Original article
A Photo-triggered and photo-calibrated nitric oxide donor: Rational design, spectral characterizations, and biological applications

https://doi.org/10.1016/j.freeradbiomed.2018.04.563Get rights and content

Highlights

  • NOD560 releases NO upon activation by green-light.

  • NOD560 is non-fluorescent with excellent chemostablity.

  • A rhodamine dye is co-generated stoichiometrically with respective to NO.

  • The potentials of NOD560 as a NO donor were showcased with MSC cells.

Abstract

Nitric oxide (NO) donors are valuable tools to probe the profound implications of NO in health and disease. The elusive nature of NO bio-relevance has largely limited the use of spontaneous NO donors and promoted the development of next generation NO donors, whose NO release is not only stimulated by a trigger, but also readily monitored via a judiciously built-in self-calibration mechanism. Light is without a doubt the most sensitive, versatile and biocompatible method of choice for both triggering and monitoring, for applications in complex biological matrices. Herein, we designed and synthesized an N-nitroso rhodamine derivative (NOD560) as a photo-triggered and photo-calibrated NO donor to address this need. NOD560 is essentially non-fluorescent. Upon irradiation by green light (532 nm), it efficiently release NO and a rhodamine dye, the dramatic fluorescence turn-on from which could be harnessed to conveniently monitor the localization, flux, and dose of NO release. The potentials of NOD560 for in vitro biological applications were also exemplified in in vitro biological models, i.e. mesenchymal stem cell (MSC) migration suppression. NOD560 is expected to complement the existing NO donors and find widespread applications in chemical biological studies.

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Introduction

The bio-relevance of nitric oxide was unambiguously established with the identification of NO as the vasodilator [1], [2], [3], [4]. This event not only rationalizes the use of NO releasing molecules (NO donors) for hypertensive emergencies [5], but also celebrates the birth of NO biology [6], [7]. Over the years, NO has been found to be produced in vivo via facile pathways and plays key roles in numerous physiological or pathological processes other than vasodilation [6], [7]. It inhibits platelet aggregation [8], [9] and is involved in wound healing [10], [11]. It is a neurotransmitter, augmenting post-synaptic plasticity and hormone secretion [12], [13]. Therefore NO is implicated in cognition [14], motor coordination [15], and sleep-wake cycle [16], etc. Recently, the involvement of NO in stem cell proliferation [17], [18], differentiation [17], [18] and migration [19] has received tremendous attentions [20]. The elusive biology of NO and profound implications in health and diseases has promoted the development of NO donors for mechanistic chemical biological studies and translational pharmaceutical therapies [21], [22], [23], [24], [25], [26].

A variety of NO-releasing compounds has been reported. Many spontaneously release NO in a biological milieu, e.g. organic nitrates, organic nitrites, nitrosothiols, diazeniumdiolates, N-nitrosamines, metal-nitrosyl complexes, (benzo-/)furoxans [24], [25], [26]. The biological outcome of NO is a function of a multitude of parameters, such as

its localization, flux, and dose [27], [28]. Lack of strict spatiotemporal control over the NO release from the spontaneous NO donors might be a contributing factor for many dichotomous reports of the NO involvement in similar biological settings. The development of light-triggered NO donors [29], [30], [31], [32], [33], [34], [35] has attracted tremendous attention, with the o-nitrobenzyl protected NONOate by Tsien et al. as the first example [29]. Other notable examples are N-nitrosamines [30], nitrobenzene [31], and metal-nitrosyl complexes [34], [35]. Currently, UV-light is required to trigger the NO release in most of these aforementioned molecular systems. Moving to the less cytotoxic longer-wavelength spectral region is keenly desired. Recent progresses in the field include visible light controlled NO donors, e.g. PPIX/AFX/Fluor-RSE[36], [37], [38], FlEt[39], BODIPY labled NONOates [33], o-trifluoromethyl nitrobenzene [40], [41] and NOBL-1[42](Fig. 1).

We recently proposed to develop NO donors with both a robust photo-trigger to render a spatiotemporal control of NO release, and a built-in calibration mechanism in form of a concomitant fluorescence turn-on to allow convenient monitoring of NO release. The first embodiment of such photo-triggered and photo-calibrated NO donors is N-nitrosated naphthalimides (NOD545)[43], whose scope of applications is however limited by the fact that a UV light at 365 nm was in need to trigger NO release. Ideally, a photo-triggered and photo-calibrated NO donor should exhibit high chemostability to avoid unintended NO release, easy photo-activation by visible light to avoid cytotoxicity, a large fluorescence turn-on ratio to facilitate monitoring of NO release, clean photo-decomposition pathway, high cell permeability, and low cytotoxicity. We wish to report our recent progress in this line of research.

Section snippets

The design rationale of NO donors

N-nitrosated secondary arylamines, especially electron-deficient arylamines, readily homolyze photolytically to release NO [44], [45], [46]. Also, dyes are typically electron-deficient scaffolds, due to intramolecular charge transfer. This inspired us to search for photo-triggered and photo-calibrated donors, from N-nitrosated push-pull dyes. Our initial success with NOD545 verifies the feasibility of this design rational (Fig. 1F). However, it requires UV-activation and precludes use in

Conclusions

We have developed a novel NO donor (NOD560) via N-nitrosation of a rhodamine dye. It is efficiently photo-activated by UV, blue, green and yellow light to release nitric oxide and a bright rhodamine dye, as evidenced by UV–Vis absorption, fluorescence emission, NMR and EPR spectra. The concomitant fluorescence turn-on can be harnessed for convenient monitoring of the flux and dose of NO release from NOD560, even in the complex biological milieu. NOD560 readily crosses the plasma membrane and is

Acknowledgment

The work is supported by the Fundamental Research Funds for the Central Universities (Nos. WY1514053, WY1516017), Shanghai Pujiang Program (16PJ1402500) and the National Natural Science Foundation of China (Nos. 21372080, 21572061, 11674101, 21236002, and 81502540).

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Supporting information

General methods, experimental, synthesis, characterizations, tabulated photophysical data, additional spectral data, crystal structure of NOD560, chemostability study, quantification of NO by DAN assay, cell cytotoxicity, 1H NMR, 13C NMR and HRMS spectra.

The following files are available free of charge.

Supporting information (PDF)

MSCs migration video (AVI)

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