A bright red-emitting flavonoid for Al³⁺ detection in live cells without quenching ICT fluorescence

Abstract

A bright red-emitting flavonoid derivative was synthesized, which exhibited a large Stokes shift (Δλ > 150 nm) and high fluorescence quantum yields (ϕ_fl = 0.10–0.35). The probe could form a stable complex with Al³⁺ in 1 : 1 binding stoichiometry, generating a large bathochromic shift in both absorption and fluorescence (Δλ ≈ 70 nm) to enable ratiometric determination of cellular Al³⁺.

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As the most abundant metal in the earth's crust, aluminium-based products are widely used in household products (e.g. cooking wares) and constructions materials.^1,2 However, a high concentration of Al³⁺ is toxic to living organisms due to its potential neurotoxicity.^3–5 Increased Al³⁺ levels in the human body can lead to severe disease conditions such as dementia, Alzheimer's disease, Parkinson's disease, Al-related bone diseases (ARBD), encephalopathy, and myopathy.^6–11 Therefore, monitoring Al³⁺ level in the human body will be useful in identifying potential risk of such disease conditions. According to recent World Health Organization (WHO) reports, the recommended daily Al³⁺ intake is 3–10 mg per day, whereas the recommended Al³⁺ concentration level in drinking water is below 200 μg L⁻¹.^12,13 Therefore, the development of highly sensitive and reliable techniques for the detection of Al³⁺ level is essential in biological research. Atomic absorption spectroscopy, mass spectrometry, voltammetry, ion selective membranes and liquid chromatography coupled mass spectrometry are commonly used for detecting Al³⁺ ions in aqueous solutions.^14–17 However, these methods are not suitable for cellular studies.

Fluorescent sensors for Al³⁺ detection in an aqueous environment have gained more attention recently, due to their high sensitivity and selectivity.^18–24 However, the most challenging step is to identify a suitable ligand for Al³⁺ binding, due to the strong hydration of the Al³⁺ cation.¹⁸ The current designs of fluorescent Al³⁺ probes utilize chelating groups such as hydrazides, Schiff bases, urea/thiourea conjugates and pyrene–amino acid conjugates for Al³⁺ binding. However, the currently reported Al³⁺ probes suffer from major drawbacks, including their characteristic blue emission, small Stokes shifts, poor chemical stability in aqueous environments, low biocompatibility and higher energy excitation, which limit their applications in biological environments.^{18–21,25–30} Therefore, it is desirable to develop red-emitting, highly biocompatible probes with large Stokes shifts for bio-imaging applications.

Flavonoids are a class of naturally occurring dyes, which often exhibit beneficial biological properties and find uses as anti-oxidants,^31–35 anti-inflammatory agents,^35,36 anti-microbial drugs,^33,37 and anti-cancer drugs.^38–42 Due to their low toxicity and environmentally sensitive fluorescence with a large Stokes shift (Δλ ≈ 100–150 nm),⁴³ flavonoids have been used in the design of fluorescent chemical sensors for proteins,⁴⁴ cations,⁴⁵ and other biological imaging applications.^44,46,47 However, the existing flavonoid probes have limited conjugation, which typically show fluorescence in the blue and green region, since many of them are based on the structure of 1. In order to overcome this barrier, we now report the synthesis of flavonoid derivative 2, in which a vinyl bond is inserted between the B and C rings for extended conjugation and intramolecular charge transfer (ICT) interaction. The result showed that the strategy effectively shifted the λ_abs and λ_em of 2 to a longer wavelength, giving bright red-emission (λ_em ≈ 610 nm in EtOH) with a large Stokes shift (Δλ ≈ 150 nm) (Scheme 1). In addition, 2 also exhibited a large spectral response upon binding with Al³⁺ in aqueous environments, showing its potential in detecting Al³⁺ in biological environments.

Scheme 1 Synthesis of probe 2 and the ESIPT/ICT process in probe 2.

Flavonoid derivative 2 was synthesized according to the previously reported procedure⁴⁷ in good yield (Scheme 1) and characterized by ¹H NMR and ¹³C NMR spectroscopy, melting point determination and high-resolution mass spectrometry (ESI,† Fig. S1 and S2).

