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Fluorescent sensors for selective detection of thiols: expanding the intramolecular displacement based mechanism to new chromophores† Li-Ya Niu,a Hai-Rong Zheng,ab Yu-Zhe Chen,a Li-Zhu Wu,a Chen-Ho Tunga and Qing-Zheng Yang*a Biological thiols, including cysteine (Cys), homocystein (Hcy) and glutathione (GSH), play crucial roles in maintaining the appropriate redox status of biological systems. An abnormal level of biothiols is associated with different diseases, therefore, the discrimination between them is of great importance. Herein, we present two fluorescent sensors for selective detection of biothiols based on our recently reported intramolecular displacement mechanism. We expanded this mechanism to commercially available chromophores, 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) and heptamethine cyanine dye IR-780. The sensors operate by undergoing displacement of chloride by thiolate. The amino groups of Cys/Hcy further replace the thiolate to form amino-substituted products, which exhibit dramatically different photophysical properties compared to sulfur-substituted products from the reaction with GSH.
Received 29th September 2013 Accepted 17th December 2013
NBD-Cl is highly selective towards Cys/Hcy and exhibits significant fluorescence enhancement. IR-780
DOI: 10.1039/c3an01849k
showed a variation in its fluorescence ratio towards Cys over other thiols. Both of the sensors can be used for live-cell imaging of Cys. The wide applicability of the mechanism may provide a powerful tool
www.rsc.org/analyst
for developing novel fluorescent sensors for selective detection of biothiols.
Introduction Low-molecular-weight biothiols, including cysteine (Cys), homocystein (Hcy) and glutathione (GSH), play crucial roles in redox-related biological processes. Despite their similar structures, alterations of the levels of the specic thiols are linked to different diseases. Cys deciency is involved in slow growth of children, liver damage, skin lesions, weakness, etc.1 Hcy is implicated in the health of the cardiovascular system. Elevated Hcy in plasma is a risk factor for cardiovascular and Alzheimer's diseases.2 GSH, as the most abundant intracellular thiols, maintains the redox activities, xenobiotic metabolism and gene regulation, and its abnormal level may lead to cancer, aging, heart problems, etc.3 Given their different roles in biological systems, the discrimination between the three thiols is of great importance. Among the various detection techniques, uorescent sensors are widely developed due to their operational simplicity, high a
Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing, 100190, P. R. China. E-mail: [email protected]; Fax: +86 10 6255 4670; Tel: +86 10 8254 3450
b
University of Chinese Academy of Sciences, Beijing, 100049, P. R. China
† Electronic supplementary information (ESI) available: Synthesis details and characterization of compounds NBD-MEA, NBD-N and NBD-S; absorption spectral changes of NBD-Cl in the presence of Cys; absorption and emission spectral changes of IR-780 in the presence of Cys, Hcy and GSH. See DOI: 10.1039/c3an01849k
This journal is © The Royal Society of Chemistry 2014
sensitivity and suitability for bioimaging in living systems.4 Most of the reported sensors for mercapto biomolecules utilized the strong nucleophilicity of thiols,5 which is involved in specic reactions, such as Michael addition,6 cleavage of sulfonamide and sulfonate ester,7 cleavage of disulphide,8 and Se–N bond,9 etc. However, most of those sensors cannot discriminate among SHcontaining molecules with their similar structures and reactivities. Strongin and co-workers reported the cyclization of Cys/Hcy with aldehydes for the selective detection of Cys/Hcy,10 which inspired the development of uorescent probes based on the selective reaction of aldehydes with Cys/Hcy over GSH.11 Later, they developed the simultaneous determination of Cys and Hcy based on different relative rates of intramolecular cyclization with acrylates.12 Based on this work, the selective detection of GSH in cetyltrimethylammonium bromide (CTAB) media was also achieved.13 Recently, we reported the ratiometric uorescent sensor for the discrimination of GSH over Cys and Hcy.14 The chlorine of the monochlorinated BODIPY can be rapidly replaced by thiolates of biothiols through thiol–halogen nucleophilic substitution. The amino groups of Cys/Hcy but not GSH further replace the thiolate via a ve- or six-membered cyclic transition state to form aminosubstituted BODIPY (Scheme 1a). Based on this work, we also successfully developed a turn-on uorescent sensor for selective detection of Cys.15 This unique and specic intramolecular displacement mechanism might be applicable to developing new chromophore based sensors for the selective detection of
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Fluorescence spectral changes of NBD-Cl upon addition of the increasing concentrations of Cys (0, 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 300, and 500 mM). Inset: the fluorescence intensity at 546 nm as a function of the concentrations of Cys. Each spectrum was recorded 2 min after Cys addition in acetonitrile–HEPES buffer (1 : 3, v/v, 20 mM, pH 7.4) at 37 C. lex ¼ 476 nm. Fig. 1
Scheme 1
Substitution reaction on different chromophores.
