FULL PAPER DOI: 10.1002/asia.201402765
Reaction-Based Fluorescent Probe for Detection of Endogenous Cyanide in Real Biological Samples Lingliang Long,*[a] Lin Wang,[a] Yanjun Wu,[a] Aihua Gong,[b] Zulin Da,[a] Chi Zhang,*[a] and Zhixiang Han[a]
Abstract: Herein, two compounds (1 a and 1 b) were rationally constructed as novel reaction-based fluorescent probes for CN by making use of the electron-withdrawing ability of the cyano group that was formed from the sensing reaction. Notably, this design strategy was first employed for the development of fluorescent CN probes. The experimental details showed that probe 1 a exhibited a fluorescence turnon response to CN, whereas other anions, biological thiols, and hydrogen sulfide gave almost no interference.
The detection limit of probe 1 a for CN was found to be 0.12 mm. The sensing reaction product of 1 a with CN was characterized by NMR spectroscopy and mass spectrometry. TDDFT calculations demonstrated that the formed cyano group drives the intramolecular charge transfer (ICT) proKeywords: cyanides · dyes/ pigments · fluorescence · fluorescent probes · intramolecular charge transfer
Introduction
ed through the consumption of certain foods and plants. Consequently, the development of efficient methods for sensing cyanide anions at environmental and biological levels is urgently required. Recently, fluorescent probes for CN detection have received great attention due to their superior features of high sensitivity, fast response time, and technical simplicity.[5] A number of small-molecule fluorescent probes for CN have been developed by use of the high binding affinity of CN to metal ions,[6] H + ,[7] or boronic acid derivatives.[8] Nevertheless, compared with binding-based probes, reaction-based probes that take advantage of the unique nucleophilic reactivity of CN display more specific selectivity and higher sensitivity. In general, the design strategy of the reactionbased probes for CN involves the nucleophilic addition reaction of CN to electrophilic C=C,[9] C=O,[10] C=N,[11] or C= N + [12] double bonds. After the addition reaction, the p conjugation of the probes is interrupted, which results in an obvious optical response. Although the reaction-based probes display good performance in sensing CN in environmental samples, when sensing endogenous CN in a biological sample the probes still confront potential interference from some biological nucleophiles, such as biological thiols and hydrogen sulfide. These biological nucleophiles could attack the electrophilic double bonds and destroy the p conjugation of a fluorophore similarly to the action of CN.[13] To avoid potential interference, there is great need for a new design strategy to develop reaction-based probes to specifically sense endogenous CN in biological samples.
Cyanide (CN) is an extremely toxic anion that can directly result in the death of human beings even in small doses.[1] However, cyanide salts are still widely used in industrial processes, such as synthetic fiber manufacture, gold mining, electroplating, and metallurgy.[2] The worldwide production of cyanide in various industries is up to 1 500 000 tons per year.[3] Thus, cyanide waste may pollute the environment and pose a severe threat to the human beings and animals. Additionally, cyanogenesis is widespread in plants. It is reported that more than 3000 plant species, including many important food crop species, such as sorghum, almonds, lima beans, white clover, and cassava, can release cyanide by enzymatic hydrolysis of cyanogenic glycosides after cell rupture.[4] Thus, a higher level of cyanide can also be accumulat[a] Dr. L. Long, L. Wang, Y. Wu, Dr. Z. Da, Prof. C. Zhang, Z. Han Scientific Research Academy & School of Chemistry and Chemical Engineering Jiangsu University Zhenjiang, Jiangsu 212013 (P. R. China) Fax: (+ 86) 511-88797815 E-mail: [email protected] [email protected] [b] Prof. A. Gong School of Medical Science and Laboratory Medicine Jiangsu University Zhenjiang, Jiangsu 212013 (P. R. China) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/asia.201402765.
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cess from coumarin dye to the cyano group and thus the original strong ICT from the coumarin dye to the 3-position pyridyl vinyl ketone substituent is weakened, which results in recovery of coumarin fluorescence. The practical utility of 1 a was also examined. By fabricating paper strips, probe 1 a can be used as a simple tool to detect CN in field measurements. Moreover, probe 1 a has been successfully applied for quantitative detection of endogenous CN from cassava root.
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Apart from the unique nucleophilic reactivity, another important feature of CN is that the cyano group formed in the nucleophilic reaction is a strong electron-withdrawing group. This electron-withdrawing group will have a significant effect on the electron distribuScheme 1. Synthesis of probes 1 a and 1 b. Reagents and conditions: i) DMF, POCl3, stirring for 12 h at 60 8C; tion of a fluorophore and, ii) 4-acetylpyridine, pyrrolidine, stirring for 24 h at RT; iii) 2-acetylpyridine, pyrrolidine, stirring for 24 h at RT. therefore, cause an optical response. We envision that this feature of CN could be employed as an alternative strategy to develop reaction-based been unambiguously characterized by using 1H NMR and 13 CN probes. It is worth noting that no reaction-based fluoC NMR spectroscopy, ESI mass spectrometry, and elemenrescent probe for CN has been developed that uses the tal analysis. The structure of probe 1 a was further confirmed by single-crystal X-ray diffraction (Figure 1). electron-withdrawing ability of the formed cyano group. The 7-diethylamino-coumarin dye is a commonly used fluorophore for developing fluorescent probes.[14] It is known that the strong ICT process from coumarin dye to the substituent linked to the 3-position of the coumarin ring causes the fluorescence of the coumarin to be quenched.[15] We anticipated that if the CN attacks the other site of the coumarin ring, the formed cyano group will force the ICT process from coumarin dye to the cyano group and thus the original strong ICT from the coumarin to the 3-position subFigure 1. ORTEP diagram of probe 1 a (ellipsoids drawn at the 30 % stituent will be weakened, which leads to recovery of the probability level). Left: Side view; right: top view. coumarin fluorescence. Bearing these considerations in mind, in this work compounds 1 a and 1 b were rationally designed as novel reacOptical Properties tion-based probes for CN by utilizing the electron-withThe optical properties of probes 1 a and 1 b were assessed in drawing ability of the formed cyano group. In compound 1 a 20 mm potassium phosphate buffer/CH3CN (1:3 v/v, pH 7.4) and 1 b, the coumarin dye was used as the fluorophore and 4- or 2-pyridyl vinyl ketone substituents were conjugated to at room temperature. As designed, the fluorescence of 1 a the 3-position of coumarin ring. Indeed, in the absence of (fluorescence quantum yield, Ff = 0.008) and 1 b (Ff = 0.072) CN, the fluorescence of 1 a and 1 b was quenched because displayed significant quenching compared with the fluorescence of compound 2 (Ff = 0.530) (Figure 2a). The dramatic of the strong ICT process from the coumarin dye to the 3position pyridyl vinyl ketone substituents. However, they exquenching fluorescence is apparently attributed to the hibited a fluorescence turn-on response to CN as a result strong ICT process from the coumarin dye to the pyridyl vinyl ketone substituents at the 3-position. Moreover, deof the formed cyano group weakening the ICT process from spite the similar structures, the extent of fluorescence coumarin to the pyridyl vinyl ketone substituents. The optiquenching for 1 a is larger than that for 1 b, which suggests cal spectroscopic studies demonstrated that the probes disa stronger ICT process occurs in 1 a. This phenomenon furplayed high selectivity for CN over other anions, biological ther reveals that minor variation in the ICT process in this thiols, and hydrogen sulfide. Moreover, probe 1 a has been probe system would afford a notable change in fluorescence successfully applied for detection of endogenous CN in an intensity. Therefore, 1 a and 1 b can be used as promising fluextract of cassava root. orescent probes for CN providing that the formed cyano group could weaken the ICT process from the coumarin dye to the pyridyl vinyl ketone substituents. The UV/Vis absorpResults and Discussion tion spectra are presented in Figure 2b, probes 1 a and 1 b Synthesis and Characterization showed intense absorption centered at l = 471 and 465 nm, respectively, which are obviously redshifted relative to comThe synthesis of the probes started from 7-diethylaminocoupound 2 (absorption centered at l = 380 nm). This drastic marin 2, which was converted to 7-diethylaminocoumarin-3redshifted absorption is clearly due to the strong ICT proaldehyde 3 according to a reported procedure.