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Construction of ratiometric fluorescent probe based on inverse electron-demand Diels–Alder reaction for pH measurement in living cells
Sensors & Actuators: B. Chemical 277 (2018) 320–327
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Construction of ratiometric fluorescent probe based on inverse electrondemand Diels–Alder reaction for pH measurement in living cells
T
Gang Feng, Boyu Zhang, Chunfei Wang, Jingyun Tan, Shichao Wang, Zhaoyang Ding, ⁎ Xuanjun Zhang Faculty of Health Science, University of Macau, Macau SAR, China
A R T I C LE I N FO
A B S T R A C T
Keywords: In situ fluorescence pH probe Ratiometric analysis Inverse electron demand Diels–Alder reaction
To endow fluorescent functions to the inverse electron demand Diels–Alder (IEDDA) reaction products, introduction of extraneous fluorophores is usually inevitable. However, this strategy is blamed for complex construction and background fluorescence. It is desirable to construct IEDDA fluorescent products in a more convenient manner. In this work, we reported the in-situ generated green fluorescence in IEDDA reaction. The fluorescence intensity of IEDDA products RA-TZ-1 and RA-TZ-2 decreased in reduced pH values (from pH 7.0–3.0). Based on this novel property, we developed a ratiometric fluorescent probe RB–RA-TZ-2 by conjugation with another probe RB-NH2 (Em = 584 nm) with inverse response toward pH values. By single excitation, the fluorescence intensity ratio (I584/I488) showed a linear response (R = 0.9797) to H+ in pH range of 3.5–5.0. Colocalization imaging with LysoTracker Green indicated the capacity of RB–RA-TZ-2 to detect lysosomal pH (Pearson’s coefficient 0.81). Confocal microscopy was applied to measure and image intracellular pH in living cells.
1. Introduction Bioorthogonal techniques such as copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC), [1,2] strain-promoted alkyne-azide cycloadditions (SPAAC) [3], photoclick reactions [4] and inverse electron-demand Diels–Alder reaction (IEDDA) [5,6] have revolutionized conjugation strategies in chemical biology. Among these techniques, IEDDA is highlighted for its excellent specificity, fast reaction rate, catalyst free and biocompatibility features [7], which has been applied in applications such as genetic code expansion [8,9], protein modification [10,11],nuclear medicine [12], and fluorescent imaging [13,14]. Usually, neither the IEDDA bioorthogonal pairs (dienophile and diene) nor the IEDDA products are fluorescent. To endow fluorescent function to IEDDA products, introduction of extraneous fluorophores is inevitable [15,16]. One classical fluorescent labeling strategy begins with decoration of a biochemical reporter with a dienophile group, and decoration of an extraneous fluorophore with 1,2,4,5-tetrazine diene group [17,18]. By means of IEDDA reaction, the biochemical reporter group successfully conjugates with the fluorophore, followed by the recovery of fluorescence results from the breakdown of 1,2,4,5-tetrazine structure. This strategy is ingenious, however has deficiencies such
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as complex construction and background fluorescence. Simple fluorescent conjugation strategy without extraneous fluorophores is ideal but challenging. Recently, several strategies are reported to meet this demand. Shang’s group endowed proteins with green fluorescence through IEDDA reaction between non-fluorescent styrene and 1,2,4,5-tetrazines [19]. Vazquez’s group reported that by combination of trans-cyclooctenes (TCOs) with 1,2,4,5-tetrazines, IEDDA products with various fluorescence were developed [20]. In this study, we began with the confirmation of in situ fluorescence from the IEDDA reaction by simply mixing Reppe anhydride with 1,2,4,5-tetrazines (Scheme 1a). This “click-fluorescence” conjugation strategy showed a promising application prospect in construction of fluorescent functional products (Scheme 1b). Moreover, IEDDA products RA-TZ-1 and RA-TZ-2 exhibited gradually decreased emission in reduced pH solutions (from pH 7.0–3.0). On the other hand, rhodamine amide derivatives were widely applied in pH sensing studies for their sensitivity to acid environment [21–23]. Importantly, it responded to reduced pH solutions (from pH 7.0–3.0) with a gradually increased fluorescence emission, which just contrasted with that of RA-TZ-1 and RA-TZ-2. It was also well known that probes with single fluorescence emission suffered low measurement accuracy
Corresponding author. E-mail address: [email protected] (X. Zhang).
https://doi.org/10.1016/j.snb.2018.09.022 Received 2 May 2018; Received in revised form 1 September 2018; Accepted 6 September 2018 Available online 07 September 2018 0925-4005/ © 2018 Elsevier B.V. All rights reserved.
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Scheme 1. (a) in situ fluorescence from the IEDDA reaction between Reppe anhydride and 1,2,4,5-tetrazines; (b) Application prospect of “click-fluorescence” conjugation strategy; (c) pH sensing mechanism of probe R B-R A-TZ-2.
2.2. Synthesis of tetrazine TZ-2
because of instrumental or operating error. However, this defect could be improved by ratiometric fluorescence strategy, whose measurement accuracy could be largely improved by means of self-calibration of two different emission bands in one individual probe [24–27]. Therefore to demonstrate the potentials of this novel “click-fluorescence” conjugation strategy in a universal manner based on the IEDDA fluorescence and rhodamine fluorescence, the construction of a concise ratiometric fluorescent pH probe was carried out. Herein, a reported rhodamine based probe RB-NH2 [28] was transformed into probe R B-R A with the ability to trigger IEDDA reaction with 1,2,4,5-tetrazines. By the IEDDA reaction between R B-R A and tetrazine TZ-2, a ratiometric fluorescent pH probe R B-R A-TZ-2 was developed. The special pH sensing mechanism of R B-R A-TZ-2 derived from the fluorescence intensity ratio between orange fluorescence (584 nm) and green fluorescence (488 nm) from rhodamine part and RA-TZ part respectively (Scheme 1c). By single excitation at 360 nm, the fluorescent intensity ratio (I584/I488) changed correspondingly in pH values. In addition, confocal microscopy was conducted to measure and image intracellular pH in living RAW 264.7 cells.
2-pyrimidinecarbonitrile (1.05 g, 10 mmol, 1.0 eq) was suspended in dry EtOH (20 mL). With the dropwise addition of hydrazine hydrate (1.56 mL, 25 mmol, 2.5 eq), the orange mixture was stirred at 85 °C with reflux under nitrogen protection for 16 h. The orange precipitate was collected by vacuum filtration and washed by EtOH. The intermediate product was suspended in acetic acid (10 mL). In fume hood, isopentyl nitrite (1 mL, 7.15 mmol) was added dropwise with stirring. The orange mixture turned purple with release of toxic gas nitrogen dioxide. After 8 h oxidation, the purple precipitate was collected and washed by EtOH. Crude purple product went for a flash chromatography in silica gel column (DCM, followed by DCM/MeOH 10:1). The purple filtrate was collected and dried to obtain TZ-2 as purple solid. (700 mg, 2.94 mmol, 70%). 1H NMR (400 MHz, DMSO-d6) δ 9.23 (d, J = 4.9 Hz, 4 H), 7.87 (t, J = 4.9 Hz, 2 H). 13C NMR (101 MHz, DMSOd6): 163.45, 159.41, 159.07, 123.67. 2.3. Synthesis of RA-TZ-1 To a solution of RA (22.2 mg, 0.11 mmol, 1.1 eq) in 30 mL DMSO, TZ-1 (28.0 mg, 0.1 mmol, 1.0 eq) was added. During the 18 h stirring at room temperature, the solution turned yellow from purple. Green fluorescence was witnessed using hand-held 365 nm UV lamp. Fresh DD water was added followed by DCM extraction to remove DMSO, the yellow crude product in DCM was dried by MgSO4. Column chromatography in silica gel column (DCM/MeOH 10:1) was conducted to afford the IEDDA product RA-TZ-1 as light-yellow solid (31.8 mg, 0.07 mmol, 70%). 1H NMR (400 MHz, DMSO-d6) δ 9.12 (d, J = 4.9 Hz, 2 H), 8.33 (d, J = 8.2 Hz, 2 H), 8.19 (d, J = 8.0 Hz, 2 H), 7.66 (t, J = 5.1 Hz, 1 H), 5.89 (q, J = 6.9, 6.2 Hz, 2 H), 4.35 (t, J = 3.9 Hz, 1 H), 4.20 (t, J = 4.1 Hz, 1 H), 3.84 (d, J = 4.9 Hz, 1 H), 3.71 (s, 1 H), 3.68 – 3.60 (m, 2 H). 13C NMR (101 MHz, DMSO-d6) δ 173.84, 173.67, 167.39, 158.77, 149.03, 145.43, 138.19, 132.92, 131.09, 130.74, 130.31, 127.96, 45.98, 45.65, 44.27, 43.76, 35.72, 35.29. ESI-MS calcd for C25H18N4O5, [M+H]+ 455.1355, found 455.1385.
2. Experimental section 2.1. Materials and instruments All commercial chemicals were purchased from commercial suppliers and used without further purification. Twice distilled water was used throughout all experiments. 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded by Bruker AV 400 MHz spectrometers. High-resolution mass spectra (HRMS) was recorded by the Waters Xevo G2-XS Q-Tof matrix-assisted time of flight mass spectrometer using high performance liquid chromatography (HPLC) and electro-spray ionization (ESI) technique. UV–vis absorption spectra were recorded by SHIMADZU UV-1800 spectrophotometer. Fluorescence spectra was recorded by HORIBA Fluorolog-3 modular spectrometer with a Xe lamp as excitation source. Absolute fluorescence quantum yield measurement was conducted by integrating sphere on HORIBA Fluorolog-3 modular spectrofluorometer [29]. Fluorescence lifetime was detected by DeltaTime TCSPC on HORIBA Fluorolog@-3 spectrometer equipped with a HORIBA NanoLED source (N-455 nm). Carl Zeiss LSM710 confocal microscope was applied for living cells fluorescent imaging. Synthesis of tetrazine TZ-1, Reppe anhydride (RA) and RA-NHS were according to published procedures [30–32].
