Article pubs.acs.org/ac
Native Chemical Ligation Combined with Spirocyclization of Benzopyrylium Dyes for the Ratiometric and Selective Fluorescence Detection of Cysteine and Homocysteine Hongmin Lv,† Xiao-Feng Yang,*,† Yaogang Zhong,‡ Yuan Guo,*,† Zheng Li,‡ and Hua Li† †
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, College of Chemistry & Materials Science, Northwest University, Xi’an, Shaanxi 710069, P. R. China ‡ College of Life Sciences, Northwest University, Xi’an, Shaanxi 710069, P. R. China S Supporting Information *
ABSTRACT: Spirocyclization of xanthene dyes has become a powerful technique for developing fluorescent probes. Herein, we extend this unique fluorescence switching mechanism to a nearinfrared (NIR) dye, 2-(7-diethylamino-2-oxo-2H-1-benzopyran-3yl)-4-(2-carboxyphenyl)-7-diethylamino-1-benzopyrylium (CB), and construct a ratiometric fluorescent probe 1 for cysteine (Cys)/homocysteine (Hcy). The ratiometric sensing of probe 1 toward Cys/Hcy is realized by utilizing a tandem native chemical ligation/spirocyclization reaction to interrupt the large π-conjugated system of CB fluorophore, thereby affording remarkable blue shifts in the spectra of sensing system (from 669 to 423 nm in absorption spectra and from 694 to 474 nm in emission spectra). Probe 1 shows a high sensitivity for Cys/Hcy, and the detection limits (3 δ) for Cys and Hcy are 1.6 × 10−7 and 1.8 × 10−7 M, respectively. Moreover, since both the sulfhydril and the adjacent amino groups are involved in the sensing process, probe 1 is selective toward Cys/Hcy over other thiols such as glutathione. All these unique features make it particularly favorable for ratiometric Cys/Hcy sensing and bioimaging applications. It has been preliminarily used for Cys detection in rabbit serum samples and the ratiometric fluorescent imaging of Cys in living HepG2 cells.
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two different emission wavelengths, and can provide a built-in correction for the environmental effects. However, these photophysical processes depend on the energy levels of molecular orbitals, which are usually difficult to accurately determine, thus leading to the poor prediction of the sensing behavior of the designed probe.12 Therefore, a new strategy for the design of ratiometric fluorescent probes for Cys/Hcy is highly desirable. Regulation of the spirocyclization of rhodamine dyes is becoming a powerful technique for the rational development of fluorescent probes.13 The spirocyclic form of rhodamine is colorless and nonfluorescent due to separation the πconjugation of the original fluorophore, whereas the ringopen form shows strong spectroscopic signals in the absorption and fluorescence spectra by retrieving the large π-conjugated system (Scheme 1a). Therefore, precise modulation of the equilibrium between the spirocyclic form and the ring-open form of rhodamines provides an ideal strategy for constructing fluorescent probes for various analytes. Later, this unique spirocyclization-based fluorescence switching mechanism was extended to some carboxylic acid-functionalized near-infrared (NIR) dyes such as Si-rhodamine and Changsha NIR,14,15 and
ysteine (Cys) and homocysteine (Hcy) play a key role in a wide range of physiological processes. Elevated levels of Cys have been associated with neurotoxicity,1 and a decreased level of Cys can cause retardation in growth, hair pigmentation, skin lesions, liver damage, edema, lethargy, and weakness.2 On the other hand, elevated Hcy in blood plasma is a well-known risk factor for cardiovascular and Alzheimer’s disease,3 neural tube defects, complications during pregnancy, inflammatory bowel disease, and osteoporosis.4 Therefore, it is of great importance to develop fluorescent probes capable of sensitive and selective detection of Cys/Hcy in biological systems. Although many fluorescent probes have been developed for the detection of biothiols, only a few can discriminate Cys/Hcy from glutathione (GSH), the most abundant intracellular nonprotein thiol (1−10 mM). To date, most reported fluorescent probes for the selective detection of Cys and/or Hcy are based on the cyclization with aldehydes,5 conjugate addition−cyclization reaction,6 the response-assisted electrostatic attraction or repulsion strategy,7 and others.8 However, most reported examples of fluorescent sensing of Cys/Hcy function by the single emission intensity changes, which tend to be influenced by some factors such as probe concentration, probe environment, and instrumental efficiency. To address this problem, ratiometric fluorescent probes for Cys/Hcy based on internal charge transfer (ICT),9 fluorescence resonance energy transfer (FRET),10 and other mechanisms have been developed recently,6e,11 which employ the ratio of the emission intensity at © 2014 American Chemical Society
Received: November 17, 2013 Accepted: December 31, 2013 Published: January 10, 2014 1800
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Scheme 1. Equilibrium between the Open Form and Spirocyclic Form of the Classic Rhodamine B (a) and Dye CE (b)
Scheme 2. Proposed Mechanism for the Ratiometric Sensing of Cys/Hcy When Using 1
NIR fluorescent probes for hypochlorous acid,14a,15 and Hg2+,14b have been developed. However, the above NIR dye scaffolds can only be used for “turn-on” fluorescent probes development. Interestingly, it was reported that 2-(7-diethylamino-2-oxo2H-1-benzopyran-3-yl)-4-(2-carboxyphenyl)-7-diethylamino-1benzopyrylium (CB), a hybrid fluorophore of coumarin and benzopyrylium, shows a NIR absorbance and fluorescence in neutral conditions but affords 7-diethylaminecoumarin spectral properties in basic solutions (pH > 9).16 This was found to be
because of intramolecular spirocyclization at its benzopyrylium C-4 atom, decreasing the π-conjugation of the original CB fluorophore (Scheme 1b). Thus, CB bears the similar fluorescence “on-off” switching mechanism as the traditional rhodamine dyes, and its carboxylic acid functional group can operate as a fluorescence switch by spirocyclization. More significantly, unlike rhodamine derivatives which are colorless and nonfluorescent in the spirocyclic form, CB retains a 7diethylaminecoumarin moiety and can afford coumarin emission even in its spirocyclic form. In other words, CB 1801
