Volume 50 Number 53 7 July 2014 Pages 6937–7076
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COMMUNICATION Jin Yong Lee, Injae Shin, Juyoung Yoon et al. Selective homocysteine turn-on fluorescent probes and their bioimaging applications
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Cite this: Chem. Commun., 2014, 50, 6967 Received 11th January 2014, Accepted 10th March 2014
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Selective homocysteine turn-on fluorescent probes and their bioimaging applications† Hye Yeon Lee,a Yoon Pyo Choi,b Sunkyung Kim,c Taejin Yoon,b Zhiqian Guo,ad Songyi Lee,a K. M. K. Swamy,ae Gyoungmi Kim,a Jin Yong Lee,*c Injae Shin*b and Juyoung Yoon*a
DOI: 10.1039/c4cc00243a www.rsc.org/chemcomm
The design and development of new pyrene-based fluorescent probes, P-Hcy-1 and P-Hcy-2, which display selective fluorescence enhancements in response to homocysteine (Hcy), are described. The distinctly different fluorescence responses of P-Hcy-1 and P-Hcy-2 to Hcy vs. Cys are explained by theoretical calculations. Finally, the results of cell experiments show that these probes can be used to selectively detect Hcy in mammalian cells.
Biological thiols (biothiols), such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), play key roles in physiological and pathological processes.1 Hcy is a risk factor for disorders including cardiovascular and Alzheimer’s diseases, pregnancy complications, neural tube defects and mental disorders.2 The normal level of homocysteine in healthy adults is in the range of 9–13 mM in the serum,3 and abnormally high levels (more than 15 mM) of homocysteine in the serum result in hyperhomocysteinemia.4 So far, methods developed for detecting Hcy mainly rely on chromatographic separation and immunoassay.5 However, because of their simplicity and high sensitivity, fluorescence-based probes serve as the most powerful tools to monitor biologically relevant species in vitro and/or in vivo. The most important advantage of the probes of this type is that they can be applied to bioimaging of biologically important species. Owing to these features, fluorescent/luminescent probes for Cys, Hcy and GSH have been devised using various molecular recognition or thiol specific reaction strategies.6
a
Department of Chemistry and Nano Science, Ewha Womans University, Seoul 120-750, Korea. E-mail: [email protected] b Department of Chemistry, Yonsei University, Seoul 120-749, Korea. E-mail: [email protected] c Department of Chemistry, Sungkyunkwan University, Suwon 440-746, Korea. E-mail: [email protected] d Key Laboratory for Advanced Materials and Institute of Fine Chemicals, East China University of Science & Technology, Shanghai 200237, P. R. China e Department of Pharmaceutical Chemistry, V. L. College of Pharmacy, Raichur-584 103, India † Electronic supplementary information (ESI) available: Experimental details and supplementary figures and characterization of compounds. See DOI: 10.1039/ c4cc00243a
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More recently, attempts to develop probes that discriminate between biothiols have encountered some success. However, as a result of the structural similarity of Cys and Hcy, i.e., they differ by only a single side chain methylene unit, discrimination between these two species using a single probe is a challenging task. Compared to those designed to detect Cys selectively,7 only a few probes selective for Hcy have been described thus far (Table S1, ESI†).8 In spite of the advances made, most biothiol probes developed to date have low selectivity toward Hcy. Therefore, it is still challenging to devise Hcy selective probes, in particular, which can be used to selectively image Hcy in cells. Therefore, the design of Hcy selective probes, in particular, those that can be used to image Hcy in cells, remains a challenge. In the current study, we developed two new turn-on fluorescent probes for Hcy and applied them in cell imaging studies. In addition, information about the source of the selective fluorescence enhancement of the probes brought about by Hcy and not Cys has been obtained from the results of theoretical calculations. The synthesis of the new Hcy selective probe P-Hcy-1 (Fig. 1) involves reaction of 1-hydroxypyrene-2-carbaldehyde (1)9 with propionyl chloride. In a similar manner, reaction of 1 with acryloyl chloride afforded the related probe, P-Hcy-2. The detailed synthetic procedures and characterizations of these probes are explained in the ESI.† As seen by inspection of the emission spectra displayed in Fig. 2a, addition of 10 equiv. of Hcy to P-Hcy-1 (10 mM) gave rise to a large increase in the fluorescence intensity at 450 nm (excitation at 350 nm). In contrast, no significant changes in emission intensity were caused by addition of Cys, GSH or other amino acids to the solution of P-Hcy-1 (Fig. 2a and Fig. S7a, ESI†). Strong blue fluorescence was observed only from the solution containing Hcy
Fig. 1
Structures of P-Hcy-1 and P-Hcy-2.