2.4. Synthesis of RA-TZ-2 To the solution of RA (22.2 mg, 0.11 mmol, 1.1 eq) in 10 mL acetonitrile, TZ-2 (23.8 mg, 0.1 mmol, 1.0 eq) was added. During the 18 h stirring at room temperature, the solution turned yellow from purple. Green fluorescence was witnessed using hand-held 365 nm UV lamp. After removal of the solvent, the crude product was purified by column chromatography in silica gel column (DCM/MeOH 20:1) to afford the 321
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acetonitrile, DIEA (51.6 mg, 0.4 mmol, 2.0 eq) was added. After stirring at 0℃ for 30 min, RB-NH2(105.4 mg, 0.2 mmol, 1.0 eq)solution in 5 mL acetonitrile was added dropwise. The mixture was given a 1 h stirring at 0℃ followed by stirring at room temperature overnight. After removal of solvent, crude product was purified by column chromatography in Al2O3 column (DCM/MeOH 10:1) to afford RB–RA as yellow solid (95.6 mg, 0.12 mmol, 60%). 1H NMR (400 MHz, Chloroform-d) δ 7.91 – 7.83 (m, 1 H), 7.66 (t, J = 5.7 Hz, 1 H), 7.54 – 7.46 (m, 2 H), 7.14 – 7.08 (m, 1 H), 6.47 – 6.29 (m, 6 H), 5.96 – 5.82 (m, 4 H), 3.59 – 3.30 (m, 15 H), 3.14 (d, J = 4.1 Hz, 2 H), 3.03 (s, 2 H), 2.84 – 2.75 (m, 6 H), 2.71 (s, 1 H), 2.24 (t, J = 7.0 Hz, 2 H), 1.19 (t, J = 7.0 Hz, 12 H). 13C NMR (101 MHz, Chloroform-d) δ 178.96, 172.13, 153.32, 148.88, 137.99, 132.63, 128.69, 128.34, 128.18, 123.89, 122.76, 108.15, 105.26, 97.70, 77.25, 58.44, 47.61, 44.38, 44.17, 43.28, 39.66, 38.84, 37.90, 36.67, 33.43, 23.92, 18.46, 12.60. ESI-MS calcd for C48H56N6O5, [M+H]+ 770.4390, found 770.4093. [M+2 H]2+ 385.7234, found 385.7182.
IEDDA product RA-TZ-2 as light-yellow solid (31.8 mg, 0.07 mmol, 70%). 1H NMR (400 MHz, Chloroform-d) δ 9.13 (s, 1 H), 8.81 (d, J = 4.8 Hz, 2 H), 8.73 (d, J = 4.8 Hz, 2 H), 7.24 (t, J = 4.9 Hz, 1 H), 7.17 (t, J = 4.9 Hz, 1 H), 6.70 – 6.64 (m, 1 H), 6.49 (t, J = 7.4 Hz, 1 H), 3.98 – 3.85 (m, 2 H), 3.50 (t, J = 3.6 Hz, 1 H), 3.31 – 3.22 (m, 2 H), 3.14 (qd, J = 8.8, 3.0 Hz, 2 H). 13C NMR (101 MHz, Chloroform-d) δ 172.76, 158.47, 157.41, 157.03, 133.33, 132.44, 131.84, 122.60, 119.83, 119.27, 47.43, 46.88, 44.08, 43.33, 41.79, 35.69, 35.15, 30.98, 29.72, 18.46. ESI-MS calcd for C22H16N6O3, [M+H]+ 413.1362, found 413.1396. 2.5. Synthesis of RB-NH2 RB-NH2 was synthesized according to a modification of a reported literature procedure [33]. To a solution of rhodamine B (1 g, 2.1 mmol, 1.0 eq) in EtOH (50 mL), diethylenetriamine (4.5 mL, 42 mmol, 20.0 eq) was added. The mixture was stirred at 85 °C under reflux for 24 h. After removal of solvent, orange residue was suspended in DCM (100 mL). DD water (100 mL) was added to extract the excess DIEA. The water layer was replaced by large amount of fresh DD water until becoming neutral. The organic layer was collected and dried to obtain RB-NH2 as orange solid (300 mg, 0.57 mmol, 27%). 1H NMR (400 MHz, Chloroform-d) δ 7.96 – 7.81 (m, 1 H), 7.43 (dd, J = 5.7, 3.1 Hz, 2 H), 7.14 – 6.99 (m, 1 H), 6.42 (d, J = 8.9 Hz, 2 H), 6.37 (d, J = 2.6 Hz, 2 H), 6.27 (d, J = 2.6 Hz, 1 H), 6.25 (d, J = 2.7 Hz, 1 H), 3.33 (q, J = 7.1 Hz, 8 H), 3.26 (t, J = 6.5 Hz, 2 H), 2.60 (t, J = 5.9 Hz, 2 H), 2.43 (t, J = 5.9 Hz, 2 H), 2.39 (t, J = 6.5 Hz, 2 H), 1.15 (t, J = 7.0 Hz, 12 H). ESI-MS calcd for C32H41N5O2, [M+H]+ 528.3339, found 528.3374.
2.7. Synthesis of R B-R A-TZ-2 To a solution of R B-R A (79.7 mg, 0.1 mmol, 1.0 eq) in 10 mL acetonitrile, tetrazine TZ-2 (26.2 mg, 0.11 mmol, 1.1 eq) was added. During the18 h stirring at room temperature, the solution turned brown from purple. Green fluorescence was witnessed by using hand-held 365 nm UV lamp. After removal of solvent, crude product was purified by column chromatography in Al2O3 column (DCM/MeOH 40:1) to afford IEDDA product RB–RA-TZ-2 as brown solid (50.3 mg,0.05 mmol, 50%). 1H NMR (400 MHz, Chloroform-d) δ 8.81 (d, J = 4.8 Hz, 2 H), 8.72 (dd, J = 4.8, 1.2 Hz, 2 H), 7.87 (s, 1 H), 7.49 – 7.38 (m, 3 H), 7.22 (t, J = 5.7 Hz, 1 H), 7.14 (d, J = 2.9 Hz, 1 H), 7.10 – 7.04 (m, 1 H), 6.49 – 6.22 (m, 8 H), 3.87 (s, 2 H), 3.82 (s, 1 H), 3.51 (s, 4 H), 3.31 (dt, J = 23.4, 7.5 Hz, 14 H), 2.82 (s, 2 H), 2.28 – 2.11 (m, 4 H), 1.90 – 1.82
2.6. Synthesis of R B-R A To a solution of RA-NHS (84.4 mg, 0.22 mmol, 1.1 eq) in 5 mL
Fig. 1. (a) Fluorescence at 518 nm intensified during the 12 h reaction between RA and tetrazine TZ-1 in DMSO; (b) Fluorescence property of RA-TZ-1 in DMSO; (c) Absorbance and (d) Emission of RA-TZ-1 in B-R buffers with 10% DMSO at various pH values.
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Scheme 2. Synthesis of (a) IEDDA product RA-TZ-1; (b) IEDDA product RA-TZ-2; (c) Fluorescent pH probe R B-R A with IEDDA capacity; (d) Ratiometric fluorescent pH probe R B-R A-TZ-2.
(m, 2 H), 1.16 (t, J = 7.0 Hz, 12 H). 13C NMR (101 MHz, Chloroform-d) δ 178.78, 168.87, 157.36, 156.96, 153.56, 153.32, 148.81, 138.00, 129.05, 128.91, 128.33, 128.05, 123.79, 122.75, 108.05, 108.02, 105.55, 105.48, 97.76, 97.67, 77.24, 67.97, 64.88, 52.26, 51.53, 48.23, 44.40, 44.20, 44.17, 43.72, 43.28, 41.01, 38.64, 36.66, 31.66, 31.40, 22.82, 22.76, 12.61. ESI-MS calcd for C58H62N12O5, [M+H]+ 1007.5044, found 1007.5079. [M + 2 H]2+ 504.2561, found 504.2583.
Green was displayed in pseudo green channel (Ex = 488 nm, Em = 530 ± 10 nm); rhodamine fluorescnece of R B-R A-TZ-2 was displayed in pseudo red channel (Ex = 543 nm, Em = 584 ± 10 nm). For the intracellular pH detecting assay with probe R B-R A-TZ-2 (5 μM), at single excitation (Ex = 405 nm), fluorescence from RA-TZ part was visualized in pseudo green channel (Em = 488 ± 20 nm), fluorescence from rhodamine part was visualized in pseudo red channel (Em = 584 ± 20 nm). Images were analyzed by Zeiss ZEN 2012 and Image J software.
2.8. Spectral measurements 3. Results and discussion Britton–Robinson (B–R) buffers with various pH values ranged from pH 3.0 to 9.0 were prepared according to standard procedures. RA-TZ1, RA-TZ-2, RB-NH2, R B-R A and R B-R A-TZ-2 were prepared at 1.0 mM in DMSO respectively as stock solutions. Samples for test were well mixed and incubated at room temperature for 60 min before measurements. For the absorption and emission spectral measurement, samples were diluted into 5 μM by B-R buffer with 10% DMSO (v/v). For the fluorescence lifetime measurement, R B-R A-TZ-2 was diluted into 5 μM in pH 4.0 and pH 8.0 B-R buffers with 10% DMSO (v/v). With excitation at 355 nm, the monitored wavelength was 488 nm. Fluorescence decay histograms were recorded using the time-correlated single photon counting technique in 4095 channels.
3.1. Confirmation of IEDDA fluorescence Reppe anhydride (RA) was introduced in DMSO solutions of tetrazine TZ-1 and TZ-2 respectively (Scheme 2). After 12 h stirring at room temperature, the solutions turned yellow from pink, which was recognized as a major IEDDA reaction feature. In both TZ-1 and TZ-2 solutions, green fluorescence (518 nm) strengthened gradually (Fig. 1a, Fig. S11a). To identify this unreported green fluorescence, IEDDA products RA-TZ-1 and RA-TZ-2 were purified. In view of the 1H NMR, 13 C NMR, mass spectra (Figs. S7, S8), the fluorescent properties of these IEDDA products were confirmed (Fig. 1b, Fig. S11b). Moreover, both RA-TZ-1 and RA-TZ-2 showed response to pH change in B-R buffers, displaying absorbance and emission decreased gradually from pH 7.0 to 3.0 (Fig. 1c, d, Fig. S11 c, d). Given this “click-fluorescence” and pH sensitivity features, together with other well-known advantages of IEDDA reaction, this novel conjugation strategy displayed a promising prospect in biochemical applications.
