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Facile preparation of naphthol AS-based fluorescent probe for highly selective detection of cysteine in aqueous solution and its imaging application in living cells
Accepted Manuscript Title: Facile preparation of naphthol AS-based fluorescent probe for highly selective detection of cysteine in aqueous solution and its imaging application in living cells Authors: Wei-Wei Ma, Meng-Yuan Wang, Di Yin, Xuan Zhang PII: DOI: Reference:
S0925-4005(17)30596-8 http://dx.doi.org/doi:10.1016/j.snb.2017.03.169 SNB 22081
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
29-11-2016 29-3-2017 31-3-2017
Please cite this article as: Wei-Wei Ma, Meng-Yuan Wang, Di Yin, Xuan Zhang, Facile preparation of naphthol AS-based fluorescent probe for highly selective detection of cysteine in aqueous solution and its imaging application in living cells, Sensors and Actuators B: Chemicalhttp://dx.doi.org/10.1016/j.snb.2017.03.169 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Facile preparation of naphthol AS-based fluorescent probe for highly selective detection of cysteine in aqueous solution and its imaging application in living cells Wei-Wei Ma, Meng-Yuan Wang, Di Yin, Xuan Zhang* College of Chemistry, Chemical Engineering & Biotechnology, Donghua University, Shanghai 201620, China. *Corresponding Author. E-mail: [email protected].
Graphical abstract
1
Highlights The fluorescent probe based on commercial naphthol AS was facilely prepared. Methacrylate group acts as an electron acceptor and a reactive site for Cys. High selectivity toward Cys over Hcy and GSH. Good water solubility and successful application in living cells imaging.
ABSTRACT A simple and new fluorescent probe (1) for selective detection of cysteine (Cys) in aqueous solution was facilely synthesized. It was developed based on the masking the phenolic OH group in naphthol AS by a methacrylate group, where the methacrylate group acts as an electron acceptor to quench fluorescence of naphthol AS as well as a reactive site for Cys. The conjugate addition/cyclization reaction of Cys toward methacrylate moiety in the probe 1 led to a cleavage of the methacrylate group and release of free naphthol AS, thereby induced a significant enhancement of fluorescence. The present fluorescent probe exhibited high selectivity toward Cys over homocysteine (Hcy) and glutathione (GSH) and worked well in a physiological aqueous medium, allowing a successful application in living cells imaging.
Keywords: Fluorescent probe; Naphthol AS; Cysteine; Living cells imaging
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1. Introduction As an essential amino acid, cysteine (Cys) is involved in many biological processes, such as protein synthesis and metabolism [1]. It has been known that the normal intracellular level of Cys is 30–200 µM, and its deficient or abundant could cause serious diseases, such as slow growth, edema, leucocyte loss, liver damage and neurotoxicity [2]. Therefore, many detection methods have been developed to detect Cys in biological systems, especially based on fluorescence technique due to its advantages of high sensitivity, rapidity, non-invasion, and capability of visual imaging in vivo [3]. These fluorescent probes were usually developed based on the intrinsic nucleophilicity of the free thiol group in Cys, even successful to differentiate Cys from other common amino acids, but quite difficult to discriminate Cys from others biothiols such as homocystein (Hcy) and glutathione (GSH) due to their similar reactivity [3, 4]. Recently, the conjugate addition/cyclization of Cys to acrylate moiety has been proven to be a promising strategy for design of Cys-selective fluorescent probe over Hcy and GSH [5]. Such strategy has been used for several fluorophores such as fluorescein [6], coumarin [7], cyanines [8], and semiheptamethine [9], to develop fluorescent probes for selective detection of Cys. However, most of these reported probes for Cys have to need sophisticated synthesis processes and involve large amounts of organic solvents as co-solvents due to poor water solubility. For example, 20–50% MeOH/EtOH [6, 7, 9], and 30–70% DMF/DMSO [4e, 10] have been usually employed as co-solvents. For practical applications in biological systems, fluorescent probes should own suitable water solubility and work in aqueous mediums containing organic solvent as less as possible. Therefore, the development of water soluble fluorescent probe that can be facilely synthesized and shows high selectivity for Cys is highly demanded. 3