[16] Then cess. In addition, 1 a displayed a greater redshift absorption probes 1 a and 1 b were achieved in about 59 and 53 % than 1 b, which is in line with the fact that a stronger ICT yields, respectively, through condensation of 3 with 4-acetylwas observed in 1 a. pyridine or 2-acetylpyridine (Scheme 1). The products have
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diately turned bright red (Figure 3a, inset). When 60 mm CN was added, the fluorescence intensity was enhanced by more than 30 fold (Ff = 0.226). Moreover, the fluorescence intensity of 1 a at l = 615 nm versus CN concentration showed a linear enhancement between 0–30 mm (Figure S1 in the Supporting Information), which indicates that probe 1 a can be useful for quantitative detection of CN. The detection limit of probe 1 a for CN was calculated to be 0.12 mm, which is far lower than the maximum level of CN (1.9 mm) permitted in drinking water by the World Health Organization (WHO).[17] Thus, probe 1 a has the ability to detect CN in natural water samples. Similar behavior was observed for probe 1 b (Figure S2 in the Supporting Information). On addition of CN, the fluorescence emission at l = 607 nm was enhanced gradually. However, due to incomplete fluorescence quenching, probe 1 b in the absence of CN still has some residual fluorescence at l = 607 nm, which is unfavorable for the fluorescence detection of CN. Accordingly, we decided to focus on probe 1 a for further optical sensing studies. The UV/Vis absorption response of probe 1 a to CN is displayed in Figure 4. Addition of CN resulted in a gradual
Figure 2. a) The fluorescence emission spectra of 1 a (&), 1 b ( ! ), and 2 (*); the excitation wavelengths were l = 540, 534, and 380 nm, respectively. b) The absorption spectra of 1 a (&), 1 b ( ! ), and 2 (*).
Optical Responses to CN The sensing response of probes 1 a and 1 b to CN was investigated in 20 mm potassium phosphate buffer/CH3CN (1:3 v/v, pH 7.4). Upon addition of an increasing amount of CN to a solution of 1 a, a new fluorescence peak at around l = 615 nm emerged and drastically enhanced (Figure 3). Correspondingly, the visual fluorescence color of 1 a imme-
Figure 4. Changes in the absorption spectra of probe 1 a (10 mm) with various concentrations of CN (0–140 mm) in 20 mm potassium phosphate buffer/CH3CN (1:3 v/v, pH 7.4) at room temperature. Inset: Visible color changes of probe 1 a (10 mm) before and after addition of CN (140 mm).
decrease in the absorption at l = 471 nm. Concomitantly, a new redshifted absorption at l = 530 nm increased. Additionally, the visible color of the solution changed from orange to red (Figure 4, inset). The presence of a well-defined isosbestic point at l = 507 nm signified that only one species was produced upon the reaction of 1 a with CN. The variation in absorption spectra implied that the original ICT process from coumarin dye to the 3-position pyridyl vinyl ketone substituent has been weakened and a new ICT process from the coumarin dye to the formed cyano group occurred. Selectivity Studies
Figure 3. a) Changes in the fluorescence emission spectra (lex = 540 nm) of probe 1 a (10 mm) in 20 mm potassium phosphate buffer/CH3CN (1:3 v/ v, pH 7.4) with various concentrations of CN (0–140 mm). Inset: Visual fluorescence color change of probe 1 a (10 mm) before and after addition of CN (140 mm). b) Changes in fluorescence intensity of probe 1 a (10 mm) at l = 615 nm with various concentrations of CN (0–140 mm).
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To inspect whether probe 1 a could specifically detect CN, we tested its ability to discriminate between CN and various relevant species. As illustrated in Figure 5a, the probe did not show evident fluorescence enhancement in the pres-
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to 10.5. However, in the presence of CN, probe 1 a exhibited a pronounced fluorescence response when the pH value was higher than 5.2. Thus, probe 1 a can be used to detect CN at physiological pH values. Reaction Products of Probe 1 a with CN To investigate the possible sensing reaction mechanism, the reaction product of probe 1 a with CN was isolated and used in a 1H NMR spectroscopy study. As shown in Figure 6, the resonance signal of proton He in 1 a disap-
Figure 5. a) Fluorescence intensity (l = 615 nm) response of probe 1 a (10 mm) to 140 mm of various species in 20 mm potassium phosphate buffer/CH3CN (1:3 v/v, pH 7.4) with excitation at l = 540 nm. b) Visual fluorescence color changes of probe 1 a (10 mm) in the presence of various species (140 mm), the photo was taken under illumination with a handheld UV lamp.
ence of other anions, including F, Cl, Br, I, HSO3, CH3COO, ClO4, H2PO4, HCO3, NO3, SCN, and N3. Moreover, the addition of biological nucleophiles, such as biological thiols (Cys, Hcy) and NaHS (a commonly used H2S donor),[13d] also gave no visible variation in the fluorescence. Obviously, only CN induced a large fluorescence enhancement. This fluorescence enhancement can also be distinguished with the naked eye, as shown in Figure 5b, which shows photos of solutions of 1 a in the absence and presence of CN under UV light (l = 365 nm) illumination. We further examined the response of probe 1 a to CN in the presence of other relevant species, and most of these species only displayed minimum interference (Figure S3 in the Supporting Information). The absorption spectra and visible color of probe 1 a also exhibited selective response to CN over other species (Figures S4 and S5 in the Supporting Information). All these results strongly suggested that probe 1 a is highly selective for CN, even in the presence of biological nucleophiles.
Figure 6. Partial 1H NMR (400 MHz) spectra of a) probe 1 a and b) the isolated product of probe 1 a + CN in CDCl3.
peared after reaction with CN. This suggested that He in 1 a was substituted by CN to produce 1 a-CN. In addition, upon reaction with CN, the resonances that corresponded to protons Hc, Hd, and Hf showed a notable upfield shift. The upfield shift is undoubtedly due to the strong electronwithdrawing ability of the formed cyano group, consistent with the aforementioned design. The formation of 1 a-CN was further confirmed by the ESI-MS spectrum, in which a peak at m/z 374.3 was assigned to [1 aCN + H] + (Figure S11 in the Supporting Information). The structure of 1 aCN was also characterized by 13C NMR spectroscopy (Figure S12 in the Supporting Information) and IR spectroscopy (Figure S13 in the Supporting Information). Moreover, the Job plot of probe 1 a with CN also supported a 1:1 adduct between probe 1 a and CN (Figure S14 in the Supporting Information). According to the reaction product, a sensing reaction mechanism of probe 1 a with CN is proposed in Figure 7.
