A novel BODIPY-Schiff base-based colorimetric and fluorometric dosimeter for Hg²⁺, Fe³⁺ and Au³⁺

Abstract

A novel Schiff base-based multi-target dosimeter for Hg²⁺, Fe³⁺ and Au³⁺ has been designed and synthesized. Upon addition of Hg²⁺, Fe³⁺ and Au³⁺ to the aqueous solution of compound BODIPY-TRIA, the dosimeter gave a rapid fluorescence response and displayed an obvious fluorescence enhancement with a blue-shift. Meanwhile, a sharp color change from purple to pale yellow occurred, which was readily detected by the naked eye. The hydrolysis of the Schiff base promoted by Hg²⁺, Fe³⁺ and Au³⁺ has been discussed, and the possible mechanism was confirmed by ¹H NMR and MS studies. The dosimeter showed high sensitivity (10 nM for Au³⁺), good stability, and excellent selectivity for Hg²⁺, Fe³⁺ and Au³⁺ with interference by other metal ions.

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1. Introduction

Selective sensing and monitoring of heavy metal ions is highly important since these metal ions have strong influence in chemical, biological and environmental sciences.¹ Significant effort has been made by scientists toward the development of highly sensitive dosimeters for metal ion recognition.^2,3 Particularly mercury (Hg²⁺) and iron ions (Fe³⁺) are of significant interest because these ions play vital roles in living systems. For example, accumulation of mercury in the human body can cause a wide variety of diseases such as prenatal brain damage, serious cognitive and motion disorders.^4–6 On the other hand, Fe³⁺ ions is an essential element in the human body, it provides the oxygen-carrying capacity of heme and acts as a cofactor in many enzymatic reactions.^7–9 However, high doses of iron ions are dangerous and can be toxic because of their ability to promote oxidation of lipids, proteins and other cellular components.^10,11

So far, several approaches including electrochemical, atomic absorption and chromatographic methods have been explored.^12–14 Comparing to other techniques, fluoresent chemosensors have attracted considerable interests because of their outstanding characteristics, such as high sensitivity, easy visualization, short response time and easy operation.^15,16 To date, a number of fluorescent chemosensors for Fe³⁺ and Hg²⁺ ions have been reported. Most of them are based on different fluorophores that included perylene bisimide,¹⁷ rhodamine,¹⁸ naphthalimide,¹⁹ boradiazaindacene (BODIPY)^20,21 and so on. Among these fluorophores, BODIPY based chemosensors have received tremendous attentions in recent years duo to their advantageous characteristics: sharp absorption with high intensity in visible to near infrared spectroscopy region, high fluorescence quantum yields and tunable redox properties.^22,23 So far, a lot of BODIPY-based fluorescence sensors for the detection of all kinds of ions, such as Hg²⁺, Cu¹⁺, Fe³⁺, Ca²⁺ and proton were developed.^24–27 For example, Xiao and his coworkers have reported a highly sensitive fluorescence dosimeters for Hg²⁺ determination and the dosimeter could be applied to detecting Hg²⁺ in living cells.²⁸ Like BODIPY-based dosimeters for Hg²⁺ ions,²⁹ many BODIPY-based fluorescent sensors for Fe³⁺ ions have been reported.^30,31 However, most of these fluorescent Fe³⁺ sensors are based on fluorescence quenching mechanisms. The BODIPY-based sensors for Fe³⁺ ions with increase in fluorescence were rare. Recently, BODIPY-based fluorescent sensors for Au³⁺ ions have also attracted the attentions of people. Several BODIPY-based fluorescent sensors for Au³⁺ ions have been reported,^32–34 which responded selectively to Au(III) ions through an irreversible C=N bond hydrolysis reaction. Most of these dosimeters were developed for sensing only one kind of ion, a few of dunal-channel fluorescent chemodosimeters were developed.^35,36

