2015, Vol.26 
b Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
Sulfur-containing compounds play vital roles in environment, industry and biological systems [1, 2, 3, 4, 5]. Consequently,the detection of these species has received growing attention. Many fluorescent and/or colorimetric probes have been developed for the determination of sulfur-containing compoundsutilizing the mechanisms of nucleophilic reaction [6, 7, 8],Michael addition [9, 10, 11, 12],reduction [13, 14, 15, 16],cleavage of 2,4-dinitrobenzenesulfonyl [17, 18, 19, 20, 21, 22] and forming complexes with metal ions [23, 24, 25]. In our previous work, we designed several probes for sulfur-containing compounds by incorporating α,β-unsaturated ketone on a coumarin fluorophore [26, 27, 28]. The experimental results revealed that a strong electrondonating group suppressed the Michael reaction. Recently,Kim, et al.,reported a coumarin-based probe with a strong electrondonating group (o-QPS) activated by an intramolecular H-bonding for the fast detection of thiols in DMSO/Hepes buffer [29]. The Michael reaction requiredmorethan one hour to be completed with a large excess of 2-mercaptoethanol (10 mmol/L). This result attracted our interest,and we repeated the experiments under the same conditions. However,we found that the spectral responses of o-QPS toward thiols at lower concentrations (less than 1 mmol/L) were quite slow (more than 10 hours for GSH) and believed that Hepes is not a good solvent for thiol detection. In addition,the spectral response of the probe toward other sulfur-containing compounds was not reported in the literature [29]. Our previous study found that sulfite and sulfide are more active nucleophilic reagents than thiols. Therefore,sulfite and sulfide are expected to form Michael addition products with o-QPS under the same experimental conditions.
Herein,we turned our attention to investigating the effects of the position of the substituent upon the Michael reaction and the nucleophilic reactivities of different sulfur-containing species. We synthesized three isomers (Scheme 1) and studied their spectral responses toward various species,including thiols,sulfite,sulfide and some other amino acids. The experimental conditions were optimized to shorten the detection time. The reaction is much faster in phosphate buffer solution than in Hepes solution taking only 10 min for o-QPS to react with Cys. To our surprise,o-QPS showed quite a different response to Cys,which allowed us to distinguish Cys from other sulfur-containing compounds.
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| Scheme. 1.The chemical structures of the compounds studied. | |
Unless otherwise specified,all the commercial reagents were of analytical grade and used without further purification. All the chemicals were purchased from Aladdin Corporation. Ultra-pure water was prepared through Sartorius Arium 611DI system.
2.2. Characterization and measurementNMR spectra were acquired on a Bruker AV-400 spectrometer (400 MHz). Mass spectra were recorded on a MA 1212 Instrument under standard conditions (ESI,70 eV). Absorption spectra were measured with an Evolution 220 UV-vis spectrophotometer (Thermo Scientific). Fluorescence spectra were recorded on a Lumina Fluorescence Spectrometer (Thermo Scientific),all the fluorescence spectra were uncorrected. The experiments were performed at 25 8C using non-degassed samples.
2.3. Absorbance and fluorescence titrationAccurately weighted amounts of the dyes were dissolved in DMF to obtain 3 × 10-3 mol/L stock solutions. The stock solution was diluted with the corresponding mediumto acquired 10 μmol/L dye solutions.
Sulfur-containing species and other amino acids were freshly prepared by dissolving in water/DMF to obtain stock solutions (20 mmol/L). A 45 μL aliquot of the above solution was added to 3mL of 1 × 10-5 mol/L dye in 2:1 DMSO/PBS (10 mmol/L,pH 7.4) to make [substrate] = 300 μmol/L.
2.4. HPLC tracesHPLC analyses were performed on an Elliot 1203 system and a Zobax C18 reversed-phase column (4.6 mm × 10 cm). The mobile phases were degassed with an ultrasonic apparatus for 10 min. Injection volume: 25 μL; mobile phase: A-0.1% TFA/water, B-acetonitrile; gradient elution: 3-15 min 5-90% B; isocratic elution: 0-3 and 18-20 min,5% B; 15-18 min,90% B; flow rate: 1.0 mL/min; detection wavelength = 430 nm.