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Fig. 2 Selective response of P-Hcy-1 to Hcy. (a) Fluorescence spectra of P-Hcy-1 (10 mM) in HEPES (0.01 M, pH 7.4) containing 10% DMSO upon addition of 10 equiv. of Hcy, Cys and GSH (excitation wavelength: 350 nm). Inset: fluorescence images of P-Hcy-1 in the presence of Hcy, Cys and GSH. (b) Fluorescence changes of P-Hcy-1 (10 mM) with Hcy in HEPES (0.01 M and pH 7.4) containing 10% DMSO.
and P-Hcy-1 (Fig. 2a). In addition, over 50-fold fluorescence enhancement occurred when 70 equiv. of Hcy were added to the solution containing P-Hcy-1 (Fig. 2b). The reaction between P-Hcy-1 and Hcy that led to the fluorescence enhancement (see below) was completed after 10 min. In contrast, Cys and GSH did not elicit a significant fluorescence response even over long time periods (Fig. S7b, ESI†). Finally, Hcy detection using P-Hcy-1 was found to have a limit of 1.94 10 6 M and a linear range between 3 and 11 mM (Fig. S8, ESI†). We anticipated that the fluorescence response of P-Hcy-1 toward Hcy could be a consequence of a reaction in which the aldehyde group in the probe reacts to generate a six-membered thiazinane.8 In order to examine the source of the selectivity of this probe, reactions of P-Hcy-1 with Hcy and Cys were explored. 1H NMR monitoring of these processes clearly shows that the aldehyde proton resonance in the spectrum of P-Hcy-1 at d 10.47 ppm disappears concomitantly with generation of the respective thiazinane and thiazolidine methine protons at ca. 5.80 ppm (DMSO-d6 : D2O = 9 : 1, v/v, Fig. S9, ESI†). Thus, although they elicit different fluorescence responses, both Hcy (Fig. S10, ESI†) and Cys react with P-Hcy-1 to form the corresponding heterocyclic adducts. To shed light on the different fluorescence responses of P-Hcy-1 toward Cys and Hcy, density functional theory (DFT) and time dependent DFT (TD-DFT) calculations were conducted at the M06/ 6-31g* level using Gaussian 09 programs.10 The optimized structures of P-Hcy-1 and the respective six-membered thiazinane and fivemembered thiazolidine adducts (Fig. S11, ESI†), P-Hcy-1 + Hcy and P-Hcy-1 + Cys, arising from ring forming reactions of Hcy and Cys, are shown in Fig. 3a. In order to analyse the emission properties of the adducts, attention was focused on the singlet excited state of the pyrene fluorophore and the nitrogen containing thiazinane and thiazolidine rings, which could serve as potential electron donors. Inspection of the frontier molecular orbitals (Fig. 3b; details in Fig. S12, ESI†) of P-Hcy-1 + Hcy shows that part (ca. 4.0%) of the HOMO 1 - LUMO + 1 transition involves intramolecular charge transfer (ICT) from the donor to the fluorophore. A similar situation exists for HOMO 1 - LUMO and HOMO 1 - LUMO + 1 excitation in P-Hcy-1 + Cys where ca. 6.8% corresponds to ICT. Additionally, ca. 10.1% of the HOMO - LUMO + 1 transition in P-Hcy-1 + Cys involves oxidative photo-induced electron transfer (PET), a kind of single electron transfer (SET), from the ring nitrogen to the pyrene fluorophore.11 This phenomenon is not seen in the
6968 | Chem. Commun., 2014, 50, 6967--6969
Fig. 3 (a) Schematic and calculated structures of P-Hcy-1 + Hcy and P-Hcy-1 + Cys. (b) Calculated frontier orbitals of an adduct generated by reaction of P-Hcy-1 + Cys relevant to the fluorescence quenching.