Recently, a new design concept of “single sensor for multiple targets” for chemosensors gets more and more attentions. Obviously, the ability to detect more than one target at the same time with multiple signal channels can shorten the analytical processing time and potentially reduce the cost. Inspired by these concepts, herein we have designed and synthesized a novel Schiff base-based BODIPY-aminophenol dosimeter BODIPY-TRIA for detecting Fe³⁺, Hg²⁺ and Au³⁺ ions in aqueous solution. The dosimeter exhibits weak fluorescence due to quenching by intramolecular charge transfer (ICT) and the non-radiative decay from –C=N isomerization and rotation. However, when Fe³⁺, Hg²⁺ and Au³⁺ ions were added the hydrolysis of Schiff base promoted by Hg²⁺, Fe³⁺ and Au³⁺ block the ICT process and result in considerable fluorescence enhancement in dosimeter BODIPY-TRIA with high selectivity and sensitivity for Hg²⁺, Fe³⁺ and Au³⁺ ions. As shown in Fig. 1, the mechanism was based on the hydrolysis reaction of Schiff base promoted by Hg²⁺, Fe³⁺ and Au³⁺, respectively.

Fig. 1 Structure of dosimeter BODIPY-TRIA.

2. Results and discussion

2.1 Synthesis of dosimeter BODIPY-TRIA

Compound BODIPY-TRIA was synthesized via simple steps, as depicted in Scheme 1. The important intermediate BODIPY-1 was prepared according to the published method. Next, it was formylated by reaction of BODIPY-1 with DMF and POCl₃ at 50 °C under a nitrogen atmosphere. Then, the formylated compound BODIPY-1 was used in the next reaction without further more purification. Compound BODIPY-OH was prepared by stirring a mixture of BODIPY-1 and 4-aminophenol in ethanol at 80 °C for 1 h, it was purified by silica gel column chromatography. At last, the target compound BODIPY-TRIA was prepared by stirring a mixture of compound BODIPY-OH (0.59 mmol), 3-butyn-1-ol (0.71 mmol), sodium ascorbate (0.023 mmol), and cupric sulfate (0.019 mmol) in a mixture of chloroform, ethanol and water (v/v/v = 12 : 3 : 2) at room temperature for 48 h. At the same time a 1,2,3-triazoles generated in the compound. As far as we know compounds containing 1,2,3-triazoles have shown a broad spectrum of biological activities such as antibacterial, herbicidal, fungicidal, antiallergic, and anti-HIV. The MS data, ¹H NMR and ¹³C NMR spectra provided in ESI† confirmed the successful synthesis of compound BODIPY-TRIA.

Scheme 1 Synthesis of compound BODIPY-TRIA.

2.2 Absorption spectra of dosimeter BODIPY-TRIA as a function of Hg²⁺, Fe³⁺ and Au³⁺ concentration

The absorption spectra of BODIPY-TRIA was recorded in THF–H₂O (1 : 9 v/v) at pH = 7.2, 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid) buffer, NaCl was used the supporting electrolyte and 0.1 M NaOH was used to adjusted pH values. Meanwhile, the absorption spectra of BODIPY-TRIA to metal ions like Li⁺, Na⁺, Ba²⁺, Mg²⁺, Al³⁺, Ca²⁺, Mn²⁺, Fe²⁺, Co²⁺, Ni²⁺, Zn²⁺, Ag⁺, Cd²⁺, Hg²⁺, Pb²⁺ and Cu²⁺ were tested. The dosimeter showed an intensity absorption bands at 495 nm in the buffer solution. No obvious changes was obtained, except for Hg²⁺, Fe³⁺ and Au³⁺.

The detection of Hg²⁺ by BODIPY-TRIA was studied by absorption spectra. Fig. 2-left shows the change in absorption of dosimeter BODIPY-TRIA in THF/H₂O solution as a function of time. The figure shows that the absorption bands at 495 nm display an continuous increase while bands at 525 nm gradually decrease, suggesting interconversion of two species which we attribute to the free BODIPY-TRIA and the Hg²⁺ ion-induced product, respectively. The ratio of absorption at 495/525 nm as a function of time is plotted in the insets figures. Dosimeter BODIPY-TRIA showed different response time for Fe³⁺ and Hg²⁺. The same results can be obtained from the measurement of Au³⁺. A similar trend of absorption spectra was observed with Fe³⁺. But here two new points at 314 and 362 nm were obtained which were no produced in compound BODIPY-TRIA with Hg²⁺ and Au³⁺. Moreover, no such significant change in the spectra of BODIPY-TRIA was observed with other tested metal cations.

Fig. 2 Absorption spectra of dosimeter BODIPY-TRIA with Hg²⁺ (left) and Fe³⁺ (right) in THF/H₂O (1 : 9, v/v) buffered with 10 mM HEPES ([BODIPY-TRIA] = 2 μM). Insets: absorption as a function of time of the dosimeter at 495/525 nm.