In this work, naphthol AS, a commercial available dye, was used to construct a selective fluorescent probe (1, Scheme 1) for Cys by facilely masking its phenolic OH with methacrylate that acts as an efficient electron acceptor to quench the native fluorescence of naphthol AS, and it was found that Cys could selectively induce a deprotection and recovery of strong fluorescence. The present fluorescent probe worked well in a physiological aqueous medium (pH 7.4, 5% DMSO), showed a high selectivity for Cys over Hcy and GSH, and was successfully applied in living cells imaging. 2. Experimental section 2.1. Materials and Methods All the reagents and solvents are analytical grade and purchased from Sinopharm Chemical Reagents Corp. (Shanghai, China). Phosphate buffered saline (PBS, pH = 7.4) was prepared from Na2HPO4 (0.1 M) and KH2PO4 (0.1 M). The stock solution of the probe 1 was prepared in DMSO and amino acids solutions were prepared in deionized water. 1H NMR and 13C NMR spectra were recorded on a Bruker AVANCE III 400 MHz spectrometer. Mass spectra were obtained on AB Sciex MALDI-TOF/TOFTMMS and Varian 310 mass spectrometers, respectively. Fluorescence spectra were measured on Edinburgh FS5 spectrofluorometer with Ex/Em slit widths of 5 nm. Confocal fluorescence imaging experiments in living Hela cells were carried out with a Carl Zeiss LSM 700 microscope. Theoretical calculations were carried out using Gaussian 09 package [11]. The ground state (S0) geometries of the compounds were optimized in the gas phase using density functional theory (DFT) at the B3LYP/6-31+G(d) level. 2.2. Synthesis Naphthol AS (2.0 mmol) was dissolved in acetone (125 mL) and methacryloyl chloride (2.0 4
mmol) was slowly added dropwise under stirring at 0 °C in the presence of K2CO3 (2.0 mmol). After completion of the reaction within 4 h, the mixture was filtrated and the filtrate was evaporated under reduced pressure. The obtained solid was then dissolved in CH2Cl2, washed with water and the organic layer was dried over MgSO4. After removing of MgSO4 by filtration, the crude product was obtained by evaporation of the solvent under reduced pressure. The product was further purified by column chromatography (silica gel, hexane/CH2Cl2 = 1:4 v/v). Yield: 33%. 1H
NMR (400 MHz, CDCl3), δ (ppm): 8.45 (s, 1H), 8.18 (s, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.84 (d,
J = 8.0 Hz, 1H), 7.65-7.53 (m, 5H), 7.37 (t, J = 8.0 Hz, 2H), 7.15 (t, J = 8.0 Hz, 1H), 6.47 (s, 1H), 5.86 (s, 1H), 2.08 (s, 3H); 13C NMR (100 MHz, CDCl3), δ (ppm): 18.47, 99.97, 120.01, 120.76, 124.63, 126.73, 127.35, 127.82, 128.35, 128.73, 129.13, 131.00, 131.20, 134.69, 135.53, 137.84, 144.85, 163.59, 166.03. MALDI-TOF-MS: m/z calcd for (M + H)+ 332.36; found 332.19. 2.3. Confocal Microscope Imaging HeLa cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C in a 95% humidity atmosphere under 5% CO2 environment. Then the cells were seeded in 6-wells glass-bottomed dishes at a density of 2 ×105 cells per dish in RPMI 1640 medium for 24 h. The cells were then incubated with probe 1 (20 µM) for 90 min at 37 °C, washed with PBS buffer (10 mM) three times to remove free probe 1, and observed under a Carl Zeiss LSM 700 fluorescence microscope. For control experiments, the cells were pretreated with N-ethylmaleimide (NEM, 1 mM) for 30 min at 37 °C, followed by washing with PBS three times, and incubated with probe 1 (20 µM) for 90 min at 37 °C; and further incubated with Cys (150 μM) for 90 min at 37 °C.
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3. Results and discussion 3.1. Design and Synthesis Naphthol AS is a commercial available dye containing an active phenolic OH group that has been usually used to develop fluorescent sensor for anions under an excited state proton transfer mechanism [12]. We envisage that the native fluorescence of naphthol AS would be quenched by masking its phenolic OH with methacrylate group, an efficient electron acceptor, due to photoinduced electron transfer (PET) process, but Cys will selectively recover its fluorescence by removal of the masking group through conjugate addition/cyclization to acrylate moiety (Scheme 1). Therefore, the probe 1 was facilely synthesized by treating naphthol AS with methacryloyl chloride in acetone as shown in Scheme 1. The chemical structure of the probe 1 was confirmed by 1H NMR, 13C NMR, and MALDI-TOF-MS (Figs. S3‒S5). 3.2. Selective response of the probe 1 toward Cys As shown in Fig. 1, the probe 1 is weakly fluorescent in PBS buffer (10 mM, pH = 7.4, 5% DMSO) probably because the quench effect of methacrylate group through a PET process [5]. Upon addition of Cys, a strong green fluorescence appeared around 530 nm, but no significant fluorescence enhancement was observed in the presence of Hcy, GSH and other species (including common amino acids, cations and anions), suggesting that the probe 1 is highly selective for Cys detection (Figs. 1 and S1). Mass spectrometry (ESI-MS) analysis of the mixture solution of the probe 1 with 20 equiv Cys reveals a strong peak at m/z = 262 corresponding to the deprotonated naphthol AS (Fig. S2), an indication of methacrylate group in the probe 1 was really removed by Cys. To gain better insight into the fluorescence behavior, the structures of probe 1 and naphthol AS were optimized and their frontier molecular orbital were analysed by using DFT calculation at 6