2.9. Cell preparation and living cell microscopy RAW 264.7 cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin solution. Cells were maintained in 5% CO2 and 37 ℃ humidified atmosphere. RAW 264.7 cells were seeded in confocal glass bottom dishes and maintained for 1 day. R B-R A-TZ-2 (5 μM) and commercial probe LysoTracker Green (2 μM) were suspended in DMEM medium with 5% DMSO. After rinsing with PBS, cells were treated with the pre-warmed experimental medium for 30 min at 37℃. After that, the medium was removed, cells were rinsed with PBS. In fresh pre-warmed DMEM medium, living cells microscopy was conducted immediately. For the colocalization assay, To avoid interference from the RA-TZ fluorescence (Em = 488 nm), LysoTracker
3.2. Probe R B-R A with IEDDA capacity As shown in Scheme 2c, a rhodamine-based probe RB-NH2 was conjugated with RA-NHS by amidation, resulting in a pH probe R B-R A with IEDDA capacity. As shown in Fig. 2 and Fig. S12, in acid media R B-R A showed similar absorbance (peak at 562 nm) as probe RB-NH2. However, in neutral and alkaline media, R B-R A displayed an extra strong absorption between 360 nm and 500 nm, which was resulted 323
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Fig. 2. Fluorescence property of probe R B-R A in B-R buffers with 10% DMSO at different pH values. (a) Absorbance; (b) Emission. Inset: photographs of R B-R A in B − R buffers (up) bright field, (down) under 365 nm irradiation.
responded to the pH change independently. As shown in Table S1, RA-TZ-1 and RA-TZ-2 maintained their emissions at 518 nm in both DMSO and B-R solutions. While R B-R ATZ-1 and R B-R A-TZ-2 exhibited solvent effect, displaying blue-shift from 518 nm (in DMSO) to 488 nm (in B-R solutions). These different response to solutions may result from the O to N conversion of RA. What is more, as shown in Fig. 3, when compared with RA-TZ-2, the RA-TZ part of probe R B-R A-TZ-2 displaying larger fluorescence intensity variation from pH 3.0 to pH 7.0. This indicated that the O to N conversion of RA may not only induced the blue-shift of emission, but also increased the sensitivity of RA-TZ part to pH changes.
from the RA decoration. In acid media, R B-R A featured an intense emission (584 nm), which was identical to probe RB-NH2. Notably, no green fluorescence was witnessed in any R B-R A samples. 3.3. Probe R B-R A-TZ-2 By simply mixing probe R B-R A with TZ-2 to triggered IEDDA reaction, products R B-R A-TZ-2 were obtained. As shown in Fig. 3a and b, in B-R solutions with reduced pH values (from pH 7.0 to 3.0), absorbance and emission (488 nm) of the RA-TZ part in R B-R A-TZ-2 decreased gradually, while the absorbance (562 nm) and emission (584 nm) of the rhodamine part in R B-R A-TZ-2 increased gradually. At pH 3.0 and pH 7.0, the absorption coefficient of R B-R A-TZ-2 was calculated to be 2.21 × 105 M−1 cm−1 and 6.20 × 103 M−1 cm−1 respectively; absolute quantum yield of fluorescence was measured as 10.96% and 1.28% respectively. In Fig. 3c, the fluorescence intensity ratio between rhodamine and RA-TZ fluorescence (I584/I488) varied from 440 folds at pH 3.0 to 0.4 folds at pH 9.0. According to the Henderson–Hasselbalch equation [34], the apparent pKa value of R B-R A-TZ-2 was determined to be 4.22. Importantly, this curve showed a good linear relation (R = 0.9797) at pH range of 3.5–5.0, which indicated the high sensitivity of probe R B-R A-TZ-2 to H+ in this pH range, enabling it to measure lysosomal pH (pH 4.5–5.5) [35,36]. In probe R B-R A-TZ-2, the emission band (peak 488 nm) of RA-TZ part partly overlapped the excitation band (peak 562 nm) of rhodamine part, which was likely to trigger Förster resonance energy transfer (FRET) [37,38]. However, fluorescence decay assay (Fig. S13) suggested that there was no obvious FERT between the RA-TZ part and the rhodamine part of R B-R A-TZ-2, indicating that these two parts
3.4. Co-location imaging in living cells To investigate the biological applications, we firstly performed the co-location assay in living RAW 264.7 cells using probe R B-R A-TZ-2 with a commercial probe LysoTracker Green as comparison. After 30 min incubation in RAW 264.7 cells, rhodamine part of R B-R A-TZ-2 displayed orange fluorescence in the pseudo red channel (Fig. 4a); LysoTracker Green displayed green fluorescence in the pseudo green channel (Fig. 4b). Fig. 4e displayed the fluorescence intensity profile of regions of interest across cells. The red curve (rhodamine part of R B-R A-TZ-2) and green curve (LysoTracker Green) exhibited a largely tendency of synchronization. In addition, the fluorescence intensity scatter plot (Fig. 4f) was shown with a Pearson’s colocalization coefficient 0.81. This result was acceptable since probe R B-R A-TZ-2 was not functioned with lysosome-targeting functional groups. The imaging data mentioned above suggested that the orange fluorescence emitted by ring-opened rhodamine part of R B-R A-TZ-2 was largely located in
Fig. 3. (a) Absorbance and (b) Emission of probe R B-R A-TZ-2 in B-R buffers at different pH values. Inset: (up) bright field, (down) under 365 nm irradiation; (c) Fluorescence intensity ratio (I584/I488) at range of pH 3.0―9.0. Inset: linear relationship between the ratio and pH values at range of pH 3.5 − 5.0. 324
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Fig. 4. Colocalization images of R B-R A-TZ-2 (5 μM) and LysoTracker Green (2 μM) in RAW 264.7 cells. (a) rhodamine fluorescence of R B-R A-TZ-2 in red channel; (b) Fluorescence image of LysoTracker Green in green channel; (c) Bright-field image; (d) Merged image of a, b and c. Yellow arrow: regions of interest across cells; (e) Fluorescence intensity profile of regions of interest with R B-R A-TZ-2 (red), and LysoTracker Green (green); (f) Fluorescence intensity scatter plot of R B-R A-TZ-2 and LysoTracker Green. Insert: absolute fluorescence intensity frequency. Scale bar: 10 μm (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
Fig. 5. Confocal fluorescent microscopy of RAW 264.7 cells incubated with R B-R A-TZ-2 (5 μM) for 30 min. With single excitation (Ex = 405 nm), (a) fluorescence of RA-TZ part was visualized in pseudo green channel (EM = 488 ± 20 nm); (b) fluorescence of rhodamine part was visualized in pseudo red channel (Em = 584 ± 20 nm); (c) bright field; (d) merged image of a, b and c; (e) merged image of a and b; (f) Regional mapping of fluorescence intensity ratio between a and b. Local pH value was indicated by the color bar. Scale bar: 5 μm (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
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lysosomes. Though lack of lysosome-targeting functional groups such as morpholine [39,40], R B-R A-TZ-2 may also be transferred into lysosomes by free diffusion, then become ring-opened form in the low-pH circumstance. Given the fact that lysosomes (pH 4.5–5.5) represented the lowest pH condition in living cells, the capacity of probe RB–RA-TZ2 for intracellular pH detection was proved.