Response Time and Effect of pH Probe 1 a remains stable in the experimental solution (Figure S6 in the Supporting Information). The time course of probe 1 a in the absence or presence of CN is displayed in Figure S7 in the Supporting Information. Upon addition of CN (140 mm) at room temperature, a dramatic enhancement in the fluorescence intensity at l = 615 nm was observed within 20 min. The responses of 1 a toward CN at different pH values were investigated (Figure S8 in the Supporting Information). In the absence of CN, there were negligible fluorescence responses within the pH range of 2
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Theoretical Calculations To gain further insight into the CN sensing mechanism of probe 1 a, density functional theory (DFT) calculations at the B3LYP/6-31 + G** level by using the Gaussian 09[18] program were carried out. The optimized geometries are shown in Figure S15 in the Supporting Information, and the optimized structure of probe 1 a is identical to the experimental crystal structures as depicted in Figure 1.
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coumarin to the pyridyl vinyl ketone substituent in 1 a-CN was significantly weakened, which in turn led to the recovery of coumarin fluorescence. The simulated absorption spectra of 1 a and 1 a-CN are also shown in Figure S16 in the Supporting Information, which is in good agreement with the experimental absorption. Thus, based on the TD-DFT calculations, the optical properties of probe 1 a and 1 a-CN have been theoretically revealed. Practical Applications Figure 7. The proposed sensing reaction mechanism of probe 1 a with CN. EWG = electron withdrawing group.
Prompted by the favorable features of probe 1 a in solution, we fabricated a simple paper strip that could be conveniently used for detection of CN. The paper strips were prepared by immersing filter papers in a solution of 1 a (1 103 m) in CH3CN and then drying them in air. Next, these paper strips were immersed for 10 s in solutions with different CN concentrations (0, 40, 80, 150 mm), then taken out of the solution. After 30 min, photographs were taken under a handheld l = 365 nm UV lamp. As expected, the red fluorescence of the paper strips gradually increased with an increasing concentration of CN in the solution (Figure 9c–e),
To study the ICT processes in 1 a and 1 a-CN, time-dependent DFT (TD-DFT) calculations were conducted. The results indicated that the electron-density distributions of the HOMO!LUMO transition (S0 !S1) for 1 a and 1 a-CN (oscillator strength f = 1.1527 and 1.1125, respectively) are allowable (Table S1 in the Supporting Information). The electron distributions in HOMO and LUMO of 1 a and 1 a-CN are displayed in Figure 8. For probe 1 a, the HOMO is dis-
Figure 9. Photographs of paper strips with probe 1 a used for detecting CN in aqueous solution at concentrations of b) 0, c) 40, d) 80, and e) 150 mm. a) A paper strip that was not immersed in the CN solution is shown for comparison.
whereas paper strips immersed into a solution with no CN exhibited no fluorescence (Figure 9b), which is similar to the strip that was not immersed into a solution (Figure 9a). Thus, these paper strips may be used as a simple tool for detecting CN in field measurements with no requirement of sophisticated equipment. Cassava root is an important source of calories for many people living in the tropics. However, the cassava is also a typical cyanogenic crop and the endogenous CN poses a potential threat to the consumer.[19] Thus, probe 1 a was applied to sense the endogenous CN level in cassava root. An extract of bitter cassava root was prepared according to a reported procedure.[20] As shown in Figure 10, addition of an increasing volume of cassava extract gave a corresponding enhancement in the fluorescent intensity, which indicated the response of probe 1 a to endogenous CN. For comparison, an extract of commercially available potato was also made. However, addition of the potato extract to a solution of 1 a exerted no visible fluorescence enhancement (Figure S17 in the Supporting Information). Thus, no endoge-
Figure 8. The HOMO and LUMO energy levels and the orbitals of a) probe 1 a and b) compound 1 a-CN.
tributed on the 7-diethylamino-coumarin moiety, whereas the LUMO is primarily delocalized over the pyridyl vinyl ketone moiety. Thus, it is clear that, upon excitation, a strong ICT process will take place from 7-diethylaminocoumarin to the pyridyl vinyl ketone substituent. It is known that strong ICT from the 7-diethylamino-coumarin to the 3postion substituent frequently serves to quench the fluorescence of coumarin.[15] Apparently, the ICT from the 7-diethylamino-coumarin to the pyridyl vinyl ketone substituent in probe 1 a causes the fluorescence quenching. However, for compound 1 a-CN, owing to the strong electron-withdrawing ability of the formed cyano group, the electrons in the coumarin ring were transferred to the cyano group in the HOMO!LUMO transition. Consequently, the ICT from
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by using a Beijing Taike XT-4 microscopy melting point apparatus, and all melting points are uncorrected. Mass spectra were recorded by using a LXQ Spectrometer (Thermo Scientific) operating in ESI mode. 1H and 13 C NMR spectra were recorded by using a Bruker Avance 400 spectrometer operating at 400 and 100 MHz respectively. Elemental (C, H, N) analysis were carried out by using Flash EA 1112 analyzer. Infrared (IR) spectra were recorded by using a Bruker VERTEX80 FT-IR spectrometer. Electronic absorption spectra were obtained by using a SHIMADZU UV-2450 spectrometer. Fluorescence spectra were measured by using a Cary Eclipse spectrometer with 5 nm excitation and emission slit widths. The pH measurements were performed by using a pH-3c digital pH meter (Shanghai ShengCi Device Works, Shanghai, China) with a combined glass-calomel electrode.
Figure 10. Changes in the fluorescence emission spectra of probe 1 a (10 mm) in 20 mm potassium phosphate buffer/CH3CN (1:3 v/v, pH 7.4) in the presence of different volumes of cassava extract (0, 10, 20, 30, and 40 mL). Inset: The linear relationship between the fluorescence intensity at l = 615 nm and the added volume of cassava extract. The excitation wavelength was l = 540 nm.
Synthesis of 7-Diethylaminocoumarin-3-aldehyde (3) 7-Diethylaminocoumarin-3-aldehyde 3 was synthesized according to a reported procedure.[16] M.p. 152–154 8C; 1H NMR (400 MHz, CDCl3): d = 10.13 (s, 1 H), 8.26 (s, 1 H), 7.41 (d, J = 8.8 Hz, 1 H), 6.64 (dd, J = 2.4, 8.8 Hz, 1 H), 6.49 (d, J = 2.4 Hz, 1 H), 3.48 (q, J = 7.2 Hz, 4 H), 1.26 ppm (t, J = 7.2 Hz, 6 H); MS (ESI): m/z: 246.1 [M+H] + .
nous CN was found in the potato extract. We further quantitatively sensed the amount of CN in cassava root by using a standard addition method (see the Supporting Information). The CN content was estimated to be 140.9 mg kg1. Thus, probe 1 a should be very useful for the quantitative detection of endogenous CN in cassava root.