biothiols. Such a suitable chromophore should meet the following two basic criteria. First, the chromophore should have a reactive nucleophilic substitution site. The high nucleophilic substitution reactivity is desired to guarantee the fast response to biothiols. Second, the photophysical properties of the chromophore should be sensitive to the variation of the substituent. In other words, its uorescence spectra could change signicantly aer the thiolate or amino substitution of the chromophore. Most importantly, the sulfur or amino substituted chromophore should exhibit different emission properties to achieve high selectivity to biothiols. With this idea in mind, in this paper, we present two uorescent sensors for selective detection of aminothiols based on commercially available chromophores, 4-chloro7-nitro-2,1,3-benzoxadiazole (NBD-Cl) and heptamethine cyanine dye IR-780 (Scheme 1b and 1c), which well meet the above requirements, indicating that the intramolecular displacement based mechanism is widely applicable to explore new chromophores for biothiol selective detection.
Results and discussion NBD-Cl has been frequently used as amine labelling agents and affixed to different biomolecules for cell imaging with favorable biocompatibility.16 NBD-Cl is non-uorescent in acetonitrile– HEPES buffer (1 : 3, v/v, 20 mM, pH 7.4). The strong electronwithdrawing nitro-group makes the C–Cl bond at the para position highly reactive for nucleophilic substitution reaction. The substitution of chlorine by nucleophiles such as amines results in uorescence enhancement, owing to the ICT process from the conjugated electron-donating to withdrawing groups.17 As designed, the treatment of Cys induced a fast response and dramatic enhancement of the uorescence intensity at 546 nm, suggesting the formation of aminosubstituted NBD.16a,18 The emission intensities at 546 nm are linearly proportional to the concentrations of Cys (0–50 mM), suggesting the potential application for quantitative
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determination of Cys (Fig. 1). The detection limit was determined to be 5.52 108 M (S/N ¼ 3). The fast response and low detection limit indicate its high sensitivity. The addition of an increasing amount of Cys to NBD-Cl also triggered gradual increase in the absorption at 476 nm, accompanied by the decrease in the absorption at 341 nm (Fig. S1 in the ESI†). The colourless solution turned to light yellow simultaneously. The large Stokes shi (70 nm) was in accordance with the typical ICT uorophore with the conjugated electron donor and receptor.19 The time course of the emission intensity of NBD-Cl at 546 nm in the presence of Cys is shown in Fig. 2. It is noteworthy that the reaction was almost nished within 1 min due to its high nucleophilic substitution reactivity, which revealed the fast response of the sensor. Most of the reported reaction-based uorescent sensors for thiols suffered from the long response time. The fast sensing behavior of NBD-Cl may facilitate its application for the imaging of biothiols in living systems. Similar absorption and uorescence responses for Hcy were also observed, although an extended reaction time was required to reach the maximum intensity. Under pseudo-rst-order kinetic conditions, the observed rate constants with Cys and
Fig. 2 The time course of the emission intensity of NBD-Cl (10 mM) at 546 nm in the presence of 100 equiv. of Cys and Hcy in acetonitrile– HEPES buffer (1 : 3, v/v, 20 mM, pH 7.4) at 37 C. lex ¼ 476 nm.