[6] C. Meyer, S. Liebscher, F. Bordusa, Selective coupling of click anchors to proteins via trypsiligase, Bioconjug. Chem. 27 (2016) 47–53, https://doi.org/10.1021/acs. bioconjchem.5b00618. [7] B.L. Oliveira, Z. Guo, G.J.L. Bernardes, Inverse electron demand Diels-Alder reactions in chemical biology, Chem. Soc. Rev. 46 (2017) 4895–4950, https://doi.org/ 10.1039/c7cs00184c. [8] T. Plass, et al., Amino acids for Diels-Alder reactions in living cells, Angew. Chem. Int. Ed. Engl. 51 (2012) 4166–4170, https://doi.org/10.1002/anie.201108231. [9] A. Dumas, L. Lercher, C.D. Spicer, B.G. Davis, Designing logical codon reassignment - expanding the chemistry in biology, Chem. Sci. 6 (2015) 50–69, https://doi.org/ 10.1039/c4sc01534g. [10] S. Chaudhuri, T. Korten, S. Diez, Tetrazine-trans-cyclooctene mediated conjugation of antibodies to microtubules facilitates subpicomolar protein detection, Bioconjug. Chem. 28 (2017) 918–922, https://doi.org/10.1021/acs.bioconjchem.7b00118. [11] J.E. Glasgow, M.L. Salit, J.R. Cochran, In vivo site-specific protein tagging with diverse amines using an engineered sortase variant, J. Am. Chem. Soc. 138 (2016) 7496–7499, https://doi.org/10.1021/jacs.6b03836. [12] J.Y. Choi, B.C. Lee, Click reaction: an applicable radiolabeling method for molecular imaging, Nucl. Med. Mol. Imaging 49 (2015) 258–267. [13] T. Peng, H.C. Hang, Site-specific bioorthogonal labeling for fluorescence imaging of intracellular proteins in living cells, J. Am. Chem. Soc. 138 (2016) 14423–14433. [14] M. Schürmann, P. Janning, S. Ziegler, H. Waldmann, Small-molecule target engagement in cells, Cell Chem. Biol. 23 (2016) 435–441. [15] L.G. Meimetis, J.C. Carlson, R.J. Giedt, R.H. Kohler, R. Weissleder, Ultrafluorogenic coumarin–tetrazine probes for real‐time biological imaging, Angew. Chemie Int. Ed. 53 (2014) 7531–7534. [16] Y. Yuan, S. Xu, X. Cheng, X. Cai, B. Liu, Bioorthogonal turn‐on probe based on aggregation‐induced emission characteristics for cancer cell imaging and ablation, Angew. Chemie Int. Ed. 55 (2016) 6457–6461. [17] H. Wu, N.K. Devaraj, Advances in Tetrazine Bioorthogonal Chemistry Driven by the Synthesis of Novel Tetrazines and Dienophiles. Accounts of Chemical Research, (2018). [18] Y. Lee, W. Cho, J. Sung, E. Kim, S.B. Park, Mono-chromophoric design strategy for tetrazine-based colorful bioorthogonal probes with a single fluorescent core skeleton, J. Am. Chem. Soc. (2017). [19] X. Shang, et al., Fluorogenic protein labeling using a genetically encoded unstrained alkene, Chem. Sci. 8 (2017) 1141–1145. [20] A. Vázquez, et al., Mechanism‐based fluorogenic trans‐cyclooctene–tetrazine cycloaddition, Angew. Chemie Int. Ed. 56 (2017) 1334–1337. [21] M. Tian, X. Peng, J. Fan, J. Wang, S. Sun, A fluorescent sensor for pH based on rhodamine fluorophore, Dye. Pigment. 95 (2012) 112–115. [22] L. Liu, et al., Fluorescent and colorimetric detection of pH by a rhodamine-based probe, Sens. Actuators B Chem. 194 (2014) 498–502. [23] Q.A. Best, R. Xu, M.E. McCarroll, L. Wang, D.J. Dyer, Design and investigation of a series of rhodamine-based fluorescent probes for optical measurements of pH, Org. Lett. 12 (2010) 3219–3221. [24] X. Liu, et al., Ratiometric fluorescent probe for lysosomal pH measurement and imaging in living cells using single-wavelength excitation, Anal. Chem. 89 (2017) 7038–7045. [25] Z. Ding, et al., Nanoscale metal–organic frameworks coated with poly (vinyl alcohol) for ratiometric peroxynitrite sensing through FRET, Chem. Sci. 8 (2017) 5101–5106. [26] S. Takahashi, et al., Development of a series of practical fluorescent chemical tools to measure pH values in living samples, J. Am. Chem. Soc. 140 (2018) 5925–5933. [27] S. Wang, et al., Elucidating the structure–reactivity correlations of phenothiazine‐based fluorescent probes toward ClO−, Chem. Eur. J. (2018). [28] M.H. Lee, et al., Two‐color probe to monitor a wide range of pH values in cells, Angew. Chemie Int. Ed. 52 (2013) 6206–6209. [29] S. Wang, et al., Activatable photoacoustic and fluorescent probe of nitric oxide for cellular and in vivo imaging, Sens. Actuators B Chem. 267 (2018) 403–411. [30] S. Jain, K. Neumann, Y. Zhang, J. Geng, M. Bradley, Tetrazine-Mediated Postpolymerization Modification, Macromolecules 49 (2016) 5438–5443. [31] R. Pipkorn, et al., Inverse‐electron‐demand Diels‐Alder reaction as a highly efficient chemoselective ligation procedure: synthesis and function of a BioShuttle for temozolomide transport into prostate cancer cells, J. Pept. Sci. 15 (2009) 235–241. [32] S. Hörner, et al., Combination of inverse electron-demand Diels–Alder reaction with highly efficient oxime ligation expands the toolbox of site-selective peptide conjugations, Chem. Commun. 51 (2015) 11130–11133. [33] K.-Y. Tan, et al., Real-time monitoring ATP in mitochondrion of living cells: a specific fluorescent probe for ATP by dual recognition sites, Anal. Chem. 89 (2017) 1749–1756. [34] G.K. Vegesna, et al., pH-activatable near-infrared fluorescent probes for detection of lysosomal pH inside living cells, J. Mater. Chem. B 2 (2014) 4500–4508. [35] S.-L. Shen, et al., A mitochondria-targeted ratiometric fluorescent probe for hypochlorite and its applications in bioimaging, J. Mater. Chem. B 5 (2017) 289–295. [36] S. Ohkuma, B. Poole, Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents, Proc. Natl. Acad. Sci. 75 (1978) 3327–3331. [37] Z. Ding, C. Wang, G. Feng, X. Zhang, Thermo-responsive fluorescent polymers with diverse LCSTs for ratiometric temperature sensing through FRET, Polymers 10 (2018) 283. [38] R. Kawagoe, I. Takashima, S. Uchinomiya, A. Ojida, Reversible ratiometric detection of highly reactive hydropersulfides using a FRET-based dual emission fluorescent probe, Chem. Sci. 8 (2017) 1134–1140. [39] J.R. Casey, S. Grinstein, J. Orlowski, Sensors and regulators of intracellular pH, Nat. Rev. Mol. Cell Biol. 11 (2010) 50.
3.5. Fluorescent imaging in living cells To evaluate the intracellular ratiometric pH detection ability of probe R B-R A-TZ-2, fluorescent microscopy in living cells was conducted. As shown in Fig. 5a, green fluorescence distributed evenly in most part of cell was regarded as the RA-TZ fluorescence, which was attributed to free diffusion of probe R B-R A-TZ2 in both cytoplasm and organelles. Differently, in Fig. 5b orange fluorescence from ring-opened rhodamine part was witnessed, which was thought to mostly locate in lysosomal site. In Fig. 5f, to measure and image the pH distribution in a ratiometric manner, regional mapping of fluorescence intensity ratio between RA-TZ fluorescence and rhodamine fluorescence was created. Once given the certain ratio (I584/I488) in a certain region of cell, the pH value in this region could be indicated by referring the ratio-pH curve (Fig. 3c). In the warmest region (red), the ratio (I584/I488) was about 40 folds, indicating the pH value to be 4.9, which was in line with real lysosomal pH; in the coolest region (blue), the ratio (I584/I488) was about 0.5 folds, indicating the pH values to be 6.5-7.0. The result mentioned above had demonstrated the capacity of probe R B-R A-TZ-2 to detect intracellular pH in a ratiometric manner. 4. Conclusion In conclusion, we reported the unique in-situ fluorescent properties of the inverse electron-demand Diels–Alder reaction products using Reppe anhydride and 1,2,4,5-tetrazines. A ratiometric fluorescent probe R B-R A-TZ-2 was constructed and applied in intracellular pH detection. Moreover, taking advantages of this “click-fluorescence” conjugation strategy, non-fluorescent antibody can be easily endowed with fluorescence, also dual-color fluorescent products can be prepared more conveniently. Efficient and flexible, this strategy is anticipated to widely contribute to biochemical applications. Acknowledgements This work was supported by the Macao Science and Technology Development Fund under Grant No.: 052/2015/A2, 082/2016/A2, and 019/2017/AMJ; the Research Grant of University of Macau under grant No.: MYRG2016-00058-FHS and MYRG2017-00066-FHS. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.snb.2018.09.022. References [1] S.I. Presolski, V. Hong, S.H. Cho, M.G. Finn, Tailored ligand acceleration of the Cucatalyzed azide-alkyne cycloaddition reaction: practical and mechanistic implications, J. Am. Chem. Soc. 132 (2010) 14570–14576, https://doi.org/10.1021/ ja105743g. [2] D. Soriano Del Amo, et al., Biocompatible copper(I) catalysts for in vivo imaging of glycans, J. Am. Chem. Soc. 132 (2010) 16893–16899, https://doi.org/10.1021/ ja106553e. [3] J. Dommerholt, F. Rutjes, F.L. van Delft, Strain-promoted 1,3-dipolar cycloaddition of cycloalkynes and organic azides, Top. Curr. Chem. (Cham) 374 (16) (2016), https://doi.org/10.1007/s41061-016-0016-4. [4] A. Herner, Q. Lin, Photo-triggered click chemistry for biological applications, Top. Curr. Chem. (Cham) 374 (1) (2016), https://doi.org/10.1007/s41061-015-0002-2. [5] L. Davis, J.W. Chin, Designer proteins: applications of genetic code expansion in cell biology, Nat. Rev. Mol. Cell Biol. 13 (2012) 168–182, https://doi.org/10.1038/ nrm3286.
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under the supervision of Prof. Yupeng Tian. At present, he is a pH.D student at Faculty of Health Sciences, University of Macau. His research interest focuses on fluorescent and photoacoustic probes.
Gang Feng received his M.E. degree in Health Science Center, Shenzhen University in 2015. He is currently a pH.D. student in Faculty of Health Sciences, University of Macau. His work mostly focuses on the application of click-reaction techniques on probing and microtubule in-vitro engineering.
Shichao Wang received his B.S. degree in 2012 and M.E. degree in 2015, both in chemistry from Anhui University. He is now a pH.D. student at Faculty of Health Sciences, University of Macau. His research interest focus on fluorescent and photoacoustic probes. Zhaoyang Ding received his PhD degree from East China University of Science and Technology, China in 2013. He worked as postdoc at Faculty of Health Sciences, University of Macau from 2016 to 2018. His research interests include hybrid smart polymeric materials for sensing applications.
Boyu Zhang received his PhD from Dalian Institute of Chemical Physics, Chinese Academy of Sciences in 2013. He worked as a research associate at Dalian Institute of Chemical Physics from 2013 to 2015 and postdoc at Faculty of Health Sciences, University of Macau from 2016 to 2018. He is currently working at Dalian Medical University.
Xuanjun Zhang received his PhD degree in Chemistry from University of Science & Technology of China in 2004. After working at Shantou University, National University of Singapore, Linköping University, and University of Washington as postdocs and visiting scientist, he started his assistant professor at Linköping University in 2011 and was promoted to Docent in 2014. He moved to University of Macau in 2015. His research interests mainly focus on molecular and nanoprobes for fluorescent and photoacoustic imaging and sensing applications.