transition of P-Hcy-1 + Hcy. Thus, the ICT and PET contributions to the singlet excited states of the probes, which could be responsible for fluorescence quenching, differ. Specifically, the fluorescence quenching related contribution to the excited state of P-Hcy-1 + Hcy is ca. 4.0%, which contrasts with the 16.9% contribution found in the excited state of P-Hcy-1 + Cys. Thus, this factor might be responsible for the much higher fluorescence efficiency of P-Hcy-1 + Hcy over that of P-Hcy-1 + Cys. It should be noted that a similar observation was made in a previous investigation.12 The heterocyclic ring forming reaction of the aldehyde group in probes is the basis of their Hcy selective fluorescence response.8 We envisaged that an a,b-unsaturated carbonyl group in a probe of this type would contribute to recognition of biothiols and perhaps the discrimination between Cys and Hcy.7a–c To test this proposal, the properties of P-Hcy-2, containing both an aldehyde and an acryloyl group, were investigated. The results show that the fluorescence responses of P-Hcy-2 to Cys, Hcy and GSH are the same as those of P-Hcy-1. The change in fluorescence efficiency caused by Hcy is not matched by Cys, GSH (Fig. S13a, ESI†) and other amino acids (Fig. S13b, ESI†). The fluorescence emission changes according to the different amounts of Hcy are shown in Fig. S14 (ESI†). The reaction between P-Hcy-2 and Hcy completed after 5 min. On the other hand, Cys and GSH did not induce significant responses over time (Fig. S15, ESI†). The detection limit of Hcy was calculated to be 1.44 10 7 M with a linear range of 600–1000 nM in HEPES containing 10% DMSO (0.01 M and pH 7.4) (Fig. S16, ESI†). To gain insight into the reaction taking place between P-Hcy-2 and Hcy, the process was monitored by using 1H NMR spectroscopy (DMSOd6 : D2O = 9 : 1, v/v). As the reaction proceeded, resonances for the aldehyde proton at 10.4 ppm and double bond protons at 6–7 ppm disappeared, being replaced by the thiazinane methine proton resonance at 5.80 ppm (Fig. S17, ESI†). These results suggest that
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Fig. 4 Fluorescence detection of Hcy in HeLa cells using P-Hcy-1. The fluorescence image of (a) cells incubated with 60 mM Hcy probes for 20 min, (b) cells treated with 20 mM Hcy for 30 min, followed by treatment with 60 mM Hcy probes for 20 min, and (c) cells treated with 20 mM Hcy for 30 min, incubated with 500 mM NEM for 20 min to remove intracellular biothiols and then stained with 60 mM Hcy probes for 20 min (scale bar = 50 mm).
Michael addition of the SH-group in Hcy to the acryloyl moiety and six-membered ring formation at the aldehyde center take place independently and with near equal rates. We next explored the ability of the Hcy probes (P-Hcy-1 and P-Hcy-2) to monitor Hcy in living cells. For this purpose, confocal microscopy analysis was carried out following incubation of HeLa cells with 60 mM of both Hcy probes for 20 min. The results show that the treated HeLa cells exhibit only weak fluorescence, presumably because they contain only ca. 1.2 nmol of Hcy per mg of cell proteins (Fig. 4 and Fig. S18, ESI†).13 Because Hcy is known to be taken up by HeLa cells,14 the cells were first incubated with 20 mM Hcy for 30 min and subsequently treated with 60 mM of the Hcy probes for 20 min. Confocal microscopy analysis of the cells revealed that