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Hcy were determined to be 4.0 102 s1 (t1/2 ¼ 17.4 s) and 0.55 102 s1 (t1/2 ¼ 126 s), respectively. The results revealed that NBD-Cl could achieve the rapid detection of Cys. We also measured the absorption and uorescence changes of NBD-Cl in the presence of GSH. Upon addition of GSH, the absorption band of free NBD-Cl centered at 341 nm decreased, and a new band centered at 419 nm emerged and increased gradually. The emission at 546 nm displayed slight enhancement, much weaker and even negligible compared to Cys/Hcy (Fig. 3). The weak uorescence of the product is in accordance with the low uorescent intensity of the reported sulfursubstituted NBD.17 The different spectral changes indicate that NBD-Cl can selectively detect Cys/Hcy over GSH. In order to verify whether the sensing mechanism is in agreement with our previous report, we tried to characterize the nal product NBD-Cys obtained by reacting NBD-Cl with Cys. Unfortunately the NBD-Cys was difficult to isolate from the reaction mixture because of its large polarity and poor solubility in organic solvents. Therefore, we used 2-mercaptoethylamine (MEA) without carboxyl groups instead of Cys to study the reaction mechanism. The product of NBD-MEA was evidenced by 1H NMR (Fig. S2†). The broad signal at 6.57 ppm and a triplet at 1.59 ppm were assigned as the exchangeable protons of the aromatic amine and SH, respectively, suggesting that MEA is attached to NBD through amino groups. Model compounds NBD-N and NBD-S were also synthesized by reacting NBD-Cl with n-butylamine and methyl mercaptoacetate under basic conditions, respectively (see ESI†). NBD-Cys, NBD-MEA and NBD-N showed similar spectroscopic properties, which further
Fig. 3 (a) Absorption and (b) emission spectral changes of NBD-Cl (10 mM) in the presence of 100 equiv. of Cys, Hcy and GSH in acetonitrile–HEPES buffer (1 : 3, v/v, 20 mM, pH 7.4) at 37 C. Each spectrum was recorded 2 min after addition. lex ¼ 476 nm.
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identied the structures of the amino-substituted NBD-Cys. The absorption and emission spectra of NBD-GSH were in accordance with NBD-S, which suggested that the reaction of NBD-Cl with GSH resulted in the sulfur-substituted product (Fig. S3†). On the basis of the above observations and our previous work,14,15 we proposed the reaction mechanism of NBD-Cl with Cys, Hcy and GSH (Scheme 2). Under physiological conditions, deprotonation of the thiol yields an active nucleophile, thiolate. The chlorine of NBD-Cl is replaced by thiolate to generate the kinetic products 1 and 4. For Cys and Hcy, the primary amine allows further intramolecular displacement of sulfur by a veor six-membered cyclic transition state 2 to yield the thermodynamic product 3. As we know, a ve-membered ring is the preferred conformation, which can explain the faster reaction rate in the presence of Cys rather than Hcy. For GSH, no favored transition state could be formed due to the unavailable adjacent amino group. As a result, product 4 is stable and maintains its structure. For better demonstration of the two-step reaction between NBD-Cl and Cys/Hcy, control reactions of NBD-Cl with N-acetylcysteine and lysine (Lys) were performed under physiological conditions. The reaction of NBD-Cl with N-acetylcysteine which has sulydryl without amino groups resulted in the formation of thioether, while the reaction of NBD-Cl with Lys which has amino without sulydryl groups cannot occur because of the protonation of amino groups at pH 7.4 (Fig. S4†). The observations provide further evidence to support our proposed reaction mechanism. To examine the selectivity, NBD-Cl was treated with other physiologically relevant amino acids (e.g., Gly, Ala, Val, Leu, Ile, Phe, Trp, Tyr, Asp, His, and Asn) under identical conditions. As shown in Fig. 4, the thiol-containing GSH induced neglectable uorescence enhancement. Besides, nearly no absorption and uorescence changes were observed in the presence of other species. The results demonstrated that NBD-Cl showed high selectivity towards Cys/Hcy. To further demonstrate the general applicability of the mechanism, heptamethine cyanine dye IR-780 was employed as another chromophore. Near-infrared (NIR) uorescent sensors in vivo have attracted great interest because of minimum photodamage to biological samples, deep tissue penetration, and
Scheme 2 Proposed mechanism for the reaction of NBD-Cl with Cys, Hcy and GSH.