Synthesis of 1 a Under a N2 atmosphere, one drop of pyrrolidine was added to a solution of 7-diethylaminocoumarin-3-aldehyde (653 mg, 2.66 mmol) and 4-acetylpyridine (645 mg, 5.33 mmol) in CH2Cl2/MeOH (1:1 v/v; 10 mL). The mixture was stirred at RT for 24 h. After the solvent was removed under reduced vacuum, the resulting residue was further purified by column chromatography on silica gel (dichloromethane/petroleum ether 1:1, v/v) to afford compound 1 a as a red solid (yield: 552.3 mg, 59.6 %). M.p. 207– 209 8C; 1H NMR (CDCl3, 400 MHz): d = 8.81 (dd, J = 2.0, 4.4 Hz, 2 H), 8.15 (d, J = 15.2 Hz, 1 H), 7.84 (dd, J = 2.0, 4.4 Hz, 2 H), 7.81 (s, 1 H), 7.67 (d, J = 15.2 Hz, 1 H), 7.34 (d, J = 8.8 Hz, 1 H), 6.62 (dd, J = 2.4, 8.8 Hz, 1 H), 6.51 (s, 1 H), 3.46 (q, J = 7.2 Hz, 4 H), 1.25 ppm (t, J = 7.2 Hz, 6 H); 13 C NMR (CDCl3, 100 MHz): d = 189.9, 160.1, 156.8, 152.3, 150.8, 147.1, 144.6, 141.8, 130.3, 121.7, 121.6, 114.4, 109.7, 108.9, 96.9, 45.1, 12.5 ppm; MS (ESI): m/z: 349.25 [M+H] + ; elemental analysis calcd (%) for C21H20N2O3 : C 72.40, H 5.79, N 8.04; found: C 72.23, H 5.83, N 7.99.
Conclusion We reported a novel strategy to develop reaction-based fluorescent probes for CN by taking advantage of the strong electron-withdrawing ability of the cyano group that was formed from the CN sensing reaction. As a proof of concept, compounds 1 a and 1 b were rationally designed and synthesized. In the absence of CN, probe 1 a and 1 b displayed fluorescence quenching, but specific reaction of the probes with CN in aqueous solutions gave a fluorescence turn-on response. TD-DFT calculations revealed that the sensing reaction formed cyano group that weakened the ICT process from coumarin dye to the pyridyl vinyl ketone substituent, which in turn led to a fluorescence turn-on response. Further optical studies established that probe 1 a could detect CN with high selectivity and sensitivity. Moreover, a paper strip based on probe 1 a was fabricated and could serve as a simple tool to detect CN in field measurements. Then, probe 1 a was successfully applied to quantitatively detect endogenous CN in cassava root.
Synthesis of 1 b The synthetic procedure for 1 b was similar to 1 a. Under a N2 atmosphere, one drop of pyrrolidine was added to a solution of 7-diethylaminocoumarin-3-aldehyde (445 mg, 1.81 mmol) and 2-acetylpyridine (439.5 mg, 3.63 mmol) in CH2Cl2/MeOH (1:1 v/v; 8 mL). The mixture was stirred at RT for 24 h. Then the solvent was removed under reduced vacuum. The resulting residue was further purified by column chromatography on silica gel (dichloromethane/petroleum ether 1:1, v/v) to afford compound 1 b as a red solid (yield: 335.5 mg, 53.2 %). M.p. 163–165 8C; 1 H NMR (CDCl3, 400 MHz): d = 8.76 (d, J = 4.0 Hz, 1 H), 8.59 (d, J = 16.0 Hz, 1 H), 8.16 (d, J = 7.6 Hz, 1 H), 7.87 (m, 3 H), 7.47 (t, J = 4.8 Hz, 1 H), 7.33 (d, J = 8.8 Hz, 1 H), 6.61 (dd, J = 2.0, 8.8 Hz, 1 H), 6.51 (s, 1 H), 3.45 (q, J = 6.8 Hz, 4 H), 1.24 ppm (t, J = 6.8 Hz, 6 H); MS (ESI): m/z: 349.16 [M+H] + ; elemental analysis calcd (%) for C21H20N2O3 : C 72.40, H 5.79, N 8.04; found: C 72.28, H 5.82, N 8.01. Synthesis of 1 a-CN Tetrabutylammonium cyanide (385 mg, 1.435 mm) was added to a solution of compound 1 a (100 mg, 0.287 mm) in CH3CN (10 mL), and the solution was stirred at RT for 1 h. Then the solvent was removed under reduced vacuum. The resulting residue was further purified by column chromatography on silica gel (dichloromethane/petroleum ether 2:1, v/v) to afford compound 1 a-CN (yield: 51.6 mg, 48.2 %). M.p. 217–219 8C; 1H NMR (CDCl3, 400 MHz): d = 8.84 (dd, J = 1.6, 4.4 Hz, 2 H), 8.44 (d, J = 15.2 Hz, 1 H), 8.02 (d, J = 15.2 Hz, 1 H), 7.86 (dd, J = 1.6, 4.4 Hz, 2 H), 7.65 (d, J = 9.2 Hz, 1 H), 6.74 (dd, J = 2.8, 9.2 Hz, 1 H), 6.51 (d, J = 2.8 Hz, 1 H), 3.50 (q, J = 7.2 Hz, 4 H), 1.28 ppm (t, J = 7.2 Hz, 6 H); 13C NMR (CDCl3, 100 MHz): d = 189.3, 158.2, 155.8, 152.9, 150.9, 143.9, 136.7, 128.5, 126.6, 126.1, 121.6, 117.6, 112.8, 110.9, 107.2, 97.1, 45.4, 12.5 ppm; MS (ESI): m/ z: 374.3 [M+H] + .
Experimental Section Materials and Instruments Unless otherwise stated, all reagents were purchased from commercial suppliers and used without further purification. Solvents were purified and dried by standard methods prior to use. Twice-distilled water was used for all experiments. The solutions of various anions were prepared from their tetrabutylammonium salt or potassium salt. TLC analyses were performed on silica gel plates and column chromatography was conducted over silica gel (mesh 200–300), both of which were obtained from Qingdao Ocean Chemicals. Melting points of compounds were measured
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Acknowledgements This research was supported by the National Natural Science Foundation of China (21202063, 21105038), the Ministry of Science and Technology of China for the International Science Linkages Program (2011DFG52970), the Ministry of Education of China for Changjiang Innovation Research Team (IRT1064), Jiangsu Innovation Research Team, the Natural Science Foundation of Jiangsu Province (BK2012281), and the Research Foundation of Jiangsu University (11JDG078).
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FULL PAPER Fluorescent Probes Lingliang Long,* Lin Wang, Yanjun Wu, Aihua Gong, Zulin Da, Chi Zhang,* Zhixiang Han &&&&—&&&&
Spotting cyanide: A novel strategy for reaction-based fluorescent probes for CN that take advantage of the strong electron-withdrawing ability of the cyano group formed from the CN sensing reaction is reported (see figure). Based on this strategy, two
probes were rationally designed and synthesized. One probe showed high sensitivity and selectivity for CN over other anions and biological nucleophiles and has been successfully applied for detection of endogenous CN in cassava root.
