A BODIPY based probe for the reversible “turn on” detection of Au(III) ions

A new “turn on” fluorescent probe for the rapid and selective detection of Au3+ ions over other metal ions was developed. The probe design was constructed on a BODIPY-2-aminopyridine skeleton showing a weak fluorescence emission signal which increased substantially after the coordination of Au3+ ions. The probe displayed remarkable sensing performances such as a low limit of detection (17 nM), a short response time (<1 min), and ability in a wide range of pH’s (6–11). The designed probe was found to have 2:1 coordination stoichiometry according to Job’s plot analysis and most importantly to interact reversibly with Au3+ ions.


General methods
All reagents were purchased from Sigma-Aldrich and used without further purification. 1H NMR and 13 C NMR were measured on a VNMRJ 600 nuclear magnetic resonance spectrometer (Varian Inc., Palo Alto, CA, USA).The mass analysis was conducted with Thermo Q-Exactive Orbitrap device (Thermo Fisher Scientific Inc., Waltham, MA, USA).The melting point was determined by using the Electrothermal Melting Point Apparatus 9200.UV-vis absorption and fluorescence emission spectra were obtained using a Shimadzu 1900i spectrophotometer and Varian Cary Eclipse fluorescence spectrophotometer (Varian Inc.), respectively.Quantum yield measurements were conducted with Hamamatsu Quantaurus-QY Absolute PL quantum yield spectrometer.The pH values were adjusted by using the HI-8014 (HANNA) pH meter.

Fluorescence measurements
The stock solution of BOD-AP (1 mM) was prepared in ethanol and diluted to proper concentrations for each measurement by using different ratios of 0.1 M phosphate buffer/ethanol (total sample volume = 2 mL).The stock solutions of metal ions (20 mM) were prepared by dissolving their nitrate and chloride salts in deionized water.For each measurement, proper volumes from the stock solution of metal ions were added to a probe solution (10 µM, 2 mL) that was contained in a quartz cuvette with 10 mm path length.Upon excitation at 460 nm, the fluorescence emission spectra were collected between 480-700 nm.In all measurements, the slit width for both excitation and emission were kept at 2.5 nm.All measurements were conducted in triplicate at least.

Synthesis of BOD-AP
Meso-chloromethyl-BODIPY (meso-BOD-Cl) was prepared as previously reported [31].The probe molecule, BOD-AP, was synthesized using a slightly modified version of protocol reported by Xu et al. [32].To a solution of meso-BOD-Cl (100 mg, 0.34 mmol) in tetrahydrofuran (50 mL) 2-aminopyridine (32 mg, 0.34 mmol), and K 2 CO 3 (64 mg, 0.4 mmol) were added, and the solution was stirred for 5 h under nitrogen atmosphere at reflux temperature.The progress of the reaction was monitored by TLC and cooled to room temperature when all starting materials consumed.THF was removed under reduced pressure and resulting mixture was extracted with dichloromethane (3 × 100 mL).The collected organic layers were combined and washed with brine, then dried over Na 2 SO 4 , filtered and solvents removed under reduced pressure.The crude mixture purified by silica gel chromatography (Hexane:DCM, 3:1, v/v) and title compound, BOD-AP, was obtained as orange solid (84.3 mg, 70%).Mp: 203-205 °C. 1