Chunfei Wang is currently a pH.D. Student in Faculty of Health Sciences, University of Macau, Macau SAR, China. His research interests include chemical sensors and biosensors. Jingyun Tan received his M.S degree of organic chemistry in Anhui University in 2015
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Construction of ratiometric fluorescent probe based on inverse electrondemand Diels–Alder reaction for pH measurement in living cells
T
Gang Feng, Boyu Zhang, Chunfei Wang, Jingyun Tan, Shichao Wang, Zhaoyang Ding, ⁎ Xuanjun Zhang Faculty of Health Science, University of Macau, Macau SAR, China
A R T I C LE I N FO
A B S T R A C T
Keywords: In situ fluorescence pH probe Ratiometric analysis Inverse electron demand Diels–Alder reaction
To endow fluorescent functions to the inverse electron demand Diels–Alder (IEDDA) reaction products, introduction of extraneous fluorophores is usually inevitable. However, this strategy is blamed for complex construction and background fluorescence. It is desirable to construct IEDDA fluorescent products in a more convenient manner. In this work, we reported the in-situ generated green fluorescence in IEDDA reaction. The fluorescence intensity of IEDDA products RA-TZ-1 and RA-TZ-2 decreased in reduced pH values (from pH 7.0–3.0). Based on this novel property, we developed a ratiometric fluorescent probe RB–RA-TZ-2 by conjugation with another probe RB-NH2 (Em = 584 nm) with inverse response toward pH values. By single excitation, the fluorescence intensity ratio (I584/I488) showed a linear response (R = 0.9797) to H+ in pH range of 3.5–5.0. Colocalization imaging with LysoTracker Green indicated the capacity of RB–RA-TZ-2 to detect lysosomal pH (Pearson’s coefficient 0.81). Confocal microscopy was applied to measure and image intracellular pH in living cells.
1. Introduction Bioorthogonal techniques such as copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC), [1,2] strain-promoted alkyne-azide cycloadditions (SPAAC) [3], photoclick reactions [4] and inverse electron-demand Diels–Alder reaction (IEDDA) [5,6] have revolutionized conjugation strategies in chemical biology. Among these techniques, IEDDA is highlighted for its excellent specificity, fast reaction rate, catalyst free and biocompatibility features [7], which has been applied in applications such as genetic code expansion [8,9], protein modification [10,11],nuclear medicine [12], and fluorescent imaging [13,14]. Usually, neither the IEDDA bioorthogonal pairs (dienophile and diene) nor the IEDDA products are fluorescent. To endow fluorescent function to IEDDA products, introduction of extraneous fluorophores is inevitable [15,16]. One classical fluorescent labeling strategy begins with decoration of a biochemical reporter with a dienophile group, and decoration of an extraneous fluorophore with 1,2,4,5-tetrazine diene group [17,18]. By means of IEDDA reaction, the biochemical reporter group successfully conjugates with the fluorophore, followed by the recovery of fluorescence results from the breakdown of 1,2,4,5-tetrazine structure. This strategy is ingenious, however has deficiencies such
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as complex construction and background fluorescence. Simple fluorescent conjugation strategy without extraneous fluorophores is ideal but challenging. Recently, several strategies are reported to meet this demand. Shang’s group endowed proteins with green fluorescence through IEDDA reaction between non-fluorescent styrene and 1,2,4,5-tetrazines [19]. Vazquez’s group reported that by combination of trans-cyclooctenes (TCOs) with 1,2,4,5-tetrazines, IEDDA products with various fluorescence were developed [20]. In this study, we began with the confirmation of in situ fluorescence from the IEDDA reaction by simply mixing Reppe anhydride with 1,2,4,5-tetrazines (Scheme 1a). This “click-fluorescence” conjugation strategy showed a promising application prospect in construction of fluorescent functional products (Scheme 1b). Moreover, IEDDA products RA-TZ-1 and RA-TZ-2 exhibited gradually decreased emission in reduced pH solutions (from pH 7.0–3.0). On the other hand, rhodamine amide derivatives were widely applied in pH sensing studies for their sensitivity to acid environment [21–23]. Importantly, it responded to reduced pH solutions (from pH 7.0–3.0) with a gradually increased fluorescence emission, which just contrasted with that of RA-TZ-1 and RA-TZ-2. It was also well known that probes with single fluorescence emission suffered low measurement accuracy
Corresponding author. E-mail address: [email protected] (X. Zhang).
https://doi.org/10.1016/j.snb.2018.09.022 Received 2 May 2018; Received in revised form 1 September 2018; Accepted 6 September 2018 Available online 07 September 2018 0925-4005/ © 2018 Elsevier B.V. All rights reserved.
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Scheme 1. (a) in situ fluorescence from the IEDDA reaction between Reppe anhydride and 1,2,4,5-tetrazines; (b) Application prospect of “click-fluorescence” conjugation strategy; (c) pH sensing mechanism of probe R B-R A-TZ-2.
2.2. Synthesis of tetrazine TZ-2
because of instrumental or operating error. However, this defect could be improved by ratiometric fluorescence strategy, whose measurement accuracy could be largely improved by means of self-calibration of two different emission bands in one individual probe [24–27]. Therefore to demonstrate the potentials of this novel “click-fluorescence” conjugation strategy in a universal manner based on the IEDDA fluorescence and rhodamine fluorescence, the construction of a concise ratiometric fluorescent pH probe was carried out. Herein, a reported rhodamine based probe RB-NH2 [28] was transformed into probe R B-R A with the ability to trigger IEDDA reaction with 1,2,4,5-tetrazines. By the IEDDA reaction between R B-R A and tetrazine TZ-2, a ratiometric fluorescent pH probe R B-R A-TZ-2 was developed. The special pH sensing mechanism of R B-R A-TZ-2 derived from the fluorescence intensity ratio between orange fluorescence (584 nm) and green fluorescence (488 nm) from rhodamine part and RA-TZ part respectively (Scheme 1c). By single excitation at 360 nm, the fluorescent intensity ratio (I584/I488) changed correspondingly in pH values. In addition, confocal microscopy was conducted to measure and image intracellular pH in living RAW 264.7 cells.
2-pyrimidinecarbonitrile (1.05 g, 10 mmol, 1.0 eq) was suspended in dry EtOH (20 mL). With the dropwise addition of hydrazine hydrate (1.56 mL, 25 mmol, 2.5 eq), the orange mixture was stirred at 85 °C with reflux under nitrogen protection for 16 h. The orange precipitate was collected by vacuum filtration and washed by EtOH. The intermediate product was suspended in acetic acid (10 mL). In fume hood, isopentyl nitrite (1 mL, 7.15 mmol) was added dropwise with stirring. The orange mixture turned purple with release of toxic gas nitrogen dioxide. After 8 h oxidation, the purple precipitate was collected and washed by EtOH. Crude purple product went for a flash chromatography in silica gel column (DCM, followed by DCM/MeOH 10:1). The purple filtrate was collected and dried to obtain TZ-2 as purple solid. (700 mg, 2.94 mmol, 70%). 1H NMR (400 MHz, DMSO-d6) δ 9.23 (d, J = 4.9 Hz, 4 H), 7.87 (t, J = 4.9 Hz, 2 H). 13C NMR (101 MHz, DMSOd6): 163.45, 159.41, 159.07, 123.67. 2.3. Synthesis of RA-TZ-1 To a solution of RA (22.2 mg, 0.11 mmol, 1.1 eq) in 30 mL DMSO, TZ-1 (28.0 mg, 0.1 mmol, 1.0 eq) was added. During the 18 h stirring at room temperature, the solution turned yellow from purple. Green fluorescence was witnessed using hand-held 365 nm UV lamp. Fresh DD water was added followed by DCM extraction to remove DMSO, the yellow crude product in DCM was dried by MgSO4. Column chromatography in silica gel column (DCM/MeOH 10:1) was conducted to afford the IEDDA product RA-TZ-1 as light-yellow solid (31.8 mg, 0.07 mmol, 70%). 1H NMR (400 MHz, DMSO-d6) δ 9.12 (d, J = 4.9 Hz, 2 H), 8.33 (d, J = 8.2 Hz, 2 H), 8.19 (d, J = 8.0 Hz, 2 H), 7.66 (t, J = 5.1 Hz, 1 H), 5.89 (q, J = 6.9, 6.2 Hz, 2 H), 4.35 (t, J = 3.9 Hz, 1 H), 4.20 (t, J = 4.1 Hz, 1 H), 3.84 (d, J = 4.9 Hz, 1 H), 3.71 (s, 1 H), 3.68 – 3.60 (m, 2 H). 13C NMR (101 MHz, DMSO-d6) δ 173.84, 173.67, 167.39, 158.77, 149.03, 145.43, 138.19, 132.92, 131.09, 130.74, 130.31, 127.96, 45.98, 45.65, 44.27, 43.76, 35.72, 35.29. ESI-MS calcd for C25H18N4O5, [M+H]+ 455.1355, found 455.1385.
2. Experimental section 2.1. Materials and instruments All commercial chemicals were purchased from commercial suppliers and used without further purification. Twice distilled water was used throughout all experiments. 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded by Bruker AV 400 MHz spectrometers. High-resolution mass spectra (HRMS) was recorded by the Waters Xevo G2-XS Q-Tof matrix-assisted time of flight mass spectrometer using high performance liquid chromatography (HPLC) and electro-spray ionization (ESI) technique. UV–vis absorption spectra were recorded by SHIMADZU UV-1800 spectrophotometer. Fluorescence spectra was recorded by HORIBA Fluorolog-3 modular spectrometer with a Xe lamp as excitation source. Absolute fluorescence quantum yield measurement was conducted by integrating sphere on HORIBA Fluorolog-3 modular spectrofluorometer [29]. Fluorescence lifetime was detected by DeltaTime TCSPC on HORIBA Fluorolog@-3 spectrometer equipped with a HORIBA NanoLED source (N-455 nm). Carl Zeiss LSM710 confocal microscope was applied for living cells fluorescent imaging. Synthesis of tetrazine TZ-1, Reppe anhydride (RA) and RA-NHS were according to published procedures [30–32].