2.5. SynthesisThree isomers were synthesized according to the following procedures (Scheme 2). Compound m1 was prepared according to Refs. [26, 27, 28, 29]. o-,m-,or p-QPS: 100 mg (0.4 mmol) of compound m1,0.72 mmol of o-,m-,or p-hydroxy-acetophenone and five drops of pyrrolidine were added to 20 mL of EtOH/DCM (1:2,v/v). After stirring at room temperature for two days,the solvent was evaporated under vacuum. Compounds o-,m- or p-QPS were purified by column chromatography (PE:DCM,5:1,v/v) to give a salmon colored solid.
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| Scheme. 2.The synthesis procedures of the three isomers. | |
o-QPS: (22.0%),1H NMR (400 MHz,DMSO-d6): δ 10.43 (s,1H), 8.46 (s,1H),8.03 (δ,1H,J = 15.4 Hz),7.96 (δ,1H,J = 8.6 Hz),7.61 (δ, 1H,J = 15.4 Hz),7.50 (δ,1H,J = 8.9 Hz),6.91 (δ,2H,J = 8.5 Hz),6.80 (δ,1H,J = 9.0 Hz),6.60 (s,1H),3.48 (t,4H,J = 6.8 Hz),1.15 (t,6H, J = 6.8 Hz). 13C NMR (100 MHz,DMSO-d6): δ 198.456,167.068, 165.196,161.870,157.414,151.498,145.618,141.318,136.137, 135.277,126.017,125.225,124.451,123.056,118.085,115.315, 113.664,101.483,49.617,17.592. HR-MS m/z: 364.1535 (M+H)+; calcd. molecular weight of C22H45NO4: 364.1549 for (M+H)+.
m-QPS: (66.0%),1H NMR (400 MHz,DMSO-d6): δ 10.43 (s,1H), 8.46 (s,1H),8.03 (δ,1H,J = 15.4 Hz),7.96 (δ,1H,J = 8.6 Hz),7.61 (δ, 1H,J = 15.4 Hz),7.50 (δ,1H,J = 8.9 Hz),6.91 (δ,2H,J = 8.5 Hz),6.80 (δ,1H,J = 9.0 Hz),6.60 (s,1H),3.48 (t,4H,J = 6.8 Hz),1.15 (t,6H, J = 6.8 Hz). 13C NMR (100 MHz,DMSO-d6): δ 194.060,165.187, 162.957,161.652,157.152,151.152,151.073,144.560,135.899, 135.141,126.182,125.269,124.266,119.627,118.405,115.160, 113.588,101.453,49.554,17.592. HR-MS m/z: 364.1544 (M+H)+; calcd. molecular weight of C22H45NO4: 364.1549 for (M+H)+.
p-QPS: (6.9%),1H NMR (400 MHz,DMSO-d6): d 9.84 (s,1H),8.47 (s,1H),7.99 (δ,1H,J = 15.4 Hz),7.64 (δ,1H,J = 15.4 Hz),7.50 (t,2H, J = 8.9 Hz),7.40-7.36 (m,2H),7.04 (dd,1H,J1 = 1.7 Hz,J2 = 7.9 Hz), 6.94 (dd,1H,J1 = 2.0 Hz,J2 = 9.0 Hz,),6.61 (δ,1H,J = 1.7 Hz,),3.49 (t, 4H,J = 6.7 Hz),1.15 (t,6H,J = 6.9 Hz). 13C NMR (100 MHz,DMSOd6): d 192.183,167.245,165.303,161.563,156.978,150.326, 143.283,135.987,135.757,134.578,126.234,120.666,118.681, 115.087,113.550,101.460,49.531,17.584. HR-MS m/z: 364.1548 (M+H)+; calcd. molecular weight of C22H45NO4: 364.1549 for (M+H)+.
3. Results and discussion 3.1. Photophysical properties of the three dyesFig. 1a shows the absorption and emission spectra of the three isomers with the absorption/emission maxima at 488 nm/573 nm, 480 nm/584 nm and 473 nm/568 nm for o-QPS,m-QPS and p-QPS, respectively. The Stokes shifts for all compounds are large enough (~100 nm) to avoid self-quenching. Their fluorescence quantum yields are 0.09,0.27 and 0.43 (coumarin 153 was employed as the reference) in 2:1 DMSO:H2O,respectively.