2.3 Kinetics of fluorescence enhancement of dosimeter BODIPY-TRIA with Hg²⁺, Fe³⁺ and Au³⁺

The detection kinetics of BODIPY-TRIA toward Hg²⁺, Fe³⁺ and Au³⁺ were studied by addition of an aliquot of Hg²⁺ stock solution (1 equivalent) to a solution of BODIPY-TRIA in HEPES buffer (10 mM with pH = 7.2) with 10% THF (Fig. 3). The emission intensity at 510 nm was continuously monitored for a duration of 60 s with an excitation at 495 nm. Fluorescence intensity appeared abruptly from a zero background. The signal enhancement was achieved in the first ca. 80 s and 300 s for Hg(II) ion and Fe(III) ion, respectively. When the Au³⁺ was added the signal enhancement was occurred at once. A slow but steady signal enhancement followed afterwards. Therefore, the different fluorescence response time of BODIPY-TRIA toward Hg²⁺, Fe³⁺ and Au³⁺ can be used to discriminate these three metal ions effectively. On the other hand, BODIPY-TRIA toward these three metal ions showed different emission intensity when excited under the same conditions and the fluorescence response were independent on their concentrations of these three ions. From Fig. 3, it shows the delayed response times of BODIPY-TRIA toward Fe³⁺, which was attributed to the different sensing mechanisms of BODIPY-TRIA toward Hg²⁺, Fe³⁺ and Au³⁺.

Fig. 3 Kinetics of fluorescence enhancement of dosimeter BODIPY-TRIA with Hg²⁺, Fe³⁺ and Au³⁺ THF–H₂O buffered with 10 mM HEPES (2 μM, λ_ex = 495 nm), NaCl was used the supporting electrolyte.

In addition, the absorption spectra of compound BODIPY-TRIA with Fe³⁺ showed two new points at 314 and 362 nm which can be designated as the characteristic absorption peaks of BODIPY-TRIA with Fe³⁺ (Fig. 4).

Fig. 4 Absorption spectra of BODIPY-TRIA with Hg²⁺, Au³⁺ and Fe³⁺ in THF–H₂O buffered with 10 mM HEPES (2 μM, λ_ex = 495 nm), NaCl was used the supporting electrolyte.

The induction period for an Fe³⁺ response is quite long compared to Hg²⁺ and Au³⁺. This could indicate the time barrier for a slower hydrolysis from pH changes. Therefore, the detection kinetics of BODIPY-TRIA in aqueous solution at different pHs were necessary. As shown in Fig. 5, compound BODIPY-TRIA shows a rapid emission enhancement with a low pH value. When the pH increases its emission intensity increases slowly. Moreover, a quick preliminary assay was took to look at the fluorescence of the dosimeter in aqueous solution (buffered with 10 mM HEPES) with Fe³⁺ (2 μM), and the fluorescence of the dosimeter after adding nearly 3 equiv. of H⁺ (Table 1). The results showed that the induction period for an Fe³⁺ response was almost consistent with 3H⁺.

Fig. 5 Fluorescence response kinetics of dosimeter BODIPY-TRIA at different pHs (2 μM, λ_ex = 495 nm), NaCl was used the supporting electrolyte and 0.1 M NaOH was used to adjusted pH values.

Table 1 Fluorescence properties of compound BODIPY-TRIA with Hg²⁺, Fe³⁺ and Hg²⁺ in THF/H₂O, at 298 K

Compounds	λab^a (nm)	λem^b (nm)	Δν1 − ν2^c (cm^−1)	Φ1^d
a Absorption maximum.b Emission maximum.c Stoke's shift = (1/λabs − 1/λem) × 10^7.d Fluorescence quantum yield, rhodamine 6G in ethanol (Φf = 0.95) was used as ref. 37.
BODIPY-TRIA	525	569	1430	0.01<
Dosimeter-Fe^3+	495	510	600	0.68
Dosimeter-Au^3+	495	510	600	0.70
Dosimeter-Hg^2+	495	510	600	0.70

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2.4 Hg²⁺, Au³⁺ and Fe³⁺ binding selectivity of dosimeter BODIPY-TRIA with naked eyes detection