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Reaction-Based Fluorescent Probe for Detection of Endogenous Cyanide in Real Biological Samples
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Reaction-Based Fluorescent Probe for Detection of Endogenous Cyanide in Real Biological Samples Lingliang Long,*[a] Lin Wang,[a] Yanjun Wu,[a] Aihua Gong,[b] Zulin Da,[a] Chi Zhang,*[a] and Zhixiang Han[a]
Abstract: Herein, two compounds (1 a and 1 b) were rationally constructed as novel reaction-based fluorescent probes for CN by making use of the electron-withdrawing ability of the cyano group that was formed from the sensing reaction. Notably, this design strategy was first employed for the development of fluorescent CN probes. The experimental details showed that probe 1 a exhibited a fluorescence turnon response to CN, whereas other anions, biological thiols, and hydrogen sulfide gave almost no interference.
The detection limit of probe 1 a for CN was found to be 0.12 mm. The sensing reaction product of 1 a with CN was characterized by NMR spectroscopy and mass spectrometry. TDDFT calculations demonstrated that the formed cyano group drives the intramolecular charge transfer (ICT) proKeywords: cyanides · dyes/ pigments · fluorescence · fluorescent probes · intramolecular charge transfer
Introduction
ed through the consumption of certain foods and plants. Consequently, the development of efficient methods for sensing cyanide anions at environmental and biological levels is urgently required. Recently, fluorescent probes for CN detection have received great attention due to their superior features of high sensitivity, fast response time, and technical simplicity.[5] A number of small-molecule fluorescent probes for CN have been developed by use of the high binding affinity of CN to metal ions,[6] H + ,[7] or boronic acid derivatives.[8] Nevertheless, compared with binding-based probes, reaction-based probes that take advantage of the unique nucleophilic reactivity of CN display more specific selectivity and higher sensitivity. In general, the design strategy of the reactionbased probes for CN involves the nucleophilic addition reaction of CN to electrophilic C=C,[9] C=O,[10] C=N,[11] or C= N + [12] double bonds. After the addition reaction, the p conjugation of the probes is interrupted, which results in an obvious optical response. Although the reaction-based probes display good performance in sensing CN in environmental samples, when sensing endogenous CN in a biological sample the probes still confront potential interference from some biological nucleophiles, such as biological thiols and hydrogen sulfide. These biological nucleophiles could attack the electrophilic double bonds and destroy the p conjugation of a fluorophore similarly to the action of CN.[13] To avoid potential interference, there is great need for a new design strategy to develop reaction-based probes to specifically sense endogenous CN in biological samples.
Cyanide (CN) is an extremely toxic anion that can directly result in the death of human beings even in small doses.[1] However, cyanide salts are still widely used in industrial processes, such as synthetic fiber manufacture, gold mining, electroplating, and metallurgy.[2] The worldwide production of cyanide in various industries is up to 1 500 000 tons per year.[3] Thus, cyanide waste may pollute the environment and pose a severe threat to the human beings and animals. Additionally, cyanogenesis is widespread in plants. It is reported that more than 3000 plant species, including many important food crop species, such as sorghum, almonds, lima beans, white clover, and cassava, can release cyanide by enzymatic hydrolysis of cyanogenic glycosides after cell rupture.[4] Thus, a higher level of cyanide can also be accumulat[a] Dr. L. Long, L. Wang, Y. Wu, Dr. Z. Da, Prof. C. Zhang, Z. Han Scientific Research Academy & School of Chemistry and Chemical Engineering Jiangsu University Zhenjiang, Jiangsu 212013 (P. R. China) Fax: (+ 86) 511-88797815 E-mail: [email protected] [email protected] [b] Prof. A. Gong School of Medical Science and Laboratory Medicine Jiangsu University Zhenjiang, Jiangsu 212013 (P. R. China) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/asia.201402765.
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cess from coumarin dye to the cyano group and thus the original strong ICT from the coumarin dye to the 3-position pyridyl vinyl ketone substituent is weakened, which results in recovery of coumarin fluorescence. The practical utility of 1 a was also examined. By fabricating paper strips, probe 1 a can be used as a simple tool to detect CN in field measurements. Moreover, probe 1 a has been successfully applied for quantitative detection of endogenous CN from cassava root.
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Apart from the unique nucleophilic reactivity, another important feature of CN is that the cyano group formed in the nucleophilic reaction is a strong electron-withdrawing group. This electron-withdrawing group will have a significant effect on the electron distribuScheme 1. Synthesis of probes 1 a and 1 b. Reagents and conditions: i) DMF, POCl3, stirring for 12 h at 60 8C; tion of a fluorophore and, ii) 4-acetylpyridine, pyrrolidine, stirring for 24 h at RT; iii) 2-acetylpyridine, pyrrolidine, stirring for 24 h at RT. therefore, cause an optical response. We envision that this feature of CN could be employed as an alternative strategy to develop reaction-based been unambiguously characterized by using 1H NMR and 13 CN probes. It is worth noting that no reaction-based fluoC NMR spectroscopy, ESI mass spectrometry, and elemenrescent probe for CN has been developed that uses the tal analysis. The structure of probe 1 a was further confirmed by single-crystal X-ray diffraction (Figure 1). electron-withdrawing ability of the formed cyano group. The 7-diethylamino-coumarin dye is a commonly used fluorophore for developing fluorescent probes.[14] It is known that the strong ICT process from coumarin dye to the substituent linked to the 3-position of the coumarin ring causes the fluorescence of the coumarin to be quenched.[15] We anticipated that if the CN attacks the other site of the coumarin ring, the formed cyano group will force the ICT process from coumarin dye to the cyano group and thus the original strong ICT from the coumarin to the 3-position subFigure 1. ORTEP diagram of probe 1 a (ellipsoids drawn at the 30 % stituent will be weakened, which leads to recovery of the probability level). Left: Side view; right: top view. coumarin fluorescence. Bearing these considerations in mind, in this work compounds 1 a and 1 b were rationally designed as novel reacOptical Properties tion-based probes for CN by utilizing the electron-withThe optical properties of probes 1 a and 1 b were assessed in drawing ability of the formed cyano group. In compound 1 a 20 mm potassium phosphate buffer/CH3CN (1:3 v/v, pH 7.4) and 1 b, the coumarin dye was used as the fluorophore and 4- or 2-pyridyl vinyl ketone substituents were conjugated to at room temperature. As designed, the fluorescence of 1 a the 3-position of coumarin ring. Indeed, in the absence of (fluorescence quantum yield, Ff = 0.008) and 1 b (Ff = 0.072) CN, the fluorescence of 1 a and 1 b was quenched because displayed significant quenching compared with the fluorescence of compound 2 (Ff = 0.530) (Figure 2a). The dramatic of the strong ICT process from the coumarin dye to the 3position pyridyl vinyl ketone substituents. However, they exquenching fluorescence is apparently attributed to the hibited a fluorescence turn-on response to CN as a result strong ICT process from the coumarin dye to the pyridyl vinyl ketone substituents at the 3-position. Moreover, deof the formed cyano group weakening the ICT process from spite the similar structures, the extent of fluorescence coumarin to the pyridyl vinyl ketone substituents. The optiquenching for 1 a is larger