From Scheme 1,it is known that the three isomers possess electronic push-pull characteristics with the N,N-diethyl amino and the carbonyl groups acting as the electron-donor and the electron-acceptor,respectively. The strong electron-donating -OH group of the appended phenyl weakens the electron push-pull property more effectively when it is linked at the ortho-position and para-position. Compared to m-QPS,evident blue-shifts in both absorption and emission spectra of o-QPS and p-QPS could be expected (Fig. 1). However,o-QPS has the longest absorption wavelength,the intramolecular H-bonding associated with the hydroxyl and the adjacent carbonyl strengthens the push-pull effect,which gives the above results.
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| Fig. 1.Normalized absorption (a) and emission (b) spectra of three dyes in DMSO:H2O (2:1, pH 6.8). [o-QPS] = [m-QPS] = [p-QPS] = 10 μmol/L, λex = 420 nm. | |
The effect of Cys on the photophysical properties of o-QPS was also studied in 2:1 DMSO:H2O. As shown in Fig. 2,the addition of Cys to the solution of o-QPS led to a decrease in the original absorption peak at 488 nm. At the same time,a new absorption at ca. 404 nm emerged and developed,which then decreased gradually along with the simultaneous recovery of the peak at 488 nm (Fig. 2a and b). An isobestic point at 430 nm was clearly observed. The solution color changed from red to yellow and then red again. Similar fluorescence changes were observed,namely, the original fluorescence at 573 nm decreased initially and then reappeared,accompanied by the development of a new emission band at 472 nm,which then decreased. A distinctly isoemissive point at 507 nm was seen (Fig. 2c and d). The dramatic blue-shifts of the absorbance and emission bands could be ascribed to the addition of Cys to the electrophilic C55C double bond in o-QPS, resulting in a shorter conjugation structure of the reaction product. The recovery of the original absorption and emission peaks might be caused by the elimination reaction.
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| Fig. 2.The time-dependent UV–vis (a, b) and fluorescence (c, d) spectra of o-QPS (10 μmol/L) in the presence of 300 μmol/L Cys in DMSO:H2O (2:1, pH 6.8) at 25 8C. λex = 430 nm. | |
Fig. 3a presents plots of the At/A0 (at 488 nm) of Cys-o-QPS vs. time in different media. From which it can be seen that the reaction is very fast in DMSO/PBS and DMSO/H2O solutions with the t1/2 ≈ 3.5 min in both solutions. After 10 min,the absorbance at 488 nm increased rapidly in DMSO/H2O,while it changed much more slowly in DMSO/PBS. In DMSO/Hepes solution,the reaction between Cys and o-QPS is quite slow and,therefore,DMSO/PBS was chosen as the reaction medium in the following experiments.
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| Fig. 3.The At/A0 plots of o-QPS (10 μmol/L) as a function of time in the presence of 30 equiv. of Cys in different systems (a) and in the presence of different additives in 2:1 DMSO-PBS (b). At and A0 are the absorbance at 488 nm at times t and 0, respectively, pH 7.4, 25 8C. | |
Fig. 3b shows the kinetic curves of o-QPS with different reactants in DMSO/PBS. It is obvious that the reaction rate decreases in the order of Cys > sulphite > sulphide > Hcy > ME > GSH. The effects of sulfur-containing species on the spectral properties of m-QPS and p-QPS in DMSO/PBS were also studied. Less than 20% decrease in the absorbance at ~483 nm was found within two hours in all cases (Fig. S4-S5 in Supporting Information),indicating that m-QPS and p-QPS are less reactive than o-QPS. The reactivity of o-QPS was speculated to be activated by intramolecular H-bonding and showed rapid spectral responses toward sulfur-containing compounds.
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| Fig. 4.HPLC traces of the reaction between o-QPS and Cys in DMSO-PBS (2:1). [o-QPS] = 10 μmol/L, [Cys] = 300 μmol/L, 25 8C. | |
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| Fig. 5.The absorption spectrum of o-QPS with different concentrations of Cys (a), and the ratios of A401/A486 (b) and I470/I570 (c) of o-QPS as a function of Cys concentration. 2:1 DMSO-PBS, equilibrated at 25 8C for 10 min, [o-QPS] = 15 mmol/L, λex = 430 nm. | |
In our previous work,we found that the nucleophilic reactivity decreases in the order: sulfite > sulfide > thiol [26]. The results in Fig. 3b demonstrate that most of the reactants are in line with this order,whereas Cys is inconsistent with that. The reaction of Cys is even faster than those of sulfide and sulfite. We assume that intermolecular hydrogen-bonding between the carboxyl in Cys and the hydroxyl and carbonyl groups in o-QPS makes the nucleophilic addition of sulfydryl to the C=C bond easier,which leads to significant blue-shifts in both absorption and emission maxima. The carboxyl group at a second Cys molecule initiates the elimination and recovery of the original spectra. The reaction procedure is illustrated in Scheme 3. In Hepes solution,Cys can form hydrogen-bonding with Hepes,which suppresses the interaction between Cys and o-QPS,and as a result,the reaction is much slower in DMSO-Hepes solution. It is worth noting that almost no spectral change of o-QPS was found with the addition of Cys stock solution prepared for several days,which is probably ascribed to the formation of hydrogen-bonding between Cys molecules.
