Tetrahedron Letters 54 (2013) 5771–5774

Contents lists available at ScienceDirect

Tetrahedron Letters

journal homepage: www.elsevier .com/ locate/ tet le t

Coumarin–TPA derivative: a reaction-based ratiometricfluorescent probe for Cu(I)

Scheme 1. Synthesis of probes.

0040-4039/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.tetlet.2013.08.046

⇑ Corresponding authors. Tel./fax: +86 28 85415886.E-mail addresses: kli@scu.edu.cn (K. Li), xqyu@scu.edu.cn (X.-Q. Yu).

Kang-Kang Yu, Kun Li ⇑, Ji-Ting Hou, Xiao-Qi Yu ⇑Key Laboratory of Green Chemistry and Technology (Ministry of Education), College of Chemistry, Sichuan University, Chengdu 610064, China

a r t i c l e i n f o

Article history:Received 7 May 2013Revised 29 July 2013Accepted 15 August 2013Available online 22 August 2013

Keywords:CoumarinReactive probeRatiometric probeCopper sensor

a b s t r a c t

A coumarin-based reactive probe 1 is reported for the highly selective detection of Cu+. The benzylic ether(C–O) bond of probe 1 can be cleaved selectively by the reaction with Cu+ under a physiological reducingenvironment, resulting in a fluorescent change. The maximum emission peak exhibited a red shift from410 nm to 472 nm, and a remarkable enhancement of emission intensity ratios (I472/I410) from 0.26 to13.82 was observed.

� 2013 Elsevier Ltd. All rights reserved.

As one of the most abundant soft transition metal elements,copper plays a very important role in the human body. A varietyof enzymes, such as tyrosinase,1 need copper to serve as an indis-pensable cofactor due to its redox-active properties. Additionally,copper is closely connected with structural elements in the humanskeleton and collagen tissue, as well as functional determinants ofthe endocrine and nervous systems. On the other hand, whenin vivo concentrations of copper exceed the normal range, the re-dox active metal becomes a biological hazard via the generationof reactive oxygen species (ROS), which can cause disruptions incellular metabolism.2,3 Alterations in copper concentration atcellular homeostasis are related to serious neurodegenerativediseases, such as Wilson and Alzheimer diseases, Menkes syn-drome, familial amyotrophic lateral sclerosis, and prion diseases.4

In consideration of the strict concentration-dependent balancefor copper between toxicity and biological, it is of great importanceto develop an exact and convenient method for the detection ofcopper in environmental and biological samples.

In recent years, more and more metal-responsive fluorescentsensors have been constructed. This method provides a convenientand effective approach for detecting metals because of its relativelow cost, high sensitivity, and logistical simplicity.5 Despite thelarge success in the development of specific fluorescent probesfor other cations, copper-selective probes are relatively rare andthe research of copper-selective probes is particularly challengingfor the dominant oxidation state in the biological system (+1).Cu+ has a paramagnetic character and is thus a potent free radical

quencher in all coordination environments.6 Most reported cop-per-specific fluorescent probes are selective for Cu2+, with onlylimited examples indicative of Cu+. More importantly, a large partof these probes relies on fluorescence intensity-based measure-ments.7 Fluorescence intensity-based detection methods are typi-cally not appropriate for complex real-world samples wheremeasuring initial probe concentration and overall excitation andemission intensity are complicated with other environmentalfactors.8 Compared to intensity-based sensors, ratiometric fluores-cence sensors show obvious advantages. They allow for the simul-taneous recording of two measurable signals in the presence and/or absence of the analyte and can, in principle, allow for accurateand quantitative readouts.9 Accordingly, it is absolutely essential

Figure 1. (A) Cation selectivity (kex = 360 nm) of probe 1 (10 lM) in PBS buffer (25 mM, pH = 7.20). (B) The bars represent the ratiometric fluorescence intensity [I472/I410] ofthe mixture, from 1 to 20: probe 1 only, Li+, Na+, K+, Ca2+, Mg2+, Ba2+, Pb2+, Al3+, Cr3+, Cd2+, Fe2+, Fe3+, Ag+, Hg2+, Ni2+, Zn2+, Cu2+, Co2+, and Cu+.

Scheme 2. The supposed mechanism of probe 1 with Cu+.