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Solvents were dried by standard procedures before use. Doubledistilled water was used throughout all experiments. Flash chromatography was performed using Qingdao Haiyang silica gel (200 - 300 mesh). The fluorescence spectra and relative fluorescence intensity were measured with a Shimadzu RF-5301 spectrofluorimeter with a 10 mm quartz cuvette at the slits of 3.0/5.0 nm. Absorption spectra were recorded using a Shimadzu UV-2550 spectrophotometer. High-resolution mass spectra were collected using a Bruker micrOTOF-Q II mass spectrometer (Bruker Daltonics Corp., USA) in electrospray ionization (ESI) mode. The 1H NMR and 13C NMR spectra were recorded at 400 and 100 MHz on an INOVA-400 spectrometer (Varian Unity) respectively, using tetramethylsilane (TMS) as the internal standard. Cell images were acquired with an Olympus BX 61 fluorescence microscope (Tokyo, Japan) at 40× magnification. The pH measurements were carried out on a Sartorius PB-10 pH meter. All the measurements were operated at room temperature (25 °C). General Procedure for the Spectra Measurement. The stock solutions of 1 and 2 (1.0 mM) were prepared in EtOAc. Solutions of various species (2.0 mM) were prepared from Cys, Hcy, Thr, Lle, Val, Tyr, Pro, Ser, Leu, Ala, Gln, Trp, Met, Glu, GSH, DTT (1,4-dithiothreitol), p-thiocresol, ascorbic acid, and glucose. Test solutions were prepared by placing 50 μL of probe 1 (1.0 mM), 4.0 mL of ethanol, 1.0 mL of phosphate buffer (0.2 M, pH 7.4), and appropriate aliquot of each analyte stock solution into a 10 mL volumetric flask, and diluting the solution to 10 mL with water. The resulting solution was kept at room temperature (25 °C) for 25 min, and then the absorption or fluorescence spectra were recorded. The fluorescence ratio of I474/I694 was measured with the excitation and emission wavelengths at 430/650 nm and 474/694 nm, respectively. Cytotoxicity Assay. HepG2 cells were seeded in 96-well plates and incubated with different concentrations of probe 1 (0−20 μM) (n = 5) in an atmosphere of 5% CO2 and 95% air at 37 °C for 12 h. Then, 20 μL of MTT solution (5.0 mg mL−1) was added to each well, followed by incubation at 37 °C for 4 h. After that, 100 μL of the supernatant was removed, and 150 μL of DMSO was added to each well to dissolve the formed formazan. The plate was shaken for 10 min, and then the absorbance was measured at 570 nm with a microplate reader (ELX 800 UV, BIO-TEK Instruments Inc.). Cell viability (VR) is calculated by the equation: VR = A/A0 × 100%, where A and A0 are the absorbance of the experimental group and control group, respectively. Cell Cultures and Fluorescence Imaging. HepG2 cells were obtained from Dr. Bian (Cell Engineering Research Centre and Department of Cell Biology, Fourth Military Medical University, Xi’an, P. R. China). 2 × 105 HepG2 cells were seeded in 6-well culture plates containing sterile coverslips and were cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS), penicillin (100 U mL−1), and streptomycin (100 μg mL−1) at 37 °C in a 95% humidity atmosphere under 5% CO2 for two days. Before imaging, the cells were washed with PBS three times and then incubated with probe 1 (5.0 μM) in a DMSO/PBS (5: 95, v/v) solution at 37 °C for 30 min. Then, the samples were rinsed with PBS three times to remove the remaining probe and observed under an Olympus BX 61 fluorescence microscope. For the control experiment, the cells were pretreated with N-ethylmaleimide (NEM, 1 mM) or Cys (600 μM) at 37 °C for 40 min. After washing with PBS three times, the cells were further incubated
exhibits NIR and visible emissions in its spirocyclic ring-open and -closed forms, respectively. Therefore, CB can serve as a broadly applicable platform to develop ratiometric probes based on altering the π-conjugation of CB derivatives by spirocyclic/ ring-open switching mechanism. Native chemical ligation (NCL) is the most powerful ligation method for synthesizing native backbone proteins or modified proteins of moderate size.17 In NCL, the thiolate group of an N-terminal cysteine residue of an unprotected peptide I attacks the C-terminal thioester of a second unprotected peptide II in an aqueous buffer. This transthioesterification step is freely reversible and leads to form a thioester intermediate III, which rearranges spontaneously by an intramolecular S, N-acyl shift to give the amide-linked product IV (Scheme S1, Supporting Information). The above NCL reaction is exquisitely regioselective and chemoselective and can proceed under biocompatible conditions. Based on the unique characters of the NCL reaction, Lin et al. have successfully developed fluorescent probes for biothiols based on FRET signaling mechanism.10,18 Since both the sulfhydril and the amino groups are involved in the above ligation reaction, the NCL reaction might show selectivity toward Cys/Hcy over other thiols. This inspired us to design a fluorescent probe for the discrimination of Cys/Hcy over GSH based on the NCL reaction. With these considerations in mind, we propose a new ratiometric fluorescent probe for Cys/Hcy by combining the NCL reaction with the spirocyclization-based fluorescence switching mechanism of CB. Our rationale is depicted in Scheme 2. In our newly designed sensing system, the thioester group is fused in dye CB as the recognition unit. Furthermore, the phenyl thioesters are selected to construct the probe because they usually performed a rapid NCL reaction than those of alkyl thioesters under identical conditions.19 We envisioned that mixture of probe 1 with Cys would afford the corresponding amide 4a via a typical NCL reaction. At physiological pH, 4a favors a spirolactam form 5a because the N atom of the amide group is more nucleophilic and may further attack the benzopyrylium C-4 atom to undergo an C−N intramolecular spirocyclization, which is quite similar to that of rhodamine amide derivatives.13a As a result, the π-conjugation system of 1 is interrupted, thereby leading to the decrease of the NIR emission. On the other hand, since 1 contains a 7diethylaminecoumarin moiety, it can afford coumarin emission even in its cyclic lactam form. Therefore, two well-resolved emission peaks before and after adding Cys would be observed due to the distinct emission between CB dye and the coumarin fluorophore. As for Hcy, a similar fluorescent response would be observed. The above “S” to “N” acyl transfer from 3a (or 3b) to 4a (or 4b) is assisted by the proximity of the amino group to the thioester functionality in compound 3a (or 3b). In the case of GSH, a similar thiol-thioester exchange reaction might occur to give the thioester 6. However, the subsequent intramolecular rearrangement cannot proceed because there is no adjacent amino group in 6 to form a favored transition state. As a result, 6 is still present in the ring-opened form and mainly emits at NIR region, thus no significant changes in the fluorescence spectra being observed. Based on the above mechanism, selective detection of Cys/Hcy over other biological thiols such as GSH might be realized.
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MATERIALS AND INSTRUMENTS Unless stated otherwise, all chemicals were obtained from commercial suppliers and used without further purification. 1802
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Scheme 3. Synthesis of Probe 1 and 2 and the Structure of Compound 9