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Fig. 4 Emission intensity at 546 nm of NBD-Cl (10 mM) upon addition of various amino acids and GSH. Each set of data was acquired 10 min after addition of different species in acetonitrile–HEPES buffer (1 : 3, v/v, 20 mM, pH 7.4) at 37 C. lex ¼ 476 nm.
minimum interference from auto-uorescence of biomolecules in the living systems.20 We performed the spectral sensing behavior of IR-780 towards thiols in methanol–HEPES buffer (5 : 95, v/v, 20 mM, pH 7.4). Free IR-780 displayed the absorption maximum at 775 nm. With addition of Cys, the absorption of IR-780 decreased sharply. When excited at 650 nm, a large hypsochromic shi in the emission spectra of IR-780 was observed. As shown in Fig. 5, with introduction of Cys, the emission at 810 nm decreased, and a new emission band at 750 nm increased gradually, suggesting that the chlorine was replaced by the amino group of Cys.21 Heptamethine cyanines with amino substituted at the central position of the polymethine bridge possess a large Stokes shi, which is attributed to an excited-state intramolecular charge transfer (ICT) between the donor and the acceptor in the dyes.20,21 The large emission shi allows the ratiometric measurements, which involve the ratio of the two signals, are independent of the sensor concentration and environment. Thus, its ratiometric mode has potential for more accurate and quantitative measurements for Cys.14,22 IR-780 was treated with Hcy, GSH and other various biologically relevant amino acids. The spectral changes of IR-780 with addition of Hcy followed the same trend with Cys, however, at a slower reaction rate. The treatment of GSH led to 10 nm red
Fig. 5 Time-dependent fluorescence spectra of IR-780 (10 mM) in the presence of 100 equiv. of Cys in methanol–HEPES buffer (5 : 95, v/v, 20 mM, pH 7.4) at 37 C. lex ¼ 650 nm.
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shis in both absorption and emission spectra. Almost no variation was observed in the presence of other amino acids under the same conditions. As shown in Fig. 6, only Cys induced a large variation in its uorescence ratio (I810/I750). The results demonstrated that IR-780 exhibited high selectivity towards Cys. Based on the above results, the mechanism for the reaction of IR-780 with Cys, Hcy and GSH was proposed and is illustrated in Scheme 3, which was in good agreement with the mechanism for the BODIPY and NBD systems. The chlorine of IR-780 was rstly replaced by thiolate. The amino groups of Cys further replaced sulfur to form an amino-substituted product via a vemembered cyclic transition state. Hcy would undergo a similar but slower procedure by six-membered cyclic transition compared to Cys. Thus, the amino-substituted product for Hcy has not been generated yet in the experimental time scale of 1 h. For GSH, a similar reaction would be difficult to occur thermodynamically and it resulted in the sulfur-substituted product. To further demonstrate the practical application of both NBD-Cl and IR-780, we tested the capability of the two sensors
Fig. 6 Ratiometric response of IR-780 (10 mM) upon addition of 100 equiv. of different analytes. Bars represent the fluorescence intensity ratio of I750/I810. Each set of data was acquired 1 h after addition of different amino acids in methanol–HEPES buffer solution (5 : 95, v/v, 20 mM, pH ¼ 7.4) at 37 C. lex ¼ 650 nm.
Proposed mechanism for the reaction of IR-780 with Cys, Hcy and GSH.