A significant feature of the sensor was its high selectivity toward Hg²⁺, Fe³⁺ and Au³⁺ over other competitive metal ions. The selectivity of the sensor BODIPY-TRIA was evaluated by observing the changes of its fluorescence emission spectra (Fig. 6). Interference experiments were performed by mixing BODIPY-TRIA (2.0 μM) with 2.0 equivalents of ions like Li⁺, Na⁺, Ba²⁺, Mg²⁺, Al³⁺, Ca²⁺, Mn²⁺, Fe²⁺, Co²⁺, Ni²⁺, Zn²⁺, Ag⁺, Cd²⁺, Hg²⁺, Pb²⁺ and Cu²⁺ respectively before Hg²⁺ and Fe³⁺ ions were added. The dosimeter BODIPY-TRIA bonding with Hg²⁺, Fe³⁺ and Au³⁺ showed obvious color change (from violet to pale yellow Fig. 7) and strong fluorescence enhancement in the presence of various other metal ions. Dosimeter BODIPY-TRIA also showed good selectivity for the detection of Fe³⁺. The results from Fig. 6 and 7 indicated that the selectivity of dosimeter BODIPY-TRIA toward Fe³⁺ and Hg²⁺ was not affected by other metal ions. Meanwhile it is a good naked eyes detection reagent with color change from violet to pale yellow.

Fig. 6 Fluorescence spectra of dosimeter BODIPY-TRIA in THF/H₂O (1 : 9, v/v) buffered with 10 mM HEPES ([BODIPY-TRIA] = 2 μM, NaCl was used the supporting electrolyte) before and after addition of Fe³⁺, Hg²⁺, Li⁺, Na⁺, Ba²⁺, Mg²⁺, Al³⁺, Ca²⁺, Mn²⁺, Fe²⁺, Co²⁺, Ni²⁺, Zn²⁺, Ag⁺, Cd²⁺, Hg²⁺, Pb²⁺ and Cu²⁺ in aqueous media.

Fig. 7 The corresponding photographs of dosimeter BODIPY-TRIA after the addition metal ions (2.0 equivalent) in aqueous media (buffered with 10 mM HEPES [BODIPY-TRIA] = 2 μM, NaCl was used the supporting electrolyte) on excitation at 365 nm using UV lamp at room temperature.

2.5 Stability of dosimeter BODIPY-TRIA in aqueous solution at pH = 7.2

As we known, the pH value of normal living cells remains a ranges from 7.2–7.4.³⁸ Therefore, the stability of dosimeter in alkaline or neutral systems becomes very important, especially for dosimeter that was used to monitor the changes of ions concentration in living cells. In order to study the stability of dosimeter BODIPY-TRIA, we have studied the changes in emission intensity of dosimeter BODIPY-TRIA at 510 nm in aqueous solution at pH = 7.0–7.2. As shown in Fig. 8, the emission intensity of dosimeter BODIPY-TRIA remain a weak change after keeping the dosimeter in a aqueous solution at pH = 7.0–7.2 for 8 days. These results indicated that dosimeter BODIPY-TRIA has a good stability in alkaline and neutral aqueous solution. So, compound BODIPY-TRIA have potential application value as fluorescent dosimeter in monitoring the changes of ions concentration in normal living cells.

Fig. 8 Changes in emission intensity of dosimeter BODIPY-TRIA at 510 nm in aqueous solution at pH = 7.0–7.2 buffered with 10 mM HEPES, NaCl was used the supporting electrolyte.

2.6 Sensing modes based on ICT mechanism

As we discussed above, the dosimeter BODIPY-TRIA displayed very weak fluorescence emission in THF–H₂O solution without any ions, but when Hg²⁺, Fe³⁺ and Au³⁺ ions was added, it showed obvious enhancement of fluorescence intensity. Here, there are two important factors that quenched the emission of dosimeter. The one was the ICT process; the other was the radiative decay from –C=N isomerization and rotation. The possible mechanism of dosimeter BODIPY-TRIA with Hg²⁺, Fe³⁺ and Au³⁺ were showed in Fig. 9. When Hg²⁺, Fe³⁺ and Au³⁺ ions was added, an irreversible C=N bond hydrolysis reaction occurred, at the same time the ICT process was destructed and the radiative decay disappeared. The hydrolysis reaction of BODIPY-TRIA with Fe³⁺ in aqueous solution was induced by H⁺: Fe³⁺ + H₂O = Fe(OH)₃ + 3H⁺. In order to eliminate the effect of water, we have also studied the fluorescence spectra of BODIPY-TRIA with Fe³⁺ in THF without water. Compound BODIPY-TRIA in THF showed an Fe³⁺ fluorescence response, which was induced by the evolution of H⁺ from the possible reaction: BODIPY-TRIA + Fe³⁺ = [Fe–O–BODIPY-TRIA]²⁺ + H⁺ as shown in Fig. S17.† The protons generated by the substitution reaction of phenolic hydroxyl suppressed the ICT process in BODIPY-TRIA. So, the radiative decay disappeared.