the cells displayed intense fluorescence in comparison with that arising from Hcy untreated cells (Fig. 4 and Fig. S18, ESI†). In contrast, when N-ethylmaleimide (NEM, 500 mM), a thiol reactive reagent, is added to Hcy-treated cells prior to incubation with 60 mM of the Hcy probes, the fluorescence intensity of the cells dramatically decreased (Fig. 4 and Fig. S18, ESI†). These results indicate that both Hcy probes can be employed to sense Hcy in living cells. Next, the possibility that Hcy probes (P-Hcy-1 and P-Hcy-2) could sense other biothiols, such as Cys and GSH, in cells was assessed. In this study, HeLa cells were pre-incubated with 500 mM NEM to remove the endogenous thiol-containing molecules, treated with 20 mM Cys, and then stained with either 60 mM Hcy probes or a 20 mM Cys probe which had been developed previously.7b Intracellular Cys was strongly detected by the Cys probe but not by Hcy probes (Fig. S18, ESI†), indicating that Hcy probes do not sense Cys in cells, a phenomena which are consistent with the observed in vitro results. It has been known that a-lipoic acid increases the level of GSH in cells.15 Thus, to test the possibility of Hcy probes to detect GSH in cells, HeLa cells were initially incubated with 250 mM a-lipoic acid for 48 h to enhance intracellular GSH levels. The incubated cells were stained with either 60 mM Hcy probes or the 20 mM thiol probe which had been exploited previously.16 The results showed that whereas strong fluorescence was observed in cells stained with the thiol probe, fluorescence intensity of cells stained with Hcy probes did not increase (Fig. S18, ESI†), indicating that Hcy probes do not respond to GSH in cells. Taken together, the Hcy sensor selectively detects Hcy over other biologically relevant thiol-containing molecules in cells. In the investigation described above, we designed the new pyrene-based fluorescent probes, P-Hcy-1 and P-Hcy-2, and
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showed that they displayed fluorescence enhancements at 450 nm in the presence of only Hcy. The selective fluorescence changes are likely a consequence of the more highly efficient ICT and PET based, pyrene singlet excited quenching by the formed thiazinane heterocyclic ring generated by the reaction of Hcy with the aldehyde moieties in the probes. Finally, the results show that the Hcy probes can be employed to selectively detect Hcy in mammalian cells. We believe that this effort has shown the utility of a new strategy to design Hcy selective probes. This study was supported by the National Research Foundation of Korea (NRF) grant (CRI No. 2012R1A3A2048814 for J.Y. and CRI No. 2010-0018272 for I.S.). The work at the Sungkyunkwan University was supported by the NRF grant (2007-0056343) funded by MEST.
Notes and references 1 S. Y. Zhang, C.-N. Ong and H.-M. Shen, Cancer Lett., 2004, 208, 143. ¨der, J. R. Harris and L. B. Poole, Trends Biochem. 2 Z. A. Wood, E. Schro Sci., 2003, 28, 32. 3 O. Nygård, S. E. Vollset, H. Refsum, I. Stensvold, A. Tverdal, J. E. Nordrehaug, P. M. Ueland and G. Kvåle, JAMA, J. Am. Med. Assoc., 1995, 274, 1526. 4 O. Nygård, J. E. Nordrehaug, H. Refsum, P. M. Ueland, M. Farstad and S. E. Vollset, N. Engl. J. Med., 1997, 337, 230. 5 O. Nekrassova, N. S. Lawrence and R. G. Compton, Talanta, 2003, 60, 1085. 