One-pot synthesis of a new rhodamine-based dually-responsive pH sensor and its application to bioimaging

Tetrahedron 70 (2014) 6974e6979

Contents lists available at ScienceDirect

Tetrahedron journal homepage: www.elsevier.com/locate/tet

One-pot synthesis of a new rhodamine-based dually-responsive pH sensor and its application to bioimaging Aifeng Liu a, b, Miaomiao Hong a, b, Wen Yang a, Shenzhou Lu c, Dongmei Xu a, b, * a

College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Ren-ai Road No.199, Suzhou Industrial Park, Suzhou, Jiangsu 215123, China b Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, Soochow University, Suzhou, Jiangsu 215123, China c College of Textile and Clothing Engineering, Soochow University, Suzhou, Jiangsu 215123, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 April 2014 Received in revised form 17 July 2014 Accepted 24 July 2014 Available online 31 July 2014

A new colorimetric and fluorescent sensor for the highly acidic pH was developed from rhodamine B, 1(2-aminoethyl)piperazine, and 4-chloro-7-nitro-2,1,3-benzoxadiazole. The sensor could be synthesized in one pot with an 82.5% yield, and for the first time N,N-diisopropylethylamine was found to be crucial for the rhodamine spirolactam formation. The sensor responded to pH rapidly, visibly, reversibly, highly selectively, and sensitively. From pH 7.77 to 2.03, the absorption and fluorescence intensity of the sensor increased by 285 and 50.3 folds, respectively. The pKa value based on the fluorescence titration was 2.87. Fluorescent imaging of living cells treated with the sensor in different pH media indicated that the sensor could provide intracellular pH information. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Rhodamine Colorimetric Fluorescent pH Sensor Bioimaging

1. Introduction It is well known that Hþ plays a key role in biological processes.1 Although the extreme acidity (pH<4) is fatal for the majority of living species, microorganisms including ‘acidophiles’, Helicobacter pylori, and enteric pathogens have evolved to live under such harsh conditions and may cause life-threatening diseases.2 Hence, measuring of the biological pH under highly acidic conditions is of great importance. Because of their high sensitivity, rapid response, operational simplicity, low cost, in situ and real time monitoring, and noninvasiveness, fluorescent sensors have attracted great attention in environmental and biological pH detection.3e6 Rhodamine and its derivatives have been widely used for fabricating fluorescence sensors due to their favorable spectroscopic properties, such as good photostability, high fluorescence quantum yield, relatively long absorption and emission wavelengths in the visible region, and especially, the great spectral changes induced by the opening and closing of the spirolactam ring.7,8 Hitherto, a lot of

* Corresponding author. Tel.: þ86 512 65882027; fax: þ86 512 65880089; e-mail address: [email protected] (D. Xu). http://dx.doi.org/10.1016/j.tet.2014.07.087 0040-4020/Ó 2014 Elsevier Ltd. All rights reserved.

rhodamine-based fluorescent sensors for metal ions9e16 and pH17e25 have been reported. The rhodamine-based sensors were usually synthesized by first esterification (or chloroformylation) then amidation, or direct amidation of the rhodamine units, perhaps followed by further reaction with aldehydes or other reagents. Hydrazine,26 ethylenediamine,27 tris-(2-aminoethyl) amine,28 and diethylenetriamine29 were often used for the amidation reaction, of which the conditions must be carefully controlled to ensure only those required among the multiple identical amino groups were reacted. While the amines like 1-(2-aminoethyl)piperazine (AEP) with different reactive amino groups can not only provide binding sites and linking groups but also facilitate the synthesis process. In our previous work, hydrazine was employed to group rhodamine B (RB) and 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl), producing a Cu2þ and Fe3þ sensor.30 To the best of our knowledge, the selectivity to analyte can be modulated through changing binding site and mode of connection in rhodamine-based sensors, and sometimes small structure modifications could confer great performance changes of the sensor. Bearing this in mind, herein, a new rhodamineenitrobenzoxadiazole (NBD) conjugated colorimetric and fluorescent sensor RPBD was constructed with AEP as the recognizing unit and simultaneously the linker. The synthesis

A. Liu et al. / Tetrahedron 70 (2014) 6974e6979

methods, the sensing behaviors, and the application of the sensor in living cell imaging were studied.