the B3LYP/6-31+G(d) level, respectively. As shown in Fig. 2, the π electrons of naphthol AS were delocalized on the whole molecular skeleton on both the lowest unoccupied molecular orbital (LUMO) and the highest occupied molecular orbital (HOMO). However, the π electrons on the LUMO of 1 were mainly localized on the methacrylate group, while the π electrons on the HOMO were distributed in the naphthol AS skeleton (Fig. 2). These results support the possible PET process in 1, where the electron transfer from the naphthol AS (electron donor) to the methacrylate group (electron acceptor) weakened the native fluorescence of the donor, but Cys-induced removal of electron acceptor recovered the fluorescence (Scheme 1). To obtain the optimal pH condition, the pH effect on fluorescence of the probe 1 in the absence and presence of Cys were then investigated. Without Cys, the probe 1 is weakly fluorescent when pH < 9, but becomes highly fluorescent when pH > 9 (Fig. 3a). This reveals that the probe 1 has been already hydrolyzed into highly fluorescent deprotonated naphthol AS on the more basic condition. However, in the presence of Cys, the probe 1 exhibits a strong fluorescence at pH 4–9, revealing Cys could induce a removal of methacylate moiety and transform 1 into free naphthol AS in a broad pH region (Fig. 3a). In this work, pH 7.4 was selected for measurements to match with the requirement in physiological environment. Moreover, it was found that the fluorescence intensity at 530 nm is continually increased with time and reaches constant after 50 min, indicating hydrolysis reaction could be finished within 50 min for Cys (Fig. 3b). On the other hand, both Hcy and GSH presented very slow reaction kinetics (Fig. 3b). The slow reaction rate of Hcy and GSH could be rationalized by considering that the conjugate addition reaction is even feasible, but the further intramolecular cyclization reaction should be kinetically disfavored relative to the formation of seven-membered ring that would result from Cys (Scheme 1) [5]. Thus 7
the difference on kinetics of intramolecular cyclization reaction could also be used to rationalize the high selectivity of the probe 1 toward Cys. Fig.4 showed the fluorescence titration of the probe 1 with Cys in a phosphate buffer (10 mM PBS containing 5% DMSO, pH = 7.4). It can be clearly seen that the green fluorescence at 530 nm was continuously increased with increasing Cys amounts up to 350 μM, and a good linear relationship (R2 = 0.9988) was obtained over the range of 1‒200 μM (Fig. 4). The detection limit was estimated to be 0.5 μM according to S/N = 3. The normal intracellular level of Cys is known to be 30–200 µM, suggesting the probe 1 is sensitive enough to satisfy Cys detection in living cells. 3.3. Fluorescence imaging in living cells To verify the potential application of 1 for Cys imaging in living cells, the HeLa cells were incubated with probe 1 at 37 ºC for 90 min and observed by confocal fluorescence microscope. As shown in Fig. 5b, a bright green fluorescence was observed inside the cells, indicating that the intracellular Cys reacts with the probe 1 led to a removal of methacylate moiety and a recovery of fluorescence. In contrast, when the cells were pretreated with a thiol-blocking agent, N-ethylmaleimides (NEM, 1 mM) prior to addition of probe 1, there is almost no detectable fluorescence (Fig. 5d); when the NEM-pretreated cells were successively incubated with the probe 1 and Cys, a bright green fluorescence was again observed inside cells (Fig. 5f). These results indicate that 1 can serve as a promising fluorescent probe for Cys imaging in living cells. 4. Conclusions A simple and new fluorescent probe 1 for selective detection of Cys was facilely synthesized based on commercial dye naphthol AS, where the methacrylate group was introduced both as an 8
electron acceptor to quench fluorescence and a reactive site for Cys. It was found that the conjugate addition/cyclization reaction of Cys toward methacrylate moiety in the probe 1 results in cleavage of the methacrylate moiety and release of free naphthol AS, thereby inducing a significant enhancement of fluorescence. The probe exhibits high selectivity toward Cys over Hcy, GSH and others species in a physiological aqueous medium. The living cells imaging results suggest that the probe 1 could be successfully applied for intracellular Cys detection. Regarding to its facile preparation, good water solubility and high selectivity, the present new probe shows a promising potential for application involving detection of Cys in biological systems.
Acknowledgment This work was financially supported by Shanghai Municipal Natural Science Foundation (16ZR1401700). We would like to appreciate Dr. Xiao-Yue Zhu and Prof. Xiang-Yang Shi for their kind assistances on cell imaging.
Supplementary data Figs. S1‒S5 are available.
References [1] (a) K.G. Reddie, K.S. Carroll, Curr. Opin. Chem. Biol. 12 (2008) 746–754; (b) N.M. Giles, A.B. Watts, G.I. Giles, F.H. Fry, J.A. Littlechild, C. Jacob, Chem. Biol. 10 (2003) 677–693. [2] (a) S. Park, J.A. Imlay, J. Bacteriol. 185 (2003) 1942–1950; (b) S. Shahrokhian, Anal. Chem. 73 (2001) 5972–5978.
9
