A low-molecular-weight fluorescent sensor with Zn2+ dependent bathochromic shift of emission wavelength and its imaging in living cells
Graphical abstract
Introduction
Zinc has a variety of essential physiological functions in living systems. For example, it influences gene expression, apoptosis, enzyme regulation, immune system response, and neurotransmission [1], [2], [3], [4], [5], [6]. Generally, Zn2+ is tightly chelated by proteins and displays structural and catalytic functions [7]. Zn2+ also exists in a free or chelatable form, and is believed to play an important role in biological systems [8], [9]. Although significant efforts to understand the role of Zn2+ in physiology and particularly in the field of neurochemistry have been put forth, the effects of free Zn2+ in terms of human health and disease remain largely unexplored.
As such, fluorescent sensors for metal ions have attracted significant attention as optical materials in biosensing due to their high sensitivity. A variety of fluorescent sensors for Zn2+ have been developed, and some have been utilized in the analysis of Zn2+ in biological samples and have provided useful information regarding zinc biology [10], [11], [12]. In order to monitor Zn2+ in living cells, fluorescent sensors should have sufficient solubility in water in addition to adequate cell permeability. Most fluorescent sensors for Zn2+ typically have large molecular weights, because they are composed of a fluorescent core and a Zn2+ binding moiety. Therefore, their aqueous solubility and cell permeability may be limited. In a previous study, we reported low-molecular-weight fluorescent sensors for Zn2+ based on a pyridine–pyridone core structure [13]. The core structure acted as the chelating functionality for Zn2+, and simultaneously functioned as the fluorescent moiety. As a result, the pyridine–pyridone derivatives exhibited good water solubility. In addition, substitution of the core with electron donating or electron withdrawing groups greatly affected the internal charge transfer (ICT) state. Accordingly, we developed fluorescence ON/OFF switching Zn2+ sensors by introducing a p-carboxyphenyl group at the 3-position of the pyridone ring [14]. However, to further understand the biological functions of Zn2+, fluorescent sensors with high sensitivity, high selectivity, and low background fluorescence, etc. are still required.
In this study, we report novel fluorescent sensors with methylene spacers of varying lengths between the aryl ring and the pyridine–pyridone core. The spacer length greatly affected the fluorescence, as some led to a Zn2+ dependent bathochromic shift of the emission spectrum in addition to enhancements in the fluorescence. This may be an advantage of this sensor as bathochromic shifts in emission spectra enable researchers to precisely detect Zn2+. Here, we describe the synthesis and fluorescence of the novel low-molecular-weight Zn2+ fluorescent sensors, and evaluate the potential of 5-benzyl-4-(methylsulfanyl)-[2,2'-bipyridin]-6(1H)-one (2) in biological systems.
Section snippets
Materials and instruments
All solvents were of analytic grade and were used as received. 1H and 13C NMR were measured on a JEOL-GX-400 (400 MHz) and a Varian Mercury-300 (300 MHz) and the chemical shifts were reported as ppm (in DMSO-d6 and CDCl3). HRMS were measured on a JMS-T100LP mass spectrometer. Mass spectra (MS) were recorded on a JEOL-DX-303 mass spectrometer and a JMS-T100LP mass spectrometer. Elemental analyses were carried out on a Perkin–Elmer instrument. Fluorescence spectra were obtained on a Jasco FP-6200
Results and discussion
We previously reported that the bipyridyl form of the pyridine–pyridone core structure interacts with Zn2+, and that the NH/OH proton of the pyridone ring is essential for chelation-enhanced fluorescence (CHEF) effects upon binding Zn2+ [13]. In addition, different phenyl groups (phenyl, p-methoxyphenyl, p-chlorophenly etc.) at the 3-position of the pyridone ring affect the fluorescence intensity upon Zn2+ binding [14]. In order to understand the role of the phenyl ring, we prepared compounds 1–
Conclusion
We designed and synthesized novel pyridine–pyridone based low-molecular-weight fluorescent sensors (1–3) with varying methylene spacer lengths between the phenyl ring and the 3-position of the pyridone ring and examined their fluorescence properties. The emission wavelength and intensity upon binding Zn2+ depended on the length of the methylene spacer. Compound 2, with a methylene spacer of n = 1, exhibited a 30 nm bathochromic shift in its emission spectrum and an 18-fold fluorescence
Acknowledgments
This work was supported by the SUNBOR GRANT from the Suntory Institute for Bioorganic Research.
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