6975

to pink, which was easily observed by naked eye (Fig. 1a). The results suggested that RPBD was a highly selective colorimetric pH sensor.

2. Results and discussion

3+

2.1. Synthesis of RPBD

Absorbance

1.2

2+

2+

0.6 H

+

0.0 400

500

600

700

Wavelength /nm (b)

+

H

5

8.0x10

FL intensity

Inspired by the literature12,14,19,28 and based on different reactivity of the amino groups in AEP, RPBD was initially designed to prepare by two-step reactions. Namely, RB was amidated with AEP to get the intermediate RAP, following which RAP was reacted with NBD-Cl to produce RPBD (Scheme 1). Unexpectedly, RAP could not be detected by LCeMS although the molar ratio of RB to AEP was changed from 1:2 to 1:10, the solvent was changed from methanol, tetrahydrofuran, ethanol, acetonitrile, 1,4-dioxane to N,N-dimethylformamide, the temperature was varied from 60 to 120  C, and the acid-capturer triethylamine20 or the dehydrant N,N0 -dicyclohexylcarbodiimide31 was adopted. Till the handy base DIPEA was tried as acid-capturer, RAP was obtained (Fig. S1) with 89.8% yield and 99.9% purity (HPLC) (Fig. S2). Na2CO3 and K2CO3 were employed to replace DIPEA, but they did not work. The reason was speculated that owing to the relatively strong alkalinity and small molecular volume, they could simultaneously change the carboxyl into carboxylate and hinder the amidation reaction when they neutralized the HCl in RB hydrochloride. The reaction between RAP and NBD-Cl was easy and afforded RPBD (Figs. S3eS6) with 83.5% yield. The total output of the two-step reactions was 75.0%. Since the intermediate RAP had good yield and purity, we further tried to add NBD-Cl and K2CO3 suspended in acetonitrile to the first step reaction system and proceeded with the second step reaction after the first step finished, the solvent was evaporated and the residue was washed with water. The result showed that RPBD could be prepared in one pot with 82.5% yield. This finding enriches the synthetic methods of rhodamine derivatives.

2+

Fe ,Cu ,Pb ,Mn , 3+ 2+ 2+ 2+ Cr ,Co ,Ca ,Hg , 2+ 2+ 2+ 2+ Zn ,Ni ,Fe ,Cd , + + 2+ Na ,K ,Mg ,none

(a)

3+

2+

2+

2+

Fe ,Cu ,Pb ,Mn , 3+ 2+ 2+ 2+ Cr ,Co ,Ca ,Hg , 2+ 2+ 2+ 2+ Zn ,Ni ,Fe ,Cd , + + 2+ Na ,K ,Mg ,none

5

4.0x10

0.0 550

600

650

700

Wavelength /nm Fig. 1. UVevis absorption (a) and fluorescence (b) spectra of RPBD and color changes in the absence and presence of various cations. Solvent: CH3CN/BeR buffer (1:1, v/v, pH 7.15) for metal ions, CH3CN/BeR buffer (1:1, v/v, pH 2.46) for Hþ. c: 20 mM for RNBD, 200 mM for metal ions. lex: 490 nm, slit width: 5 nm.

Similarly, the CH3CN/BeR buffer (1:1, v/v, pH 7.15) solution of RPBD was almost non-fluorescent and various metal ions showed almost no effects. However, the decrease of pH value to 2.46 caused pronounced fluorescence changes of RPBD. An emission band centered at 580 nm with a shoulder peak at 536 nm appeared. Correspondingly, the fluorescence images of the RPBD solutions at pH 7.15 and pH 2.46 were black and red when excited with green light on an OLYMPUS U-LH100HG fluorescence microscope (Fig. 1b). These results indicated that the fluorescence of RPBD was highly selective to pH. Scheme 1. Synthetic route of RPBD.

