An efficient pyrene-Schiff base fluorescent sensor PySb was synthesized and evaluated for its fluorescence response to metal ions. Sensor PySb exhibits an “off-on-type” mode with high selectivity to Zn 2+ and Al 3+ in ethanol (470 nm) and in dimethyl sulfoxide (458 nm) respectively. The originally non-fluorescent PySb, due to photo-induced electron transfer (PET) from imine moiety, is turned on after binding with the cations. The stoichiometric ratio between PySb and Zn 2+ is 1:2; moreover, the limit of detection (LOD) and bonding constant were 2.39 × 10 -8 M and 2 × 10 9 M -1 respectively, as obtained from titration experiments.
Zinc ion has been known as the second most abundant transition metal ion in human body. While most zinc ions are tightly bound in proteins, a small amount of free Zn2+ ions is presented in various human tissues [
Schiff base moieties have been extensively applied as ionophores in fluorescent sensors due to their ability to form coordination complexes with metal ions [
In this work, a novel fluorescent sensor PySb was synthesized and characterized. Fluorescent pyrene moiety was selected as fluorophore to enhance sensitivity. 2-amino-2-(hydroxymethyl)propane-1,3-diol was chosen as part of binding moiety. By forming imine via reaction with 2-hydroxybenzaldehyde, the three alcoholic -OH along with phenolic -OH should be able to interact with multiple metal ions [
All the reagents and solvents were purchased from commercial sources and were used without further purification. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) was dissolved in water (2.5 × 10−4 M) which was used as buffer solution. Nitrate salts of Na+, K+, Ca2+, Cu2+, Ni2+, Co2+, Zn2+, Pb2+, Fe3+, Cr3+ and Al3+ were dissolved in the HEPES(aq) buffer to prepare their stock solutions (10−2 M). PySb was dissolved in ethanol, DMF, or DMSO; the concentration was 2 × 10−5 M. 1H NMR spectra with the chemical shifts reported in ppm were recorded on a Bruker AMX-600 MHz spectrometer, using tetramethylsilane (TMS) as an internal standard. Elemental analysis (EA) of carbon, hydrogen, and nitrogen were measured on a Heraus CHN-Rapid elemental analyzer. The UV-Vis absorption spectra were recorded on a Jasco V-550 spectrophotometer using a quartz cuvette (path length = 1 cm). Photoluminescence (PL) spectra were recorded on a fluorescence spectrophotometer (Hitachi F-4500) in mixtures of water with appropriate organic solvents.
To a sodium carbonate solution (0.6 M, 2.8 ml) in 50-mL glass reactor was added with 5-bromo-2-hydroxybenzaldehyde (1: 0.264 g, 1.32 mmol), 2,7-bis(4,4,5, 5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrene (2:0.2 g, 0.44 mmol), and tri-tert-
Scheme 1. Synthesis of fluorescent sensor PySb.
butylphosphonium tetrafluoroborate (0.032 g, 0.11 mmol). Tris(dibenzylideneacetone)dipalladium(0) (Pd2(dba)3: 57 mg, 0.06 mmol) was dissolved in THF (18 ml) and added into the reactor by a syringe. The mixture was degassed by the freeze-pump-thaw cycle for three times. Next, the mixture was stirred at 65˚C for two days; then it was poured into water and extracted with dichloromethane. The combined organic layer was concentrated under reduced pressure, poured into n-hexane to obtain precipitate as crude product. The crude product was purified by flash column chromatography (eluent: dichloromethane) to afford 5,5’-(pyrene-2,7-diyl)bis(2-hydroxybenzaldehyde) (3) (yield: 0.11 g, 56.4%). 1H NMR (600 MHz, DMSO-d6, TMS, 25˚C): δ 10.80 - 11.03 (s, 2H, -CHO), 10.47 - 10.36 (s, 2H, -OH), 8.62 - 8.57 (s, 4H, Ar-H), 8.31 - 8.26 (s, 6H,Ar-H), 8.23 - 8.13 (d, 2H, Ar-H), 7.28 - 7.20 (d, 2H, Ar-H).
A mixture of 3 (0.15 g, 0.39 mmol), 2-amino-2-(hydroxymethyl)propane- 1,3-diol (4:0.41 g, 3.9 mmol) and methanol (150 ml) was stirred at 65˚C for two days. Methanol was evaporated by a rotavapor. The crude product was washed by water and acetone several times to obtain PySb (yield: 0.20 g, 90.8%). 1H NMR (600 MHz, DMSO-d6, TMS, 25˚C): δ 14.79 - 14.64 (s, 2H, phenolic OH), 8.80 - 8.71 (s, 2H, CNH), 8.63 - 8.55 (s, 4H, Ar-H), 8.28 - 8.19 (s, 4H, Ar-H), 8.17 - 8.09 (s, 2H, Ar-H), 8.03 - 7.95 (d, 2H, Ar-H), 7.01 - 6.93 (d, 2H, Ar-H), 4.90 - 4.78 (s, 6H, OH), 3.71 - 3.63 (d, 12H, CH2). Anal. Calcd. for C38H36N2O8 (%): C, 70.36; H, 5.59; N, 4.32. Found: C, 69.47; H, 5.56; N, 4.20.