Results and discussion
The title compound, BOD-AP was obtained by following the synthetic route outlined in Scheme 1.The meso-BOD-Cl was prepared as previously reported [31] and treated with the commercially available 2-aminopyridine to give BOD-AP as an orange solid with a good reaction yield (70%).The structure of BOD-AP was confirmed by 1 H and 13 C NMR spectroscopy and HRMS analysis.
The aromatic protons resonated in the range of 8.11 ppm and 6.10 ppm in the 1 H NMR spectra proved the presence of a pyridine ring on the probe structure.The NH proton showed a broad singlet peak centred at 5.18 ppm, and the methylene protons resonated at 4.79 ppm which is split into a doublet due to the low exchange rate of proton on the neighbouring nitrogen proton [33,34].The obtained spectral profile clearly demonstrated that the nucleophilic substitution Scheme 1. Synthetic route to BOD-AP. of 2-aminopyridine to the BODIPY core took place at the 3-methyl position instead of at the meso position.As reported in the literature, four methyl groups of meso substituted products must have appeared as two singlets (each peak 6H) [32].However, in obtained 1 H NMR spectra, the presence of four singlet peaks for methyl groups (each centred at 2.58 ppm, 2.54 ppm, 2.42 ppm, 2.37 ppm) was clear evidence of the position that the nucleophile substituted at 3-position.
The optimization studies of sensing conditions for Au 3+ ion detection were performed by the investigation of the effect of several parameters including a solvent/buffer type, water ratio, pH, and time.Owing to the hydrophobic core structure of BOD-AP, the addition of an organic cosolvent was required to prevent any aggregation in the solution.In this regard, EtOH-H 2 O (1/1, v/v) and CH 3 CN-H 2 O (1/1, v/v) mixtures buffered at pH 7.0 by HEPES (HEP), phosphate buffer (PB), or PBS were screened.As shown in Figure 1a, the addition of Au 3+ (2 equiv.) to the BOD-AP solutions (10 µM in EtOH-H 2 O and CH 3 CN-H 2 O mixtures buffered at pH 7.0 by phosphate buffer (0.1 M)) resulted in the same fold increase in the fluorescence signal at 511 nm.Because of its low toxicity and commercial availability, ethanol was selected as a cosolvent to perform further fluorescence measurements.In addition, the effect of the water percentage was investigated where 1:1 (v/v) EtOH/H 2 O ratio was determined as optimal condition (pH 7.0, 0.1 M phosphate buffer) (Figure 1b).
The receptor unit is prone to protonation under acidic conditions (pH 2-5).Therefore, BOD-AP exhibited a strong fluorescence signal (protonation cancels the effect of PET quenching) at this pH range and a negligible increase was observed in the presence of Au 3+ ions (2 equiv.).At physiological or higher pH values, the receptor unit enables the quench fluorophore over PET mechanism and the addition of Au 3+ ions, thus resulting in a remarkable increase in fluorescence emission (Figure 1c).After the careful examination of all parameters, the optimal sensing conditions were established as 10 µM BOD-AP in 0.1 M phosphate buffer/EtOH (pH 7.0, 1:1, v/v).The spectral characteristics of BOD-AP were investigated by using UV-vis and fluorescence spectroscopy.BOD-AP has an absorption band at 497 nm which remained unchanged after the addition of Au 3+ ions (Figure 2).As expected, the probe showed a very weak fluorescence emission (F F = 0.002) centred at 509 nm due to the PET quenching process.Upon the addition of increasing concentrations of Au 3+ ions (0.2 to 20 µM) a linear increase in fluorescence emission with a very small red shift (λ em = 511 nm) was observed (Figure 3).The saturation point in the fluorescence was achieved by the addition of 2 equiv. of Au 3+ with a 29-fold signal enhancement (F F = 0.88).The fluorescence titration experiment revealed that the minimum detectable amount of Au 3+ ions is 17 nM based on the signal-to-noise ratio (S/N = 3) (Figure S1).It is also important to note that the spectral response of the probe towards Au 3+ ions was extremely fast, reaching maximum levels within only a min (Figure 1d).To evaluate the selectivity profile of the probe, excess amounts of other metal ions (including Na + , K + , Li + , Ca 2+ , Mg 2+ , Ba 2+ , Ag + , Hg 2+ , Zn 2+ , Pb 2+ , Ni 2+ , Cd 2+ , Fe 2+ , Cr 3+ , Ce 3+ , and Al 3+ ) were added to the BOD-AP solution (10 μM) and then the changes in the fluorescence signals were recorded.BOD-AP did not show any significant response towards the addition of excess amounts (100 μM, 10 equiv.) of any other metal cations (Figure 4a).
Moreover, the possible interference of these species was investigated by the addition of Au 3+ ions (2 equiv.) to the solution of BOD-AP (10 μM) treated by an excess of other metal cations (100 μM, 10 equiv.).The performed study resulted in only negligible changes in the fluorometric response of the probe to Au 3+ ions (Figure 4b).These results clearly demonstrate that the probe allows the selective detection of Au 3+ ions in the presence of any other competitive species.Furthermore, to investigate the applicability of the sensing system in the more complex sensing media, the combination of the potentially interfering metal cations was added into the probe solution.The probe remained silent in the presence of these cations and signal enhancement was only observed upon after the addition of Au 3+ ions (2 equiv.)(Figure S4).
The mechanism and stoichiometry of the sensing event were explored by a reversibility experiment and Job's plot analysis (Scheme 2).As shown in Figure S2, the treatment of BOD-AP with 2 equiv. of Au 3+ ions reduced the effect of the PET mechanism, and a strong fluorescence emission band at 511 nm was observed.To reverse this, Na 2 S (2 equiv.)which has a high affinity to Au 3+ ions was added to the BOD-AP + Au 3+ solution.The immediate decrease in the fluorescence signal down to its original intensity confirmed the reversibility of the sensing event.In addition, Job's plot analysis based on fluorescence titration experiment results revealed the 2:1 stoichiometry between BOD-AP and Au 3+ ions for the complexation process (Figure S3).In conclusion, synthesis, characterization, and spectral behaviours of a new fluorescent chemosensor that enables the detection of Au 3+ ions in an aqueous environment were reported.The probe displayed a rapid (<1 min) "turn on" fluorescence response towards Au 3+ ions with high selectivity and sensitivity (17 nm LoD).In addition, the designed probe showed 2:1 coordination stoichiometry and most importantly reversible interaction with gold ions.