Fig. 9 The possible mechanism of BODIPY-TRIA toward Hg²⁺, Au³⁺ and Fe³⁺ in THF–H₂O, buffered with 10 mM HEPES, NaCl was used the supporting electrolyte.

The coordination modes were further supported by ¹H-NMR studies. As shown in Fig. 10, after adding 1.0 equivalent of Hg²⁺, the proton signals of CH̲=N (at 9.38 ppm) and ph–OH̲ (at 8.50 ppm) shifted downfield. At the same time, the new peak of CH̲O (at 9.94 ppm) and ph–OH̲ (at 5.73 ppm) appeared, indicating that charge transfer from the benzene to the BODIPY groups was blocked and ICT disappeared. In addition, the bond breaking mechanism was further confirmed by the appearance of a peak at m/z 522.2480, assignable to [BODIPY-CHO]⁺ in the ESI/MS (Fig. 11). The possible bonding behavior of dosimeter BODIPY-TRIA with Fe³⁺ ions in THF–H₂O was also studied by ESI/MS. As shown in Fig. 12-left, the key intermediate compound [BODIPY-CHO]⁺ (m/z 522.2480) and [BODIPY-TRIA]⁺ (m/z = 613.2905) were obtained. Moreover, the peaks of ionic fragments in mass spectra [Fe–O–Ph–NH₂]⁻ (m/z = 160.8796) and [Cl–Fe–O–Ph–NH₂]⁻ (m/z = 197.8485) further proved the possible coordination modes of BODIPY-TRIA with Fe³⁺ (Fig. 12 right). From the results of the ¹H NMR spectroscopic titration and mass spectra, it can be proven that the reactions between BODIPY-TRIA, Hg²⁺, Fe³⁺ and Au³⁺ have took place.

Fig. 10 ¹H-NMR shifts of BODIPY-TRIA and with an addition of 1.0 equivalent of Hg²⁺ in DMSO recorded using a 300 MHz Bruker NMR spectrometer.

Fig. 11 Mass spectrum of compound BODIPY-TRIA with Hg²⁺.

Fig. 12 Mass spectrum of compound BODIPY-TRIA with Fe³⁺.

3. Conclusion

In conclusion, a novel Schiff base-based dosimeter for Hg²⁺, Fe³⁺ and Au³⁺ ions has been synthesized. Upon addition of Hg²⁺, Au³⁺ and Fe³⁺ ions to the aqueous solution of dosimeter BODIPY-TRIA, the hydrolysis reaction of Schiff base occurred immediately accompanying with a sharp color change from purple to pale yellow, which was readily detected by the naked eye. Dosimeter BODIPY-TRIA also gave a rapid fluorescence response and displayed obvious fluorescence enhancement with a blue shift. The possible mechanism has been confirmed by ¹H NMR and MS studies. Meanwhile, the dosimeter showed high sensitivity for Hg²⁺, Fe³⁺ and Au³⁺ (with a detection limit of 12 nM for Hg²⁺, 18 nM for Fe³⁺ and 10 nM for Au³⁺), good stability in alkaline and neutral systems, and high selectivity over other metal ions including Na⁺, K⁺, Ca²⁺, Mg²⁺, Co²⁺, Fe²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Mg²⁺, Pd²⁺, Pb²⁺, Mn²⁺ and Al³⁺.