with probe 1 at 37 °C for 30 min. The cell images were then acquired after washing the cells with PBS three times. Synthesis. Synthesis of Probe 1 and 2 (Scheme 3). CB was synthesized according to the reported procedure.20 To a mixture of CB (50 mg, 0.093 mmol), 3,5-bis(trifluoromethyl)benzenethiol (20 μL, 0.12 mmol), N-(3-dimethylaminopropyl)N′-ethylcarbodiimide hydrochloride (EDC) (17.2 mg, 0.09 mmol), and 4-dimethylaminopyridine (DMAP) (1.22 mg, 0.01 mmol) was added dry CH2Cl2 (5.0 mL). The reaction solution was stirred at room temperature overnight. The solvent was then removed under reduced pressure, and the resulted residue was purified by silica gel flash chromatography (ethyl acetate/ petroleum ether = 1:1 → CH2Cl2/CH3OH = 100:1, v/v) to afford compound 1 as a dark blue solid (27.04 mg, 38% yield). 1H NMR (CDCl3, 400 MHz): δ ppm 9.48 (s, 1H), 8.34 (s, 1H), 8.13−8.19 (m, 2H), 7.77−7.86 (m, 4H), 7.67 (d, J = 2.0 Hz, 1H), 7.49 (d, J = 7.2 Hz, 1H), 7.31 (d, J = 9.6 Hz, 1H), 6.99 (d, J = 9.6 Hz, 1H), 6.77 (d, J = 9.2 Hz, 1H), 6.45 (s, 1H), 3.66 (bs, 4H), 3.52 (q, J = 6.8 Hz, 4H), 1.35 (bs, 6H), 1.28 (t, J = 6.8 Hz, 6H); 13C NMR (CDCl3, 100 MHz): 188.74, 162.54, 159.34, 158.92, 158.73, 158.53, 155.54, 154.88, 148.02, 136.05, 134.89, 134.67, 133.91, 133.56, 132.93, 132.59, 132.26, 131.92, 130.90, 130.77, 130.06, 129.15, 123.99, 123.58, 121.27, 116.67, 115.64, 112.02, 111.55, 110.65, 105.63, 97.83, 96.23, 45.69, 12.540; ESI-HRMS: [M] + m/z 765.2250, calcd for C41H35F6N2O4S 765.2216. Probe 2 was obtained by condensation of CB with pthiocresol using the same procedure described above. Yield 23.9 mg, 40%. 1H NMR (CDCl3, 400 MHz): δ ppm 9.45 (s, 1H), 8.30 (s, 1H), 8.23 (d, J = 7.6 Hz, 1H), 8.14 (d, J = 9.2 Hz, 1H), 7.70−7.77 (m, 2H), 7.62 (d, J = 2.0 Hz, 1H), 7.38 (d, J = 7.2 Hz, 1H), 7.23 (s, 1H), 7.16 (dd, J1 = 8.0 Hz, J2 = 18.4 Hz, 4H), 6.93 (d, J = 9.6 Hz, 1H), 6.76 (dd, J1 = 2.0 Hz, J2 = 9.2 Hz, 1H), 6.43 (d, J = 2.0 Hz, 1H), 3.66−3.73 (bs, 4H), 3.50 (q, J = 7.20 Hz, 4H), 2.29 (s, 3H), 1.33 (bs, 6H), 1.26 (t, J = 7.20 Hz, 6H); 13 C NMR (CDCl3, 100 MHz): 190.77, 162.56, 158.75, 158.69, 158.45, 155.45, 154.73, 147.93, 136.57, 133.84, 130.54, 129.36,
122.91, 115.95, 110.55, 105.85, 97.67, 96.22, 46.25, 45.66, 21.31, 12.58; ESI-HRMS: [M]+ m/z 643.2621, calcd for C40H39N2O4S 643.2625. Synthesis of Compound 9. To the solution of 1 (40 mg, 0.052 mmol) in CH3OH (8 mL) was added cysteamine hydrochloride (11.8 mg, 0.104 mmol) and tributylphosphine (13 μL, 0.052 mmol) (Scheme S2, Supporting Information). The mixture was stirred at room temperature for 6 h. The solvent was removed under reduced pressure to give the crude product, which was purified by silica gel flash chromatography (petroleum ether/ethyl acetate, 1:1, v/v) to afford compound 9 as a orange yellow solid (11 mg, 35% yield). 1H NMR (CDCl3, 400 MHz): δ ppm 8.31 (s, 1H), 7.86 (d, J = 6.8 Hz, 1H), 7.46 (m, 3H), 7.21 (d, J = 6.8 Hz, 1H), 6.64 (d, J = 7.6 Hz, 1H), 6.50 (s, 1H), 6.42 (m, 2H), 6.31 (d, J = 7.6 Hz, 1H), 6.24 (s, 1H), 3.50−3.54 (m, 2H), 3.44 (q, J = 6.4 Hz, 4H), 3.36 (q, J = 6.4 Hz, 4H), 2.57 (m, 2H), 1.71 (t, J = 6.4 Hz, 1H), 1.23 (t, J = 6.8 Hz, 6H), 1.19 (t, J = 8.4 Hz, 6H); 13C NMR (CDCl3, 100 MHz): 167.62, 159.49, 156.15, 152.05, 151.31, 148.73, 146.55, 139.59, 132.29, 129.85, 128.30, 128.23, 124.15, 123.93, 122.96, 111.61, 109.36, 108.98, 108.17, 101.81, 97.66, 96.63, 63.36, 44.91, 44.32, 43.70, 23.15, 12.59, 12.46. ESI-HRMS: [M + H]+ m/z 596.2581, calcd for C35H38N3O4S 596.2578.
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RESULTS AND DISCUSSION Effect of Substituent on the Reactivity of the Probe. In this study, the effect of substituent on the reactivity of the probe was investigated by comparing two thiophenylester derivatives with different substituents on the thiophenyl ring. The reactivity of probe 1 and 2 was studied by mixing them with Cys (or Hcy) in ethanol/phosphate buffer (40:60 v/v, 20 mM, pH 7.4), respectively. As shown in Figure S1 (Supporting Information), the emission ratio (I474/I694) of 1 increases dramatically upon mixing with Cys (or Hcy), whereas the intensity ratio of 2 gives a very limited increase under identical conditions. The significant difference between them lies in the substituents on the thiophenol moiety. As for 1, it contains a 1803
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moiety. The distinct gap between the two emission bands is up to 220 nm, which are much larger than those of FRET or TBET (through-bond energy transfer)-based rhodamine systems.21 This facilitates the dual emission ratiometric sensing owing to the minimum overlap between the two emission bands. Similar spectral changes were observed for Hcy under the same reaction conditions (Figure S2, Supporting Information). The fluorescence intensity ratios at 694 and 474 nm (I474/I694) increase from 0.028 to 16.15 upon Cys treatment, and the final enhancement factor is over 576-fold. Furthermore, the intensity ratios (I474/I694) were plotted as a function of Cys (or Hcy) concentration and a typical calibration graph was obtained as shown in Figure 2 and
thioester leaving group with more strong electron-withdrawing substituents (−CF3), which can significantly increase the rate of NCL.17 Therefore, compound 1 was identified as a better probe than compound 2, due to its higher sensitivity and faster reaction with Cys, and was used for further studies. Sensing of Probe 1 to Cys/Hcy. Initially, the sensing behavior of 1 was examined with UV−vis absorption spectroscopy. Upon introducing increasing concentrations of Cys (0−250 μM) to the solution of 1 in ethanol/phosphate buffer (40:60 v/v, 20 mM, pH 7.4) at room temperature (25 °C). The absorption peak of probe 1 at 669 nm gradually decreases with the concomitant growth of a new absorption band centered at around 423 nm (Figure 1a). A well-defined
Figure 2. Linear plot of the emission ratio (I474/I694) against Cys concentration (2−100 μM) when using 1 (5 μM). The inset shows the emission ratio (I474/I694) as a function of Cys concentration.
Figure S3 (Supporting Information). The I474/I694 value is linearly related to the concentration of Cys from 2 to 100 μM and 3 to 100 μM for Hcy. The detection limits (3 δ) for Cys and Hcy are 1.6 × 10−7 and 1.8 × 10−7 M, respectively. In addition, the ratiometric sensing can also be built based on the synchronous fluorescence spectroscopy by setting Δλ at 44 nm (Figure S4, Supporting Information). These results demonstrate that probe 1 can detect Cys/Hcy quantitatively. Mechanism Studies. To gain insight into the above proposed sensing mechanism, the reaction product 9 of probe 1 with cysteamine (cysteamine was selected instead of Cys because 9 was easy to isolate from the mixture) was isolated and characterized by 1H NMR, 13C NMR, and HRMS. In the 1 H NMR spectrum of 9 (Figure S21, Supporting Information), the resonance signal corresponding to the thiol proton at 1.71 ppm appeared, which proves that NCL reaction indeed occurs between probe 1 and cysteamine. Furthermore, the spirocyclic structure of 9 is confirmed by its 13C NMR spectrum (Figure S22, Supporting Information), and a peak at δ 63.36 ppm assigned to the spiro carbon in compound 9 is clearly observed.22 The structure of 9 was further characterized by HRMS, and a prominent peak at m/z 596.2581 corresponding to [9 + H]+ (calcd. 596.2578 for C35H38N3O4S) is observed in the HRMS data (Figure S23, Supporting Information). In addition, the above mechanism is confirmed by comparison the absorption and emission spectra of 9 with CB and 8. As shown in Figure S5 (Supporting Information), compound 9 affords no CB spectroscopic characteristics but displays the absorption and emission spectra (λabs, max = 427 nm; λem, max = 474 nm) similar to that of compound 8, confirming the disruption of the
Figure 1. Absorption (a) and fluorescence (b) spectra of probe 1 (5 μM) upon addition of increasing concentrations of Cys (0−250 μM) in ethanol/phosphate buffer (40:60 v/v, 20 mM, pH 7.4) for 25 min. The inset shows the color changes (a) and fluorescence changes (b) of 1 in the absence and presence of Cys under visible or UV light at 365 nm.