than that for 1 b, which suggests cal spectroscopic studies demonstrated that the probes disa stronger ICT process occurs in 1 a. This phenomenon furplayed high selectivity for CN over other anions, biological ther reveals that minor variation in the ICT process in this thiols, and hydrogen sulfide. Moreover, probe 1 a has been probe system would afford a notable change in fluorescence successfully applied for detection of endogenous CN in an intensity. Therefore, 1 a and 1 b can be used as promising fluextract of cassava root. orescent probes for CN providing that the formed cyano group could weaken the ICT process from the coumarin dye to the pyridyl vinyl ketone substituents. The UV/Vis absorpResults and Discussion tion spectra are presented in Figure 2b, probes 1 a and 1 b Synthesis and Characterization showed intense absorption centered at l = 471 and 465 nm, respectively, which are obviously redshifted relative to comThe synthesis of the probes started from 7-diethylaminocoupound 2 (absorption centered at l = 380 nm). This drastic marin 2, which was converted to 7-diethylaminocoumarin-3redshifted absorption is clearly due to the strong ICT proaldehyde 3 according to a reported procedure.[16] Then cess. In addition, 1 a displayed a greater redshift absorption probes 1 a and 1 b were achieved in about 59 and 53 % than 1 b, which is in line with the fact that a stronger ICT yields, respectively, through condensation of 3 with 4-acetylwas observed in 1 a. pyridine or 2-acetylpyridine (Scheme 1). The products have
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diately turned bright red (Figure 3a, inset). When 60 mm CN was added, the fluorescence intensity was enhanced by more than 30 fold (Ff = 0.226). Moreover, the fluorescence intensity of 1 a at l = 615 nm versus CN concentration showed a linear enhancement between 0–30 mm (Figure S1 in the Supporting Information), which indicates that probe 1 a can be useful for quantitative detection of CN. The detection limit of probe 1 a for CN was calculated to be 0.12 mm, which is far lower than the maximum level of CN (1.9 mm) permitted in drinking water by the World Health Organization (WHO).[17] Thus, probe 1 a has the ability to detect CN in natural water samples. Similar behavior was observed for probe 1 b (Figure S2 in the Supporting Information). On addition of CN, the fluorescence emission at l = 607 nm was enhanced gradually. However, due to incomplete fluorescence quenching, probe 1 b in the absence of CN still has some residual fluorescence at l = 607 nm, which is unfavorable for the fluorescence detection of CN. Accordingly, we decided to focus on probe 1 a for further optical sensing studies. The UV/Vis absorption response of probe 1 a to CN is displayed in Figure 4. Addition of CN resulted in a gradual
Figure 2. a) The fluorescence emission spectra of 1 a (&), 1 b ( ! ), and 2 (*); the excitation wavelengths were l = 540, 534, and 380 nm, respectively. b) The absorption spectra of 1 a (&), 1 b ( ! ), and 2 (*).
Optical Responses to CN The sensing response of probes 1 a and 1 b to CN was investigated in 20 mm potassium phosphate buffer/CH3CN (1:3 v/v, pH 7.4). Upon addition of an increasing amount of CN to a solution of 1 a, a new fluorescence peak at around l = 615 nm emerged and drastically enhanced (Figure 3). Correspondingly, the visual fluorescence color of 1 a imme-
Figure 4. Changes in the absorption spectra of probe 1 a (10 mm) with various concentrations of CN (0–140 mm) in 20 mm potassium phosphate buffer/CH3CN (1:3 v/v, pH 7.4) at room temperature. Inset: Visible color changes of probe 1 a (10 mm) before and after addition of CN (140 mm).
decrease in the absorption at l = 471 nm. Concomitantly, a new redshifted absorption at l = 530 nm increased. Additionally, the visible color of the solution changed from orange to red (Figure 4, inset). The presence of a well-defined isosbestic point at l = 507 nm signified that only one species was produced upon the reaction of 1 a with CN. The variation in absorption spectra implied that the original ICT process from coumarin dye to the 3-position pyridyl vinyl ketone substituent has been weakened and a new ICT process from the coumarin dye to the formed cyano group occurred. Selectivity Studies
Figure 3. a) Changes in the fluorescence emission spectra (lex = 540 nm) of probe 1 a (10 mm) in 20 mm potassium phosphate buffer/CH3CN (1:3 v/ v, pH 7.4) with various concentrations of CN (0–140 mm). Inset: Visual fluorescence color change of probe 1 a (10 mm) before and after addition of CN (140 mm). b) Changes in fluorescence intensity of probe 1 a (10 mm) at l = 615 nm with various concentrations of CN (0–140 mm).
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To inspect whether probe 1 a could specifically detect CN, we tested its ability to discriminate between CN and various relevant species. As illustrated in Figure 5a, the probe did not show evident fluorescence enhancement in the pres-
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to 10.5. However, in the presence of CN, probe 1 a exhibited a pronounced fluorescence response when the pH value was higher than 5.2. Thus, probe 1 a can be used to detect CN at physiological pH values. Reaction Products of Probe 1 a with CN To investigate the possible sensing reaction mechanism, the reaction product of probe 1 a with CN was isolated and used in a 1H NMR spectroscopy study. As shown in Figure 6, the resonance signal of proton He in 1 a disap-
Figure 5. a) Fluorescence intensity (l = 615 nm) response of probe 1 a (10 mm) to 140 mm of various species in 20 mm potassium phosphate buffer/CH3CN (1:3 v/v, pH 7.4) with excitation at l = 540 nm. b) Visual fluorescence color changes of probe 1 a (10 mm) in the presence of various species (140 mm), the photo was taken under illumination with a handheld UV lamp.
ence of other anions, including F, Cl, Br, I, HSO3, CH3COO, ClO4, H2PO4, HCO3, NO3, SCN, and N3. Moreover, the addition of biological nucleophiles, such as biological thiols (Cys, Hcy) and NaHS (a commonly used H2S donor),[13d] also gave no visible variation in the fluorescence. Obviously, only CN induced a large fluorescence enhancement. This fluorescence enhancement can also be distinguished with the naked eye, as shown in Figure 5b, which shows photos of solutions of 1 a in the absence and presence of CN under UV light (l = 365 nm) illumination. We further examined the response of probe 1 a to CN in the presence of other relevant species, and most of these species only displayed minimum interference (Figure S3 in the Supporting Information). The absorption spectra and visible color of probe 1 a also exhibited selective response to CN over other species (Figures S4 and S5 in the Supporting Information). All these results strongly suggested that probe 1 a is highly selective for CN, even in the presence of biological nucleophiles.
Figure 6. Partial 1H NMR (400 MHz) spectra of a) probe 1 a and b) the isolated product of probe 1 a + CN in CDCl3.
peared after reaction with CN. This suggested that He in 1 a was substituted by CN to produce 1 a-CN. In addition, upon reaction with CN, the resonances that corresponded to protons Hc, Hd, and Hf showed a notable upfield shift. The upfield shift is undoubtedly due to the strong electronwithdrawing ability of the formed cyano group, consistent with the aforementioned design. The formation of 1 a-CN was further confirmed by the ESI-MS spectrum, in which a peak at m/z 374.3 was assigned to [1 aCN + H] + (Figure S11 in the Supporting Information). The structure of 1 aCN was also characterized by 13C NMR spectroscopy (Figure S12 in the Supporting Information) and IR spectroscopy (Figure S13 in the Supporting Information). Moreover, the Job plot of probe 1 a with CN also supported a 1:1 adduct between probe 1 a and CN (Figure S14 in the Supporting Information). According to the reaction product, a sensing reaction mechanism of probe 1 a with CN is proposed in Figure 7.