Scheme 3
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NEM to the cell culture prior to the incubation of IR-780 only showed clear uorescence at emission collection windows of 750–800 nm. The ratio image resulted in the emission ratio of
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Cite this: DOI: 10.1039/c3an01849k
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Fluorescent sensors for selective detection of thiols: expanding the intramolecular displacement based mechanism to new chromophores† Li-Ya Niu,a Hai-Rong Zheng,ab Yu-Zhe Chen,a Li-Zhu Wu,a Chen-Ho Tunga and Qing-Zheng Yang*a Biological thiols, including cysteine (Cys), homocystein (Hcy) and glutathione (GSH), play crucial roles in maintaining the appropriate redox status of biological systems. An abnormal level of biothiols is associated with different diseases, therefore, the discrimination between them is of great importance. Herein, we present two fluorescent sensors for selective detection of biothiols based on our recently reported intramolecular displacement mechanism. We expanded this mechanism to commercially available chromophores, 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) and heptamethine cyanine dye IR-780. The sensors operate by undergoing displacement of chloride by thiolate. The amino groups of Cys/Hcy further replace the thiolate to form amino-substituted products, which exhibit dramatically different photophysical properties compared to sulfur-substituted products from the reaction with GSH.
Received 29th September 2013 Accepted 17th December 2013
NBD-Cl is highly selective towards Cys/Hcy and exhibits significant fluorescence enhancement. IR-780
DOI: 10.1039/c3an01849k
showed a variation in its fluorescence ratio towards Cys over other thiols. Both of the sensors can be used for live-cell imaging of Cys. The wide applicability of the mechanism may provide a powerful tool
www.rsc.org/analyst
for developing novel fluorescent sensors for selective detection of biothiols.
Introduction Low-molecular-weight biothiols, including cysteine (Cys), homocystein (Hcy) and glutathione (GSH), play crucial roles in redox-related biological processes. Despite their similar structures, alterations of the levels of the specic thiols are linked to different diseases. Cys deciency is involved in slow growth of children, liver damage, skin lesions, weakness, etc.1 Hcy is implicated in the health of the cardiovascular system. Elevated Hcy in plasma is a risk factor for cardiovascular and Alzheimer's diseases.2 GSH, as the most abundant intracellular thiols, maintains the redox activities, xenobiotic metabolism and gene regulation, and its abnormal level may lead to cancer, aging, heart problems, etc.3 Given their different roles in biological systems, the discrimination between the three thiols is of great importance. Among the various detection techniques, uorescent sensors are widely developed due to their operational simplicity, high a
Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing, 100190, P. R. China. E-mail: [email protected]; Fax: +86 10 6255 4670; Tel: +86 10 8254 3450
b
University of Chinese Academy of Sciences, Beijing, 100049, P. R. China
† Electronic supplementary information (ESI) available: Synthesis details and characterization of compounds NBD-MEA, NBD-N and NBD-S; absorption spectral changes of NBD-Cl in the presence of Cys; absorption and emission spectral changes of IR-780 in the presence of Cys, Hcy and GSH. See DOI: 10.1039/c3an01849k
This journal is © The Royal Society of Chemistry 2014
sensitivity and suitability for bioimaging in living systems.4 Most of the reported sensors for mercapto biomolecules utilized the strong nucleophilicity of thiols,5 which is involved in specic reactions, such as Michael addition,6 cleavage of sulfonamide and sulfonate ester,7 cleavage of disulphide,8 and Se–N bond,9 etc. However, most of those sensors cannot discriminate among SHcontaining molecules with their similar structures and reactivities. Strongin and co-workers reported the cyclization of Cys/Hcy with aldehydes for the selective detection of Cys/Hcy,10 which inspired the development of uorescent probes based on the selective reaction of aldehydes with Cys/Hcy over GSH.11 Later, they developed the simultaneous determination of Cys and Hcy based on different relative rates of intramolecular cyclization with acrylates.12 Based on this work, the selective detection of GSH in cetyltrimethylammonium bromide (CTAB) media was also achieved.13 Recently, we reported the ratiometric uorescent sensor for the discrimination of GSH over Cys and Hcy.14 The chlorine of the monochlorinated BODIPY can be rapidly replaced by thiolates of biothiols through thiol–halogen nucleophilic substitution. The amino groups of Cys/Hcy but not GSH further replace the thiolate via a ve- or six-membered cyclic transition state to form aminosubstituted BODIPY (Scheme 1a). Based on this work, we also successfully developed a turn-on uorescent sensor for selective detection of Cys.15 This unique and specic intramolecular displacement mechanism might be applicable to developing new chromophore based sensors for the selective detection of
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Fluorescence spectral changes of NBD-Cl upon addition of the increasing concentrations of Cys (0, 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 300, and 500 mM). Inset: the fluorescence intensity at 546 nm as a function of the concentrations of Cys. Each spectrum was recorded 2 min after Cys addition in acetonitrile–HEPES buffer (1 : 3, v/v, 20 mM, pH 7.4) at 37 C. lex ¼ 476 nm. Fig. 1
Scheme 1
Substitution reaction on different chromophores.