isosbestic point is observed at 455 nm, which may indicate that a new species was formed during the sensing process. Meanwhile, a prominent solution color change from dark blue to yellow-green was observed, thereby suggesting Cys can be detected with the“naked-eye” when using probe 1. Furthermore, the fluorescence sensing behavior of 1 toward Cys was examined under the same conditions. Two different absorption wavelengths corresponding to probe 1 (650 nm) and the spirolactam product 5a (430 nm) were selected as the excitation wavelengths. As shown in Figure 1b, the free probe gives only fluorescence emission at 694 nm (λex = 650 nm). However, addition of an increasing amount of Cys to the solution of 1 elicits a gradual decrease of the fluorescence intensity at around 694 nm and a progressive increase of new emission band at 474 nm simultaneously, indicating the interruption of the π-conjugation of 1 at its benzopyrylium 1804
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π-conjugation in probe 1 caused by spirocyclization. These data are in good agreement with the proposed sensing mechanism shown in Scheme 2. Kinetic Studies. The time course of the emission ratio (I474/I694) of probe 1 (5 μM) in the absence and presence of Cys or Hcy (20 equiv) was studied, and the results are shown in Figure 3. The emission ratio (I474/I694) of probe 1 itself
Figure 4. The emission ratio (I474/I694) of 1 (5 μM) in the presence of various analytes (10 equiv) in ethanol/phosphate buffer (40:60 v/v, 20 mM, pH 7.4) for 25 min. A) blank; B) Cys; C) Hcy; D) GSH; E) Thr; F) Lle; G) Val; H) Tyr; I) Pro; J) Ser; K) Leu; L) Ala; M) Gln; N) ascorbic acid; O) glucose; P) Trp; Q) Met; R) DTT; S) Glu; T) pthiocresol. The inset shows the fluorescence changes of 1 upon addition of various species under UV light at 365 nm. Figure 3. Time-dependent the emission ratio (I474/I694) of probe 1 (5 μM) in the absence (blank) and presence of Cys or Hcy (20 equiv) in ethanol/phosphate buffer (40:60 v/v, 20 mM, pH 7.4).
(Figure S10, Supporting Information), which further proves the high selectivity of probe 1 toward Cys/Hcy over other thiols. Applications of Probe 1 in Biological Systems. In order to show its application in biology, probe 1 was employed to detect Cys in rabbit serum samples (although Hcy gives similar response to Cys, Hcy would not significantly interfere with Cys assay because the total concentration of Hcy in serum is much lower than that of Cys).25 The serum sample was treated with triphenylphosphine to release the free Cys, according to the previous reported procedure.26 Different amounts of the reduced rabbit serum sample were then added to the solution of probe 1 in ethanol/phosphate buffer (40:60 v/v, 20 mM, pH 7.4). As shown in Figure S11, a linear relationship between the emission ratio (I474/I694) and the volume of reduced rabbit serum was noted, thus indicating that probe 1 is capable of detecting Cys in biological fluids. Furthermore, we proceeded to investigate the capability of 1 to image Cys in living HepG2 cells. First, the cytotoxicity of probe 1 was evaluated by the MTT assays, and the results showed that the cellular viability was about 80% even with 10 μM 1 at 37 °C for 12 h, indicating the low cytotoxicity of the probe (Figure S12, Supporting Information). Thus, a 5 μM of 1 was selected for further cell image experiments. HepG2 cells were incubated with 1 (5 μM) at 37 °C for 30 min, and we could observe fluorescence in both blue (Figure 5B) and red channels (Figure 5C). Furthermore, in a control experiment, the cells were pretreated with 1.0 mM NEM (a thiol blocking reagent) to reduce the concentration of intracellular thiols prior to incubation with probe 1. A marked fluorescence quenching in the blue channel (Figure 5E) and a fluorescence increase in the red channel were observed (Figure 5F). However, when the cells were pretreated with Cys (600 μM) and then incubated with probe 1, we could note bright blue fluorescence (Figure 5H) but almost no red fluorescence (Figure 5I). The above image variations are inconsistent with Cys-induced ratiometric fluorescence response. Thus, these results revealed that 1 is cellpermeable and can be used as a ratiometric fluorescent probe for bioimaging of Cys/Hcy in living cells.
exhibits no noticeable changes in ethanol/phosphate buffer (40:60 v/v, 20 mM, pH 7.4). By contrast, upon addition of Cys, the I474/I694 value of probe 1 shows a rapid increase at first, and the emission ratio essentially reaches a maximum in 25 min. A similar emission ratio increase was observed for Hcy. The calculated pseudo-first-order rate constants (k) for Cys and Hcy are 0.139 ± 0.011 and 0.113 ± 0.007 min−1, respectively (Figure S6, Supporting Information).23 The reaction rate for Hcy is slightly lower than that of Cys because the intramolecular attack by the amino group via a six-membered intermediate for Hcy is less favored than the facile fivemembered ring mediated for Cys in the second step of NCL.24 Selectivity Studies. To further verify the selectivity, probe 1 was incubated with the representative biologically relevant species in ethanol/phosphate buffer (40:60 v/v, 20 mM, pH 7.4). As shown in Figure 4 and Figure S7, only Cys/Hcy induces a dramatic increment of the emission ratio, while other thiol-containing compounds (GSH, DTT, p-thiocresol), amino acids (Thr, Lle, Val, Tyr, Pro, Ser, Leu, Ala, Gln, Trp, Met, Glu), metal ions (Zn2+, Ag+, Hg2+, Ca2+, Cd2+, Mg2+, K+) (Figure S8, Supporting Information), ascorbic acid, and glucose trigger no or very minor changes. The high selectivity of 1 toward Cys/Hcy can also be observed by the naked eye, and only Cys/Hcy induces an obvious color change under visible or UV light (Figure 4 and Figure S9). Notably, only very limited changes in the emission ratio were observed upon addition of other thiol-containing compounds. This can be explained by the fact that although transthioesterification can occur between 1 and common thiols, the ensuing intramolecular S to N acyl shift of thioester intermediate to give the final amide cannot proceed due to the lack of amino group in proximity, which further confirms that both the sulfhydril and the adjacent amino groups contribute to the sensing process. In addition, the above transthioesterification is reversible and would not consume a large amount of the probe if no further intramolecular S, N-acyl transfer occurs. Therefore, probe 1 can still retain its sensing response to Cys even in the presence of a large amount of GSH 1805
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Figure 5. Fluorescence images of HepG2 cells treated with probe 1. (A) Bright field image of cells incubated with 1 (5 μM) for 30 min; (B) fluorescence image (A) from blue channel; (C) fluorescence image (A) from red channel. (D) Bright field image of cells incubated with NEM (1.0 mM) for 40 min and then further incubation with 1 (5 μM) for 30 min; (E) fluorescence image (D) from blue channel; (F) fluorescence image (D) from red channel. (G) Bright field image of cells incubated with Cys (600 μM) for 40 min and then further incubation with 1 (5 μM) for 30 min; (H) fluorescence image (G) from blue channel; (I) fluorescence image (G) from red channel.
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*E-mail: [email protected] (Y.G.).
CONCLUSION In summary, we have developed a ratiometric fluorescent probe for the selective detection of Cys/Hcy. The ratiometric sensing is realized via interrupting the large π-conjugated system of CB fluorophore by a tandem NCL/spirocyclization reaction. Notably, significant ratiometric fluorescent response coupling with a large emission blue shift (Δλ ≈ 220 nm) allows for convenient sensing of Cys/Hcy. Because both the sulfhydril and the adjacent amino groups contribute to the sensing process, the probe displays excellent selectivity toward Cys/ Hcy over other thiols. Importantly, preliminary studies show that probe 1 is low cytotoxic and cell-permeable and can be employed for the ratiometric fluorescent imaging of Cys/Hcy in living cells. Further, modulation of the π-conjugated system in benzopyrylium dyes by spirocyclization provides a promising methodology for constructing various ratiometric fluorescent probes, and this research work is still under way in our laboratory.
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by the National Natural Science Foundation of China (No. 21275117, 21375105), the Science & Technology Department (No. 2012JM2004), and the Education Department (No. 12JK0518) of Shaanxi Province of China.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in the text (Figures S1−S12, Scheme S1, and Scheme S2); 1H NMR, 13C NMR, and HRMS data of 1, 2, and 9. This material is available free of charge via the Internet at http://pubs.acs.org.