2.2. Sensing behaviors of RPBD 2.2.1. The selectivity of RPBD to pH. The absorption spectra of RPBD (20 mM) in CH3CN/BrittoneRobinson (BeR) buffer (1:1, v/v, pH 7.15) solution exhibited very weak bands over 500 nm, which indicated RPBD in spirolactam form. A large number of cations, Naþ, Kþ, Mg2þ, Ca2þ, Fe2þ, Mn2þ, Cd2þ, Cr3þ, Co2þ, Ni2þ, Cu2þ, Pb2þ, Zn2þ, Fe3þ, and Hg2þ (200 mM), had no obvious effects on the spectra. However, when the pH value decreased from 7.15 to 2.46, the absorption centered at 490 nm blueshifted to 467 nm and reduced slightly, and a new band centered at 560 nm emerged indicating the opening of spirolactam ring. The remarkable absorption spectra variation was accompanied by a dramatic color change from yellow

2.2.2. The sensitivity of RPBD to pH. Moreover, the relationship between the spectra of RPBD and the pH value was studied. When the pH value varied from 7.77 to 2.03, the absorbance at 560 nm and the fluorescence intensity at 580 nm were greatly strengthened with the maximal increase of 285 and 50.3 folds, respectively (Fig. 2). Furthermore, the absorbance and the fluorescence intensity increased almost linearly from pH 3.70 to 2.03. Therefore, RPBD could be used as a highly sensitively colorimetric and fluorescent pH indicator. The pKa value of RPBD based on the fluorimetric titration (Fig. 2b) was 2.87. This value implies that RPBD can be used for the study of strong acidic environment. 2.2.3. Effects of coexistent ions. Competitive ions Naþ, Kþ, Mg2þ, Ca2þ, Cr3þ, Mn2þ, Fe2þ, Fe3þ, Co2þ, Ni2þ, Cu2þ, Zn2þ, Cd2þ, Hg2þ, and Pb2þ (200 mM) were added into the CH3CN/BeR buffer (1:1, v/v, pH

A. Liu et al. / Tetrahedron 70 (2014) 6974e6979

the maximal signal within 5 min, and kept stable over 1 h. Therefore, RPBD is a fast responsive and reliable colorimetric and fluorescent pH sensor.

0.3 0.0

0.6

4 6 pH

8

Absorbance

2

0.0 400

500

600

b a

0.30

5

4x10

0.15

700

0

0.00

Wavelength /nm

0

20

FL intensity

1.6x10

1.6x10

(b)

FL intensity

0.0

5

8.0x10

2

4

6

8

pH

0.0

600

660

720 5

8x10

5

4x10

0.15

0

pH=7.15

0.00 0.0

0.5

1.0

1.5

2.0

Cycle Index Fig. 5. Reversibility of the absorption (square and solid line) and fluorescence (circle and short dash line) detection of pH with RPBD (20 mM). Solvent: CH3CN/BeR buffer (1:1, v/v). lmax: 560 nm; lex: 490 nm; lF: 580 nm; slit width: 5 nm.

6

f

Absorbance

2.46 or 3.70) solution of RPBD (20 mM) together, and they had no significant influence on the UVevis absorption and fluorescence spectra of the solutions (Fig. 3). Similar results were obtained when the above competitive ions were added into the solutions individually, which indicated that RPBD displayed an excellent selectivity toward Hþ.

e

pH=2.46

0.30

Fig. 2. Dependence of UVevis absorption (a) and fluorescence (b) spectra of RPBD (20 mM) on pH in CH3CN/BeR buffer (1:1, v/v). lex: 490 nm, slit width: 5 nm. From top to bottom, pH: 2.03, 2.46, 2.94, 3.41, 3.70, 3.98, 4.20, 4.88, 5.00, 6.44, 7.15, and 7.77. Insets: plot of maximal absorbance at 560 nm and fluorescence intensity at 580 nm depending on the pH values.

b

80

2.2.5. Reversibility of the sensor. To investigate the reversibility of the sensor, HCl and NaOH alternately added experiments were conducted. The absorption and emission were turned on when HCl was added, while switched off when NaOH was added (Fig. 5), accompanied by vivid color changes. The result showed that RPBD reversibly coordinated with Hþ (Scheme 2), as depicted in the literature.18,20,32

Wavelength /nm

0.4

60

Fig. 4. Time response of the absorption (a) and fluorescence (b) intensity of RPBD (20 mM) to Hþ. Solvent: CH3CN/BeR buffer (1:1, v/v, pH 2.46). lmax: 560 nm; lex: 490 nm; lF: 580 nm; slit width: 5 nm.