biothiols. Such a suitable chromophore should meet the following two basic criteria. First, the chromophore should have a reactive nucleophilic substitution site. The high nucleophilic substitution reactivity is desired to guarantee the fast response to biothiols. Second, the photophysical properties of the chromophore should be sensitive to the variation of the substituent. In other words, its uorescence spectra could change signicantly aer the thiolate or amino substitution of the chromophore. Most importantly, the sulfur or amino substituted chromophore should exhibit different emission properties to achieve high selectivity to biothiols. With this idea in mind, in this paper, we present two uorescent sensors for selective detection of aminothiols based on commercially available chromophores, 4-chloro7-nitro-2,1,3-benzoxadiazole (NBD-Cl) and heptamethine cyanine dye IR-780 (Scheme 1b and 1c), which well meet the above requirements, indicating that the intramolecular displacement based mechanism is widely applicable to explore new chromophores for biothiol selective detection.
Results and discussion NBD-Cl has been frequently used as amine labelling agents and affixed to different biomolecules for cell imaging with favorable biocompatibility.16 NBD-Cl is non-uorescent in acetonitrile– HEPES buffer (1 : 3, v/v, 20 mM, pH 7.4). The strong electronwithdrawing nitro-group makes the C–Cl bond at the para position highly reactive for nucleophilic substitution reaction. The substitution of chlorine by nucleophiles such as amines results in uorescence enhancement, owing to the ICT process from the conjugated electron-donating to withdrawing groups.17 As designed, the treatment of Cys induced a fast response and dramatic enhancement of the uorescence intensity at 546 nm, suggesting the formation of aminosubstituted NBD.16a,18 The emission intensities at 546 nm are linearly proportional to the concentrations of Cys (0–50 mM), suggesting the potential application for quantitative
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determination of Cys (Fig. 1). The detection limit was determined to be 5.52 108 M (S/N ¼ 3). The fast response and low detection limit indicate its high sensitivity. The addition of an increasing amount of Cys to NBD-Cl also triggered gradual increase in the absorption at 476 nm, accompanied by the decrease in the absorption at 341 nm (Fig. S1 in the ESI†). The colourless solution turned to light yellow simultaneously. The large Stokes shi (70 nm) was in accordance with the typical ICT uorophore with the conjugated electron donor and receptor.19 The time course of the emission intensity of NBD-Cl at 546 nm in the presence of Cys is shown in Fig. 2. It is noteworthy that the reaction was almost nished within 1 min due to its high nucleophilic substitution reactivity, which revealed the fast response of the sensor. Most of the reported reaction-based uorescent sensors for thiols suffered from the long response time. The fast sensing behavior of NBD-Cl may facilitate its application for the imaging of biothiols in living systems. Similar absorption and uorescence responses for Hcy were also observed, although an extended reaction time was required to reach the maximum intensity. Under pseudo-rst-order kinetic conditions, the observed rate constants with Cys and
Fig. 2 The time course of the emission intensity of NBD-Cl (10 mM) at 546 nm in the presence of 100 equiv. of Cys and Hcy in acetonitrile– HEPES buffer (1 : 3, v/v, 20 mM, pH 7.4) at 37 C. lex ¼ 476 nm.