4. Experimental

4.1 Materials and methods

¹H NMR and ¹³C NMR spectra were recorded on a Bruker DMX 300 NMR spectrometer and a Bruker ADVANCE 500 NMR spectrometer in CDCl₃ with tetramethylsilane (TMS) as internal standard. Chemical shifts are given in parts per million (ppm). Mass spectra were recorded on Agilent Technologies 6530 Accurate-Mass Q-TOF LC/MS. FT-IR spectra were recorded on a Nicolet 750 series in the region of 4000–400 cm⁻¹ using KBr pellets. All reagents used were purchased from Aldrich, Fluka or Alfa Aesar and used without further purification. All solvents used in spectroscopic measurements were of analytical grade. Reactions were monitored by thin layer chromatography using Merck TLC Silica gel 60 F254. Silica gel column chromatography was performed over Merck Silica gel 60.

UV-visible absorption spectra were determined on a Shimadzu UV-3600 spectrophotometer. Fluorescence spectra were measured on a HORIBA FL-4 Max spectrometer. 1 × 1 × 3 cm quartz cuvettes were used for absorption and emission spectral titration. The absorption and fluorescence spectral titrations of dosimeter BODIPY-TRIA were prepared in THF–H₂O, the solutions of guest cations were prepared in H₂O in the order of 10⁻⁶ M. Absorption and fluorescence spectra were recorded after mixing dosimeter BODIPY-TRIA and metal ions like Li⁺, Na⁺, Ba²⁺, Mg²⁺, Al³⁺, Ca²⁺, Mn²⁺, Fe²⁺, Co²⁺, Ni²⁺, Zn²⁺, Ag⁺, Cd²⁺, Hg²⁺, Pb²⁺, and Cu²⁺.

Hela cell were cultured in media RPMI 1640 supplemented with 10% PBS, 100 units per mL of penicillin and 100 units per mL of streptomycin at 37 °C in a humidified incubator. The culture media were replaced with fresh media every day. The cells were treated with 2 μM BODIPY-TRIA in culture media for 30 min at 37 °C and then washed with phosphate buffered saline (PBS) before experiment.

For the MTT assay, to derive viability/proliferation curves of long-term inhibitor-treated cells. This procedure is for cells in 96 well plates. A series of solutions of BODIPY-TRIA (100, 20, 4, 0.8, 0.16 μM) were cultured with cells for 24 h. Make a solution of 5 mg mL⁻¹ MTT dissolved in 10% PBS and filter sterilise through a 0.2 μM filter and stored at 2–8 °C. 5 hours before the end of the incubation, add 20 μL of MTT solution from step one to each well containing cells. Incubate the plate at 37 °C for 4–5 hours. Remove media with needle and syringe. Add 200 μL of DMSO to each well and pipette up and down to dissolve crystals. Put plate into the incubator for 5 min to dissolve air bubbles. Transfer to plate reader and measure absorption at wavelength 570 nm. Each test was repeated at least four times. The data was processed using the modified Käber equation. The results showed that compound BODIPY-TRIA has low toxicity.

4.2 Synthesis

The detailed synthesis methods of compound CHO-Br and compound CHO-N₃ were in the ESI.† The synthetic routes adopted for preparation of compound BODIPY and BODIPY-TRIA were shown in Scheme 1.

Compound diethyl 3,5-dimethyl-1H-pyrrole-2,4-dicarboxylate was prepared according to the literature.³⁹

Compound 2,4-dimethylpyrrole. A mixture of diethyl 3,5-dimethyl-1H-pyrrole-2,4-dicarboxylate (3.00 g, 14 mmol) and potassium hydroxide (4.06 g, 72 mmol) in ethylene glycol (10 mL) was heated at 160 °C for 4 h. After completion of the reaction, the solution was extracted with chloroform, dried with anhydrous sodium sulfate and then concentrated to give the crude product. The crude product was distilled in vacuum to give the pure product 0.96 g, yield: 81%. The ¹H NMR data are identical to the data in the literature.⁴⁰

Compound CHO-Br. A mixture of 4-(2-bromoethyloxy)benzaldehyde (26.3 mmol, 3.2 g) and 1,3-dibromopropane (0.078 mol, 15.6 g) in acetone (10 mL) was heated at 62 °C for 8 h. After completion of the reaction, water (80 mL) and dichloromethane (100 mL) was added, the organic phase was washed with water and dried over Na₂SO₄. The crude product was purified by silica gel column chromatography using dichloromethane as eluent to give an oily compound CHO-Br 4.09 g, yield: 76%. ¹H NMR (CDCl₃, ppm): δ 9.86 (s, 1H, PhCH̲O), 7.82–7.80 (d, 2H, J = 6 Hz, Ph), 6.99–6.97 (d, 2H, J = 6 Hz, Ph), 4.13–4.09 (t, 2H, J = 6 Hz, Ph–CH̲₂–CH₂–), 3.54–3.49 (t, 2H, J = 9 Hz, –CH̲₂–N₃), 2.11–2.02 (m, 2H, CH₂CH̲₂CH₂). ¹³C NMR (CDCl₃, ppm): δ 190.32, 163.35, 131.59, 129.77, 114.43, 68.34, 64.64, 47.71, 36.03, 28.24. TOF-MS-ES: m/z. Calculated: ([M + Na])⁺ = 243.0, found: 243.0.