6 (a) H. S. Jung, X. Chen, J. S. Kim and J. Yoon, Chem. Soc. Rev., 2013, ´n ˜ez, Y. Yang, 42, 6019; (b) C. Yin, F. Huo, J. Zhang, R. Martı´nez-Ma H. Lv and S. Li, Chem. Soc. Rev., 2013, 42, 6032. 7 (a) X. Yang, Y. Guo and R. Strongin, Angew. Chem., Int. Ed., 2011, 50, 10690; (b) Z. Guo, S. Nam, S. Park and J. Yoon, Chem. Sci., 2012, 3, 2760; (c) H. Wang, G. Zhou, H. Gai and X. Chen, Chem. Commun., 2012, 48, 8341; (d) J. H. Lee, C. S. Lim, Y. S. Tian, J. H. Han and B. R. Cho, J. Am. Chem. Soc., 2010, 132, 1216; (e) H. S. Jung, J. H. Han, T. Pradhan, S. Kim, S. W. Lee, J. L. Sessler, T. W. Kim, C. Kang and J. S. Kim, Biomaterials, 2012, 33, 945; ( f ) H. Li, J. Fan, J. Wang, M. Tian, J. Du, S. Sun, P. Sun and X. Peng, Chem. Commun., 2009, 5904; (g) L. Yuan, W. Lin and Y. Yang, Chem. Commun., 2011, 47, 6275; (h) J. Shao, H. Sun, H. Guo, S. Ji, J. Zhao, W. Wu, X. Yuan, C. Zhang and T. D. James, Chem. Sci., 2012, 3, 1049; (i) L. Long, W. Lin, B. Chen, W. Gao and L. Yuan, Chem. Commun., 2011, 47, 893; ( j) H. Kwon, K. Lee and H.-J. Kim, Chem. Commun., 2011, 47, 1773; (k) Y.-K. Yang, S. Shim and J. Tae, Chem. Commun., 2010, 46, 7766. 8 (a) J. O. Escobedo, W. Wang and R. M. Strongin, Nat. Protocols, 2006, 1, 2759; (b) T.-K. Kim, D.-N. Lee and H.-J. Kim, Tetrahedron Lett., 2008, 49, 4879; (c) W. Wang, J. O. Escobedo, C. M. Lawrence and R. M. Strongin, J. Am. Chem. Soc., 2004, 126, 3400; (d) H. Chen, Q. Zhao, Y. Wu, F. Li, H. Yang, T. Yi and C. Huang, Inorg. Chem., 2007, 46, 11075; (e) K.-S. Lee, T.-K. Kim, J. H. Lee, H.-J. Kim and J.-I. Hong, Chem. Commun., 2008, 6173; ( f ) S.-K. Sun, H.-F. Wang and X.-P. Yan, Chem. Commun., 2011, 47, 3817. 9 (a) P. Demerseman, J. Einhorn, J. F. Gourvest and R. J. Royer, Heterocycl. Chem., 1985, 22, 39; (b) Y. Zhou, F. Wang, Y. Kim, S.-J. Kim and J. Yoon, Org. Lett., 2009, 11, 4442. 10 M. J. Frisch, et al., GAUSSIAN 09 (Revision B.01), Gaussian, Inc., Wallingford CT, 2009. 11 F. Wang, J. H. Moon, R. Nandhakumar, B. Kang, D. Kim, K. M. Kim, J. Y. Lee and J. Yoon, Chem. Commun., 2013, 49, 7228. 12 H. S. Jung, P.-S. Kwon, J. W. Lee, J. I. Kim, C. S. Hong, J. Kim, S. Yan, J. Y. Lee, J. H. Lee, T. Joo and J. S. Kim, J. Am. Chem. Soc., 2009, 131, 2008. 13 B. Hultberg, A. Andersson and A. Isaksson, Clin. Chem. Lab. Med., 2000, 38, 1243. 14 B. Hultberg, Clin. Chim. Acta, 2003, 330, 151. 15 L. Packer, Drug Metab. Rev., 1998, 30, 245. 16 X. Chen, S.-K. Ko, M. J. Kim, I. Shin and J. Yoon, Chem. Commun., 2010, 46, 2751.
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ISSN 1359-7345
COMMUNICATION Jin Yong Lee, Injae Shin, Juyoung Yoon et al. Selective homocysteine turn-on fluorescent probes and their bioimaging applications
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Selective homocysteine turn-on fluorescent probes and their bioimaging applications† Hye Yeon Lee,a Yoon Pyo Choi,b Sunkyung Kim,c Taejin Yoon,b Zhiqian Guo,ad Songyi Lee,a K. M. K. Swamy,ae Gyoungmi Kim,a Jin Yong Lee,*c Injae Shin*b and Juyoung Yoon*a
DOI: 10.1039/c4cc00243a www.rsc.org/chemcomm
The design and development of new pyrene-based fluorescent probes, P-Hcy-1 and P-Hcy-2, which display selective fluorescence enhancements in response to homocysteine (Hcy), are described. The distinctly different fluorescence responses of P-Hcy-1 and P-Hcy-2 to Hcy vs. Cys are explained by theoretical calculations. Finally, the results of cell experiments show that these probes can be used to selectively detect Hcy in mammalian cells.