[3] (a) L.-Y Niu, Y.-Z. Chen, H.-R. Zheng, L.-Z. Wu, C.-H. Tung, Q.-Z. Yang, Chem. Soc. Rev. 44 (2015) 6143–6160; (b) H. Chen, Y. Tang, W. Lin, Trend. Anal. Chem. 76 (2016) 166–181; (c) X. Chen, Y. Zhou, X. Peng, J. Yoon, Chem. Soc. Rev. 39 (2010) 2120–2135; (d) J. Chan, S. C. Dodani, C. J. Chang, Nat. Chem. 4 (2012) 973–984; (e) Y. Yang, Q. Zhao, W. Feng, F. Li, Chem. Rev. 113 (2013) 192–270; (f) X. Li, X. Gao, W. Shi, H. Ma, Chem. Rev. 114 (2014) 590–659. [4] (a) D. Gong, Y. Tian, C. Yang, A. Iqbal, Z. Wang, W. Liu, W. Qin, X. Zhu, H. Guo, Biosens. Bioelectron. 85 (2016) 178–183; (b) C. Yang, X. Wang, L. Shen, W. Deng, H. Liu, S. Ge, M. Yan, X. Song, Biosens. Bioelectron. 80 (2016) 17–23; (c) Y. Liu, X. Lv, M. Hou, Y. Shi, W. Guo, Anal. Chem. 87 (2015) 11475–11483; (d) D. Lee, G. Kim, J. Yin, J. Yoon, Chem. Commun. 51 (2015) 6518–6520; (e) X.-F. Yang, Q. Huang, Y. Zhong, Z. Li, H. Li, M. Lowry, J. O. Escobedo, R. M. Strongin, Chem. Sci. 5 (2014) 2177–2183; (f) H. Lv, X.-F. Yang, Y. Zhong, Y. Guo, Z. Li, H. Li, Anal. Chem. 86 (2014) 1800–1807; (g) X. Gao, X. Li, L. Li, J. Zhou, H. Ma, Chem. Commun. 51 (2015) 9388–9390; (h) Y. Yang, F. Huo, C. Yin, J. Chao, Y. Zhang, Dyes Pigm. 114 (2015) 105–109; (i) J. Liu, Y.-Q. Sun, Y. Huo, H. Zhang, L. Wang, P. Zhang, D. Song, Y. Shi, W. Guo, J. Am. Chem. Soc. 136 (2014) 574−577; (j) T. Liu, F. Huo, J. Li, J. Chao, Y. Zhang, C. Yin, Sens. Actuators B 237 (2016) 127–132; (k) J. Xu, J. Pan, Y. Zhang, J. Liu, L. Zeng, X. Liu, Sens. Actuators B 238 (2017) 58–65; 10
(l) Z. Yao, H. Bai, C. Li, G. Shi, Chem. Commun. 47 (2011) 7431–7433; (m) X. Zhang, J.-Y. Liu, W.-W. Ma, M.-L. Yang, J. Mater. Chem. B 4 (2016) 6662–6669; (n) L. Bu, J. Chen, X. Wei, X. Li, H. Ågren, Y. Xie, Dyes Pigm. 136 (2017) 724–731. [5] X.-F. Yang, Y.-X. Guo, R.M. Strongin, Angew. Chem. Int. Ed. 50 (2011) 10690–0693. [6] H. Wang, G. Zhou, H. Gai, X. Chen, Chem. Commun. 48 (2012) 8341–8343. [7] (a) X. Liu, D. Yang, W. Chen, L. Yang, F. Qi, X. Song, Sens. Actuators B 234(2016) 27–33; (b) Y.-C. Liao, P. Venkatesan, L.-F. Wei, S.-P. Wu, Sens. Actuators B 232 (2016) 732–737. [8] (a) Z. Guo, S. Nam, S. Park, J. Yoon, Chem. Sci. 3 (2012) 2760–2765; (b) J. Zhang, J. Wang, J. Liu, L. Ning, X. Zhu, B. Yu, X. Liu, X. Yao, H. Zhang, Anal. Chem. 87 (2015) 4856–4863. [9] C. Han, H. Yang, M. Chen, Q. Su, W. Feng, F. Li, ACS Appl. Mater. Interfaces 7 (2015) 27968−27975. [10] (a) H.-M. Lv, D.-H. Yuan, W. Liu, Y. Chen, C.-T. Au, S.-F. Yin, Sens. Actuators B 233 (2016) 173–179; (b) X. Xiong, F. Song, G. Chen, W. Sun, J. Wang, P. Gao, Y. Zhang, B. Qiao, W. Li, S. Sun, J. Fan, X. Peng, Chem. Eur. J. 19 (2013) 6538–6545. [11] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G.A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H.P. Hratchian, A.F. Izmaylov, J. Bloino, G. Zheng, J.L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J.A. Montgomery, J.E. Peralta, F. Ogliaro, M. Bearpark, J.J. Heyd, E. Brothers, K.N. Kudin, V.N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J.C. 11
Burant, S.S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J.M. Millam, M. Klene, J.E. Knox, J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, R.L. Martin, K. Morokuma, V.G. Zakrzewski, G.A. Voth, P. Salvador, J.J. Dannenberg, S. Dapprich, A.D. Daniels, O. Farkas, J.B. Foresman, J.V. Ortiz, J. Cioslowski, D.J. Fox, Gaussian 09, Revision C.01, Gaussian Inc., Wallingford, CT, 2009. [12] (a) X. Zhang, L. Guo, F.-Y. Wu, Y.-B. Jiang, Org. Lett. 15 (2003) 2667−2670; (b) X. Zhang, Y. Shiraishi, T. Hirai, Tetrahedron Lett. 48 (2007) 8803–8806; (c) X. Zhang, J.-Y. Liu, Dyes Pigm. 125 (2016) 80–88; (d) X. Peng, Y. Wu, J. Fan, M. Tian, K. Han, J. Org. Chem. 70 (2005) 10524–10531; (e) K. Choi, A.D. Hamilton, Angew. Chem. Inter. Ed. 40 (2001) 3912–3915.
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Biographies
Wei-Wei Ma, Meng-Yuan Wang and Di Yin are master students of College of Chemistry, Chemical Engineering& Biotechnology, Donghua University. Their current research interests are focused on the fluorescent probes. Xuan Zhang received his Ph.D. degree in 2003 from Xiamen University, China. He worked as JSPS fellow and postdoc in Japan during 2004-2011. Then he got back to China and was appointed as an associate professor of Chemistry in College of Chemistry, Chemical Engineering& Biotechnology, Donghua University. His current research interests are mainly focused on the fluorescent and electrocatalytic materials.
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Figure Captions:
Fig. 1. Fluorescence spectra of 1 (10 μM, pH = 7.4 PBS containing 5% DMSO) in the presence of 20 equiv Cys, Hcy, GSH, Asp, Asn, Ser, Pro, Ala, Gly, Val, Leu, Ile, Thr, Arg, Glu, Gln, Tyr, His, Met, Phe, Trp, Lys, Tau, Na2S, HSCH2COOH, glucose, K+, Na+, Ca2+, Mg2+, Cl−, NO3−, SO42−, PO42−. Excitation wavelength is 400 nm. Inset is photograph of 1 before and after addition of Cys under a UV lamp (365 nm) irradiation.
Fig. 2. HOMO and LUMO orbitals of 1 and naphthol AS. Calculations were based on ground state geometry by DFT at the B3LYP/6-31+G(d) level.
Fig. 3. Time- and pH-dependent fluorescence intensity change (λem = 530 nm, λex = 400 nm,) of 1 (10 μM, pH = 7.4 PBS containing 5% DMSO) in the presence of 20 equiv Cys.
Fig. 4. Change of fluorescence spectra of 1 (10 μM, pH = 7.4 PBS containing 5% DMSO) upon addition of various amounts of Cys (0–350 μM) (a), and the corresponding linear relationship between the fluorescence intensity at 530 nm and concentration of Cys (1‒200 μM) (b). Fig. 5. The bright-field images (a, c, e) and fluorescence images (b, d, f) of living HeLa cells incubated with the probe 1 (20 μM) for 90 min at 37 ºC: cells without treatment (a, b), cells pretreated with NEM (1 mM) (c, d), and cells pretreated with NEM (1 mM) and addition of 150 μM Cys (e, f). Scheme 1. Synthesis of the probe 1 and proposed sensing mechanism toward Cys.