Compound CHO-N₃. A mixture of 4-(2-bromoethyloxy)benzaldehyde CHO-Br (10.3 mmol, 2.5 g) and sodium azide (15 mmol, 2.01 g) in DMF (10 mL) was stirred at room temperature for 24 h. After completion of the reaction, dichloromethane (100 mL) and water (100 mL) was added and the organic phase was washed with water and dried over Na₂SO₄. Removing of the excess solvent a colorless oily compound got 2.06 g, yield: 98%. ¹H NMR (CDCl₃, ppm): δ 9.86 (s, 1H, PhCH̲O), 7.82–7.80 (d, 2H, J = 6 Hz, Ph), 6.99–6.97 (d, 2H, J = 6 Hz, Ph), 4.13–4.09 (t, 2H, J = 6 Hz, Ph–CH̲₂–CH₂–), 3.54–3.49 (t, 2H, J = 9 Hz, –CH̲₂–N₃), 2.11–2.02 (m, 2H, CH₂CH̲₂CH₂). ¹³C NMR (CDCl₃, ppm): δ 190.46, 163.48, 131.78, 129.96, 114.64, 68.83, 65.54, 34.73, 31.92, 29.52. TOF-MS-ES: m/z. Calculated: ([M + Na])⁺ = 228.0, found: 228.0.

Compound BODIPY-1. 2,4-Dimethyl pyrrole (2.01 g, 21 mmol) and compound CHO-N₃ (2.15 g, 10.5 mmol) were dissolved in dry dichloromethane (300 mL) under a nitrogen atmosphere. One drop of trifluoroacetic acid (TFA) was added to the solution. The reaction mixture was stirred at room temperature for 24 h. After disappearance of the aldehyde, a solution of DDQ (0.013 mmol, 2.6 g) in dichloromethane was added. Absolute triethylamine (10 mL) was then added to the mixture. At last BF₃·OEt₂ (10 mL) was added dropwise at 0 °C. The mixture was stirred 2 h again and then the reaction mixture was washed with water for several times and extracted with dichloromethane. The separated organic phase was dried over Na₂SO₄. The solvent was evaporated and the residue was purified by silica gel column chromatography using dichloromethane as eluent to obtain a red solid 2 g, yield: 25%. ¹H NMR (CDCl₃, ppm): δ 7.18–7.15 (d, 2H, J = 9 Hz, c), 7.01–7.98 (d, 2H, J = 6 Hz, c′), 5.93 (s, 2H, g), 4.13–4.09 (t, 2H, J = 6 Hz, Ph–O–CH̲₂–CH₂–), 3.57–3.53 (t, 2H, J = 6 Hz, –CH̲₂–N₃), 2.55 (s, 6H), 2.12–2.07 (m, 2H, CH₂CH̲₂CH₂), 1.43 (s, 6H). ¹³C NMR (CDCl₃, ppm): δ 190.46, 163.48, 131.78, 114.62, 68.83, 65.54, 34.73, 31.92, 29.52. TOF-MS-ES: m/z. Calculated: ([M + Na])⁺ = 446.2, found: 446.2, ([M])⁺ = 424.2, found: 424.2.

Compound BODIPY-CHO-N₃. A mixture of DMF (6 mL) and POCl₃ (6 mL) was stirred in an ice bath for 10 min. After removing the ice bath and warming to room temperature, BODIPY-1 (0.50 g, 0.80 mmol) in 1,2-dichloroethane (30 mL) was added, which was then heated for 2 h at 50 °C. When the reaction mixture was cooled to room temperature, it was slowly poured into a saturated NaHCO₃ aqueous solution (100 mL) with an ice bath. The mixture was warmed to room temperature, further stirred for 30 min, and washed with water twice (50 × 2 mL). The organic layers were collected and combined, dried over anhydrous Na₂SO₄ and evaporated in vacuo. The crude product was used in the next reaction without purification.