Biological thiols (biothiols), such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), play key roles in physiological and pathological processes.1 Hcy is a risk factor for disorders including cardiovascular and Alzheimer’s diseases, pregnancy complications, neural tube defects and mental disorders.2 The normal level of homocysteine in healthy adults is in the range of 9–13 mM in the serum,3 and abnormally high levels (more than 15 mM) of homocysteine in the serum result in hyperhomocysteinemia.4 So far, methods developed for detecting Hcy mainly rely on chromatographic separation and immunoassay.5 However, because of their simplicity and high sensitivity, fluorescence-based probes serve as the most powerful tools to monitor biologically relevant species in vitro and/or in vivo. The most important advantage of the probes of this type is that they can be applied to bioimaging of biologically important species. Owing to these features, fluorescent/luminescent probes for Cys, Hcy and GSH have been devised using various molecular recognition or thiol specific reaction strategies.6
a
Department of Chemistry and Nano Science, Ewha Womans University, Seoul 120-750, Korea. E-mail: [email protected] b Department of Chemistry, Yonsei University, Seoul 120-749, Korea. E-mail: [email protected] c Department of Chemistry, Sungkyunkwan University, Suwon 440-746, Korea. E-mail: [email protected] d Key Laboratory for Advanced Materials and Institute of Fine Chemicals, East China University of Science & Technology, Shanghai 200237, P. R. China e Department of Pharmaceutical Chemistry, V. L. College of Pharmacy, Raichur-584 103, India † Electronic supplementary information (ESI) available: Experimental details and supplementary figures and characterization of compounds. See DOI: 10.1039/ c4cc00243a
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More recently, attempts to develop probes that discriminate between biothiols have encountered some success. However, as a result of the structural similarity of Cys and Hcy, i.e., they differ by only a single side chain methylene unit, discrimination between these two species using a single probe is a challenging task. Compared to those designed to detect Cys selectively,7 only a few probes selective for Hcy have been described thus far (Table S1, ESI†).8 In spite of the advances made, most biothiol probes developed to date have low selectivity toward Hcy. Therefore, it is still challenging to devise Hcy selective probes, in particular, which can be used to selectively image Hcy in cells. Therefore, the design of Hcy selective probes, in particular, those that can be used to image Hcy in cells, remains a challenge. In the current study, we developed two new turn-on fluorescent probes for Hcy and applied them in cell imaging studies. In addition, information about the source of the selective fluorescence enhancement of the probes brought about by Hcy and not Cys has been obtained from the results of theoretical calculations. The synthesis of the new Hcy selective probe P-Hcy-1 (Fig. 1) involves reaction of 1-hydroxypyrene-2-carbaldehyde (1)9 with propionyl chloride. In a similar manner, reaction of 1 with acryloyl chloride afforded the related probe, P-Hcy-2. The detailed synthetic procedures and characterizations of these probes are explained in the ESI.† As seen by inspection of the emission spectra displayed in Fig. 2a, addition of 10 equiv. of Hcy to P-Hcy-1 (10 mM) gave rise to a large increase in the fluorescence intensity at 450 nm (excitation at 350 nm). In contrast, no significant changes in emission intensity were caused by addition of Cys, GSH or other amino acids to the solution of P-Hcy-1 (Fig. 2a and Fig. S7a, ESI†). Strong blue fluorescence was observed only from the solution containing Hcy
Fig. 1
Structures of P-Hcy-1 and P-Hcy-2.
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Fig. 2 Selective response of P-Hcy-1 to Hcy. (a) Fluorescence spectra of P-Hcy-1 (10 mM) in HEPES (0.01 M, pH 7.4) containing 10% DMSO upon addition of 10 equiv. of Hcy, Cys and GSH (excitation wavelength: 350 nm). Inset: fluorescence images of P-Hcy-1 in the presence of Hcy, Cys and GSH. (b) Fluorescence changes of P-Hcy-1 (10 mM) with Hcy in HEPES (0.01 M and pH 7.4) containing 10% DMSO.
