Elsevier

Talanta

Volume 224, 1 March 2021, 121902
Talanta

A novel benzo[a]phenazin-based fluorescence probe for selective detection of cysteine with anti-cancer potency

https://doi.org/10.1016/j.talanta.2020.121902Get rights and content

Highlights

  • Benzo[a]phenazin moiety as a basic fluorophore for developing specific fluorescent Cys probe.

  • Introducing anti-cancer potency to initial the linkage between diagnostic and therapeutic concerns.

  • Enzyme inhibition tests and docking simulation for revealing effect on Topoisomerase I/II.

  • Practical for monitoring the level of Cys in living cancer cells.

Abstract

Among the physiological and pathological sulfur-containing species, cysteine (Cys) is the most typical one which is an important component of the REDOX system in vivo. Monitoring the level of Cys from other competing species seems quite important in pre-clinical diagnosis and therapeutic evaluation. Herein, we developed a selective fluorescent probe, BPCys, for Cys from the benzo[a]phenazin backbone which had the potential of anti-cancer potency. BPCys suggested advantages including high specificity (40 fold over other species), high sensitivity (detection limit: 18 nM), wide pH adaptability (6.0–11.0) and in particular, the anti-cancer effect. Biological assays and in silico simulation hinted the potency of the detecting product on Topoisomerase I/II. In brief, this study raised a practical strategy for monitoring the Cys level in living cells, especially in cancer models with its anti-cancer potential, thus opened the mind of exploring more specific tool for specific applications.

Graphical abstract

We developed a selective fluorescent probe, BPCys, for Cys from the benzo[a]phenazin backbone which had the potential of anti-cancer potency. BPCys suggested advantages including high specificity, high sensitivity, wide pH adaptability and the anti-cancer effect. Biological assays and in silico simulation hinted the potency of the detecting product on Topoisomerase I/II. In brief, this work raised a practical strategy for monitoring the Cys level in living cells, especially in cancer models with its anti-cancer potential, thus opened the mind of exploring more specific tool for specific applications.

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Introduction

Within the physiological and pathological procedures of organism, the circulatory system of sulfur element seems an indispensable component [1,2], which contains the conformations of sulfur dioxide (SO2), hydrogen sulfide (H2S), glutathione (GSH), cysteine (Cys), homocysteine (Hcy), thiophenol (PhSH) and so on according to specific oxido-reduction [[3], [4], [5], [6], [7], [8]]. Cys, as one of the sulfur-containing species, is typical and significant in both composition and function [9,10]. It is commonly generated from serine and methionine by methionine adenosyltransferase, cystathionine-β-synthase and cystathionine-γ-lyase [[11], [12], [13]]. The physiological level of Cys is 30–200 μM in human [14], while the abnormal Cys level is connected with cardiovascular diseases, neurological disorders, liver damage, and growth retardation [[15], [16], [17], [18]]. In tumor, high GSH level (over 1 mM and even up to 10 mM) could significantly cause a consequent increase in endogenous Cys level [19,20]. Thus, considering concentration is essential for revealing key interactions and parameters in pre-clinical diagnose [21].

There are a number of methods currently used to detect cysteine including high performance liquid chromatography [22], mass spectrometry [23], capillary electrophoresis [24], and fluorescent probes which brought the pre-clinical convenience from high cost and sophisticated operations [25]. With various fluorophores, the Cys-recognition groups involved N-ethylmaleimide [26], chloracetate ester [27], α,β-unsaturated ketone [28], nitrobenzoxadiazole [29], and of course the most applicative acrylate [[30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46]]. Acrylate usually act as the pioneer during the attemption of novel ideas such as sulobility optimization [47] and optical regulation [48]. Here we attempted to develop a selective fluorescent probe for Cys with anti-cancer potency, thus to initial the linkage between diagnostic and therapeutic concerns. There seemed several logistic requirements: 1) high selectivity for detecting target (Cys here) in the scene (tumor cells here); 2) the detecting procedure which was beneficial for the therapeutic purpose; 3) the therapeutic strategy which was free from interfering the detecting accuracy. All of them have been rigorously validated.

Among the fluorophores, benzo[a]phenazin (“BP” for short in this work) moiety was reported to show anti-cancer potency [49,50]. This kind of small molecules performed anti-proliferation effect on cancer cells via nucleic acid-related mechanisms. Different from interrupting protein-protein interactions [49], inhibiting Topoisomerase I/II was a more determined mechanism with derived substrates and established evaluating protocol [50]. Accordingly, benzo[a]phenazin could act as an anti-cancer agent.

In this work, we selected benzo[a]phenazin backbone to act as the origin of fluorophore due to its reported optical properties as well as anti-cancer potency [49,50]. The obtained probe, BPCys, was designed to fulfill the requirements above (Fig. 1). Acrylate was anchored to BP to realize the high selectivity. While the anti-cancer potential of the detecting procedure was investigated, the interference onto the accuracy of the detection was preliminarily evaluated. Therefore, a practical strategy for monitoring the Cys level in living cells, especially in cancer models with its anti-cancer potency, was established. To our knowledge, for small molecular fluorescent probes, this might be the first trial to combine the selective detection with anti-cancer potency.

Section snippets

Materials and methods

For chemicals from commercial sources, they were used directly with no further purification. NMR data were from Bruker DRX-600 spectrometer, while Mass data were from Agilent 6540 UHD Accurate Mass Q-TOF LC/MS. The pH values were determined by PHS-25 pH-meter. The fluorescence-associated experiments were performed on Hitachi F-7000 Fluorescence Spectrophotometer. The biological imaging was performed on Olympus FV1000 confocal fluorescent microscope.

The probe BPCys was prepared in the stock

Synthesis of the probe BPCys

BPCys was acquired as shown in Fig. 2. The characteristics were confirmed by satisfactory spectroscopic data (1H NMR, 13C NMR and HRMS, Figs. S1–6, Supporting Information (SI)). Notably, potential substitutes can be introduced from either reactant if necessary. According to the differences in HRMS spectra of BPCys and the detecting product BPCys-Prod, the responding mechanism could be indicated.

Fluorescent responses towards Cys

In preliminary tests, the probe BPCys indicated a negligible fluorescence at 525 nm when being

Conclusion

To summarize, from the benzo[a]phenazin backbone showing the potential of anti-cancer potency, we developed a specifically fluorescent probe BPCys. Compared with the most recent reports (Tables S2 and SI), the basic advantages of this probe included high specificity (40 fold over other species), high sensitivity (detection limit: 18 nM), wide pH adaptability (6.0–11.0). In particular, BPCys indicated the anti-cancer effect during the detection procedure, which was possibly caused by the

Credit author statement

Bing Rui: Methodology, Writing - Original Draft, Validation; Yangrui Feng: Formal analysis, Validation, Visualization; Lan Luo: Conceptualization, Writing - Review & Editing, Supervision.

Declaration of competing interest

All authors declare that there are no conflicts of interest.

Acknowledgments

We thank Dr. Zhimin Yin for helpful discussions and critical reading of the manuscript.

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