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REFERENCES
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AUTHOR INFORMATION
Corresponding Authors
*Phone: 86-29-88302604. Fax: 86-29-88303798. E-mail: [email protected] (X.-F.Y.). 1806
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Native Chemical Ligation Combined with Spirocyclization of Benzopyrylium Dyes for the Ratiometric and Selective Fluorescence Detection of Cysteine and Homocysteine Hongmin Lv,† Xiao-Feng Yang,*,† Yaogang Zhong,‡ Yuan Guo,*,† Zheng Li,‡ and Hua Li† †
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, College of Chemistry & Materials Science, Northwest University, Xi’an, Shaanxi 710069, P. R. China ‡ College of Life Sciences, Northwest University, Xi’an, Shaanxi 710069, P. R. China S Supporting Information *
ABSTRACT: Spirocyclization of xanthene dyes has become a powerful technique for developing fluorescent probes. Herein, we extend this unique fluorescence switching mechanism to a nearinfrared (NIR) dye, 2-(7-diethylamino-2-oxo-2H-1-benzopyran-3yl)-4-(2-carboxyphenyl)-7-diethylamino-1-benzopyrylium (CB), and construct a ratiometric fluorescent probe 1 for cysteine (Cys)/homocysteine (Hcy). The ratiometric sensing of probe 1 toward Cys/Hcy is realized by utilizing a tandem native chemical ligation/spirocyclization reaction to interrupt the large π-conjugated system of CB fluorophore, thereby affording remarkable blue shifts in the spectra of sensing system (from 669 to 423 nm in absorption spectra and from 694 to 474 nm in emission spectra). Probe 1 shows a high sensitivity for Cys/Hcy, and the detection limits (3 δ) for Cys and Hcy are 1.6 × 10−7 and 1.8 × 10−7 M, respectively. Moreover, since both the sulfhydril and the adjacent amino groups are involved in the sensing process, probe 1 is selective toward Cys/Hcy over other thiols such as glutathione. All these unique features make it particularly favorable for ratiometric Cys/Hcy sensing and bioimaging applications. It has been preliminarily used for Cys detection in rabbit serum samples and the ratiometric fluorescent imaging of Cys in living HepG2 cells.
C
two different emission wavelengths, and can provide a built-in correction for the environmental effects. However, these photophysical processes depend on the energy levels of molecular orbitals, which are usually difficult to accurately determine, thus leading to the poor prediction of the sensing behavior of the designed probe.12 Therefore, a new strategy for the design of ratiometric fluorescent probes for Cys/Hcy is highly desirable. Regulation of the spirocyclization of rhodamine dyes is becoming a powerful technique for the rational development of fluorescent probes.13 The spirocyclic form of rhodamine is colorless and nonfluorescent due to separation the πconjugation of the original fluorophore, whereas the ringopen form shows strong spectroscopic signals in the absorption and fluorescence spectra by retrieving the large π-conjugated system (Scheme 1a). Therefore, precise modulation of the equilibrium between the spirocyclic form and the ring-open form of rhodamines provides an ideal strategy for constructing fluorescent probes for various analytes. Later, this unique spirocyclization-based fluorescence switching mechanism was extended to some carboxylic acid-functionalized near-infrared (NIR) dyes such as Si-rhodamine and Changsha NIR,14,15 and
ysteine (Cys) and homocysteine (Hcy) play a key role in a wide range of physiological processes. Elevated levels of Cys have been associated with neurotoxicity,1 and a decreased level of Cys can cause retardation in growth, hair pigmentation, skin lesions, liver damage, edema, lethargy, and weakness.2 On the other hand, elevated Hcy in blood plasma is a well-known risk factor for cardiovascular and Alzheimer’s disease,3 neural tube defects, complications during pregnancy, inflammatory bowel disease, and osteoporosis.4 Therefore, it is of great importance to develop fluorescent probes capable of sensitive and selective detection of Cys/Hcy in biological systems. Although many fluorescent probes have been developed for the detection of biothiols, only a few can discriminate Cys/Hcy from glutathione (GSH), the most abundant intracellular nonprotein thiol (1−10 mM). To date, most reported fluorescent probes for the selective detection of Cys and/or Hcy are based on the cyclization with aldehydes,5 conjugate addition−cyclization reaction,6 the response-assisted electrostatic attraction or repulsion strategy,7 and others.8 However, most reported examples of fluorescent sensing of Cys/Hcy function by the single emission intensity changes, which tend to be influenced by some factors such as probe concentration, probe environment, and instrumental efficiency. To address this problem, ratiometric fluorescent probes for Cys/Hcy based on internal charge transfer (ICT),9 fluorescence resonance energy transfer (FRET),10 and other mechanisms have been developed recently,6e,11 which employ the ratio of the emission intensity at © 2014 American Chemical Society
Received: November 17, 2013 Accepted: December 31, 2013 Published: January 10, 2014 1800
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Scheme 1. Equilibrium between the Open Form and Spirocyclic Form of the Classic Rhodamine B (a) and Dye CE (b)
Scheme 2. Proposed Mechanism for the Ratiometric Sensing of Cys/Hcy When Using 1
NIR fluorescent probes for hypochlorous acid,14a,15 and Hg2+,14b have been developed. However, the above NIR dye scaffolds can only be used for “turn-on” fluorescent probes development. Interestingly, it was reported that 2-(7-diethylamino-2-oxo2H-1-benzopyran-3-yl)-4-(2-carboxyphenyl)-7-diethylamino-1benzopyrylium (CB), a hybrid fluorophore of coumarin and benzopyrylium, shows a NIR absorbance and fluorescence in neutral conditions but affords 7-diethylaminecoumarin spectral properties in basic solutions (pH > 9).16 This was found to be
because of intramolecular spirocyclization at its benzopyrylium C-4 atom, decreasing the π-conjugation of the original CB fluorophore (Scheme 1b). Thus, CB bears the similar fluorescence “on-off” switching mechanism as the traditional rhodamine dyes, and its carboxylic acid functional group can operate as a fluorescence switch by spirocyclization. More significantly, unlike rhodamine derivatives which are colorless and nonfluorescent in the spirocyclic form, CB retains a 7diethylaminecoumarin moiety and can afford coumarin emission even in its spirocyclic form. In other words, CB 1801