The new fluorescent sensor PySb was synthesized in two steps as shown in Scheme 1. First, the bis(2-hydroxybenzaldehyde) derivative (3) of pyrene was synthesized from 5-bromo-2-hydroxybenzaldehyde (1) and diboronate derivative of pyrene (2) via the Suzuki coupling reaction (
The absorption spectra of PySb with various metal ions in ethanol buffer solutions
are depicted in
PySb itself exhibits very weak fluorescence in ethanol buffer solution (
The binding stoichiometry between PySb and Zn2+ was determined by the Job plot. By plotting the fluorescence intensities versus the molar fraction of Zn2+, two regression lines are obtained and they intersect at about 0.66 (
To further investigate the sensitivity of this fluorescence enhancement, PL spectra of PySb were measured with increasing amount of Zn2+ (from 0.1 to 20 equivalents). As depicted in
For real-time application, it is important to evaluate the interference from other metal ions. As shown in
paramagnetic nature of Cu2+ [
In preparation of PySb solutions for fluorescence experiments, we observed fluorescence intensity changes of PySb in different solvents. PySb-Zn2+ exhibited stronger fluorescence intensity in ethanol than in DMF. However, ethanol was a poor solvent for PySb while DMF was a good solvent for PySb. Therefore, an experiment was carried out to investigate this interesting phenomenon. The variations of fluorescence spectra of PySb were monitored in mixture solvents of DMF and ethanol. As presented in
It is noteworthy that although ethanol is a poor solvent, ratiometric detection toward Zn2+ as discussed previously still shows good accuracy. The reason can be explained as following: The poor solubility of PySb is mainly attributed to its
pyrene core, whereas the binding site in 2-iminophenol group contains four polar -OH groups. Therefore, the binding ability toward Zn2+ is not greatly influenced in ethanol solution, and therefore data with good consistency and accuracy were obtained.
In
Next, we investigated the photophysical properties of PySb in DMSO solution. The absorption spectra [
generated strong fluorescence enhancement. Cr3+ also induced weak fluorescence enhancement but with a deeper blue color. Therefore, it appears that PySb can serve as Al3+ sensor in DMSO solution.
The sensitivity of PySb toward Al3+ in DMSO solution was investigated by titration experiment as depicted in
The formation of PySb-Zn2+ complex was further investigated by 1H NMR spectra in deuterated N,N-dimethylformamide (DMF-d7,
PySb-Zn2+ complex might involve an additional hydroxide ion (OH−). The disappearance of signals of alcoholic (h) and alcoholic protons (a) after addition of D2O confirms the presence of these exchangeable protons.
1H NMR spectra of PySb-Al3+ complex in DMSO (
An efficient fluorescent sensor PySb comprising of pyrene moiety as the fluorophore, benzene ring as the spacer, and 2-(hydroxymethyl)propane-1,3-diol as the ionophore was successfully synthesized and characterized. The PySb itself exhibited weak fluorescence due to PET mechanism; however, the fluorescence
was turned on by Zn2+ in ethanol solution (λem = 470 nm) and by Al3+ in DMSO solution (λem = 458 nm). The complexes of PySb-Zn2+ and PySb-Al3+ were further confirmed by 1H NMR spectra. The stoichiometric ratio between PySb and Zn2+ was 1:2, as obtained from the Job plot. Based on the titration experiment, limit of detection (LOD) and binding constant toward Zn2+ were 2.39 × 10−8 M and 2 × 109 M−1, respectively. The PySb-Zn2+ complex in DMF solution showed aggregation-induced emission enhancement with increasing content of ethanol. It can be utilized as a fluorescent sensor in a wide range of pH (3 - 11). Green emission of PySb (λem = 515 nm) was observed when pH was higher than 12 due to ICT mechanism. Current results indicate that PySb is a promising fluorescent “turn on” sensor for Zn2+ and Al3+ in ethanol and DMSO, respectively.
Authors are thankful to Ministry of Science and Technology, Taiwan for financial support through grant MOST 105-2221-E-006-250. This research was partially funded by the Headquarters of University Advancement at National Cheng Kung University, under the sponsorship of the Ministry of Education, Taiwan.
Hsu, P.-F. and Chen, Y. (2018) Synthesis of a Pyrene- Derived Schiff Base and Its Selective Fluorescent Enhancement by Zinc and Aluminum Ions. International Journal of Organic Chemistry, 8, 207-228. https://doi.org/10.4236/ijoc.2018.82016
Binding Constant
The binding constant can be calculated according to Benesi-Hildebrand equation by measuring the fluorescence intensity change upon addition of various concentrations of Zn2+. The equation is depicted as following [
Assuming 1:2 stoichiometry for association between PySb and Zn2+
1 F − F 0 = 1 Δ F = 1 F max − F 0 + 1 K a × ( F max − F 0 ) × 1 [ Zn 2 + ] 2
F: Observed fluorescence
F0: Fluorescence of free PySb
Fmax: Saturated fluorescence of PySb and [Zn2+] complex
Ka: Binding constant of PySb and [Zn2+]
As illustrated in
K a = A B = 2 × 10 9 M − 1
Limit of Detection (LOD)
Detection limit was calculated based on titration experiment, using the equation as following [
LOD = 3 σ m
σ = standard deviation of the blank solution, calculated from 10 blank solutions.
m = slope of fluorescence intensity versus Zn2+ concentration.
σ = 0.558
LOD = 3 × 0.558 7 × 10 7 = 2.39 × 10 − 8 M
The detection limit of PySb toward Zn2+ was 2.39 × 10 − 8 M, which is much higher than the World Health Organization guideline for maximum acceptable concentration in drinking water: 76 μM [