and P-Hcy-1 (Fig. 2a). In addition, over 50-fold fluorescence enhancement occurred when 70 equiv. of Hcy were added to the solution containing P-Hcy-1 (Fig. 2b). The reaction between P-Hcy-1 and Hcy that led to the fluorescence enhancement (see below) was completed after 10 min. In contrast, Cys and GSH did not elicit a significant fluorescence response even over long time periods (Fig. S7b, ESI†). Finally, Hcy detection using P-Hcy-1 was found to have a limit of 1.94 10 6 M and a linear range between 3 and 11 mM (Fig. S8, ESI†). We anticipated that the fluorescence response of P-Hcy-1 toward Hcy could be a consequence of a reaction in which the aldehyde group in the probe reacts to generate a six-membered thiazinane.8 In order to examine the source of the selectivity of this probe, reactions of P-Hcy-1 with Hcy and Cys were explored. 1H NMR monitoring of these processes clearly shows that the aldehyde proton resonance in the spectrum of P-Hcy-1 at d 10.47 ppm disappears concomitantly with generation of the respective thiazinane and thiazolidine methine protons at ca. 5.80 ppm (DMSO-d6 : D2O = 9 : 1, v/v, Fig. S9, ESI†). Thus, although they elicit different fluorescence responses, both Hcy (Fig. S10, ESI†) and Cys react with P-Hcy-1 to form the corresponding heterocyclic adducts. To shed light on the different fluorescence responses of P-Hcy-1 toward Cys and Hcy, density functional theory (DFT) and time dependent DFT (TD-DFT) calculations were conducted at the M06/ 6-31g* level using Gaussian 09 programs.10 The optimized structures of P-Hcy-1 and the respective six-membered thiazinane and fivemembered thiazolidine adducts (Fig. S11, ESI†), P-Hcy-1 + Hcy and P-Hcy-1 + Cys, arising from ring forming reactions of Hcy and Cys, are shown in Fig. 3a. In order to analyse the emission properties of the adducts, attention was focused on the singlet excited state of the pyrene fluorophore and the nitrogen containing thiazinane and thiazolidine rings, which could serve as potential electron donors. Inspection of the frontier molecular orbitals (Fig. 3b; details in Fig. S12, ESI†) of P-Hcy-1 + Hcy shows that part (ca. 4.0%) of the HOMO 1 - LUMO + 1 transition involves intramolecular charge transfer (ICT) from the donor to the fluorophore. A similar situation exists for HOMO 1 - LUMO and HOMO 1 - LUMO + 1 excitation in P-Hcy-1 + Cys where ca. 6.8% corresponds to ICT. Additionally, ca. 10.1% of the HOMO - LUMO + 1 transition in P-Hcy-1 + Cys involves oxidative photo-induced electron transfer (PET), a kind of single electron transfer (SET), from the ring nitrogen to the pyrene fluorophore.11 This phenomenon is not seen in the
6968 | Chem. Commun., 2014, 50, 6967--6969
Fig. 3 (a) Schematic and calculated structures of P-Hcy-1 + Hcy and P-Hcy-1 + Cys. (b) Calculated frontier orbitals of an adduct generated by reaction of P-Hcy-1 + Cys relevant to the fluorescence quenching.
transition of P-Hcy-1 + Hcy. Thus, the ICT and PET contributions to the singlet excited states of the probes, which could be responsible for fluorescence quenching, differ. Specifically, the fluorescence quenching related contribution to the excited state of P-Hcy-1 + Hcy is ca. 4.0%, which contrasts with the 16.9% contribution found in the excited state of P-Hcy-1 + Cys. Thus, this factor might be responsible for the much higher fluorescence efficiency of P-Hcy-1 + Hcy over that of P-Hcy-1 + Cys. It should be noted that a similar observation was made in a previous investigation.12 The heterocyclic ring forming reaction of the aldehyde group in probes is the basis of their Hcy selective fluorescence response.8 We envisaged that an a,b-unsaturated carbonyl group in a probe of this type would contribute to recognition of biothiols and perhaps the discrimination between Cys and Hcy.7a–c To test this proposal, the properties of P-Hcy-2, containing both an aldehyde and an acryloyl group, were investigated. The results show that the fluorescence responses of P-Hcy-2 to Cys, Hcy and GSH are the same as those of P-Hcy-1. The change in fluorescence efficiency caused by Hcy is not matched by Cys, GSH (Fig. S13a, ESI†) and other amino acids (Fig. S13b, ESI†). The fluorescence emission changes according to the different amounts of Hcy are shown in Fig. S14 (ESI†). The reaction between P-Hcy-2 and Hcy completed after 5 min. On the other hand, Cys and GSH did not induce significant responses over time (Fig. S15, ESI†). The detection limit of Hcy was calculated to be 1.44 10 7 M with a linear range of 600–1000 nM in HEPES containing 10% DMSO (0.01 M and pH 7.4) (Fig. S16, ESI†). To gain insight into the reaction taking place between P-Hcy-2 and Hcy, the process was monitored by using 1H NMR spectroscopy (DMSOd6 : D2O = 9 : 1, v/v). As the reaction proceeded, resonances for the aldehyde proton at 10.4 ppm and double bond protons at 6–7 ppm disappeared, being replaced by the thiazinane methine proton resonance at 5.80 ppm (Fig. S17, ESI†). These results suggest that
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Fig. 4 Fluorescence detection of Hcy in HeLa cells using P-Hcy-1. The fluorescence image of (a) cells incubated with 60 mM Hcy probes for 20 min, (b) cells treated with 20 mM Hcy for 30 min, followed by treatment with 60 mM Hcy probes for 20 min, and (c) cells treated with 20 mM Hcy for 30 min, incubated with 500 mM NEM for 20 min to remove intracellular biothiols and then stained with 60 mM Hcy probes for 20 min (scale bar = 50 mm).
