Elsevier

Analytical Biochemistry

Volume 655, 15 October 2022, 114855
Analytical Biochemistry

A fluorescence “turn-on” probe for Cu () based on flavonoid intermediates generated by copper-induced oxidative cyclization and its fluorescence imaging in living cells

https://doi.org/10.1016/j.ab.2022.114855Get rights and content

Highlights

  • A Cu2+ ions biosensor was constructed by the condensation reaction of coumaraldehyde and 1-hydroxy-2-acetylnaphthalene.

  • The probe exhibited high selectivity for Cu2+ and could not be disturbed even if high concentration of GSH existed.

  • The mechanism probably involved the procedure of copper-mediated cyclization to generate the flavonoid intermediate.

  • The probe can be used to detect Cu2+ ions in physiological environment proven by the cell image experiments.

Abstract

A fluorescence “turn-on” probe for Cu (Ⅱ) ions was prepared based on the condensation reaction of coumaraldehyde and 1-hydroxy-2-acetylnaphthalene. A strong fluorescent flavonoid intermediate was formed and verified by the NMR and ESI-MS experiments. The water-soluble and pH dependence experiments were performed to confirm the optimal solvent condition (CH3CN: HEPES = 1:1, v/v, pH = 7.2–7.4). The dynamic experiments indicated that the formation process of the intermediate catalyzed by Cu(Ⅱ) ions was probably pseudo-first-order reaction process. The probe showed good selectivity toward copper ions and almost no interference except Ag+ ions by the selectivity and competitive experiments. The HeLa cells were used in the cell fluorescence imaging tests and it was demonstrated that the probe could be used in the phycological condition and showed weak cytotoxicity by the MTT experiments.

Introduction

Copper (Cu) is an essential trace element in the human body, and plays a pivotal role in many metabolic processes. Mainly in the form of metal enzymes to participate in a series of processes such as electron transport, small molecule activation, substrate oxidation, signal transduction, etc [[1], [2], [3], [4]]. Cu deficiency in the daily diet can lead to the oxidative damage of DNA, lipids and proteins directly or indirectly, and even lead to some cardiovascular diseases, such as increment of blood pressure, weak antioxidant defense system, structural changes to the heart and blood vessels, an intensification of inflammation, lipid peroxidation, impaired glucose metabolism and so on [5]. However, excessive intake of copper can also lead to drastic consequences for the human body. For instance, long time exposure to excessive copper ions will induce some neurodegenerative diseases including Alzheimer's disease, Wilson's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS) and so on [[6], [7], [8], [9]]. In addition, copper is one of the most widely used and most common heavy metals in industrial production activities. The discharge of the wastewater containing copper can not only cause serious waste of resources, but also cause great pollution to the environment. Therefore, it is of great significance to detect copper ion in situ with high selectivity and sensitivity in the field of life science and environment.

Nowadays, fluorescent probe as a non-invasive method with high sensitivity and selectivity has been widely used in the biomedical field to detect metal ions, active substances, biological small molecules and other substances in organisms [[10], [11], [12], [13], [14]]. Many probes based on copper activity had been designed and produced to detect various bioactive substances, such as amino acids, mercaptan, active nitrogen or reactive oxygen species in organisms [15,16]. Their design was mainly based on the coordination ability and catalytic ability of copper ions [17,18]. For example, the Copper ions might induce the catalytic hydrolysis of hydrazides, esters, lactones, amides, lactams and other substances [[19], [20], [21]]. In addition, the probes based on the copper-mediated oxidation procession had also been reported, such as oxidative dehydrogenation reaction [22], oxidative cyclization [23], oxidation of organic amines [24], benzyl ether C–O bond cleavage reaction [25], etc. Wherein only a few probes based on the copper-mediated oxidative cyclization procession were reported so far, such as the oxidative cyclization to form oxadiazole, benzotriazole and quinine compounds were reported [23,26,27]. The probe based on copper-mediated oxidative cyclization to generate the flavonoid intermediate has not been reported yet.

In this paper, a new “turn on” probe with good photostability and high quantum yield was prepared based on the condensation reaction of coumaraldehyde and 1-hydroxy-2-acetylnaphthalene. As we all know, coumarin and naphthalene with excellent fluorescence quantum yields, good chemical stability and photostability, low cytotoxicity, and desirable compatibility to biological systems had been used in many probes [[28], [29], [30], [31], [32]]. In our probe a double bond between the coumarin fluorophore and naphthalene derivatives was formed to further extend its conjugated system, which might induce the probe to produce larger Stokes shift to avoid biological self-fluorescence. In addition, the probe itself showed weak fluorescence characteristics, however obvious fluorescence enhancement could be observed after the addition of copper ions. The probable mechanism might involve a process of copper-mediated oxidative cyclization to generate the flavonoid intermediate. More details were described in the following texts.

Section snippets

Experimental reagents and instruments

The reagents used in the experiments were analytically pure and used directly after purchase. 7-(diethylamino) coumarin, 1-hydroxy-2-acetylnaphthalene and other chemical reagents were purchased from Energy Chemical Reagent Company as raw materials. Stock perchlorate solutions (2.0 × 10−2 M) of various metal ions were prepared in aqueous solutions. In the spectral measurements, the stock solutions of probe 1 were prepared in CH3CN: HEPES (1:1, v/v, pH = 7.2–7.4) solutions. A 3 mL solution of

The water-soluble and pH dependence experiments

The excellent water solubility of the probe is a prerequisite for its application in vivo. Thus, we firstly tested the variation of fluorescence intensity of the probe in different ratio of acetonitrile and HEPES buffer solutions to screen out an appropriate solvent system. These results indicated that in different ratios of acetonitrile and HEPES solutions only weak fluorescence could be observed for the probe 1 (10.0 μM, pH = 7.2–7.4, Ex = 440 nm, Em = 540 nm) and no significant fluorescent

Conclusions

Generally, a new “turn on” fluorescent probe for Cu2+ was designed and synthesized based on the condensation reaction of coumaraldehyde and 1-hydroxy-2-acetylnaphthalene. As a reactive probe, in the present of Cu2+ ions its fluorescence enhancement might involve a process of copper-mediated oxidative cyclization to generate the flavonoid intermediate. This probe had good water solubility and pH adaptability, so it could be used as a biocompatible probe. The selectivity experiments showed that

Author contributions

Liguo Ji: Data curation, Writing- original draft, Preparation, Writing-reviewing and editing, Yutian Fu: Synthetic experiments, Spectrum measurement, Nan Yang: Cell experiments, Meifei Wang: Software, Linlin Yang: Conceptualization, Methodology, Qingzhi Wang: Supervision. Wanbing Shang: Validation, Guangjie He: Visualization, Investigation.

Declaration of competing interest

There are no conflicts to declare.

Acknowledgements

This work was supported by Key Scientific Research Projects of Higher Education Institutions of Henan Province (No. 19A150007) and the Doctoral Scientific Research Initiation Foundation of Xinxiang Medical University (XYBSKYZZ201649).

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