5

8.0x10

540

40 Time /min

6

6

5

8x10

FL intensity

0.6

FL intensity

(a)

1.2 Absorbance

Absorbance

6976

1x10

5

5x10

0.2 g c

0.0 2.46

h

0

d

3.70

FL intensity

Absorbance

a

pH

2.46

3.70

Fig. 3. Effects of coexisting ions on the absorption at 560 nm (aed) and fluorescence maxima at 580 nm (eeh) of RPBD at pH 2.46 and 3.70. a, c, e, and g: without metal ions, b, d, f, and h: with Naþ, Kþ, Mg2þ, Ca2þ, Fe2þ, Mn2þ, Cd2þ, Cr3þ, Co2þ, Ni2þ, Cu2þ, Pb2þ, Zn2þ, Fe3þ, and Hg2þ. Solvent: CH3CN/BeR buffer (1:1, v/v); c: 20 mM for RPBD, 200 mM for metal ions. lex: 490 nm; slit width: 5 nm.

2.2.4. Time response of the sensor. In addition, time response of the UVevis absorption and fluorescence spectra of RPBD was recorded (Fig. 4). It can be seen that RPBD responded to Hþ instantly, reached

Scheme 2. Proposed sensing mechanism of RPBD for pH.

To verify the proposed sensing mechanism, LCeMS of the CH3CN/BeR buffer (1:1, v/v, pH 2.46) solution of RPBD was carried out and the result was shown in Fig. 6. The clearly observed

A. Liu et al. / Tetrahedron 70 (2014) 6974e6979

molecular ion peak of [open-loop architecture of RPBD]þ, [openloop architecture of RPBDþH]þ, and [open-loop architecture of RPBDþH]2þ/2 at m/e 717.3659, 718.3514, and 359.1824, respectively, in Fig. 6 well supported the proposed sensing mechanism, as well as confirmed the structure of RPBD in extreme acidity (pH<4).

6977

stain living cells and provide intracellular pH information for studying physiological and pathological processes. 3. Conclusions In summary, a novel dually-responsive pH sensor (RPBD) from AEP, NBD-Cl, and RB was designed and synthesized via two-step reactions in one pot in the presence of the essential DIPEA. RPBD can highly selectively and sensitively detect pH with naked-eye observable vivid color changes and has potential use in strong acidic environment and living cell imaging. The rapid and reversible response of RPBD to pH can be attributed to the protontriggered ring-opening of the rhodamine spirolactam. 4. Experimental 4.1. Materials and instruments

Fig. 6. LCeMS of RPBD (20 mM) in CH3CN/BeR buffer (1:1, v/v, pH 2.46) recorded by a Brukermicro TOF-QIII LC/MS (Bruker Daltonics Co., Germany).

2.3. Living cell imaging with RPBD The application of RPBD in bioimaging was explored. L929 cells were incubated with RPBD (15 mM) in DMEM (pH 7.4) for 30 min at 37  C and then washed three times with PBS at pH 2.5. Intracellular fluorescence was monitored by fluorescence microscopy. The control group without RPBD and the group incubated with RPBD (15 mM) in DMEM at pH 7.4 were non-fluorescent (Fig. 7b and d), while the group loaded with RPBD and rinsed with PBS at pH 2.5 containing 1 mg/mL of nigericin emitted notable red fluorescence (Fig. 7f). The normal shape of the cells in bright field images (Fig. 7c and e) confirmed that the cells were viable after the sensor incubation. These results were consistent with the spectral data shown in Fig. 2b, indicate that RPBD can permeate cell membrane,