5772 K.-K. Yu et al. / Tetrahedron Letters 54 (2013) 5771–5774

Figure 2. (A) Cation selectivity (kex = 360 nm) of probe 4 (10 lM) in PBS buffer (25 mM, pH 7.20). (B) The bars represent the fluorescence intensity at 410 nm of the [1-Cu+].From 1 to 19: probe 4 only, Li+, Na+, K+, Ca2+, Mg2+, Ba2+, Pb2+, Al3+, Cr3+, Fe2+, Fe3+, Ag+, Hg2+, Ni2+, Zn2+, Cu2+, Co2+, and Cu+.

K.-K. Yu et al. / Tetrahedron Letters 54 (2013) 5771–5774 5773

to design a novel ratiometric fluorescence sensor for Cu+. Herein,we present a reaction-based ratiometric probe 1 for Cu+ under aphysiological reducing environment.

Coumarin was selected as the fundamental platform, because itis known to be a strongly fluorescent compound, and it is relativelyeasy to synthesize a large variety of structural varients.10 Couma-rin-based probes are widely used in various biological assays.11

Recently, Taki et al. reported a reaction-based turn-on sensing ofCu+ by using a tetradentate ligand tris[(2-pyridyl)-methyl]amine(TPA).1a Inspired by this work, probe 1 was prepared by combiningthe coumarin platform (compound 2) with a tetradentate moietythrough an oxidatively-cleavable benzyl ether bond (Scheme 1).Compound 2 and probe 1 exhibited native blue-green and violetfluorescence, respectively. Probe 1 was designed so that in thepresence of Cu+ the reaction between the probe and the redox-ac-tive cation would selectively remove TPA from probe 1 by oxida-tively cleaving the benzylic ether bond, resulting in the release ofthe bright blue-green coumarin fluorophore with a resultantincrease in the quantum yield12 and a large Stokes shift in theobserved fluorescence emission.

The target compound (probe 1) could be synthesized easily via atwo-step nucleophilic substitution with a moderate overall yield of54%. Meanwhile, pyridylmethylene ether 4 was also synthesized toserve as a control compound. The probes and intermediates allwere characterized by 1H NMR, 13C NMR, and MS (Supplementarydata).

The fluorescence responses of probe 1 to a range of physiologi-cally and environmentally relevant metal cations were measuredin an aqueous buffer solution (25 mM PBS, pH 7.20), containing2 mM glutathione (GSH) for maintaining the dominant oxidationstate of Cu+. As exhibited in Figure 1, the addition of 10 equiv ofLi+, Na+, K+, Ca2+, Mg2+, Ba2+, Pb2+, Al3+, Cr3+, Cd2+, Fe2+, Fe3+, Ag+,Hg2+, Ni2+, Zn2+, Cu2+, and Co2+, did not cause any significant changein the fluorescence spectra of probe 1, while Zn2+, Cd2+ could lead toa moderate fluorescence enhancement at 410 nm. However, uponaddition of 10 equiv Cu+, a large red shift of probe 1 from 410 to472 nm (with a corresponding color change from violet to blue-green under a 365 nm UV lamp, Fig. S1) was observed, in addition,a remarkable enhancement in emission intensity ratios (I472/I410)from 0.26 to 13.82 was found. Meanwhile, the response of probe 1(10 lM) to 10 equiv of Cu+ in the presence of 10 equiv of differentcompeting metal ions was also investigated (Fig. S2). The resultsindicate that Zn2+, Ni2+ and Cd2+ greatly reduced the ability of probe1 to detect Cu+ due to the strong coordination abilities of thesecompeting cations for the TPA moiety.

The emission intensity ratios (I472/I410) of Cu2+ and Cu+ are 0.42and 13.82, respectively. Obviously, probe 1 is capable of discrimi-nating between Cu+ and Cu2+ and other metals. Additionally, thesignificant red-shift indicated that the benzylic ether (C–O) linkageindeed was cleaved, accompanied by the release of the coumarinmoiety. Notably, the cleavage of C–O bond only requires approxi-mately 20 min (Fig. S3); we use a 30 min mixing time to ensurea complete reaction. In order to provide support for the proposedmechanism, various ESI-MS spectra of the reaction between probe1 and a Cu+ solution were collected at different times (Scheme 2).In the absence of Cu+, the peak at m/z 544.1 was recognized as cor-responding to probe 1 (Fig. S4, Supplementary data); after Cu+ wasadded, peaks m/z 554.75 and 602.72, corresponding to [1-Cu+] and[1-Cu+-H2O] intermediates, respectively, were found immediately(Fig. S5). After 30 min the peak for probe 1 and [1-Cu+] disappearedand a new peak at m/z 396.1 was detected. This new peak corre-sponds to the complex between Cu2+ and TPA (Fig. S6). These re-sults provide strong evidence for the Cu+ catalyzed oxidativecleavage of the benzylic C–O bond of probe 1 in the presence ofO2. From these results, we infer that Cu+ first coordinates withprobe 1 and water. This complex is then oxidized by O2 resultingeventually in cleavage of the C–O bond and formation of a stabi-lized Cu2+–TPA complex.3a,13

