluorophore owing to its large Stokes shift and near infrared emission, which could potentially provide high sensitivity and minimal autofluorescence background in biological matrix [27]. Then, DCX-TPP was obtained by
Conclusions
In summary, we have developed a novel NIR fluorescent turn-on probe for the detection and imaging of exogenous and endogenous HNO in living cells. The probe was composed of a NIR fluorophore with a large Stokes shift of 160 nm, which was derived with a diphenylphosphinobenzoyl moiety as the HNO-responsive unit. The probe was non-emissive in the absence of HNO. However, there was a rapid increase (within 600 s) in fluorescence upon addition of HNO. The probe was demonstrated to exhibit excellent
Acknowledgements
This work was supported by the National Natural Science Foundation of China (Grants 21527810, 91753107, and 21205034).
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