RB was bought from Shanghai SSS Reagent Co., Ltd. AEP (99%) and NBD-Cl (98%) were provided by Yake Chemical Reagent Co., Ltd. N,N-Diisopropylethylamine (DIPEA) (99%) was purchased from Jiande New Delhi Chemical Co., Ltd. Nigericin (Aldrich) was purchased from J&K Scientific Co., Ltd. HCl (ca. 37% aqueous solution), NaOH, Na2CO3, K2CO3, triethylamine, the solvents, and the metal cation sources, NaCl, KCl, CaCl2, MgCl2, FeCl3$6H2O, CuSO4$5H2O, Zn(NO3)2$6H2O, CrCl3$6H2O, Pb(NO3)2, Ni(NO3)2$6H2O, FeCl2$7H2O, MnSO4$H2O, HgCl2, CoCl2$6H2O, and CdCl2$2.5H2O were provided by Sinopharm Chemical Reagent Co., Ltd. All the reagents were used as received. The solvents used in synthesis were analytical grade and others were spectroscopic grade. Infrared (IR) spectra were recorded on a Nicolet Magan-550 spectrometer (Nicolet Co., USA). LCeMS was performed on an Agilent 6120 Quadruple LC/MS (Agilent Co., USA) unless otherwise specified. High performance liquid chromatography (HPLC) was carried out on an Angilent 1260 Liquid Chromatograph (2504.6 mm C18 column, CH3CN/H2O, 50:50, v/v. flow rate: 1 mL/ min, detection wavelength: 254 nm, temperature: 30  C) (Agilent Co., USA). 1H NMR and 13C NMR spectra were carried out on

Fig. 7. Images of L929 cells incubated in different media. (a), (c), and (e) are corresponding bright field images of fluorescence images (b), (d), and (f), respectively. (b) without RPBD at pH 7.4; (d) with RPBD (15 mM) at pH 7.4; (f) with RPBD (15 mM) at pH 2.5. lex: 500e530 nm.