Furthermore, to verify that cleavage of the benzylic C–O bondonly occurs after the [1-Cu+] complex is generated, the fluorescentresponse of compound 4 was investigated under the same condi-tions as with probe 1. As shown in Figure 2, the emission intensityof 4 showed no obvious change upon the addition of the testedions, including Cu+. These results indicate the importance of theTPA moiety. With the replacement of TPA by pyridine, Cu+ couldnot effectively complex with 4, and the C–O bond of the ethercould not be broken, resulting in the maintenance of the fluores-cence. That is to say, the benzylic ether (C–O) linkage is cleavedonly after Cu+ selectivity coordinates with the tetradentate moietyof probe 1.

The fluorescence titration of probe 1 (10 lM) toward Cu+ wasconducted in a buffered solution (25 mM PBS, pH 7.20) in the pres-ence of 2 mM glutathione (GSH). With increasing concentrations ofCu+, the fluorescence of probe 1 at 410 nm decreased gradually,accompanied by the appearance of a new peak at 472 nm(Fig. 3). Additionally, the emission intensity ratios (I472/I410) in-creased from 0.23 to 7.66 when 5 lM Cu+ was added and the detec-tion limit of probe 1 was calculated to 2.29 � 10�7 M14 (Fig. S7).Moreover, the effect of pH on the cleavage of the benzylic ether(C–O) linkage was investigated and summarized in Figure 4.

Figure 4. Ratiometric fluorescence intensity [I471/I410] as Cu+ (100 lM) was addedin different pH of probe 1 (10 lM, 25 mM PBS buffer). Black: probe 1 only; red:probe 1 with Cu+ (2 nm GSH).

Figure 3. Fluorescence spectra (kex = 360 nm) of probe 1 (10 lM) in PBS buffer(25 mM, pH 7.20) upon addition of Cu+ (0–5 lM). Inset: Ratiometric fluorescenceintensity [I472/I410] as a function of Cu+.

5774 K.-K. Yu et al. / Tetrahedron Letters 54 (2013) 5771–5774

Throughout the biologically relevant pH range of 4.50–10.50, probe1 could detect Cu+ by the mechanism we mentioned (Scheme 2).Accordingly, there is no need to worry about interference frompH effects in the detection of Cu+ with probe 1, except possiblyfor strongly basic conditions (pH >10.50) which may result inhydrolytic ring-opening of the coumarin moiety.

In conclusion, a reaction-based ratiometric chemosensor is re-ported for the selective detection of Cu+ in aqueous solutions.Probe 1 exhibited high selectivity toward Cu+ over other metals

with a detecting limit of 2.29 � 10�7 M. ESI-MS results indicatedthat the oxidative properties of Cu+ are responsible for the mecha-nism, which resulted in cleavage of the benzylic ether (C–O) bond.This unique chemosensor is an important and valuable new ad-vance for the ratiometric detection of Cu+.

Acknowledgments

This work was financially supported by the National Programon Key Basic Research Project of China (973 Program,2012CB720603 and 2013CB328900) and the National ScienceFoundation of China (Nos. 21232005 and 21001077). We alsothank Analytical & Testing Center of Sichuan University for NMRanalysis. K.L. thanks Professor Jason J. Chruma (Sichuan University)for assistance with document preparation.

Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.tetlet.2013.08.046.

References and notes

1. (a) Taki, M.; Iyoshi, S.; Ojida, A.; Hamachi, I.; Yamamoto, Y. J. Am. Chem. Soc.2010, 132, 5938; (b) Domaille, D. W.; Que, E. L.; Chang, C. J. Nat. Chem. Biol.2008, 4, 168; (c) Zeng, L.; Miller, E. W.; Pralle, A.; Isacoff, E. Y.; Chang, C. J. J. Am.Chem. Soc. 2006, 128, 10.