2.4. Synthesis of RA-TZ-2 To the solution of RA (22.2 mg, 0.11 mmol, 1.1 eq) in 10 mL acetonitrile, TZ-2 (23.8 mg, 0.1 mmol, 1.0 eq) was added. During the 18 h stirring at room temperature, the solution turned yellow from purple. Green fluorescence was witnessed using hand-held 365 nm UV lamp. After removal of the solvent, the crude product was purified by column chromatography in silica gel column (DCM/MeOH 20:1) to afford the 321
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acetonitrile, DIEA (51.6 mg, 0.4 mmol, 2.0 eq) was added. After stirring at 0℃ for 30 min, RB-NH2(105.4 mg, 0.2 mmol, 1.0 eq)solution in 5 mL acetonitrile was added dropwise. The mixture was given a 1 h stirring at 0℃ followed by stirring at room temperature overnight. After removal of solvent, crude product was purified by column chromatography in Al2O3 column (DCM/MeOH 10:1) to afford RB–RA as yellow solid (95.6 mg, 0.12 mmol, 60%). 1H NMR (400 MHz, Chloroform-d) δ 7.91 – 7.83 (m, 1 H), 7.66 (t, J = 5.7 Hz, 1 H), 7.54 – 7.46 (m, 2 H), 7.14 – 7.08 (m, 1 H), 6.47 – 6.29 (m, 6 H), 5.96 – 5.82 (m, 4 H), 3.59 – 3.30 (m, 15 H), 3.14 (d, J = 4.1 Hz, 2 H), 3.03 (s, 2 H), 2.84 – 2.75 (m, 6 H), 2.71 (s, 1 H), 2.24 (t, J = 7.0 Hz, 2 H), 1.19 (t, J = 7.0 Hz, 12 H). 13C NMR (101 MHz, Chloroform-d) δ 178.96, 172.13, 153.32, 148.88, 137.99, 132.63, 128.69, 128.34, 128.18, 123.89, 122.76, 108.15, 105.26, 97.70, 77.25, 58.44, 47.61, 44.38, 44.17, 43.28, 39.66, 38.84, 37.90, 36.67, 33.43, 23.92, 18.46, 12.60. ESI-MS calcd for C48H56N6O5, [M+H]+ 770.4390, found 770.4093. [M+2 H]2+ 385.7234, found 385.7182.
IEDDA product RA-TZ-2 as light-yellow solid (31.8 mg, 0.07 mmol, 70%). 1H NMR (400 MHz, Chloroform-d) δ 9.13 (s, 1 H), 8.81 (d, J = 4.8 Hz, 2 H), 8.73 (d, J = 4.8 Hz, 2 H), 7.24 (t, J = 4.9 Hz, 1 H), 7.17 (t, J = 4.9 Hz, 1 H), 6.70 – 6.64 (m, 1 H), 6.49 (t, J = 7.4 Hz, 1 H), 3.98 – 3.85 (m, 2 H), 3.50 (t, J = 3.6 Hz, 1 H), 3.31 – 3.22 (m, 2 H), 3.14 (qd, J = 8.8, 3.0 Hz, 2 H). 13C NMR (101 MHz, Chloroform-d) δ 172.76, 158.47, 157.41, 157.03, 133.33, 132.44, 131.84, 122.60, 119.83, 119.27, 47.43, 46.88, 44.08, 43.33, 41.79, 35.69, 35.15, 30.98, 29.72, 18.46. ESI-MS calcd for C22H16N6O3, [M+H]+ 413.1362, found 413.1396. 2.5. Synthesis of RB-NH2 RB-NH2 was synthesized according to a modification of a reported literature procedure [33]. To a solution of rhodamine B (1 g, 2.1 mmol, 1.0 eq) in EtOH (50 mL), diethylenetriamine (4.5 mL, 42 mmol, 20.0 eq) was added. The mixture was stirred at 85 °C under reflux for 24 h. After removal of solvent, orange residue was suspended in DCM (100 mL). DD water (100 mL) was added to extract the excess DIEA. The water layer was replaced by large amount of fresh DD water until becoming neutral. The organic layer was collected and dried to obtain RB-NH2 as orange solid (300 mg, 0.57 mmol, 27%). 1H NMR (400 MHz, Chloroform-d) δ 7.96 – 7.81 (m, 1 H), 7.43 (dd, J = 5.7, 3.1 Hz, 2 H), 7.14 – 6.99 (m, 1 H), 6.42 (d, J = 8.9 Hz, 2 H), 6.37 (d, J = 2.6 Hz, 2 H), 6.27 (d, J = 2.6 Hz, 1 H), 6.25 (d, J = 2.7 Hz, 1 H), 3.33 (q, J = 7.1 Hz, 8 H), 3.26 (t, J = 6.5 Hz, 2 H), 2.60 (t, J = 5.9 Hz, 2 H), 2.43 (t, J = 5.9 Hz, 2 H), 2.39 (t, J = 6.5 Hz, 2 H), 1.15 (t, J = 7.0 Hz, 12 H). ESI-MS calcd for C32H41N5O2, [M+H]+ 528.3339, found 528.3374.
2.7. Synthesis of R B-R A-TZ-2 To a solution of R B-R A (79.7 mg, 0.1 mmol, 1.0 eq) in 10 mL acetonitrile, tetrazine TZ-2 (26.2 mg, 0.11 mmol, 1.1 eq) was added. During the18 h stirring at room temperature, the solution turned brown from purple. Green fluorescence was witnessed by using hand-held 365 nm UV lamp. After removal of solvent, crude product was purified by column chromatography in Al2O3 column (DCM/MeOH 40:1) to afford IEDDA product RB–RA-TZ-2 as brown solid (50.3 mg,0.05 mmol, 50%). 1H NMR (400 MHz, Chloroform-d) δ 8.81 (d, J = 4.8 Hz, 2 H), 8.72 (dd, J = 4.8, 1.2 Hz, 2 H), 7.87 (s, 1 H), 7.49 – 7.38 (m, 3 H), 7.22 (t, J = 5.7 Hz, 1 H), 7.14 (d, J = 2.9 Hz, 1 H), 7.10 – 7.04 (m, 1 H), 6.49 – 6.22 (m, 8 H), 3.87 (s, 2 H), 3.82 (s, 1 H), 3.51 (s, 4 H), 3.31 (dt, J = 23.4, 7.5 Hz, 14 H), 2.82 (s, 2 H), 2.28 – 2.11 (m, 4 H), 1.90 – 1.82
2.6. Synthesis of R B-R A To a solution of RA-NHS (84.4 mg, 0.22 mmol, 1.1 eq) in 5 mL
Fig. 1. (a) Fluorescence at 518 nm intensified during the 12 h reaction between RA and tetrazine TZ-1 in DMSO; (b) Fluorescence property of RA-TZ-1 in DMSO; (c) Absorbance and (d) Emission of RA-TZ-1 in B-R buffers with 10% DMSO at various pH values.
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Scheme 2. Synthesis of (a) IEDDA product RA-TZ-1; (b) IEDDA product RA-TZ-2; (c) Fluorescent pH probe R B-R A with IEDDA capacity; (d) Ratiometric fluorescent pH probe R B-R A-TZ-2.
(m, 2 H), 1.16 (t, J = 7.0 Hz, 12 H). 13C NMR (101 MHz, Chloroform-d) δ 178.78, 168.87, 157.36, 156.96, 153.56, 153.32, 148.81, 138.00, 129.05, 128.91, 128.33, 128.05, 123.79, 122.75, 108.05, 108.02, 105.55, 105.48, 97.76, 97.67, 77.24, 67.97, 64.88, 52.26, 51.53, 48.23, 44.40, 44.20, 44.17, 43.72, 43.28, 41.01, 38.64, 36.66, 31.66, 31.40, 22.82, 22.76, 12.61. ESI-MS calcd for C58H62N12O5, [M+H]+ 1007.5044, found 1007.5079. [M + 2 H]2+ 504.2561, found 504.2583.
Green was displayed in pseudo green channel (Ex = 488 nm, Em = 530 ± 10 nm); rhodamine fluorescnece of R B-R A-TZ-2 was displayed in pseudo red channel (Ex = 543 nm, Em = 584 ± 10 nm). For the intracellular pH detecting assay with probe R B-R A-TZ-2 (5 μM), at single excitation (Ex = 405 nm), fluorescence from RA-TZ part was visualized in pseudo green channel (Em = 488 ± 20 nm), fluorescence from rhodamine part was visualized in pseudo red channel (Em = 584 ± 20 nm). Images were analyzed by Zeiss ZEN 2012 and Image J software.
2.8. Spectral measurements 3. Results and discussion Britton–Robinson (B–R) buffers with various pH values ranged from pH 3.0 to 9.0 were prepared according to standard procedures. RA-TZ1, RA-TZ-2, RB-NH2, R B-R A and R B-R A-TZ-2 were prepared at 1.0 mM in DMSO respectively as stock solutions. Samples for test were well mixed and incubated at room temperature for 60 min before measurements. For the absorption and emission spectral measurement, samples were diluted into 5 μM by B-R buffer with 10% DMSO (v/v). For the fluorescence lifetime measurement, R B-R A-TZ-2 was diluted into 5 μM in pH 4.0 and pH 8.0 B-R buffers with 10% DMSO (v/v). With excitation at 355 nm, the monitored wavelength was 488 nm. Fluorescence decay histograms were recorded using the time-correlated single photon counting technique in 4095 channels.
3.1. Confirmation of IEDDA fluorescence Reppe anhydride (RA) was introduced in DMSO solutions of tetrazine TZ-1 and TZ-2 respectively (Scheme 2). After 12 h stirring at room temperature, the solutions turned yellow from pink, which was recognized as a major IEDDA reaction feature. In both TZ-1 and TZ-2 solutions, green fluorescence (518 nm) strengthened gradually (Fig. 1a, Fig. S11a). To identify this unreported green fluorescence, IEDDA products RA-TZ-1 and RA-TZ-2 were purified. In view of the 1H NMR, 13 C NMR, mass spectra (Figs. S7, S8), the fluorescent properties of these IEDDA products were confirmed (Fig. 1b, Fig. S11b). Moreover, both RA-TZ-1 and RA-TZ-2 showed response to pH change in B-R buffers, displaying absorbance and emission decreased gradually from pH 7.0 to 3.0 (Fig. 1c, d, Fig. S11 c, d). Given this “click-fluorescence” and pH sensitivity features, together with other well-known advantages of IEDDA reaction, this novel conjugation strategy displayed a promising prospect in biochemical applications.