6978

A. Liu et al. / Tetrahedron 70 (2014) 6974e6979

a 300 MHz Varian Unity Inova spectrometer (Varian Co., USA). Elemental analysis was tested on a Carlo-Erba EA1110 CHNO-S (CarloErba Co., Italy). UVevis spectra were recorded on a U-3900 spectrophotometer (PerkineElmer Co., USA). Fluorescence spectra were taken on a Fluoromax-4 spectrofluorometer (HORIBA Jobin Yvon Co., France). Fluorescence pictures were taken on an OLYMPUS ULH100HG fluorescence microscope (Olympus Co., Japan). Cell images were taken on Nikon IX71 fluorescence inverted system microscope (Nikon Co., Japan). The pH values were measured with a Mettler-Toledo FE20 pH meter (Mettler-Toledo Co., USA). All the pH and the spectra were measured at 25  C except special instructions. 4.2. Synthetic procedures and characterization data for RPBD The first step reaction: under the protection of nitrogen, AEP (1 mL, 7.63 mmol) and DIPEA (2 mL, 12.11 mmol) were dissolved in acetonitrile (CH3CN) (2 mL) in a 100 mL three-necked flask equipped with a magnetic stirrer, a condenser pipe, and a thermometer. Then acetonitrile solution (10 mL) of RB (0.5 g, 1.04 mmol) was added dropwise. After stirring for 30 min, the mixture was heated under reflux for 12 h then, the solvent was removed by evaporation under reduced pressure and the residue was washed with water (20 mL3), filtered, and dried in vacuum to afford a pale yellow solid. The yield: 0.5184 g (89.8%). LC-mass analysis of the solid showed that only two peaks at m/e 554.3 and 576.3 appeared, which matched the molecular ion peaks of [RAPþH]þ and [RAPþNa]þ (Fig. S1). HPLC analysis showed that the RAP content was 99.9% (Fig. S2). The second step reaction: RAP (0.1 g, 0.18 mmol) and K2CO3 (25 mg, 0.18 mmol) were dispersed in acetonitrile (8 mL) in a 100 mL three-necked flask equipped with a magnetic stirrer, a condenser pipe, and a thermometer. After stirring for 10 min, acetonitrile solution (2 mL) of NBD-Cl (36 mg, 0.18 mmol) was added gradually. The reaction mixture was stirred at room temperature for 30 min, and then the solvent was removed on a rotary evaporator. The remains were separated by column chromatography on silica with ethylacetate/petroleum ether (2:1, v/v) as eluent to afford RPBD as an orange red solid. The yield: 0.0910 g (83.5%). The total output of the two-step reactions was 75.0%. In the one-pot synthesis method, after the first step reaction was finished, the solvent was removed and the residue was washed by water (20 mL3). Acetonitrile (10 mL) and K2CO3 (0.144 g, 1.04 mmol) were added successively and the mixture was stirred for 10 min. Next, an acetonitrile solution (5 mL) of NBD-Cl (0.2 g, 1.04 mmol) was added gradually and the reaction was carried out under the same conditions as the second step of the aforementioned reactions to afford RPBD. The yield: 0.6164 g (82.5%, solid). IR (KBr) cm1: 2971, 2927 (CH3, CH2), 1688 (C]O), 1620, 1514, 1436 (benzene ring), 1541 (NO2), 1235, 1301 (C]N), 1118 (CeOeC) (Fig. S3). 1H NMR (CDCl3, 400 MHz): d 1.16 (t, J¼6.8 Hz, 12H), 2.17 (t, J¼6.8 Hz, 2H), 2.50 (t, J¼7.2 Hz, 4H), 3.33 (m, 10H), 3.97 (t, J¼6.8 Hz, 4H), 6.26 (m, 3H), 6.39 (d, J¼8.0 Hz, 2H), 6.45 (d, J¼8.8 Hz, 2H), 7.10 (t, J¼7.6 Hz, 1H), 7.44 (m, 2H), 7.90 (d, J¼8.2 Hz, 1H), 8.40 (d, J¼8.8 Hz, 1H) (Fig. S4). 13C NMR (CDCl3, 300 MHz): d 168.30, 153.51, 148.38, 145.15, 144.74, 135.16, 132.38, 131.17, 129.09, 128.04, 123.79, 123.06, 122.64, 108.40, 105.65, 102.18, 97.53, 64.79, 55.10, 52.38, 49.26, 44.22,37.21, 12.70 (Fig. S5). LCeMS: m/z calcd for C40H44N8O5þHþ ([MþHþ]), 717.8; Found, 717.3 (Fig. S6). Anal. Calcd for C40H44N8O5 (716.83): C, 67.02; H, 6.19; N, 15.63%. Found: C, 67.00; H, 6.21; N, 15.30%.

deionized water (H2O) separately to get 10 mM stock solutions each. When studying the selectivity of RPBD to Hþ, 100 mL stock solution of RPBD was put into each of the 17 volumetric flasks (sample 1e17), respectively. Next, 100 mL deionized water were added in flask 1 and 17. For flasks 2e16, 200 mL of a metal ion stock solution (Naþ, Kþ, Mg2þ, Ca2þ, Cr3þ, Mn2þ, Fe2þ, Fe3þ, Co2þ, Ni2þ, Cu2þ, Zn2þ, Pb2þ, Cd2þ or Hg2þ), and 100 mL acetonitrile were added, respectively. Then, the sample 1 was diluted with CH3CN/ BeR buffer (1:1, v/v, pH 2.46) solution, and the others were diluted with CH3CN/BeR buffer (1:1, v/v, pH 7.15) solution to 10 mL to get test solutions No. 1e17. The UVevis absorption and fluorescence spectra were assayed after 30 min at 25  C unless otherwise specified. In the UVevis absorption and fluorescence titration, the test solutions were prepared by the method similar to No. 17 with BeR buffer solutions at pH 2.03, 2.46, 2.94, 3.41, 3.70, 3.98, 4.20, 4.88, 5.00, 6.44, 7.15, and 7.77, respectively. In the time response experiments, the UVevis absorption and fluorescence spectra of test solution No. 1 were observed immediately after it was prepared and the time lasted for 70 min. To exam the effects of the competitive ions, 100 mL stock solution of RPBD was put into a 10 mL volumetric flask, mixed individually or together with 200 mL of each stock solution of the metal ions, Naþ, Kþ, Mg2þ, Ca2þ, Cr3þ, Mn2þ, Fe2þ, Fe3þ, Co2þ, Ni2þ, Cu2þ, Zn2þ, Pb2þ, Cd2þ, and Hg2þ, and diluted with 4.9 mL acetonitrile and BeR buffer solution at pH 2.46 and 3.70, respectively. In the study of reversibility, 10 M HCl (50 mL) and 10 M NaOH (50 mL) aqueous solutions were added alternatively to the test solution No. 17. The UVevis absorption and fluorescence spectra of the resulted solutions were recorded. 4.3.2. Acidity constant. The acidity constant pKa was calculated by HendersoneHasselbalch Eq. 118,19,23,25 based on the fluorimetric titration data, where I is the observed fluorescence intensity at a fixed wavelength, Imax and Imin are the maximal and minimal fluorescence intensity, respectively.