Spectroscopic properties. The spectroscopic properties of 2 were examined in different solvents (Table 1 and ESI,† Fig. S3). The absorption λ_abs of 2 was affected slightly by solvent polarity (Δλ ≈ 14 nm, Table 1 and ESI,† Fig. S3), in sharp contrast to 1 whose absorption λ_abs was affected by Δλ ≈ 45 nm.⁴⁷ However, the emission spectra of 2 displayed a large solvatochromic effect, with emission λ_em shifting from 528 nm (in toluene) to 640 nm (in water). This can be explained by the relative stability of the polar ICT-transition state complex of probe 2 in different solvents. On the basis of the observed large Stokes shift (e.g. Δλ ≈ 175 nm in EtOH), the emission was assumed to arise from the ESIPT process that was coupled with ICT (Scheme 2). Interestingly, the emission of 2 was very weak in aqueous solutions (ϕ_fl = 0.008) but became strong in non-aqueous solutions (ϕ_fl ≈ 0.34 in acetonitrile). Therefore, probe 2 retained the valuable environmentally sensitive fluorescence of 1, while extending the emission to a longer wavelength.

Table 1 Spectroscopic properties of probe 2

Solvent	λ abs (nm)	λ em (nm)	ϕ fl	Δλ (cm^−1)	Δλ (nm)	ε (M^−1 cm^−1)
Toluene	437	528	0.11	3944	91	27 260
DCM	440	567	0.14	5091	127	28 235
Acetonitrile	432	588	0.37	6141	156	26 100
THF	431	561	0.21	5377	130	24 395
DMSO	444	595	0.33	5716	151	28 543
DMF	438	589	0.32	5853	151	29 173
EtOH	438	613	0.24	6518	175	29 460
MeOH	439	621	0.24	6676	182	27 316
Water	430	640	0.008	7631	210	16 306

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Scheme 2 Proposed formation of the Al³⁺ complex with 2. Structure 3 shows enhanced ICT in the complex.

Low temperature fluorescence. In order to evaluate the extent of the ICT effect, a sample of 2 (in EtOH) was frozen quickly by immersing it into liquid nitrogen in a quartz Dewar. At an extremely low temperature (i.e., −189 °C), the molecule of 2 was frozen in a rigid solvent matrix (m.p. of EtOH: −112 °C), which restricted the molecular motion and bond changes that are associated with the ESIPT/ICT process. As a consequence, the emission λ_em was observed at 541 nm (Fig. 1), which could be attributed to its locally excited state. When the temperature was increased to room temperature, the emission peak was red-shifted towards 612 nm, as molecular motion became possible, enabling the ESIPT/ICT process. The observed large spectral shift (Δλ_em ≈ 70 nm from 541 nm to ∼612 nm) provided experimental evidence, supporting the assumption that the ESIPT/ICT process played an important role in the emission of 2 occurring at a longer wavelength.

Fig. 1 Excitation (broken line) and fluorescence (solid line) spectra of 2 (1 × 10⁻⁶ M) in EtOH at different temperatures.

Al ³⁺ sensing in solution. When the solution of 2 (in acetonitrile) was titrated with an Al³⁺ (1 mM in aqueous) solution, a new absorption band was observed at ∼507 nm, whose intensity gradually increased with the Al³⁺ concentration (Fig. 2a). The absorbance at 507 nm exhibited a good linear correlation with the Al³⁺ concentration up to 1 equivalent. Probe 2 was excited at either 430 nm or 507 nm to record the emission spectra of the resulting Al³⁺ complex. When 2 was excited at 430 nm, the emission peak at 588 nm gradually decreased with increasing Al³⁺ concentration (ESI,† Fig. S4). However, excitation at 507 nm revealed the emission only from the resulting Al³⁺ complex (λ_em ≈ 658 nm), which gradually increased with the Al³⁺ concentration (Fig. 2b). The emission spectra remained unchanged after the addition of 1 equivalent of Al³⁺ cation, indicating 1 : 1 binding stoichiometry for the probe 2-Al³⁺ complex. The 1 : 1 ligand-to-metal ratio for the Al³⁺ complex was determined by Job's plot (ESI,† Fig. S8). Also, the limit of detection (LOD) for probe 2 was found to be 0.05 μM. The calculated binding constant (log K) for the 2-Al complex was found to be 6.77, which further revealed the stronger binding properties of the 2-Al complex (ESI,† Fig. S9).

Fig. 2 Absorption (a) and emission (b) spectra recorded for probe 2 (1 × 10⁻⁵ M) in acetonitrile upon spectrometric titration with Al³⁺ (1 mM in water) at room temperature. The 2-Al complex was excited at 507 nm to obtain the emission spectra.