Compound BODIPY-OH. A mixture of compound BODIPY-CHO-N₃ (0.50 g, 1.1 mmol) and 4-aminophenol (0.14 g, 1.3 mmol) in ethanol (30 mL) was heated at 80 °C for 30 min. After completion of the reaction, water (50 mL) and dichloromethane (100 mL) was added, the organic phase was washed with water and dried over Na₂SO₄. The crude product was purified by silica gel column chromatography (CH₂CH₂/methanol = 9 : 1). A purple solid got 0.48 g, yield: 80%. ¹H NMR (CDCl₃, ppm): δ 9.38 (s, 1H), 8.48 (s, 1H), 7.34–7.31 (d, 2H, J = 9 Hz), 7.18–7.15 (d, 2H, J = 9 Hz), 7.11–7.08 (d, 2H, J = 9 Hz), 6.78–6.75 (d, 2H, J = 9 Hz), 6.31 (s, 1H), 4.16–4.13 (m, 2H, OCH̲₂CH₂CH₂N₃), 3.58–3.54 (t, 2H, OCH₂CH̲₂CH₂N₃), 2.52 (s, 6H), 2.06–2.02 (m, 2H, CH₂CH₂CH̲₂N₃), 1.69 (s, 3H), 1.44 (s, 3H). ¹³C NMR (CDCl₃, ppm): δ 159.01, 158.72, 157.50, 155.62, 154.17, 151.92, 145.30, 144.60, 128.92, 122.01, 121.60, 115.72, 114.80, 64.27, 56.10, 47.76, 29.24, 28.33, 27.93, 14.42, 13.51, 11.99. TOF-MS-ES: m/z. Calculated: ([M + H])⁺ = 543.3, found: 543.3.

Compound BODIPY-TRIA. A solution of compound BODIPY-OH (0.59 mmol, 0.32 g), 3-butyn-1-ol (0.71 mmol, 0.05 g), sodium ascorbate (0.023 mmol, 0.0045 g), and cupric sulfate (0.019 mmol, 0.003 g) in a mixture of chloroform, ethanol and water (v/v/v = 12 : 3 : 2) was stirred at room temperature for 48 h. The solvents were removed under reduced pressure and the crude product was purified by column chromatography (CHCl₃/methanol = 10 : 1). A purple solid got 0.31 g, yield: 65% (mp 118–120 °C). FT-IR (KBr) cm⁻¹: 3424 (νNH); 2964–2870 (νCH); 1696 (νasN–C=O), 1659 (νsN–C=O), 1551 (νN–C=O). ¹H NMR (CDCl₃, ppm): δ 8.40 (s, 1H), 7.42 (s, 1H), 7.19–7.16 (d, 2H, J = 9 Hz), 7.03–6.98 (m, 4H), 6.87–6.84 (d, 2H, J = 9 Hz), 6.05 (s, 1H), 4.61–4.57 (m, 2H, PhOCH̲₂CH₂CH₂–), 4.06–4.02 (t, 2H, PhOCH₂CH₂CH̲₂–), 3.95–3.91 (t, 2H, HOCH̲₂CH₂C–), 2.98 (s, CH₂OH̲ 1H), 2.93 (m, 2H, HOCH̲₂CH₂CH₂–OPh), 2.85 (s, 3H), 2.57 (s, 3H), 2.45–2.41 (m, 2H, CH₂CH̲₂CH₂–OPh), 1.67 (s, 3H), 1.44 (s, 3H). ¹³C NMR (CDCl₃, ppm): δ 192.13, 165.26, 165.2, 156.69, 144.47, 143.12, 133.41, 131.47, 128.70, 122.51, 116.43, 78.85, 66.34, 31.10, 30.59. TOF-MS-ES: m/z. Calculated: [M + H]⁺ = 613.2905, found: 613.2901, [M + Na]⁺ = 635.2724, found: 635.2721.

Acknowledgements

We sincerely acknowledge Miss H. L. Liu and Mr Y. Yang for the confocal fluorescence images. This work was Financially supported by the Fundamental Research Funds for the National Natural Science Foundation of China (No. 61178057) and the Central Universities (No. CXLX12_0085).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra15546k

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