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Solvents were dried by standard procedures before use. Doubledistilled water was used throughout all experiments. Flash chromatography was performed using Qingdao Haiyang silica gel (200 - 300 mesh). The fluorescence spectra and relative fluorescence intensity were measured with a Shimadzu RF-5301 spectrofluorimeter with a 10 mm quartz cuvette at the slits of 3.0/5.0 nm. Absorption spectra were recorded using a Shimadzu UV-2550 spectrophotometer. High-resolution mass spectra were collected using a Bruker micrOTOF-Q II mass spectrometer (Bruker Daltonics Corp., USA) in electrospray ionization (ESI) mode. The 1H NMR and 13C NMR spectra were recorded at 400 and 100 MHz on an INOVA-400 spectrometer (Varian Unity) respectively, using tetramethylsilane (TMS) as the internal standard. Cell images were acquired with an Olympus BX 61 fluorescence microscope (Tokyo, Japan) at 40× magnification. The pH measurements were carried out on a Sartorius PB-10 pH meter. All the measurements were operated at room temperature (25 °C). General Procedure for the Spectra Measurement. The stock solutions of 1 and 2 (1.0 mM) were prepared in EtOAc. Solutions of various species (2.0 mM) were prepared from Cys, Hcy, Thr, Lle, Val, Tyr, Pro, Ser, Leu, Ala, Gln, Trp, Met, Glu, GSH, DTT (1,4-dithiothreitol), p-thiocresol, ascorbic acid, and glucose. Test solutions were prepared by placing 50 μL of probe 1 (1.0 mM), 4.0 mL of ethanol, 1.0 mL of phosphate buffer (0.2 M, pH 7.4), and appropriate aliquot of each analyte stock solution into a 10 mL volumetric flask, and diluting the solution to 10 mL with water. The resulting solution was kept at room temperature (25 °C) for 25 min, and then the absorption or fluorescence spectra were recorded. The fluorescence ratio of I474/I694 was measured with the excitation and emission wavelengths at 430/650 nm and 474/694 nm, respectively. Cytotoxicity Assay. HepG2 cells were seeded in 96-well plates and incubated with different concentrations of probe 1 (0−20 μM) (n = 5) in an atmosphere of 5% CO2 and 95% air at 37 °C for 12 h. Then, 20 μL of MTT solution (5.0 mg mL−1) was added to each well, followed by incubation at 37 °C for 4 h. After that, 100 μL of the supernatant was removed, and 150 μL of DMSO was added to each well to dissolve the formed formazan. The plate was shaken for 10 min, and then the absorbance was measured at 570 nm with a microplate reader (ELX 800 UV, BIO-TEK Instruments Inc.). Cell viability (VR) is calculated by the equation: VR = A/A0 × 100%, where A and A0 are the absorbance of the experimental group and control group, respectively. Cell Cultures and Fluorescence Imaging. HepG2 cells were obtained from Dr. Bian (Cell Engineering Research Centre and Department of Cell Biology, Fourth Military Medical University, Xi’an, P. R. China). 2 × 105 HepG2 cells were seeded in 6-well culture plates containing sterile coverslips and were cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS), penicillin (100 U mL−1), and streptomycin (100 μg mL−1) at 37 °C in a 95% humidity atmosphere under 5% CO2 for two days. Before imaging, the cells were washed with PBS three times and then incubated with probe 1 (5.0 μM) in a DMSO/PBS (5: 95, v/v) solution at 37 °C for 30 min. Then, the samples were rinsed with PBS three times to remove the remaining probe and observed under an Olympus BX 61 fluorescence microscope. For the control experiment, the cells were pretreated with N-ethylmaleimide (NEM, 1 mM) or Cys (600 μM) at 37 °C for 40 min. After washing with PBS three times, the cells were further incubated
exhibits NIR and visible emissions in its spirocyclic ring-open and -closed forms, respectively. Therefore, CB can serve as a broadly applicable platform to develop ratiometric probes based on altering the π-conjugation of CB derivatives by spirocyclic/ ring-open switching mechanism. Native chemical ligation (NCL) is the most powerful ligation method for synthesizing native backbone proteins or modified proteins of moderate size.17 In NCL, the thiolate group of an N-terminal cysteine residue of an unprotected peptide I attacks the C-terminal thioester of a second unprotected peptide II in an aqueous buffer. This transthioesterification step is freely reversible and leads to form a thioester intermediate III, which rearranges spontaneously by an intramolecular S, N-acyl shift to give the amide-linked product IV (Scheme S1, Supporting Information). The above NCL reaction is exquisitely regioselective and chemoselective and can proceed under biocompatible conditions. Based on the unique characters of the NCL reaction, Lin et al. have successfully developed fluorescent probes for biothiols based on FRET signaling mechanism.10,18 Since both the sulfhydril and the amino groups are involved in the above ligation reaction, the NCL reaction might show selectivity toward Cys/Hcy over other thiols. This inspired us to design a fluorescent probe for the discrimination of Cys/Hcy over GSH based on the NCL reaction. With these considerations in mind, we propose a new ratiometric fluorescent probe for Cys/Hcy by combining the NCL reaction with the spirocyclization-based fluorescence switching mechanism of CB. Our rationale is depicted in Scheme 2. In our newly designed sensing system, the thioester group is fused in dye CB as the recognition unit. Furthermore, the phenyl thioesters are selected to construct the probe because they usually performed a rapid NCL reaction than those of alkyl thioesters under identical conditions.19 We envisioned that mixture of probe 1 with Cys would afford the corresponding amide 4a via a typical NCL reaction. At physiological pH, 4a favors a spirolactam form 5a because the N atom of the amide group is more nucleophilic and may further attack the benzopyrylium C-4 atom to undergo an C−N intramolecular spirocyclization, which is quite similar to that of rhodamine amide derivatives.13a As a result, the π-conjugation system of 1 is interrupted, thereby leading to the decrease of the NIR emission. On the other hand, since 1 contains a 7diethylaminecoumarin moiety, it can afford coumarin emission even in its cyclic lactam form. Therefore, two well-resolved emission peaks before and after adding Cys would be observed due to the distinct emission between CB dye and the coumarin fluorophore. As for Hcy, a similar fluorescent response would be observed. The above “S” to “N” acyl transfer from 3a (or 3b) to 4a (or 4b) is assisted by the proximity of the amino group to the thioester functionality in compound 3a (or 3b). In the case of GSH, a similar thiol-thioester exchange reaction might occur to give the thioester 6. However, the subsequent intramolecular rearrangement cannot proceed because there is no adjacent amino group in 6 to form a favored transition state. As a result, 6 is still present in the ring-opened form and mainly emits at NIR region, thus no significant changes in the fluorescence spectra being observed. Based on the above mechanism, selective detection of Cys/Hcy over other biological thiols such as GSH might be realized.
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MATERIALS AND INSTRUMENTS Unless stated otherwise, all chemicals were obtained from commercial suppliers and used without further purification. 1802
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Scheme 3. Synthesis of Probe 1 and 2 and the Structure of Compound 9
