A rhodamine chromene-based turn-on fluorescence probe for selectively imaging Cu²⁺ in living cell

Abstract

We describe the development of a rhodamine chromene-based turn-on fluorescence probe to monitor the intracellular Cu²⁺ level in living cells. The new fluorescent probe with a chlorine group in chromene moiety exhibits good membrane-permeable property than previous reported because the predicted lipophilicity of present probe 4 is stronger than that of methoxyl substituted probe in our previous work (CLogP of 4: 8.313, CLogP of methoxyl substituted probe: 7.706), and a fluorescence response toward Cu²⁺ under physiological conditions with high sensitivity and selectivity, and facilitates naked-eye detection of Cu²⁺. The fluorescence intensity was remarkably increased upon the addition of Cu²⁺ within 1 or 2 min, while the other sixteen metal ions caused no significant effect.

Introduction

Copper, after zinc and iron, ranks third in abundance in human bodies among the essential heavy metals [1]. Copper plays a critical role in the physiology of living organisms and has multiple functions, as iron absorption, hemopoiesis, and diverse enzyme activities and in the redox processes. However, alteration in the cellular homeostasis of copper ions is connected to various severe neurodegenerative diseases such as Menkes syndrome, Wilson’s disease, amyotrophic lateral sclerosis, and Alzheimer’s disease [2]. Furthermore, a higher level of Cu²⁺ is detected in tumors with a possible role in promoting angiogenesis (new blood vessel growth) [3]. Moreover, with excessive loading, Cu²⁺ is highly toxic to organisms [4]. Therefore, the quantitative detection of intracellular Cu²⁺ is of great importance for elucidating its complex physiological and pathological roles.

The design and synthesis of chemosensors for copper ions has become a very highly focused area of research, as a result of the demand for more sensitive and selective chemosensors for imaging in living cells and in vivo purposes [5]. A number of fluorescent probes based on coumarin [6], 1,3,5-triphenylbenzene fluorophore [7], N-(quinolin-8-yl)acetamide [8], dansyl-anthracene dyads [9], nanoparticle [10], polymer-based [11] with high sensitivity and specificity toward Cu²⁺ have been developed.

It is known that the rhodamine framework is an ideal mode to construct fluorescent chemosensors, because the spirolactam ring-opening process leads to a turn-on fluorescence change with excellent photophysical properties, such as long absorption and emission wavelengths, large absorption coefficient, and high fluorescence quantum yield [12]. An additional advantage of such a rhodamine-based sensing system is that the ring-opening process is also accompanied by a vivid color change from colorless to pink, thus enabling the metal detection with the naked eye [13].

Although a number of rhodamine-based fluorescent probes for transition metals including zinc, iron, chromium and mercury have been developed over the past decades [14], [15], [16], [17], relatively few copper-selective probes have been reported [5](a), [18]. Among these probes, membrane-permeable chemosensors that allow monitoring of intracellular copper are rare [5](a), [5](b), [18](b), [18](c), [18](d). Thus, the development of turn-on fluorescent probes with high sensitivity and selectivity for monitoring Cu²⁺ in living cells remains a significant challenge. Due to the good stability of Schiff base ligands with Cu²⁺ to form complexes [19], rhodamine Schiff base ligands based fluorescence probe have attracted a great deal of attention in recent years [14](a), [17](a), [18](a), [18](e), [20]. In our previous paper, we described a rhodamine chromene-based fluorescence probe to monitor the intracellular Cu²⁺ level in living cells [21]. Due to the less membrane permeation ability of the probe, the incubation of the probe needs 5 h to take fluorescence microscope images of living HeLa cells. Hence, in order to increase the membrane permeation ability of the probe, we modified the structure of the probe by introducing a chlorine atom to chromene moiety because the predicted lipophilicity of the probe with chlorine instead of methoxyl group is stronger. In addition, a smaller size of chlorine atom compared to methoxyl group was expected to improve the cell permeability of the probe. The results showed that the modified probe not only possess higher fluorescence intensity but also have favorable membrane permeation ability for the detection of the intracellular Cu²⁺ level in living cells.