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Fig. 1
Fig. 2 15
Fig. 3
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Fig. 4
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Fig.5
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Scheme 1
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S0925-4005(17)30596-8 http://dx.doi.org/doi:10.1016/j.snb.2017.03.169 SNB 22081
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
29-11-2016 29-3-2017 31-3-2017
Please cite this article as: Wei-Wei Ma, Meng-Yuan Wang, Di Yin, Xuan Zhang, Facile preparation of naphthol AS-based fluorescent probe for highly selective detection of cysteine in aqueous solution and its imaging application in living cells, Sensors and Actuators B: Chemicalhttp://dx.doi.org/10.1016/j.snb.2017.03.169 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Facile preparation of naphthol AS-based fluorescent probe for highly selective detection of cysteine in aqueous solution and its imaging application in living cells Wei-Wei Ma, Meng-Yuan Wang, Di Yin, Xuan Zhang* College of Chemistry, Chemical Engineering & Biotechnology, Donghua University, Shanghai 201620, China. *Corresponding Author. E-mail: [email protected].
Graphical abstract
1
Highlights The fluorescent probe based on commercial naphthol AS was facilely prepared. Methacrylate group acts as an electron acceptor and a reactive site for Cys. High selectivity toward Cys over Hcy and GSH. Good water solubility and successful application in living cells imaging.
ABSTRACT A simple and new fluorescent probe (1) for selective detection of cysteine (Cys) in aqueous solution was facilely synthesized. It was developed based on the masking the phenolic OH group in naphthol AS by a methacrylate group, where the methacrylate group acts as an electron acceptor to quench fluorescence of naphthol AS as well as a reactive site for Cys. The conjugate addition/cyclization reaction of Cys toward methacrylate moiety in the probe 1 led to a cleavage of the methacrylate group and release of free naphthol AS, thereby induced a significant enhancement of fluorescence. The present fluorescent probe exhibited high selectivity toward Cys over homocysteine (Hcy) and glutathione (GSH) and worked well in a physiological aqueous medium, allowing a successful application in living cells imaging.
Keywords: Fluorescent probe; Naphthol AS; Cysteine; Living cells imaging
2
1. Introduction As an essential amino acid, cysteine (Cys) is involved in many biological processes, such as protein synthesis and metabolism [1]. It has been known that the normal intracellular level of Cys is 30–200 µM, and its deficient or abundant could cause serious diseases, such as slow growth, edema, leucocyte loss, liver damage and neurotoxicity [2]. Therefore, many detection methods have been developed to detect Cys in biological systems, especially based on fluorescence technique due to its advantages of high sensitivity, rapidity, non-invasion, and capability of visual imaging in vivo [3]. These fluorescent probes were usually developed based on the intrinsic nucleophilicity of the free thiol group in Cys, even successful to differentiate Cys from other common amino acids, but quite difficult to discriminate Cys from others biothiols such as homocystein (Hcy) and glutathione (GSH) due to their similar reactivity [3, 4]. Recently, the conjugate addition/cyclization of Cys to acrylate moiety has been proven to be a promising strategy for design of Cys-selective fluorescent probe over Hcy and GSH [5]. Such strategy has been used for several fluorophores such as fluorescein [6], coumarin [7], cyanines [8], and semiheptamethine [9], to develop fluorescent probes for selective detection of Cys. However, most of these reported probes for Cys have to need sophisticated synthesis processes and involve large amounts of organic solvents as co-solvents due to poor water solubility. For example, 20–50% MeOH/EtOH [6, 7, 9], and 30–70% DMF/DMSO [4e, 10] have been usually employed as co-solvents. For practical applications in biological systems, fluorescent probes should own suitable water solubility and work in aqueous mediums containing organic solvent as less as possible. Therefore, the development of water soluble fluorescent probe that can be facilely synthesized and shows high selectivity for Cys is highly demanded. 3
In this work, naphthol AS, a commercial available dye, was used to construct a selective fluorescent probe (1, Scheme 1) for Cys by facilely masking its phenolic OH with methacrylate that acts as an efficient electron acceptor to quench the native fluorescence of naphthol AS, and it was found that Cys could selectively induce a deprotection and recovery of strong fluorescence. The present fluorescent probe worked well in a physiological aqueous medium (pH 7.4, 5% DMSO), showed a high selectivity for Cys over Hcy and GSH, and was successfully applied in living cells imaging. 2. Experimental section 2.1. Materials and Methods All the reagents and solvents are analytical grade and purchased from Sinopharm Chemical Reagents Corp. (Shanghai, China). Phosphate buffered saline (PBS, pH = 7.4) was prepared from Na2HPO4 (0.1 M) and KH2PO4 (0.1 M). The stock solution of the probe 1 was prepared in DMSO and amino acids solutions were prepared in deionized water. 1H NMR and 13C NMR spectra were recorded on a Bruker AVANCE III 400 MHz spectrometer. Mass spectra were obtained on AB Sciex MALDI-TOF/TOFTMMS and Varian 310 mass spectrometers, respectively. Fluorescence spectra were measured on Edinburgh FS5 spectrofluorometer with Ex/Em slit widths of 5 nm. Confocal fluorescence imaging experiments in living Hela cells were carried out with a Carl Zeiss LSM 700 microscope. Theoretical calculations were carried out using Gaussian 09 package [11]. The ground state (S0) geometries of the compounds were optimized in the gas phase using density functional theory (DFT) at the B3LYP/6-31+G(d) level. 2.2. Synthesis Naphthol AS (2.0 mmol) was dissolved in acetone (125 mL) and methacryloyl chloride (2.0 4