with probe 1 at 37 °C for 30 min. The cell images were then acquired after washing the cells with PBS three times. Synthesis. Synthesis of Probe 1 and 2 (Scheme 3). CB was synthesized according to the reported procedure.20 To a mixture of CB (50 mg, 0.093 mmol), 3,5-bis(trifluoromethyl)benzenethiol (20 μL, 0.12 mmol), N-(3-dimethylaminopropyl)N′-ethylcarbodiimide hydrochloride (EDC) (17.2 mg, 0.09 mmol), and 4-dimethylaminopyridine (DMAP) (1.22 mg, 0.01 mmol) was added dry CH2Cl2 (5.0 mL). The reaction solution was stirred at room temperature overnight. The solvent was then removed under reduced pressure, and the resulted residue was purified by silica gel flash chromatography (ethyl acetate/ petroleum ether = 1:1 → CH2Cl2/CH3OH = 100:1, v/v) to afford compound 1 as a dark blue solid (27.04 mg, 38% yield). 1H NMR (CDCl3, 400 MHz): δ ppm 9.48 (s, 1H), 8.34 (s, 1H), 8.13−8.19 (m, 2H), 7.77−7.86 (m, 4H), 7.67 (d, J = 2.0 Hz, 1H), 7.49 (d, J = 7.2 Hz, 1H), 7.31 (d, J = 9.6 Hz, 1H), 6.99 (d, J = 9.6 Hz, 1H), 6.77 (d, J = 9.2 Hz, 1H), 6.45 (s, 1H), 3.66 (bs, 4H), 3.52 (q, J = 6.8 Hz, 4H), 1.35 (bs, 6H), 1.28 (t, J = 6.8 Hz, 6H); 13C NMR (CDCl3, 100 MHz): 188.74, 162.54, 159.34, 158.92, 158.73, 158.53, 155.54, 154.88, 148.02, 136.05, 134.89, 134.67, 133.91, 133.56, 132.93, 132.59, 132.26, 131.92, 130.90, 130.77, 130.06, 129.15, 123.99, 123.58, 121.27, 116.67, 115.64, 112.02, 111.55, 110.65, 105.63, 97.83, 96.23, 45.69, 12.540; ESI-HRMS: [M] + m/z 765.2250, calcd for C41H35F6N2O4S 765.2216. Probe 2 was obtained by condensation of CB with pthiocresol using the same procedure described above. Yield 23.9 mg, 40%. 1H NMR (CDCl3, 400 MHz): δ ppm 9.45 (s, 1H), 8.30 (s, 1H), 8.23 (d, J = 7.6 Hz, 1H), 8.14 (d, J = 9.2 Hz, 1H), 7.70−7.77 (m, 2H), 7.62 (d, J = 2.0 Hz, 1H), 7.38 (d, J = 7.2 Hz, 1H), 7.23 (s, 1H), 7.16 (dd, J1 = 8.0 Hz, J2 = 18.4 Hz, 4H), 6.93 (d, J = 9.6 Hz, 1H), 6.76 (dd, J1 = 2.0 Hz, J2 = 9.2 Hz, 1H), 6.43 (d, J = 2.0 Hz, 1H), 3.66−3.73 (bs, 4H), 3.50 (q, J = 7.20 Hz, 4H), 2.29 (s, 3H), 1.33 (bs, 6H), 1.26 (t, J = 7.20 Hz, 6H); 13 C NMR (CDCl3, 100 MHz): 190.77, 162.56, 158.75, 158.69, 158.45, 155.45, 154.73, 147.93, 136.57, 133.84, 130.54, 129.36,
122.91, 115.95, 110.55, 105.85, 97.67, 96.22, 46.25, 45.66, 21.31, 12.58; ESI-HRMS: [M]+ m/z 643.2621, calcd for C40H39N2O4S 643.2625. Synthesis of Compound 9. To the solution of 1 (40 mg, 0.052 mmol) in CH3OH (8 mL) was added cysteamine hydrochloride (11.8 mg, 0.104 mmol) and tributylphosphine (13 μL, 0.052 mmol) (Scheme S2, Supporting Information). The mixture was stirred at room temperature for 6 h. The solvent was removed under reduced pressure to give the crude product, which was purified by silica gel flash chromatography (petroleum ether/ethyl acetate, 1:1, v/v) to afford compound 9 as a orange yellow solid (11 mg, 35% yield). 1H NMR (CDCl3, 400 MHz): δ ppm 8.31 (s, 1H), 7.86 (d, J = 6.8 Hz, 1H), 7.46 (m, 3H), 7.21 (d, J = 6.8 Hz, 1H), 6.64 (d, J = 7.6 Hz, 1H), 6.50 (s, 1H), 6.42 (m, 2H), 6.31 (d, J = 7.6 Hz, 1H), 6.24 (s, 1H), 3.50−3.54 (m, 2H), 3.44 (q, J = 6.4 Hz, 4H), 3.36 (q, J = 6.4 Hz, 4H), 2.57 (m, 2H), 1.71 (t, J = 6.4 Hz, 1H), 1.23 (t, J = 6.8 Hz, 6H), 1.19 (t, J = 8.4 Hz, 6H); 13C NMR (CDCl3, 100 MHz): 167.62, 159.49, 156.15, 152.05, 151.31, 148.73, 146.55, 139.59, 132.29, 129.85, 128.30, 128.23, 124.15, 123.93, 122.96, 111.61, 109.36, 108.98, 108.17, 101.81, 97.66, 96.63, 63.36, 44.91, 44.32, 43.70, 23.15, 12.59, 12.46. ESI-HRMS: [M + H]+ m/z 596.2581, calcd for C35H38N3O4S 596.2578.
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RESULTS AND DISCUSSION Effect of Substituent on the Reactivity of the Probe. In this study, the effect of substituent on the reactivity of the probe was investigated by comparing two thiophenylester derivatives with different substituents on the thiophenyl ring. The reactivity of probe 1 and 2 was studied by mixing them with Cys (or Hcy) in ethanol/phosphate buffer (40:60 v/v, 20 mM, pH 7.4), respectively. As shown in Figure S1 (Supporting Information), the emission ratio (I474/I694) of 1 increases dramatically upon mixing with Cys (or Hcy), whereas the intensity ratio of 2 gives a very limited increase under identical conditions. The significant difference between them lies in the substituents on the thiophenol moiety. As for 1, it contains a 1803
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moiety. The distinct gap between the two emission bands is up to 220 nm, which are much larger than those of FRET or TBET (through-bond energy transfer)-based rhodamine systems.21 This facilitates the dual emission ratiometric sensing owing to the minimum overlap between the two emission bands. Similar spectral changes were observed for Hcy under the same reaction conditions (Figure S2, Supporting Information). The fluorescence intensity ratios at 694 and 474 nm (I474/I694) increase from 0.028 to 16.15 upon Cys treatment, and the final enhancement factor is over 576-fold. Furthermore, the intensity ratios (I474/I694) were plotted as a function of Cys (or Hcy) concentration and a typical calibration graph was obtained as shown in Figure 2 and
thioester leaving group with more strong electron-withdrawing substituents (−CF3), which can significantly increase the rate of NCL.17 Therefore, compound 1 was identified as a better probe than compound 2, due to its higher sensitivity and faster reaction with Cys, and was used for further studies. Sensing of Probe 1 to Cys/Hcy. Initially, the sensing behavior of 1 was examined with UV−vis absorption spectroscopy. Upon introducing increasing concentrations of Cys (0−250 μM) to the solution of 1 in ethanol/phosphate buffer (40:60 v/v, 20 mM, pH 7.4) at room temperature (25 °C). The absorption peak of probe 1 at 669 nm gradually decreases with the concomitant growth of a new absorption band centered at around 423 nm (Figure 1a). A well-defined
Figure 2. Linear plot of the emission ratio (I474/I694) against Cys concentration (2−100 μM) when using 1 (5 μM). The inset shows the emission ratio (I474/I694) as a function of Cys concentration.
Figure S3 (Supporting Information). The I474/I694 value is linearly related to the concentration of Cys from 2 to 100 μM and 3 to 100 μM for Hcy. The detection limits (3 δ) for Cys and Hcy are 1.6 × 10−7 and 1.8 × 10−7 M, respectively. In addition, the ratiometric sensing can also be built based on the synchronous fluorescence spectroscopy by setting Δλ at 44 nm (Figure S4, Supporting Information). These results demonstrate that probe 1 can detect Cys/Hcy quantitatively. Mechanism Studies. To gain insight into the above proposed sensing mechanism, the reaction product 9 of probe 1 with cysteamine (cysteamine was selected instead of Cys because 9 was easy to isolate from the mixture) was isolated and characterized by 1H NMR, 13C NMR, and HRMS. In the 1 H NMR spectrum of 9 (Figure S21, Supporting Information), the resonance signal corresponding to the thiol proton at 1.71 ppm appeared, which proves that NCL reaction indeed occurs between probe 1 and cysteamine. Furthermore, the spirocyclic structure of 9 is confirmed by its 13C NMR spectrum (Figure S22, Supporting Information), and a peak at δ 63.36 ppm assigned to the spiro carbon in compound 9 is clearly observed.22 The structure of 9 was further characterized by HRMS, and a prominent peak at m/z 596.2581 corresponding to [9 + H]+ (calcd. 596.2578 for C35H38N3O4S) is observed in the HRMS data (Figure S23, Supporting Information). In addition, the above mechanism is confirmed by comparison the absorption and emission spectra of 9 with CB and 8. As shown in Figure S5 (Supporting Information), compound 9 affords no CB spectroscopic characteristics but displays the absorption and emission spectra (λabs, max = 427 nm; λem, max = 474 nm) similar to that of compound 8, confirming the disruption of the
Figure 1. Absorption (a) and fluorescence (b) spectra of probe 1 (5 μM) upon addition of increasing concentrations of Cys (0−250 μM) in ethanol/phosphate buffer (40:60 v/v, 20 mM, pH 7.4) for 25 min. The inset shows the color changes (a) and fluorescence changes (b) of 1 in the absence and presence of Cys under visible or UV light at 365 nm.