Response Time and Effect of pH Probe 1 a remains stable in the experimental solution (Figure S6 in the Supporting Information). The time course of probe 1 a in the absence or presence of CN is displayed in Figure S7 in the Supporting Information. Upon addition of CN (140 mm) at room temperature, a dramatic enhancement in the fluorescence intensity at l = 615 nm was observed within 20 min. The responses of 1 a toward CN at different pH values were investigated (Figure S8 in the Supporting Information). In the absence of CN, there were negligible fluorescence responses within the pH range of 2
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Theoretical Calculations To gain further insight into the CN sensing mechanism of probe 1 a, density functional theory (DFT) calculations at the B3LYP/6-31 + G** level by using the Gaussian 09[18] program were carried out. The optimized geometries are shown in Figure S15 in the Supporting Information, and the optimized structure of probe 1 a is identical to the experimental crystal structures as depicted in Figure 1.
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coumarin to the pyridyl vinyl ketone substituent in 1 a-CN was significantly weakened, which in turn led to the recovery of coumarin fluorescence. The simulated absorption spectra of 1 a and 1 a-CN are also shown in Figure S16 in the Supporting Information, which is in good agreement with the experimental absorption. Thus, based on the TD-DFT calculations, the optical properties of probe 1 a and 1 a-CN have been theoretically revealed. Practical Applications Figure 7. The proposed sensing reaction mechanism of probe 1 a with CN. EWG = electron withdrawing group.
Prompted by the favorable features of probe 1 a in solution, we fabricated a simple paper strip that could be conveniently used for detection of CN. The paper strips were prepared by immersing filter papers in a solution of 1 a (1 103 m) in CH3CN and then drying them in air. Next, these paper strips were immersed for 10 s in solutions with different CN concentrations (0, 40, 80, 150 mm), then taken out of the solution. After 30 min, photographs were taken under a handheld l = 365 nm UV lamp. As expected, the red fluorescence of the paper strips gradually increased with an increasing concentration of CN in the solution (Figure 9c–e),
To study the ICT processes in 1 a and 1 a-CN, time-dependent DFT (TD-DFT) calculations were conducted. The results indicated that the electron-density distributions of the HOMO!LUMO transition (S0 !S1) for 1 a and 1 a-CN (oscillator strength f = 1.1527 and 1.1125, respectively) are allowable (Table S1 in the Supporting Information). The electron distributions in HOMO and LUMO of 1 a and 1 a-CN are displayed in Figure 8. For probe 1 a, the HOMO is dis-
Figure 9. Photographs of paper strips with probe 1 a used for detecting CN in aqueous solution at concentrations of b) 0, c) 40, d) 80, and e) 150 mm. a) A paper strip that was not immersed in the CN solution is shown for comparison.
whereas paper strips immersed into a solution with no CN exhibited no fluorescence (Figure 9b), which is similar to the strip that was not immersed into a solution (Figure 9a). Thus, these paper strips may be used as a simple tool for detecting CN in field measurements with no requirement of sophisticated equipment. Cassava root is an important source of calories for many people living in the tropics. However, the cassava is also a typical cyanogenic crop and the endogenous CN poses a potential threat to the consumer.[19] Thus, probe 1 a was applied to sense the endogenous CN level in cassava root. An extract of bitter cassava root was prepared according to a reported procedure.[20] As shown in Figure 10, addition of an increasing volume of cassava extract gave a corresponding enhancement in the fluorescent intensity, which indicated the response of probe 1 a to endogenous CN. For comparison, an extract of commercially available potato was also made. However, addition of the potato extract to a solution of 1 a exerted no visible fluorescence enhancement (Figure S17 in the Supporting Information). Thus, no endoge-
Figure 8. The HOMO and LUMO energy levels and the orbitals of a) probe 1 a and b) compound 1 a-CN.
tributed on the 7-diethylamino-coumarin moiety, whereas the LUMO is primarily delocalized over the pyridyl vinyl ketone moiety. Thus, it is clear that, upon excitation, a strong ICT process will take place from 7-diethylaminocoumarin to the pyridyl vinyl ketone substituent. It is known that strong ICT from the 7-diethylamino-coumarin to the 3postion substituent frequently serves to quench the fluorescence of coumarin.[15] Apparently, the ICT from the 7-diethylamino-coumarin to the pyridyl vinyl ketone substituent in probe 1 a causes the fluorescence quenching. However, for compound 1 a-CN, owing to the strong electron-withdrawing ability of the formed cyano group, the electrons in the coumarin ring were transferred to the cyano group in the HOMO!LUMO transition. Consequently, the ICT from
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by using a Beijing Taike XT-4 microscopy melting point apparatus, and all melting points are uncorrected. Mass spectra were recorded by using a LXQ Spectrometer (Thermo Scientific) operating in ESI mode. 1H and 13 C NMR spectra were recorded by using a Bruker Avance 400 spectrometer operating at 400 and 100 MHz respectively. Elemental (C, H, N) analysis were carried out by using Flash EA 1112 analyzer. Infrared (IR) spectra were recorded by using a Bruker VERTEX80 FT-IR spectrometer. Electronic absorption spectra were obtained by using a SHIMADZU UV-2450 spectrometer. Fluorescence spectra were measured by using a Cary Eclipse spectrometer with 5 nm excitation and emission slit widths. The pH measurements were performed by using a pH-3c digital pH meter (Shanghai ShengCi Device Works, Shanghai, China) with a combined glass-calomel electrode.
Figure 10. Changes in the fluorescence emission spectra of probe 1 a (10 mm) in 20 mm potassium phosphate buffer/CH3CN (1:3 v/v, pH 7.4) in the presence of different volumes of cassava extract (0, 10, 20, 30, and 40 mL). Inset: The linear relationship between the fluorescence intensity at l = 615 nm and the added volume of cassava extract. The excitation wavelength was l = 540 nm.
Synthesis of 7-Diethylaminocoumarin-3-aldehyde (3) 7-Diethylaminocoumarin-3-aldehyde 3 was synthesized according to a reported procedure.[16] M.p. 152–154 8C; 1H NMR (400 MHz, CDCl3): d = 10.13 (s, 1 H), 8.26 (s, 1 H), 7.41 (d, J = 8.8 Hz, 1 H), 6.64 (dd, J = 2.4, 8.8 Hz, 1 H), 6.49 (d, J = 2.4 Hz, 1 H), 3.48 (q, J = 7.2 Hz, 4 H), 1.26 ppm (t, J = 7.2 Hz, 6 H); MS (ESI): m/z: 246.1 [M+H] + .
nous CN was found in the potato extract. We further quantitatively sensed the amount of CN in cassava root by using a standard addition method (see the Supporting Information). The CN content was estimated to be 140.9 mg kg1. Thus, probe 1 a should be very useful for the quantitative detection of endogenous CN in cassava root.