mmol) was slowly added dropwise under stirring at 0 °C in the presence of K2CO3 (2.0 mmol). After completion of the reaction within 4 h, the mixture was filtrated and the filtrate was evaporated under reduced pressure. The obtained solid was then dissolved in CH2Cl2, washed with water and the organic layer was dried over MgSO4. After removing of MgSO4 by filtration, the crude product was obtained by evaporation of the solvent under reduced pressure. The product was further purified by column chromatography (silica gel, hexane/CH2Cl2 = 1:4 v/v). Yield: 33%. 1H
NMR (400 MHz, CDCl3), δ (ppm): 8.45 (s, 1H), 8.18 (s, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.84 (d,
J = 8.0 Hz, 1H), 7.65-7.53 (m, 5H), 7.37 (t, J = 8.0 Hz, 2H), 7.15 (t, J = 8.0 Hz, 1H), 6.47 (s, 1H), 5.86 (s, 1H), 2.08 (s, 3H); 13C NMR (100 MHz, CDCl3), δ (ppm): 18.47, 99.97, 120.01, 120.76, 124.63, 126.73, 127.35, 127.82, 128.35, 128.73, 129.13, 131.00, 131.20, 134.69, 135.53, 137.84, 144.85, 163.59, 166.03. MALDI-TOF-MS: m/z calcd for (M + H)+ 332.36; found 332.19. 2.3. Confocal Microscope Imaging HeLa cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C in a 95% humidity atmosphere under 5% CO2 environment. Then the cells were seeded in 6-wells glass-bottomed dishes at a density of 2 ×105 cells per dish in RPMI 1640 medium for 24 h. The cells were then incubated with probe 1 (20 µM) for 90 min at 37 °C, washed with PBS buffer (10 mM) three times to remove free probe 1, and observed under a Carl Zeiss LSM 700 fluorescence microscope. For control experiments, the cells were pretreated with N-ethylmaleimide (NEM, 1 mM) for 30 min at 37 °C, followed by washing with PBS three times, and incubated with probe 1 (20 µM) for 90 min at 37 °C; and further incubated with Cys (150 μM) for 90 min at 37 °C.
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3. Results and discussion 3.1. Design and Synthesis Naphthol AS is a commercial available dye containing an active phenolic OH group that has been usually used to develop fluorescent sensor for anions under an excited state proton transfer mechanism [12]. We envisage that the native fluorescence of naphthol AS would be quenched by masking its phenolic OH with methacrylate group, an efficient electron acceptor, due to photoinduced electron transfer (PET) process, but Cys will selectively recover its fluorescence by removal of the masking group through conjugate addition/cyclization to acrylate moiety (Scheme 1). Therefore, the probe 1 was facilely synthesized by treating naphthol AS with methacryloyl chloride in acetone as shown in Scheme 1. The chemical structure of the probe 1 was confirmed by 1H NMR, 13C NMR, and MALDI-TOF-MS (Figs. S3‒S5). 3.2. Selective response of the probe 1 toward Cys As shown in Fig. 1, the probe 1 is weakly fluorescent in PBS buffer (10 mM, pH = 7.4, 5% DMSO) probably because the quench effect of methacrylate group through a PET process [5]. Upon addition of Cys, a strong green fluorescence appeared around 530 nm, but no significant fluorescence enhancement was observed in the presence of Hcy, GSH and other species (including common amino acids, cations and anions), suggesting that the probe 1 is highly selective for Cys detection (Figs. 1 and S1). Mass spectrometry (ESI-MS) analysis of the mixture solution of the probe 1 with 20 equiv Cys reveals a strong peak at m/z = 262 corresponding to the deprotonated naphthol AS (Fig. S2), an indication of methacrylate group in the probe 1 was really removed by Cys. To gain better insight into the fluorescence behavior, the structures of probe 1 and naphthol AS were optimized and their frontier molecular orbital were analysed by using DFT calculation at 6
the B3LYP/6-31+G(d) level, respectively. As shown in Fig. 2, the π electrons of naphthol AS were delocalized on the whole molecular skeleton on both the lowest unoccupied molecular orbital (LUMO) and the highest occupied molecular orbital (HOMO). However, the π electrons on the LUMO of 1 were mainly localized on the methacrylate group, while the π electrons on the HOMO were distributed in the naphthol AS skeleton (Fig. 2). These results support the possible PET process in 1, where the electron transfer from the naphthol AS (electron donor) to the methacrylate group (electron acceptor) weakened the native fluorescence of the donor, but Cys-induced removal of electron acceptor recovered the fluorescence (Scheme 1). To obtain the optimal pH condition, the pH effect on fluorescence of the probe 1 in the absence and