isosbestic point is observed at 455 nm, which may indicate that a new species was formed during the sensing process. Meanwhile, a prominent solution color change from dark blue to yellow-green was observed, thereby suggesting Cys can be detected with the“naked-eye” when using probe 1. Furthermore, the fluorescence sensing behavior of 1 toward Cys was examined under the same conditions. Two different absorption wavelengths corresponding to probe 1 (650 nm) and the spirolactam product 5a (430 nm) were selected as the excitation wavelengths. As shown in Figure 1b, the free probe gives only fluorescence emission at 694 nm (λex = 650 nm). However, addition of an increasing amount of Cys to the solution of 1 elicits a gradual decrease of the fluorescence intensity at around 694 nm and a progressive increase of new emission band at 474 nm simultaneously, indicating the interruption of the π-conjugation of 1 at its benzopyrylium 1804
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π-conjugation in probe 1 caused by spirocyclization. These data are in good agreement with the proposed sensing mechanism shown in Scheme 2. Kinetic Studies. The time course of the emission ratio (I474/I694) of probe 1 (5 μM) in the absence and presence of Cys or Hcy (20 equiv) was studied, and the results are shown in Figure 3. The emission ratio (I474/I694) of probe 1 itself
Figure 4. The emission ratio (I474/I694) of 1 (5 μM) in the presence of various analytes (10 equiv) in ethanol/phosphate buffer (40:60 v/v, 20 mM, pH 7.4) for 25 min. A) blank; B) Cys; C) Hcy; D) GSH; E) Thr; F) Lle; G) Val; H) Tyr; I) Pro; J) Ser; K) Leu; L) Ala; M) Gln; N) ascorbic acid; O) glucose; P) Trp; Q) Met; R) DTT; S) Glu; T) pthiocresol. The inset shows the fluorescence changes of 1 upon addition of various species under UV light at 365 nm. Figure 3. Time-dependent the emission ratio (I474/I694) of probe 1 (5 μM) in the absence (blank) and presence of Cys or Hcy (20 equiv) in ethanol/phosphate buffer (40:60 v/v, 20 mM, pH 7.4).
(Figure S10, Supporting Information), which further proves the high selectivity of probe 1 toward Cys/Hcy over other thiols. Applications of Probe 1 in Biological Systems. In order to show its application in biology, probe 1 was employed to detect Cys in rabbit serum samples (although Hcy gives similar response to Cys, Hcy would not significantly interfere with Cys assay because the total concentration of Hcy in serum is much lower than that of Cys).25 The serum sample was treated with triphenylphosphine to release the free Cys, according to the previous reported procedure.26 Different amounts of the reduced rabbit serum sample were then added to the solution of probe 1 in ethanol/phosphate buffer (40:60 v/v, 20 mM, pH 7.4). As shown in Figure S11, a linear relationship between the emission ratio (I474/I694) and the volume of reduced rabbit serum was noted, thus indicating that probe 1 is capable of detecting Cys in biological fluids. Furthermore, we proceeded to investigate the capability of 1 to image Cys in living HepG2 cells. First, the cytotoxicity of probe 1 was evaluated by the MTT assays, and the results showed that the cellular viability was about 80% even with 10 μM 1 at 37 °C for 12 h, indicating the low cytotoxicity of the probe (Figure S12, Supporting Information). Thus, a 5 μM of 1 was selected for further cell image experiments. HepG2 cells were incubated with 1 (5 μM) at 37 °C for 30 min, and we could observe fluorescence in both blue (Figure 5B) and red channels (Figure 5C). Furthermore, in a control experiment, the cells were pretreated with 1.0 mM NEM (a thiol blocking reagent) to reduce the concentration of intracellular thiols prior to incubation with probe 1. A marked fluorescence quenching in the blue channel (Figure 5E) and a fluorescence increase in the red channel were observed (Figure 5F). However, when the cells were pretreated with Cys (600 μM) and then incubated with probe 1, we could note bright blue fluorescence (Figure 5H) but almost no red fluorescence (Figure 5I). The above image variations are inconsistent with Cys-induced ratiometric fluorescence response. Thus, these results revealed that 1 is cellpermeable and can be used as a ratiometric fluorescent probe for bioimaging of Cys/Hcy in living cells.
exhibits no noticeable changes in ethanol/phosphate buffer (40:60 v/v, 20 mM, pH 7.4). By contrast, upon addition of Cys, the I474/I694 value of probe 1 shows a rapid increase at first, and the emission ratio essentially reaches a maximum in 25 min. A similar emission ratio increase was observed for Hcy. The calculated pseudo-first-order rate constants (k) for Cys and Hcy are 0.139 ± 0.011 and 0.113 ± 0.007 min−1, respectively (Figure S6, Supporting Information).23 The reaction rate for Hcy is slightly lower than that of Cys because the intramolecular attack by the amino group via a six-membered intermediate for Hcy is less favored than the facile fivemembered ring mediated for Cys in the second step of NCL.24 Selectivity Studies. To further verify the selectivity, probe 1 was incubated with the representative biologically relevant species in ethanol/phosphate buffer (40:60 v/v, 20 mM, pH 7.4). As shown in Figure 4 and Figure S7, only Cys/Hcy induces a dramatic increment of the emission ratio, while other thiol-containing compounds (GSH, DTT, p-thiocresol), amino acids (Thr, Lle, Val, Tyr, Pro, Ser, Leu, Ala, Gln, Trp, Met, Glu), metal ions (Zn2+, Ag+, Hg2+, Ca2+, Cd2+, Mg2+, K+) (Figure S8, Supporting Information), ascorbic acid, and glucose trigger no or very minor changes. The high selectivity of 1 toward Cys/Hcy can also be observed by the naked eye, and only Cys/Hcy induces an obvious color change under visible or UV light (Figure 4 and Figure S9). Notably, only very limited changes in the emission ratio were observed upon addition of other thiol-containing compounds. This can be explained by the fact that although transthioesterification can occur between 1 and common thiols, the ensuing intramolecular S to N acyl shift of thioester intermediate to give the final amide cannot proceed due to the lack of amino group in proximity, which further confirms that both the sulfhydril and the adjacent amino groups contribute to the sensing process. In addition, the above transthioesterification is reversible and would not consume a large amount of the probe if no further intramolecular S, N-acyl transfer occurs. Therefore, probe 1 can still retain its sensing response to Cys even in the presence of a large amount of GSH 1805
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Figure 5. Fluorescence images of HepG2 cells treated with probe 1. (A) Bright field image of cells incubated with 1 (5 μM) for 30 min; (B) fluorescence image (A) from blue channel; (C) fluorescence image (A) from red channel. (D) Bright field image of cells incubated with NEM (1.0 mM) for 40 min and then further incubation with 1 (5 μM) for 30 min; (E) fluorescence image (D) from blue channel; (F) fluorescence image (D) from red channel. (G) Bright field image of cells incubated with Cys (600 μM) for 40 min and then further incubation with 1 (5 μM) for 30 min; (H) fluorescence image (G) from blue channel; (I) fluorescence image (G) from red channel.
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*E-mail: [email protected] (Y.G.).
CONCLUSION In summary, we have developed a ratiometric fluorescent probe for the selective detection of Cys/Hcy. The ratiometric sensing is realized via interrupting the large π-conjugated system of CB fluorophore by a tandem NCL/spirocyclization reaction. Notably, significant ratiometric fluorescent response coupling with a large emission blue shift (Δλ ≈ 220 nm) allows for convenient sensing of Cys/Hcy. Because both the sulfhydril and the adjacent amino groups contribute to the sensing process, the probe displays excellent selectivity toward Cys/ Hcy over other thiols. Importantly, preliminary studies show that probe 1 is low cytotoxic and cell-permeable and can be employed for the ratiometric fluorescent imaging of Cys/Hcy in living cells. Further, modulation of the π-conjugated system in benzopyrylium dyes by spirocyclization provides a promising methodology for constructing various ratiometric fluorescent probes, and this research work is still under way in our laboratory.
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by the National Natural Science Foundation of China (No. 21275117, 21375105), the Science & Technology Department (No. 2012JM2004), and the Education Department (No. 12JK0518) of Shaanxi Province of China.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in the text (Figures S1−S12, Scheme S1, and Scheme S2); 1H NMR, 13C NMR, and HRMS data of 1, 2, and 9. This material is available free of charge via the Internet at http://pubs.acs.org.
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*Phone: 86-29-88302604. Fax: 86-29-88303798. E-mail: [email protected] (X.-F.Y.). 1806
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