2. (a) Dodani, S. C.; Leary, S. C.; Cobine, P. A.; Winge, D. R.; Chang, C. J. J. Am. Chem.Soc. 2011, 133, 8606; (b) Haas, K. L.; Franz, K. J. Chem. Rev. 2009, 109, 4921.

3. (a) Maity, D.; Kumar, V.; Govindaraju, T. Org. Lett. 2012, 14, 6008; (b) Brown, D.R.; Kozlowski, H. Dalton Trans. 2004, 1907; (c) Bruijn, L. I.; Miller, T. M.;Cleveland, D. W. Annu. Rev. Neurosci. 2004, 27, 723; (d) Valentine, J. S.; Hart, P. J.Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 3617.

4. (a) Hung, Y. H.; Bush, A. I.; Cherny, R. A. J. Biol. Inorg. Chem. 2010, 15, 61; (b)Tapiero, H.; Townsend, D. M.; Tew, K. D. Biomed. Pharmacother. 2003, 57, 386;(c) Vulpe, C.; Levinson, B.; Whitney, S.; Packman, S.; Gitschier, J. Nat. Genet.1993, 3, 7.

5. Hargrove, A. E.; Nieto, S.; Zhang, T.; Sessler, J. L.; Anslyn, E. V. Chem. Rev. 2011,111, 6603.

6. Au-Yeung, H. Y.; New, E. J.; Chang, C. J. Chem. Commun. 2012, 48, 5268.7. (a) Cody, J.; Fahrni, C. J. Tetrahedron 2004, 60, 11099; (b) Miller, E. W.; Zeng, L.;

Domaille, D. W.; Chang, C. J. Nat. Protoc. 2006, 1, 824; (c) Domaille, D. W.; Zeng,L.; Chang, C. J. J. Am. Chem. Soc. 2010, 132, 1194.

8. Zhou, Y.; Yoon, J. Chem. Soc. Rev. 2012, 41, 52.9. (a) Albers, A. E.; Okreglak, V. S.; Chang, C. J. J. Am. Chem. Soc. 2006, 128, 9640; (b)

Srikun, D.; Miller, S. E.; Domaille, D. W.; Chang, C. J. J. Am. Chem. Soc. 2008, 130,4596; (c) Peng, X.; Du, J.; Fan, J.; Wang, J.; Wu, Y.; Zhao, J.; Sun, S.; Xu, T. J. Am.Chem. Soc. 2007, 129, 1500; (d) Zhang, X. L.; Xiao, Y.; Qian, X. H. Angew. Chem.,Int. Ed. 2008, 47, 8025; (e) Yuan, L.; Lin, W.; Yang, Y.; Song, J.; Wang, J. Org. Lett.2011, 13, 3730; (f) Yuan, L.; Lin, W.; Chen, B.; Xie, Y. Org. Lett. 2012, 14, 432.

10. (a) Komatsu, K.; Urano, Y.; Kojima, H.; Nagano, T. J. Am. Chem. Soc. 2007, 129,13447; (b) Roussakis, E.; Pergantis, S. A.; Katerinopoulos, H. E. Chem. Commun.2008, 6221; (c) Upadhyay, K. K.; Mishra, R. K.; Kumar, V.; Roychowdhury, P. K.Talanta 2010, 82, 312; (d) Helal, A.; Rashid, M. H. O.; Choi, C. H.; Kim, H. S.Tetrahedron 2011, 67, 2794.

11. (a) Katerinopoulos, H. E. Curr. Pharm. Des. 2004, 10, 3835; (b) Sheng, R.; Wang,P.; Gao, Y.; Wu, Y.; Liu, W.; Ma, J.; Li, H.; Wu, S. Org. Lett. 2008, 10, 5015; (c) Lv,X.; Liu, J.; Liu, Y.; Zhao, Y.; Chen, M.; Wang, P.; Guo, W. Org. Biomol. Chem. 2011,9, 4954.

12. Hou, J. T.; Li, K.; Yu, K. K.; Wu, M. Y.; Yu, X. Q. Qrg. Biomol. Chem. 2013, 11, 717.13. (a) Vad, M. S.; Nielsen, A.; Lennartson, A.; Bond, A. D.; McGrady, J. E.; McKenzie,

C. J. Dalton Trans. 2011, 40, 10698; (b) Bayer, A.; Villiger, V. Ber. Dtsch. Chem.Ges. 1899, 32, 3625.

14. Shortreed, M.; Kopelman, R.; Kuhn, M.; Hoyland, B. Anal. Chem. 1996, 68, 1414.