presence of Cys were then investigated. Without Cys, the probe 1 is weakly fluorescent when pH < 9, but becomes highly fluorescent when pH > 9 (Fig. 3a). This reveals that the probe 1 has been already hydrolyzed into highly fluorescent deprotonated naphthol AS on the more basic condition. However, in the presence of Cys, the probe 1 exhibits a strong fluorescence at pH 4–9, revealing Cys could induce a removal of methacylate moiety and transform 1 into free naphthol AS in a broad pH region (Fig. 3a). In this work, pH 7.4 was selected for measurements to match with the requirement in physiological environment. Moreover, it was found that the fluorescence intensity at 530 nm is continually increased with time and reaches constant after 50 min, indicating hydrolysis reaction could be finished within 50 min for Cys (Fig. 3b). On the other hand, both Hcy and GSH presented very slow reaction kinetics (Fig. 3b). The slow reaction rate of Hcy and GSH could be rationalized by considering that the conjugate addition reaction is even feasible, but the further intramolecular cyclization reaction should be kinetically disfavored relative to the formation of seven-membered ring that would result from Cys (Scheme 1) [5]. Thus 7
the difference on kinetics of intramolecular cyclization reaction could also be used to rationalize the high selectivity of the probe 1 toward Cys. Fig.4 showed the fluorescence titration of the probe 1 with Cys in a phosphate buffer (10 mM PBS containing 5% DMSO, pH = 7.4). It can be clearly seen that the green fluorescence at 530 nm was continuously increased with increasing Cys amounts up to 350 μM, and a good linear relationship (R2 = 0.9988) was obtained over the range of 1‒200 μM (Fig. 4). The detection limit was estimated to be 0.5 μM according to S/N = 3. The normal intracellular level of Cys is known to be 30–200 µM, suggesting the probe 1 is sensitive enough to satisfy Cys detection in living cells. 3.3. Fluorescence imaging in living cells To verify the potential application of 1 for Cys imaging in living cells, the HeLa cells were incubated with probe 1 at 37 ºC for 90 min and observed by confocal fluorescence microscope. As shown in Fig. 5b, a bright green fluorescence was observed inside the cells, indicating that the intracellular Cys reacts with the probe 1 led to a removal of methacylate moiety and a recovery of fluorescence. In contrast, when the cells were pretreated with a thiol-blocking agent, N-ethylmaleimides (NEM, 1 mM) prior to addition of probe 1, there is almost no detectable fluorescence (Fig. 5d); when the NEM-pretreated cells were successively incubated with the probe 1 and Cys, a bright green fluorescence was again observed inside cells (Fig. 5f). These results indicate that 1 can serve as a promising fluorescent probe for Cys imaging in living cells. 4. Conclusions A simple and new fluorescent probe 1 for selective detection of Cys was facilely synthesized based on commercial dye naphthol AS, where the methacrylate group was introduced both as an 8
electron acceptor to quench fluorescence and a reactive site for Cys. It was found that the conjugate addition/cyclization reaction of Cys toward methacrylate moiety in the probe 1 results in cleavage of the methacrylate moiety and release of free naphthol AS, thereby inducing a significant enhancement of fluorescence. The probe exhibits high selectivity toward Cys over Hcy, GSH and others species in a physiological aqueous medium. The living cells imaging results suggest that the probe 1 could be successfully applied for intracellular Cys detection. Regarding to its facile preparation, good water solubility and high selectivity, the present new probe shows a promising potential for application involving detection of Cys in biological systems.
Acknowledgment This work was financially supported by Shanghai Municipal Natural Science Foundation (16ZR1401700). We would like to appreciate Dr. Xiao-Yue Zhu and Prof. Xiang-Yang Shi for their kind assistances on cell imaging.
Supplementary data Figs. S1‒S5 are available.
References [1] (a) K.G. Reddie, K.S. Carroll, Curr. Opin. Chem. Biol. 12 (2008) 746–754; (b) N.M. Giles, A.B. Watts, G.I. Giles, F.H. Fry, J.A. Littlechild, C. Jacob, Chem. Biol. 10 (2003) 677–693. [2] (a) S. Park, J.A. Imlay, J. Bacteriol. 185 (2003) 1942–1950; (b) S. Shahrokhian, Anal. Chem. 73 (2001) 5972–5978.
9
[3] (a) L.-Y Niu, Y.-Z. Chen, H.-R. Zheng, L.-Z. Wu, C.-H. Tung, Q.-Z. Yang, Chem. Soc. Rev. 44 (2015) 6143–6160; (b) H. Chen, Y. Tang, W. Lin, Trend. Anal. Chem. 76 (2016) 166–181; (c) X. Chen, Y. Zhou, X. Peng, J. Yoon, Chem. Soc. Rev. 39 (2010) 2120–2135; (d) J. Chan, S. C. Dodani, C. J. Chang, Nat. Chem. 4 (2012) 973–984; (e) Y. Yang, Q. Zhao, W. Feng, F. Li, Chem. Rev. 113 (2013) 192–270; (f) X. Li, X. Gao, W. Shi, H. Ma, Chem. Rev. 114 (2014) 590–659. [4] (a) D. Gong, Y. Tian, C. Yang, A. Iqbal, Z. Wang, W. Liu, W. Qin, X. Zhu, H. Guo, Biosens. Bioelectron. 85 (2016) 178–183; (b) C. Yang, X. Wang, L. Shen, W. Deng, H. Liu, S. Ge, M. Yan, X. Song, Biosens. Bioelectron. 80 (2016) 17–23; (c) Y. Liu, X. Lv, M. Hou, Y. Shi, W. Guo, Anal. Chem. 87 (2015) 11475–11483; (d) D. Lee, G. Kim, J. Yin, J. Yoon, Chem. Commun. 51 (2015) 6518–6520; (e) X.-F. Yang, Q. Huang, Y. Zhong, Z. Li, H. Li, M. Lowry, J. O. Escobedo, R. M. Strongin, Chem. Sci. 5 (2014) 2177–2183; (f) H. Lv, X.-F. Yang, Y. Zhong, Y. Guo, Z. Li, H. Li, Anal. Chem. 86 (2014) 1800–1807; (g) X. Gao, X. Li, L. Li, J. Zhou, H. Ma, Chem. Commun. 51 (2015) 9388–9390; (h) Y. Yang, F. Huo, C. Yin, J. Chao, Y. Zhang, Dyes Pigm. 114 (2015) 105–109; (i) J. Liu, Y.-Q. Sun, Y. Huo, H. Zhang, L. Wang, P. Zhang, D. Song, Y. Shi, W. Guo, J. Am. Chem. Soc. 136 (2014) 574−577; (j) T. Liu, F. Huo, J. Li, J. Chao, Y. Zhang, C. Yin, Sens. Actuators B 237 (2016) 127–132; (k) J. Xu, J. Pan, Y. Zhang, J. Liu, L. Zeng, X. Liu, Sens. Actuators B 238 (2017) 58–65; 10