2.9. Cell preparation and living cell microscopy RAW 264.7 cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin solution. Cells were maintained in 5% CO2 and 37 ℃ humidified atmosphere. RAW 264.7 cells were seeded in confocal glass bottom dishes and maintained for 1 day. R B-R A-TZ-2 (5 μM) and commercial probe LysoTracker Green (2 μM) were suspended in DMEM medium with 5% DMSO. After rinsing with PBS, cells were treated with the pre-warmed experimental medium for 30 min at 37℃. After that, the medium was removed, cells were rinsed with PBS. In fresh pre-warmed DMEM medium, living cells microscopy was conducted immediately. For the colocalization assay, To avoid interference from the RA-TZ fluorescence (Em = 488 nm), LysoTracker
3.2. Probe R B-R A with IEDDA capacity As shown in Scheme 2c, a rhodamine-based probe RB-NH2 was conjugated with RA-NHS by amidation, resulting in a pH probe R B-R A with IEDDA capacity. As shown in Fig. 2 and Fig. S12, in acid media R B-R A showed similar absorbance (peak at 562 nm) as probe RB-NH2. However, in neutral and alkaline media, R B-R A displayed an extra strong absorption between 360 nm and 500 nm, which was resulted 323
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Fig. 2. Fluorescence property of probe R B-R A in B-R buffers with 10% DMSO at different pH values. (a) Absorbance; (b) Emission. Inset: photographs of R B-R A in B − R buffers (up) bright field, (down) under 365 nm irradiation.
responded to the pH change independently. As shown in Table S1, RA-TZ-1 and RA-TZ-2 maintained their emissions at 518 nm in both DMSO and B-R solutions. While R B-R ATZ-1 and R B-R A-TZ-2 exhibited solvent effect, displaying blue-shift from 518 nm (in DMSO) to 488 nm (in B-R solutions). These different response to solutions may result from the O to N conversion of RA. What is more, as shown in Fig. 3, when compared with RA-TZ-2, the RA-TZ part of probe R B-R A-TZ-2 displaying larger fluorescence intensity variation from pH 3.0 to pH 7.0. This indicated that the O to N conversion of RA may not only induced the blue-shift of emission, but also increased the sensitivity of RA-TZ part to pH changes.
from the RA decoration. In acid media, R B-R A featured an intense emission (584 nm), which was identical to probe RB-NH2. Notably, no green fluorescence was witnessed in any R B-R A samples. 3.3. Probe R B-R A-TZ-2 By simply mixing probe R B-R A with TZ-2 to triggered IEDDA reaction, products R B-R A-TZ-2 were obtained. As shown in Fig. 3a and b, in B-R solutions with reduced pH values (from pH 7.0 to 3.0), absorbance and emission (488 nm) of the RA-TZ part in R B-R A-TZ-2 decreased gradually, while the absorbance (562 nm) and emission (584 nm) of the rhodamine part in R B-R A-TZ-2 increased gradually. At pH 3.0 and pH 7.0, the absorption coefficient of R B-R A-TZ-2 was calculated to be 2.21 × 105 M−1 cm−1 and 6.20 × 103 M−1 cm−1 respectively; absolute quantum yield of fluorescence was measured as 10.96% and 1.28% respectively. In Fig. 3c, the fluorescence intensity ratio between rhodamine and RA-TZ fluorescence (I584/I488) varied from 440 folds at pH 3.0 to 0.4 folds at pH 9.0. According to the Henderson–Hasselbalch equation [34], the apparent pKa value of R B-R A-TZ-2 was determined to be 4.22. Importantly, this curve showed a good linear relation (R = 0.9797) at pH range of 3.5–5.0, which indicated the high sensitivity of probe R B-R A-TZ-2 to H+ in this pH range, enabling it to measure lysosomal pH (pH 4.5–5.5) [35,36]. In probe R B-R A-TZ-2, the emission band (peak 488 nm) of RA-TZ part partly overlapped the excitation band (peak 562 nm) of rhodamine part, which was likely to trigger Förster resonance energy transfer (FRET) [37,38]. However, fluorescence decay assay (Fig. S13) suggested that there was no obvious FERT between the RA-TZ part and the rhodamine part of R B-R A-TZ-2, indicating that these two parts
3.4. Co-location imaging in living cells To investigate the biological applications, we firstly performed the co-location assay in living RAW 264.7 cells using probe R B-R A-TZ-2 with a commercial probe LysoTracker Green as comparison. After 30 min incubation in RAW 264.7 cells, rhodamine part of R B-R A-TZ-2 displayed orange fluorescence in the pseudo red channel (Fig. 4a); LysoTracker Green displayed green fluorescence in the pseudo green channel (Fig. 4b). Fig. 4e displayed the fluorescence intensity profile of regions of interest across cells. The red curve (rhodamine part of R B-R A-TZ-2) and green curve (LysoTracker Green) exhibited a largely tendency of synchronization. In addition, the fluorescence intensity scatter plot (Fig. 4f) was shown with a Pearson’s colocalization coefficient 0.81. This result was acceptable since probe R B-R A-TZ-2 was not functioned with lysosome-targeting functional groups. The imaging data mentioned above suggested that the orange fluorescence emitted by ring-opened rhodamine part of R B-R A-TZ-2 was largely located in
Fig. 3. (a) Absorbance and (b) Emission of probe R B-R A-TZ-2 in B-R buffers at different pH values. Inset: (up) bright field, (down) under 365 nm irradiation; (c) Fluorescence intensity ratio (I584/I488) at range of pH 3.0―9.0. Inset: linear relationship between the ratio and pH values at range of pH 3.5 − 5.0. 324
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Fig. 4. Colocalization images of R B-R A-TZ-2 (5 μM) and LysoTracker Green (2 μM) in RAW 264.7 cells. (a) rhodamine fluorescence of R B-R A-TZ-2 in red channel; (b) Fluorescence image of LysoTracker Green in green channel; (c) Bright-field image; (d) Merged image of a, b and c. Yellow arrow: regions of interest across cells; (e) Fluorescence intensity profile of regions of interest with R B-R A-TZ-2 (red), and LysoTracker Green (green); (f) Fluorescence intensity scatter plot of R B-R A-TZ-2 and LysoTracker Green. Insert: absolute fluorescence intensity frequency. Scale bar: 10 μm (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
Fig. 5. Confocal fluorescent microscopy of RAW 264.7 cells incubated with R B-R A-TZ-2 (5 μM) for 30 min. With single excitation (Ex = 405 nm), (a) fluorescence of RA-TZ part was visualized in pseudo green channel (EM = 488 ± 20 nm); (b) fluorescence of rhodamine part was visualized in pseudo red channel (Em = 584 ± 20 nm); (c) bright field; (d) merged image of a, b and c; (e) merged image of a and b; (f) Regional mapping of fluorescence intensity ratio between a and b. Local pH value was indicated by the color bar. Scale bar: 5 μm (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
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lysosomes. Though lack of lysosome-targeting functional groups such as morpholine [39,40], R B-R A-TZ-2 may also be transferred into lysosomes by free diffusion, then become ring-opened form in the low-pH circumstance. Given the fact that lysosomes (pH 4.5–5.5) represented the lowest pH condition in living cells, the capacity of probe RB–RA-TZ2 for intracellular pH detection was proved.