Michael addition of the SH-group in Hcy to the acryloyl moiety and six-membered ring formation at the aldehyde center take place independently and with near equal rates. We next explored the ability of the Hcy probes (P-Hcy-1 and P-Hcy-2) to monitor Hcy in living cells. For this purpose, confocal microscopy analysis was carried out following incubation of HeLa cells with 60 mM of both Hcy probes for 20 min. The results show that the treated HeLa cells exhibit only weak fluorescence, presumably because they contain only ca. 1.2 nmol of Hcy per mg of cell proteins (Fig. 4 and Fig. S18, ESI†).13 Because Hcy is known to be taken up by HeLa cells,14 the cells were first incubated with 20 mM Hcy for 30 min and subsequently treated with 60 mM of the Hcy probes for 20 min. Confocal microscopy analysis of the cells revealed that the cells displayed intense fluorescence in comparison with that arising from Hcy untreated cells (Fig. 4 and Fig. S18, ESI†). In contrast, when N-ethylmaleimide (NEM, 500 mM), a thiol reactive reagent, is added to Hcy-treated cells prior to incubation with 60 mM of the Hcy probes, the fluorescence intensity of the cells dramatically decreased (Fig. 4 and Fig. S18, ESI†). These results indicate that both Hcy probes can be employed to sense Hcy in living cells. Next, the possibility that Hcy probes (P-Hcy-1 and P-Hcy-2) could sense other biothiols, such as Cys and GSH, in cells was assessed. In this study, HeLa cells were pre-incubated with 500 mM NEM to remove the endogenous thiol-containing molecules, treated with 20 mM Cys, and then stained with either 60 mM Hcy probes or a 20 mM Cys probe which had been developed previously.7b Intracellular Cys was strongly detected by the Cys probe but not by Hcy probes (Fig. S18, ESI†), indicating that Hcy probes do not sense Cys in cells, a phenomena which are consistent with the observed in vitro results. It has been known that a-lipoic acid increases the level of GSH in cells.15 Thus, to test the possibility of Hcy probes to detect GSH in cells, HeLa cells were initially incubated with 250 mM a-lipoic acid for 48 h to enhance intracellular GSH levels. The incubated cells were stained with either 60 mM Hcy probes or the 20 mM thiol probe which had been exploited previously.16 The results showed that whereas strong fluorescence was observed in cells stained with the thiol probe, fluorescence intensity of cells stained with Hcy probes did not increase (Fig. S18, ESI†), indicating that Hcy probes do not respond to GSH in cells. Taken together, the Hcy sensor selectively detects Hcy over other biologically relevant thiol-containing molecules in cells. In the investigation described above, we designed the new pyrene-based fluorescent probes, P-Hcy-1 and P-Hcy-2, and
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showed that they displayed fluorescence enhancements at 450 nm in the presence of only Hcy. The selective fluorescence changes are likely a consequence of the more highly efficient ICT and PET based, pyrene singlet excited quenching by the formed thiazinane heterocyclic ring generated by the reaction of Hcy with the aldehyde moieties in the probes. Finally, the results show that the Hcy probes can be employed to selectively detect Hcy in mammalian cells. We believe that this effort has shown the utility of a new strategy to design Hcy selective probes. This study was supported by the National Research Foundation of Korea (NRF) grant (CRI No. 2012R1A3A2048814 for J.Y. and CRI No. 2010-0018272 for I.S.). The work at the Sungkyunkwan University was supported by the NRF grant (2007-0056343) funded by MEST.
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