(l) Z. Yao, H. Bai, C. Li, G. Shi, Chem. Commun. 47 (2011) 7431–7433; (m) X. Zhang, J.-Y. Liu, W.-W. Ma, M.-L. Yang, J. Mater. Chem. B 4 (2016) 6662–6669; (n) L. Bu, J. Chen, X. Wei, X. Li, H. Ågren, Y. Xie, Dyes Pigm. 136 (2017) 724–731. [5] X.-F. Yang, Y.-X. Guo, R.M. Strongin, Angew. Chem. Int. Ed. 50 (2011) 10690–0693. [6] H. Wang, G. Zhou, H. Gai, X. Chen, Chem. Commun. 48 (2012) 8341–8343. [7] (a) X. Liu, D. Yang, W. Chen, L. Yang, F. Qi, X. Song, Sens. Actuators B 234(2016) 27–33; (b) Y.-C. Liao, P. Venkatesan, L.-F. Wei, S.-P. Wu, Sens. Actuators B 232 (2016) 732–737. [8] (a) Z. Guo, S. Nam, S. Park, J. Yoon, Chem. Sci. 3 (2012) 2760–2765; (b) J. Zhang, J. Wang, J. Liu, L. Ning, X. Zhu, B. Yu, X. Liu, X. Yao, H. Zhang, Anal. Chem. 87 (2015) 4856–4863. [9] C. Han, H. Yang, M. Chen, Q. Su, W. Feng, F. Li, ACS Appl. Mater. Interfaces 7 (2015) 27968−27975. [10] (a) H.-M. Lv, D.-H. Yuan, W. Liu, Y. Chen, C.-T. Au, S.-F. Yin, Sens. Actuators B 233 (2016) 173–179; (b) X. Xiong, F. Song, G. Chen, W. Sun, J. Wang, P. Gao, Y. Zhang, B. Qiao, W. Li, S. Sun, J. Fan, X. Peng, Chem. Eur. J. 19 (2013) 6538–6545. [11] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G.A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H.P. Hratchian, A.F. Izmaylov, J. Bloino, G. Zheng, J.L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J.A. Montgomery, J.E. Peralta, F. Ogliaro, M. Bearpark, J.J. Heyd, E. Brothers, K.N. Kudin, V.N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J.C. 11
Burant, S.S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J.M. Millam, M. Klene, J.E. Knox, J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, R.L. Martin, K. Morokuma, V.G. Zakrzewski, G.A. Voth, P. Salvador, J.J. Dannenberg, S. Dapprich, A.D. Daniels, O. Farkas, J.B. Foresman, J.V. Ortiz, J. Cioslowski, D.J. Fox, Gaussian 09, Revision C.01, Gaussian Inc., Wallingford, CT, 2009. [12] (a) X. Zhang, L. Guo, F.-Y. Wu, Y.-B. Jiang, Org. Lett. 15 (2003) 2667−2670; (b) X. Zhang, Y. Shiraishi, T. Hirai, Tetrahedron Lett. 48 (2007) 8803–8806; (c) X. Zhang, J.-Y. Liu, Dyes Pigm. 125 (2016) 80–88; (d) X. Peng, Y. Wu, J. Fan, M. Tian, K. Han, J. Org. Chem. 70 (2005) 10524–10531; (e) K. Choi, A.D. Hamilton, Angew. Chem. Inter. Ed. 40 (2001) 3912–3915.
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Biographies
Wei-Wei Ma, Meng-Yuan Wang and Di Yin are master students of College of Chemistry, Chemical Engineering& Biotechnology, Donghua University. Their current research interests are focused on the fluorescent probes. Xuan Zhang received his Ph.D. degree in 2003 from Xiamen University, China. He worked as JSPS fellow and postdoc in Japan during 2004-2011. Then he got back to China and was appointed as an associate professor of Chemistry in College of Chemistry, Chemical Engineering& Biotechnology, Donghua University. His current research interests are mainly focused on the fluorescent and electrocatalytic materials.
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Figure Captions:
Fig. 1. Fluorescence spectra of 1 (10 μM, pH = 7.4 PBS containing 5% DMSO) in the presence of 20 equiv Cys, Hcy, GSH, Asp, Asn, Ser, Pro, Ala, Gly, Val, Leu, Ile, Thr, Arg, Glu, Gln, Tyr, His, Met, Phe, Trp, Lys, Tau, Na2S, HSCH2COOH, glucose, K+, Na+, Ca2+, Mg2+, Cl−, NO3−, SO42−, PO42−. Excitation wavelength is 400 nm. Inset is photograph of 1 before and after addition of Cys under a UV lamp (365 nm) irradiation.
Fig. 2. HOMO and LUMO orbitals of 1 and naphthol AS. Calculations were based on ground state geometry by DFT at the B3LYP/6-31+G(d) level.
Fig. 3. Time- and pH-dependent fluorescence intensity change (λem = 530 nm, λex = 400 nm,) of 1 (10 μM, pH = 7.4 PBS containing 5% DMSO) in the presence of 20 equiv Cys.
Fig. 4. Change of fluorescence spectra of 1 (10 μM, pH = 7.4 PBS containing 5% DMSO) upon addition of various amounts of Cys (0–350 μM) (a), and the corresponding linear relationship between the fluorescence intensity at 530 nm and concentration of Cys (1‒200 μM) (b). Fig. 5. The bright-field images (a, c, e) and fluorescence images (b, d, f) of living HeLa cells incubated with the probe 1 (20 μM) for 90 min at 37 ºC: cells without treatment (a, b), cells pretreated with NEM (1 mM) (c, d), and cells pretreated with NEM (1 mM) and addition of 150 μM Cys (e, f). Scheme 1. Synthesis of the probe 1 and proposed sensing mechanism toward Cys.
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Fig. 1
Fig. 2 15
Fig. 3
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Fig. 4
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Fig.5
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Scheme 1
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