[6] C. Meyer, S. Liebscher, F. Bordusa, Selective coupling of click anchors to proteins via trypsiligase, Bioconjug. Chem. 27 (2016) 47–53, https://doi.org/10.1021/acs. bioconjchem.5b00618. [7] B.L. Oliveira, Z. Guo, G.J.L. Bernardes, Inverse electron demand Diels-Alder reactions in chemical biology, Chem. Soc. Rev. 46 (2017) 4895–4950, https://doi.org/ 10.1039/c7cs00184c. [8] T. Plass, et al., Amino acids for Diels-Alder reactions in living cells, Angew. Chem. Int. Ed. Engl. 51 (2012) 4166–4170, https://doi.org/10.1002/anie.201108231. [9] A. Dumas, L. Lercher, C.D. Spicer, B.G. Davis, Designing logical codon reassignment - expanding the chemistry in biology, Chem. Sci. 6 (2015) 50–69, https://doi.org/ 10.1039/c4sc01534g. [10] S. Chaudhuri, T. Korten, S. Diez, Tetrazine-trans-cyclooctene mediated conjugation of antibodies to microtubules facilitates subpicomolar protein detection, Bioconjug. Chem. 28 (2017) 918–922, https://doi.org/10.1021/acs.bioconjchem.7b00118. [11] J.E. Glasgow, M.L. Salit, J.R. Cochran, In vivo site-specific protein tagging with diverse amines using an engineered sortase variant, J. Am. Chem. Soc. 138 (2016) 7496–7499, https://doi.org/10.1021/jacs.6b03836. [12] J.Y. Choi, B.C. Lee, Click reaction: an applicable radiolabeling method for molecular imaging, Nucl. Med. Mol. Imaging 49 (2015) 258–267. [13] T. Peng, H.C. Hang, Site-specific bioorthogonal labeling for fluorescence imaging of intracellular proteins in living cells, J. Am. Chem. Soc. 138 (2016) 14423–14433. [14] M. Schürmann, P. Janning, S. Ziegler, H. Waldmann, Small-molecule target engagement in cells, Cell Chem. Biol. 23 (2016) 435–441. [15] L.G. Meimetis, J.C. Carlson, R.J. Giedt, R.H. Kohler, R. Weissleder, Ultrafluorogenic coumarin–tetrazine probes for real‐time biological imaging, Angew. Chemie Int. Ed. 53 (2014) 7531–7534. [16] Y. Yuan, S. Xu, X. Cheng, X. Cai, B. Liu, Bioorthogonal turn‐on probe based on aggregation‐induced emission characteristics for cancer cell imaging and ablation, Angew. Chemie Int. Ed. 55 (2016) 6457–6461. [17] H. Wu, N.K. Devaraj, Advances in Tetrazine Bioorthogonal Chemistry Driven by the Synthesis of Novel Tetrazines and Dienophiles. Accounts of Chemical Research, (2018). [18] Y. Lee, W. Cho, J. Sung, E. Kim, S.B. Park, Mono-chromophoric design strategy for tetrazine-based colorful bioorthogonal probes with a single fluorescent core skeleton, J. Am. Chem. Soc. (2017). [19] X. Shang, et al., Fluorogenic protein labeling using a genetically encoded unstrained alkene, Chem. Sci. 8 (2017) 1141–1145. [20] A. Vázquez, et al., Mechanism‐based fluorogenic trans‐cyclooctene–tetrazine cycloaddition, Angew. Chemie Int. Ed. 56 (2017) 1334–1337. [21] M. Tian, X. Peng, J. Fan, J. Wang, S. Sun, A fluorescent sensor for pH based on rhodamine fluorophore, Dye. Pigment. 95 (2012) 112–115. [22] L. Liu, et al., Fluorescent and colorimetric detection of pH by a rhodamine-based probe, Sens. Actuators B Chem. 194 (2014) 498–502. [23] Q.A. Best, R. Xu, M.E. McCarroll, L. Wang, D.J. Dyer, Design and investigation of a series of rhodamine-based fluorescent probes for optical measurements of pH, Org. Lett. 12 (2010) 3219–3221. [24] X. Liu, et al., Ratiometric fluorescent probe for lysosomal pH measurement and imaging in living cells using single-wavelength excitation, Anal. Chem. 89 (2017) 7038–7045. [25] Z. Ding, et al., Nanoscale metal–organic frameworks coated with poly (vinyl alcohol) for ratiometric peroxynitrite sensing through FRET, Chem. Sci. 8 (2017) 5101–5106. [26] S. Takahashi, et al., Development of a series of practical fluorescent chemical tools to measure pH values in living samples, J. Am. Chem. Soc. 140 (2018) 5925–5933. [27] S. Wang, et al., Elucidating the structure–reactivity correlations of phenothiazine‐based fluorescent probes toward ClO−, Chem. Eur. J. (2018). [28] M.H. Lee, et al., Two‐color probe to monitor a wide range of pH values in cells, Angew. Chemie Int. Ed. 52 (2013) 6206–6209. [29] S. Wang, et al., Activatable photoacoustic and fluorescent probe of nitric oxide for cellular and in vivo imaging, Sens. Actuators B Chem. 267 (2018) 403–411. [30] S. Jain, K. Neumann, Y. Zhang, J. Geng, M. Bradley, Tetrazine-Mediated Postpolymerization Modification, Macromolecules 49 (2016) 5438–5443. [31] R. Pipkorn, et al., Inverse‐electron‐demand Diels‐Alder reaction as a highly efficient chemoselective ligation procedure: synthesis and function of a BioShuttle for temozolomide transport into prostate cancer cells, J. Pept. Sci. 15 (2009) 235–241. [32] S. Hörner, et al., Combination of inverse electron-demand Diels–Alder reaction with highly efficient oxime ligation expands the toolbox of site-selective peptide conjugations, Chem. Commun. 51 (2015) 11130–11133. [33] K.-Y. Tan, et al., Real-time monitoring ATP in mitochondrion of living cells: a specific fluorescent probe for ATP by dual recognition sites, Anal. Chem. 89 (2017) 1749–1756. [34] G.K. Vegesna, et al., pH-activatable near-infrared fluorescent probes for detection of lysosomal pH inside living cells, J. Mater. Chem. B 2 (2014) 4500–4508. [35] S.-L. Shen, et al., A mitochondria-targeted ratiometric fluorescent probe for hypochlorite and its applications in bioimaging, J. Mater. Chem. B 5 (2017) 289–295. [36] S. Ohkuma, B. Poole, Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents, Proc. Natl. Acad. Sci. 75 (1978) 3327–3331. [37] Z. Ding, C. Wang, G. Feng, X. Zhang, Thermo-responsive fluorescent polymers with diverse LCSTs for ratiometric temperature sensing through FRET, Polymers 10 (2018) 283. [38] R. Kawagoe, I. Takashima, S. Uchinomiya, A. Ojida, Reversible ratiometric detection of highly reactive hydropersulfides using a FRET-based dual emission fluorescent probe, Chem. Sci. 8 (2017) 1134–1140. [39] J.R. Casey, S. Grinstein, J. Orlowski, Sensors and regulators of intracellular pH, Nat. Rev. Mol. Cell Biol. 11 (2010) 50.
3.5. Fluorescent imaging in living cells To evaluate the intracellular ratiometric pH detection ability of probe R B-R A-TZ-2, fluorescent microscopy in living cells was conducted. As shown in Fig. 5a, green fluorescence distributed evenly in most part of cell was regarded as the RA-TZ fluorescence, which was attributed to free diffusion of probe R B-R A-TZ2 in both cytoplasm and organelles. Differently, in Fig. 5b orange fluorescence from ring-opened rhodamine part was witnessed, which was thought to mostly locate in lysosomal site. In Fig. 5f, to measure and image the pH distribution in a ratiometric manner, regional mapping of fluorescence intensity ratio between RA-TZ fluorescence and rhodamine fluorescence was created. Once given the certain ratio (I584/I488) in a certain region of cell, the pH value in this region could be indicated by referring the ratio-pH curve (Fig. 3c). In the warmest region (red), the ratio (I584/I488) was about 40 folds, indicating the pH value to be 4.9, which was in line with real lysosomal pH; in the coolest region (blue), the ratio (I584/I488) was about 0.5 folds, indicating the pH values to be 6.5-7.0. The result mentioned above had demonstrated the capacity of probe R B-R A-TZ-2 to detect intracellular pH in a ratiometric manner. 4. Conclusion In conclusion, we reported the unique in-situ fluorescent properties of the inverse electron-demand Diels–Alder reaction products using Reppe anhydride and 1,2,4,5-tetrazines. A ratiometric fluorescent probe R B-R A-TZ-2 was constructed and applied in intracellular pH detection. Moreover, taking advantages of this “click-fluorescence” conjugation strategy, non-fluorescent antibody can be easily endowed with fluorescence, also dual-color fluorescent products can be prepared more conveniently. Efficient and flexible, this strategy is anticipated to widely contribute to biochemical applications. Acknowledgements This work was supported by the Macao Science and Technology Development Fund under Grant No.: 052/2015/A2, 082/2016/A2, and 019/2017/AMJ; the Research Grant of University of Macau under grant No.: MYRG2016-00058-FHS and MYRG2017-00066-FHS. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.snb.2018.09.022. References [1] S.I. Presolski, V. Hong, S.H. Cho, M.G. Finn, Tailored ligand acceleration of the Cucatalyzed azide-alkyne cycloaddition reaction: practical and mechanistic implications, J. Am. Chem. Soc. 132 (2010) 14570–14576, https://doi.org/10.1021/ ja105743g. [2] D. Soriano Del Amo, et al., Biocompatible copper(I) catalysts for in vivo imaging of glycans, J. Am. Chem. Soc. 132 (2010) 16893–16899, https://doi.org/10.1021/ ja106553e. [3] J. Dommerholt, F. Rutjes, F.L. van Delft, Strain-promoted 1,3-dipolar cycloaddition of cycloalkynes and organic azides, Top. Curr. Chem. (Cham) 374 (16) (2016), https://doi.org/10.1007/s41061-016-0016-4. [4] A. Herner, Q. Lin, Photo-triggered click chemistry for biological applications, Top. Curr. Chem. (Cham) 374 (1) (2016), https://doi.org/10.1007/s41061-015-0002-2. [5] L. Davis, J.W. Chin, Designer proteins: applications of genetic code expansion in cell biology, Nat. Rev. Mol. Cell Biol. 13 (2012) 168–182, https://doi.org/10.1038/ nrm3286.
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G. Feng et al. [40] H. Yu, Y. Xiao, L. Jin, A lysosome-targetable and two-photon fluorescent probe for monitoring endogenous and exogenous nitric oxide in living cells, J. Am. Chem. Soc. 134 (2012) 17486–17489.
under the supervision of Prof. Yupeng Tian. At present, he is a pH.D student at Faculty of Health Sciences, University of Macau. His research interest focuses on fluorescent and photoacoustic probes.
Gang Feng received his M.E. degree in Health Science Center, Shenzhen University in 2015. He is currently a pH.D. student in Faculty of Health Sciences, University of Macau. His work mostly focuses on the application of click-reaction techniques on probing and microtubule in-vitro engineering.
Shichao Wang received his B.S. degree in 2012 and M.E. degree in 2015, both in chemistry from Anhui University. He is now a pH.D. student at Faculty of Health Sciences, University of Macau. His research interest focus on fluorescent and photoacoustic probes. Zhaoyang Ding received his PhD degree from East China University of Science and Technology, China in 2013. He worked as postdoc at Faculty of Health Sciences, University of Macau from 2016 to 2018. His research interests include hybrid smart polymeric materials for sensing applications.
Boyu Zhang received his PhD from Dalian Institute of Chemical Physics, Chinese Academy of Sciences in 2013. He worked as a research associate at Dalian Institute of Chemical Physics from 2013 to 2015 and postdoc at Faculty of Health Sciences, University of Macau from 2016 to 2018. He is currently working at Dalian Medical University.
Xuanjun Zhang received his PhD degree in Chemistry from University of Science & Technology of China in 2004. After working at Shantou University, National University of Singapore, Linköping University, and University of Washington as postdocs and visiting scientist, he started his assistant professor at Linköping University in 2011 and was promoted to Docent in 2014. He moved to University of Macau in 2015. His research interests mainly focus on molecular and nanoprobes for fluorescent and photoacoustic imaging and sensing applications.
Chunfei Wang is currently a pH.D. Student in Faculty of Health Sciences, University of Macau, Macau SAR, China. His research interests include chemical sensors and biosensors. Jingyun Tan received his M.S degree of organic chemistry in Anhui University in 2015
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