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Hcy were determined to be 4.0 102 s1 (t1/2 ¼ 17.4 s) and 0.55 102 s1 (t1/2 ¼ 126 s), respectively. The results revealed that NBD-Cl could achieve the rapid detection of Cys. We also measured the absorption and uorescence changes of NBD-Cl in the presence of GSH. Upon addition of GSH, the absorption band of free NBD-Cl centered at 341 nm decreased, and a new band centered at 419 nm emerged and increased gradually. The emission at 546 nm displayed slight enhancement, much weaker and even negligible compared to Cys/Hcy (Fig. 3). The weak uorescence of the product is in accordance with the low uorescent intensity of the reported sulfursubstituted NBD.17 The different spectral changes indicate that NBD-Cl can selectively detect Cys/Hcy over GSH. In order to verify whether the sensing mechanism is in agreement with our previous report, we tried to characterize the nal product NBD-Cys obtained by reacting NBD-Cl with Cys. Unfortunately the NBD-Cys was difficult to isolate from the reaction mixture because of its large polarity and poor solubility in organic solvents. Therefore, we used 2-mercaptoethylamine (MEA) without carboxyl groups instead of Cys to study the reaction mechanism. The product of NBD-MEA was evidenced by 1H NMR (Fig. S2†). The broad signal at 6.57 ppm and a triplet at 1.59 ppm were assigned as the exchangeable protons of the aromatic amine and SH, respectively, suggesting that MEA is attached to NBD through amino groups. Model compounds NBD-N and NBD-S were also synthesized by reacting NBD-Cl with n-butylamine and methyl mercaptoacetate under basic conditions, respectively (see ESI†). NBD-Cys, NBD-MEA and NBD-N showed similar spectroscopic properties, which further
Fig. 3 (a) Absorption and (b) emission spectral changes of NBD-Cl (10 mM) in the presence of 100 equiv. of Cys, Hcy and GSH in acetonitrile–HEPES buffer (1 : 3, v/v, 20 mM, pH 7.4) at 37 C. Each spectrum was recorded 2 min after addition. lex ¼ 476 nm.
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identied the structures of the amino-substituted NBD-Cys. The absorption and emission spectra of NBD-GSH were in accordance with NBD-S, which suggested that the reaction of NBD-Cl with GSH resulted in the sulfur-substituted product (Fig. S3†). On the basis of the above observations and our previous work,14,15 we proposed the reaction mechanism of NBD-Cl with Cys, Hcy and GSH (Scheme 2). Under physiological conditions, deprotonation of the thiol yields an active nucleophile, thiolate. The chlorine of NBD-Cl is replaced by thiolate to generate the kinetic products 1 and 4. For Cys and Hcy, the primary amine allows further intramolecular displacement of sulfur by a veor six-membered cyclic transition state 2 to yield the thermodynamic product 3. As we know, a ve-membered ring is the preferred conformation, which can explain the faster reaction rate in the presence of Cys rather than Hcy. For GSH, no favored transition state could be formed due to the unavailable adjacent amino group. As a result, product 4 is stable and maintains its structure. For better demonstration of the two-step reaction between NBD-Cl and Cys/Hcy, control reactions of NBD-Cl with N-acetylcysteine and lysine (Lys) were performed under physiological conditions. The reaction of NBD-Cl with N-acetylcysteine which has sulydryl without amino groups resulted in the formation of thioether, while the reaction of NBD-Cl with Lys which has amino without sulydryl groups cannot occur because of the protonation of amino groups at pH 7.4 (Fig. S4†). The observations provide further evidence to support our proposed reaction mechanism. To examine the selectivity, NBD-Cl was treated with other physiologically relevant amino acids (e.g., Gly, Ala, Val, Leu, Ile, Phe, Trp, Tyr, Asp, His, and Asn) under identical conditions. As shown in Fig. 4, the thiol-containing GSH induced neglectable uorescence enhancement. Besides, nearly no absorption and uorescence changes were observed in the presence of other species. The results demonstrated that NBD-Cl showed high selectivity towards Cys/Hcy. To further demonstrate the general applicability of the mechanism, heptamethine cyanine dye IR-780 was employed as another chromophore. Near-infrared (NIR) uorescent sensors in vivo have attracted great interest because of minimum photodamage to biological samples, deep tissue penetration, and
Scheme 2 Proposed mechanism for the reaction of NBD-Cl with Cys, Hcy and GSH.