Synthesis of 1 a Under a N2 atmosphere, one drop of pyrrolidine was added to a solution of 7-diethylaminocoumarin-3-aldehyde (653 mg, 2.66 mmol) and 4-acetylpyridine (645 mg, 5.33 mmol) in CH2Cl2/MeOH (1:1 v/v; 10 mL). The mixture was stirred at RT for 24 h. After the solvent was removed under reduced vacuum, the resulting residue was further purified by column chromatography on silica gel (dichloromethane/petroleum ether 1:1, v/v) to afford compound 1 a as a red solid (yield: 552.3 mg, 59.6 %). M.p. 207– 209 8C; 1H NMR (CDCl3, 400 MHz): d = 8.81 (dd, J = 2.0, 4.4 Hz, 2 H), 8.15 (d, J = 15.2 Hz, 1 H), 7.84 (dd, J = 2.0, 4.4 Hz, 2 H), 7.81 (s, 1 H), 7.67 (d, J = 15.2 Hz, 1 H), 7.34 (d, J = 8.8 Hz, 1 H), 6.62 (dd, J = 2.4, 8.8 Hz, 1 H), 6.51 (s, 1 H), 3.46 (q, J = 7.2 Hz, 4 H), 1.25 ppm (t, J = 7.2 Hz, 6 H); 13 C NMR (CDCl3, 100 MHz): d = 189.9, 160.1, 156.8, 152.3, 150.8, 147.1, 144.6, 141.8, 130.3, 121.7, 121.6, 114.4, 109.7, 108.9, 96.9, 45.1, 12.5 ppm; MS (ESI): m/z: 349.25 [M+H] + ; elemental analysis calcd (%) for C21H20N2O3 : C 72.40, H 5.79, N 8.04; found: C 72.23, H 5.83, N 7.99.
Conclusion We reported a novel strategy to develop reaction-based fluorescent probes for CN by taking advantage of the strong electron-withdrawing ability of the cyano group that was formed from the CN sensing reaction. As a proof of concept, compounds 1 a and 1 b were rationally designed and synthesized. In the absence of CN, probe 1 a and 1 b displayed fluorescence quenching, but specific reaction of the probes with CN in aqueous solutions gave a fluorescence turn-on response. TD-DFT calculations revealed that the sensing reaction formed cyano group that weakened the ICT process from coumarin dye to the pyridyl vinyl ketone substituent, which in turn led to a fluorescence turn-on response. Further optical studies established that probe 1 a could detect CN with high selectivity and sensitivity. Moreover, a paper strip based on probe 1 a was fabricated and could serve as a simple tool to detect CN in field measurements. Then, probe 1 a was successfully applied to quantitatively detect endogenous CN in cassava root.
Synthesis of 1 b The synthetic procedure for 1 b was similar to 1 a. Under a N2 atmosphere, one drop of pyrrolidine was added to a solution of 7-diethylaminocoumarin-3-aldehyde (445 mg, 1.81 mmol) and 2-acetylpyridine (439.5 mg, 3.63 mmol) in CH2Cl2/MeOH (1:1 v/v; 8 mL). The mixture was stirred at RT for 24 h. Then the solvent was removed under reduced vacuum. The resulting residue was further purified by column chromatography on silica gel (dichloromethane/petroleum ether 1:1, v/v) to afford compound 1 b as a red solid (yield: 335.5 mg, 53.2 %). M.p. 163–165 8C; 1 H NMR (CDCl3, 400 MHz): d = 8.76 (d, J = 4.0 Hz, 1 H), 8.59 (d, J = 16.0 Hz, 1 H), 8.16 (d, J = 7.6 Hz, 1 H), 7.87 (m, 3 H), 7.47 (t, J = 4.8 Hz, 1 H), 7.33 (d, J = 8.8 Hz, 1 H), 6.61 (dd, J = 2.0, 8.8 Hz, 1 H), 6.51 (s, 1 H), 3.45 (q, J = 6.8 Hz, 4 H), 1.24 ppm (t, J = 6.8 Hz, 6 H); MS (ESI): m/z: 349.16 [M+H] + ; elemental analysis calcd (%) for C21H20N2O3 : C 72.40, H 5.79, N 8.04; found: C 72.28, H 5.82, N 8.01. Synthesis of 1 a-CN Tetrabutylammonium cyanide (385 mg, 1.435 mm) was added to a solution of compound 1 a (100 mg, 0.287 mm) in CH3CN (10 mL), and the solution was stirred at RT for 1 h. Then the solvent was removed under reduced vacuum. The resulting residue was further purified by column chromatography on silica gel (dichloromethane/petroleum ether 2:1, v/v) to afford compound 1 a-CN (yield: 51.6 mg, 48.2 %). M.p. 217–219 8C; 1H NMR (CDCl3, 400 MHz): d = 8.84 (dd, J = 1.6, 4.4 Hz, 2 H), 8.44 (d, J = 15.2 Hz, 1 H), 8.02 (d, J = 15.2 Hz, 1 H), 7.86 (dd, J = 1.6, 4.4 Hz, 2 H), 7.65 (d, J = 9.2 Hz, 1 H), 6.74 (dd, J = 2.8, 9.2 Hz, 1 H), 6.51 (d, J = 2.8 Hz, 1 H), 3.50 (q, J = 7.2 Hz, 4 H), 1.28 ppm (t, J = 7.2 Hz, 6 H); 13C NMR (CDCl3, 100 MHz): d = 189.3, 158.2, 155.8, 152.9, 150.9, 143.9, 136.7, 128.5, 126.6, 126.1, 121.6, 117.6, 112.8, 110.9, 107.2, 97.1, 45.4, 12.5 ppm; MS (ESI): m/ z: 374.3 [M+H] + .
Experimental Section Materials and Instruments Unless otherwise stated, all reagents were purchased from commercial suppliers and used without further purification. Solvents were purified and dried by standard methods prior to use. Twice-distilled water was used for all experiments. The solutions of various anions were prepared from their tetrabutylammonium salt or potassium salt. TLC analyses were performed on silica gel plates and column chromatography was conducted over silica gel (mesh 200–300), both of which were obtained from Qingdao Ocean Chemicals. Melting points of compounds were measured
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Acknowledgements This research was supported by the National Natural Science Foundation of China (21202063, 21105038), the Ministry of Science and Technology of China for the International Science Linkages Program (2011DFG52970), the Ministry of Education of China for Changjiang Innovation Research Team (IRT1064), Jiangsu Innovation Research Team, the Natural Science Foundation of Jiangsu Province (BK2012281), and the Research Foundation of Jiangsu University (11JDG078).
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E. Mirione, M. Ernesto, F. Massaza, J. Cliff, M. R. Haque, J. H. Bradbury, J. Food Compos. Anal. 2005, 18, 451 – 460; d) W. L. B. White, D. I. Arias-Garzon, J. M. McMahon, R. T. Sayre, Plant Physiol. 1998, 116, 1219 – 1225. [20] C. Mnnel-Crois , B. Probst, F. Zelder, Anal. Chem. 2009, 81, 9493 – 9498. Received: July 1, 2014 Published online: && &&, 0000
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2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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FULL PAPER Fluorescent Probes Lingliang Long,* Lin Wang, Yanjun Wu, Aihua Gong, Zulin Da, Chi Zhang,* Zhixiang Han &&&&—&&&&
Spotting cyanide: A novel strategy for reaction-based fluorescent probes for CN that take advantage of the strong electron-withdrawing ability of the cyano group formed from the CN sensing reaction is reported (see figure). Based on this strategy, two
probes were rationally designed and synthesized. One probe showed high sensitivity and selectivity for CN over other anions and biological nucleophiles and has been successfully applied for detection of endogenous CN in cassava root.
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These are not the final page numbers! ÞÞ
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Reaction-Based Fluorescent Probe for Detection of Endogenous Cyanide in Real Biological Samples
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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