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| Scheme. 3.Proposed reaction procedure between Cys and o-QPS. | |
To understand the sensing mechanism of Cys,HPLC traces of the reaction processeswere conducted (Fig. 4a and b). The peak of o-QPS was at 17.01 min,after reacting with Cys,the original peak decayed and two new peaks at 10.95min and 11.32 min emerged and developed. After 24 h,the peaks at 10.95 min and 11.32 min disappeared accompanied by the appearance of a third,new peak at 15.66 min. The peaks at 10.95 min and 11.32min are conjectured to be the Michael addition products,and that at 15.66 min is supposed to be the final product as proposed in Scheme 3.
3.5. Spectral responses of o-QPS toward other sulfur-containing compoundsOther sulfur-containing species including sulfite,sulfide, 2-mercaptoethanol,GSH and Hcy induced similar spectral responses of o-QPS: the absorption peak shifted from 488 nm to 404 nm accompanied by a ca. 100 nm blue shift in the emission band (Fig. S6 in Supporting information). During the experimental period (two hours),the original peaks were not found to reappear. Therefore,Cys can be discriminated from other sulfur-containing compounds with o-QPS as the probe. In addition,the non-thiol amino acids did not trigger apparent spectral changes of o-QPS, indicating that o-QPS could be employed as a probe for sulfurcontaining nucleophilic reagents.
3.6. Quantitative detection of Cys with o-QPSTo evaluate the feasibility of quantitatively detecting Cys with o-QPS,the selectivity and competition of o-QPS toward Cys over other biologically relevant species including amino acids and sulfur-containing compounds were investigated. As shown in Fig. S9 (in Supporting information),Cys induced a dramatic increase in the absorbance at 401 nm and about 50% fluorescence quenching at 570 nm,while other additives except for sulfurcontaining compounds did not cause noticeable spectral changes of the probe. Hcy,MEA,GSH and MAP triggered much smaller spectral changes of o-QPS. Fig. S10 (in Supporting information) shows the effects of the other additives on the detection of Cys. No remarkable interference in the determination of Cys was found in the presence of one equiv. of the additives. Thus,o-QPS exhibits excellent selectivity and competition for Cys.
Then the absorbance ratio of A401 to A486 and the fluorescence intensity ratio of I470 to I570 were used to quantitatively detect Cys. Good linear relationship between the ratio of A401/A488 (I470/I570) and Cys concentration was found in the range of 0-400 μmol/L (Fig. 5). ThedetectionlimitsofCysobtainedbyUV-vis andemissionmethods were calculated to be 3.0 × 10-6 mol/L and 2.3 × 10-6 mol/L, respectively. The detection limit of Cys in this work is much lower than reported in Ref. [29] (3 μmol/L compared to 37 μmol/L).
4. Conclusion Three fluorescent isomers were synthesized for detection of sulfur-containing species in aqueous solution based on the Michael addition activated by the intramolecular hydrogen-bonding. Compared to Heper buffer,PBS can accelerate the reaction between o-QPS and Cys,which allows the rapid measurement and much lower detection limit of Cys. An unexpected elimination reaction of the addition product of o-QPS with Cys enables us to discriminate Cys from other sulfur-containing compounds. This work may inspire the design of new fluorescent probes for sensitive identification of sulphur compounds. AcknowledgmentsThis work was financially supported by National 973 Program (No. 2011CB910403),Shanghai Municipal Natural Science Foundation (No. 15ZR1409000) and the open fund of Shanghai Key Laboratory of Chemical Biology (No. SKLCB-2013-03).
Appendix A. Supplementary dataSupplementary data associated with this article can be found,in the online version,at http://dx.doi.org/10.1016/j.cclet.2015.06.016.
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