pH  pKa ¼ log½ðImax  IÞ=ðI  Imin Þ 4.3.3. Cell culture and imaging. L929 cells were cultured in Dulbecco modified Eagle medium (DMEM, Gibco) containing 10% (v/v) calf bovine serum (HyClone, USA) at 37  C in humidified air with 5% CO2. For fluorescence imaging, the cells (5104 cells/mL) were seeded into 24-well plates and separated into three groups. The group 1 was left as the control. The others were incubated with 15 mM of RPBD for 30 min, and then one of the two was washed three times with phosphate buffer solution (PBS) at pH 2.5. Nigericin (1 mg/mL) was added to the PBS medium to induce a rapid exchange of Kþ for Hþ for a fast equilibration of external and internal pH.33,34 All cells were imaged under a Nikon IX71 fluorescence inverted system microscope, excited with a green light (500e530 nm).

Acknowledgements This project was supported by the National Natural Science Foundation of China (21074085) and the Priority Academic Program Development of Jiangsu Higher Education Institutions.

4.3. Testing and calculating methods

Supplementary data

4.3.1. General procedure for pH detection. RPBD was dissolved in CH3CN to form a 2 mM stock solution. Metal salts were dissolved in

Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.tet.2014.07.087.

A. Liu et al. / Tetrahedron 70 (2014) 6974e6979

References and notes 1. Tan, Y.; Yu, J.; Gao, J.; Cui, Y.; Wang, Z.; Yang, Y.; Qian, G. RSC Adv. 2013, 3, 4872e4875. 2. Yang, M.; Song, Y.; Zhang, M.; Lin, S.; Hao, Z.; Liang, Y.; Zhang, D.; Chen, P. Angew. Chem., Int. Ed. 2012, 51, 7674e7679. 3. Ma, L.; Qian, J.; Tian, H.; Lan, M.; Zhang, W. Analyst 2012, 137, 5046e5050. 4. Kaur, K.; Saini, R.; Kumar, A.; Luxami, V.; Kaur, N.; Singh, P.; Kumar, S. Coord. Chem. Rev. 2012, 256, 1992e2028. 5. Santos-Figueroa, L. E.; Moragues, M. E.; Climent, E.; Agostini, A.; MartinezManez, R.; Sancenon, F. Chem. Soc. Rev. 2013, 42, 3489e3613. 6. Achilefu, S. Chem. Rev. 2010, 110, 2709e2728. 7. Kim, H. N.; Lee, M. H.; Kim, H. J.; Kim, J. S.; Yoon, J. Chem. Soc. Rev. 2008, 37, 1465e1472. 8. Chen, X.; Pradhan, T.; Wang, F.; King, J. S.; Yoon, J. Chem. Rev. 2012, 112, 1910e1956. 9. Beija, M.; Afonso, C. A. M.; Martinho, J. M. G. Chem. Soc. Rev. 2009, 38, 2410e2433. 10. Kim, H. N.; Ren, W. X.; Kim, J. S.; Yoon, J. Chem. Soc. Rev. 2012, 41, 3210e3244. 11. Wang, L.; Yan, J. X.; Qin, W.; Liu, W.; Wang, R. Dyes Pigm. 2012, 92, 1083e1090. 12. Zhang, D.; Li, M.; Wang, M.; Wang, J.; Yang, X.; Ye, Y.; Zhao, Y. Sens. Actuators, B 2013, 177, 997e1002. 13. Yu, C.; Wang, T.; Xu, K.; Zhao, J.; Li, M.; Weng, S.; Zhang, J. Dyes Pigm. 2013, 96, 38e44. 14. Wang, J.; Long, L.; Xie, D.; Song, X. Sens. Actuators, B 2013, 177, 27e33. 15. Zhou, Y.; Zhang, J.; Zhang, L.; Zhang, Q.; Ma, T.; Niu, J. Dyes Pigm. 2013, 97, 148e154. 16. Meng, Q.; Zhang, Y.; Hou, D.; Xin, G.; Li, T.; He, C.; Duan, C. Tetrahedron 2013, 69, 636e641.