The spectral evidence clearly indicated that the chelation of ligand 2 with the Al³⁺ cation was sufficiently strong to replace the water on the metal cation, forming the 2-Al complex. When the concentration of Al³⁺ was less than 0.5 equiv., the equilibrium could also include some 2-Al-2, in addition to the major product 2-Al complex. This assumption could account for the higher response in the absorbance in the presence of 0–0.5 equiv. of Al³⁺ (inset in Fig. 2a). Since Al³⁺ was more reactive compared to 2-Al (bearing two positive charge), the reaction of “2-Al-2 + Al³⁺ → 2 (2-Al) “occurred when the concentration of the Al³⁺ cation was higher than 0.5 equiv., as the Job plot shows. This assumption also explained the lack of an ideal isobestic point in the absorption spectra (Fig. 2a). However, the fluorescence response of 2 at 658 nm was almost linear (inset in Fig. 2b). The ability of 2 to show a large spectral shift upon binding to Al³⁺ makes it a potentially useful method for ratiometric determination (I₆₅₈/I₅₈₈) of the Al³⁺ concentration in the solution (ESI,† Fig. S6 and S7).

The proposed formation for Al³⁺ binding was further studied by NMR spectroscopic analysis in deuterated DMSO (Fig. 3). Addition of Al³⁺ (1 eq.) into probe 2 resulted in the disappearance of the –OH proton signal (H_a) at 9.34 ppm, which is in agreement with the proposed formation of the 2-Al complex (Scheme 2). The aromatic proton H_c signal (on the B ring of 2) at 6.75 ppm disappeared and shifted notably downfield, indicating that ligand 2 was binding to the cation when one equiv. of Al³⁺ was used. The noticeable downfield shift in the ¹H NMR signal also indicated the reduced electron density on the B ring of 2, which is in agreement with the enhanced ICT interaction upon Al³⁺ binding (shown in 3 in Scheme 2).

Fig. 3 ¹H NMR spectra of probe 2 in the absence (bottom) and presence (top) of one equiv. of Al³⁺ (prepared from Al(NO₃)₃·9H₂O dissolved in acetonitrile-d₃) in deuterated DMSO-d₆.

Probe 2 was tested against other cationic species to investigate its selective interaction with metal cations. Interestingly, 2 did not show any noticeable optical response towards any other cationic species except for Al³⁺ (ESI,† Fig. S11). Therefore, it could be used for selective identification of Al³⁺ in the presence of other metal ion species.

Al ³⁺ detection in live cells. The attractive photophysical properties, in addition to its selectivity towards Al³⁺, led us to investigate the potential use of 2 for Al³⁺ detection in live cells. Thus, progenitor oligodendrocytes (MO3.13) were pre-incubated with an aqueous Al³⁺ solution (1 μM) for 30 minutes and then incubated with probe 2 (1 μM) for another 30 minute period. As a control experiment, another batch of MO3.13 cells was incubated with probe 2 (1 μM) in the absence of Al³⁺ in the media. Surprisingly, the cells pre-incubated with Al³⁺ showed bright red fluorescence confocal images upon exciting probe 2 with a 561 nm laser line (Fig. 4c and d). In sharp contrast, the cells incubated with probe 2 (1 μM) only (i.e., absence of Al³⁺) did not reveal any noticeable fluorescence signal (Fig. 4a and b) upon 561 nm laser excitation. However, probe 2 (1 μM) showed weak red emission upon excitation with a 488 nm laser (ESI,† Fig. S12). This result illustrated that probe 2 could be a reliable tool for determination of Al³⁺ toxicity in live cells. It is also important to notice that the probe showed bright confocal microscopy images with Al³⁺ concentrations as low as 1 μM, which is a significant improvement in comparison to previously reported Al³⁺ sensing fluorescent probes.

Fig. 4 Fluorescence confocal microscopy images of the MO3.13 cells stained with probe 2 (1 μM) for 30 minutes at 60× oil magnification. Images (a) and (b) represent the control experiment carried out in the absence of Al³⁺ in the media. Images (c) and (d) represent the cells pre-incubated with a 1 μM solution of Al³⁺ for 30 minutes before introducing probe 2. The cells were excited using a 561 nm laser line and the emission was recorded in the 580–700 nm range.

In conclusion, flavonoid derivative 2 was synthesized in good yield, which exhibited excellent selectivity towards Al³⁺. Probe 2 exhibited a large Stokes shift (Δλ > 150 nm) due to Al³⁺ binding enhanced ICT. Bright red emission (λ_em ≈ 640 nm) could be generated upon binding with Al³⁺ due to the formation of the 2-Al complex, with a 1 : 1 ligand-to-metal ratio. Very good response towards Al³⁺, while being silent to other metal ions, indicated that probe 2 could be a potentially useful sensor for tracking Al³⁺ concentrations in biological cells.

We acknowledge the Coleman endowment from the University of Akron. We also thank Dr Leah Shriver from the University of Akron for the generous gift of the MO3.13 cell line, Dr Michael Konopka from the University of Akron for assistance in bio-imaging, and Nicolas Alexander for providing mass spectrometry data.

Conflicts of interest

There is no conflict of interest to declare.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c9cc02322d

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