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Fig. 4 Emission intensity at 546 nm of NBD-Cl (10 mM) upon addition of various amino acids and GSH. Each set of data was acquired 10 min after addition of different species in acetonitrile–HEPES buffer (1 : 3, v/v, 20 mM, pH 7.4) at 37 C. lex ¼ 476 nm.
minimum interference from auto-uorescence of biomolecules in the living systems.20 We performed the spectral sensing behavior of IR-780 towards thiols in methanol–HEPES buffer (5 : 95, v/v, 20 mM, pH 7.4). Free IR-780 displayed the absorption maximum at 775 nm. With addition of Cys, the absorption of IR-780 decreased sharply. When excited at 650 nm, a large hypsochromic shi in the emission spectra of IR-780 was observed. As shown in Fig. 5, with introduction of Cys, the emission at 810 nm decreased, and a new emission band at 750 nm increased gradually, suggesting that the chlorine was replaced by the amino group of Cys.21 Heptamethine cyanines with amino substituted at the central position of the polymethine bridge possess a large Stokes shi, which is attributed to an excited-state intramolecular charge transfer (ICT) between the donor and the acceptor in the dyes.20,21 The large emission shi allows the ratiometric measurements, which involve the ratio of the two signals, are independent of the sensor concentration and environment. Thus, its ratiometric mode has potential for more accurate and quantitative measurements for Cys.14,22 IR-780 was treated with Hcy, GSH and other various biologically relevant amino acids. The spectral changes of IR-780 with addition of Hcy followed the same trend with Cys, however, at a slower reaction rate. The treatment of GSH led to 10 nm red
Fig. 5 Time-dependent fluorescence spectra of IR-780 (10 mM) in the presence of 100 equiv. of Cys in methanol–HEPES buffer (5 : 95, v/v, 20 mM, pH 7.4) at 37 C. lex ¼ 650 nm.
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shis in both absorption and emission spectra. Almost no variation was observed in the presence of other amino acids under the same conditions. As shown in Fig. 6, only Cys induced a large variation in its uorescence ratio (I810/I750). The results demonstrated that IR-780 exhibited high selectivity towards Cys. Based on the above results, the mechanism for the reaction of IR-780 with Cys, Hcy and GSH was proposed and is illustrated in Scheme 3, which was in good agreement with the mechanism for the BODIPY and NBD systems. The chlorine of IR-780 was rstly replaced by thiolate. The amino groups of Cys further replaced sulfur to form an amino-substituted product via a vemembered cyclic transition state. Hcy would undergo a similar but slower procedure by six-membered cyclic transition compared to Cys. Thus, the amino-substituted product for Hcy has not been generated yet in the experimental time scale of 1 h. For GSH, a similar reaction would be difficult to occur thermodynamically and it resulted in the sulfur-substituted product. To further demonstrate the practical application of both NBD-Cl and IR-780, we tested the capability of the two sensors
Fig. 6 Ratiometric response of IR-780 (10 mM) upon addition of 100 equiv. of different analytes. Bars represent the fluorescence intensity ratio of I750/I810. Each set of data was acquired 1 h after addition of different amino acids in methanol–HEPES buffer solution (5 : 95, v/v, 20 mM, pH ¼ 7.4) at 37 C. lex ¼ 650 nm.
Proposed mechanism for the reaction of IR-780 with Cys, Hcy and GSH.
Scheme 3
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NEM to the cell culture prior to the incubation of IR-780 only showed clear uorescence at emission collection windows of 750–800 nm. The ratio image resulted in the emission ratio of