6979

17. Han, J.; Burgess, K. Chem. Rev. 2010, 110, 2709e2728. 18. Tian, M.; Peng, X.; Fan, J.; Wang, J.; Sun, S. Dyes Pigm. 2012, 95, 112e115. 19. Ma, Q.-J.; Li, H.-P.; Yang, F.; Zhang, J.; Wu, X.-F.; Bai, Y.; Li, X.-F. Sens. Actuators, B 2012, 166e167, 68e74. 20. Yapici, N. B.; Mandalapu, S. R.; Chew, T.-L.; Khuon, S.; Bi, L. Bioorg. Med. Chem. Lett. 2012, 22, 2440e2443. 21. Hu, Z.-Q.; Li, M.; Liu, M.-D.; Zhuang, W.-M.; Li, G.-K. Dyes Pigm. 2013, 96, 71e75. 22. Zhang, W.; Tang, B.; Liu, X.; Liu, Y.; Xu, K.; Ma, J.; Tong, L.; Yang, G. Analyst 2009, 134, 367e371. 23. Lv, H.-S.; Liu, J.; Zhao, J.; Zhao, B.-X.; Miao, J.-Y. Sens. Actuators, B 2013, 77, 956e963. 24. Lv, H.-S.; Huang, S.-Y.; Zhao, B.-X.; Miao, J.-Y. Anal. Chim. Acta 2013, 788, 177e182. 25. Liu, L.; Guo, P.; Lu, C.; Shi, Q.; Xu, B.; Yuan, J.; Wang, X.; Shi, X.; Zhang, W. Sens. Actuators, B 2014, 194, 498e502. 26. Dujols, V.; Ford, F.; Czarnik, A. W. J. Am. Chem. Soc. 1997, 119, 7386e7387. 27. Zhang, L.; Wang, J.; Fan, J.; Guo, K.; Peng, X. Bioorg. Med. Chem. Lett. 2011, 21, 5413e5416. 28. Lee, M. H.; Kim, H. J.; Yoon, S.; Park, N.; Kim, J. S. Org. Lett. 2008, 10, 213e216. 29. Mao, J.; Heb, Q.; Liu, W. Talanta 2010, 80, 2093e2098. 30. Liu, A.; Yang, L.; Zhang, Z.; Zhang, Z.; Xu, D. Dyes Pigm. 2013, 99, 472e479. 31. Yuan, L.; Lin, W.; Chen, B.; Xie, Y. Org. Lett. 2012, 14, 432e435. 32. Kaoutit, H. E.; Estevez, P.; Ibeas, S.; Garcia, F. C.; Serna, F.; Benabdelouahab, F. B.; Garcia, J. M. Dyes Pigm. 2013, 96, 414e423. 33. Zhu, S.; Lin, W.; Yuan, L. Dyes Pigm. 2013, 99, 465e471. 34. Han, J.; Loudet, A.; Barhoumi, R.; Burghardt, R. C.; Burgess, K. J. Am. Chem. Soc. 2009, 131, 1642e1643.