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DOI: 10.1002/ajoc.201200034

Synthesis of p-Extended Coumarins and Evaluation of Their Precursors as Reactive Fluorescent Probes for Mercury Ions
Inae Kim, Dokyoung Kim, Sunderraman Sambasivan, and Kyo Han Ahn*[a]
Abstract: An efficient synthetic route to p-extended coumarin derivatives and their precursors that contain amino substituents has been developed. The synthesis involves monosubstitution of 2,7-dihydroxynaphthalene with dimethylamine through the Bucherer reaction. The extended coumarins emit at much longer wavelengths and with higher quantum yields relative to a related coumarin. Two vinyl ethers, precursors of the extended coumarins, were prepared and evaluated as reactive probes for Hg2 + ions. The vinyl ethers undergo hydrolysis promoted by Hg2 + ions to produce the extended coumarins at physiological pH (pH 7.4) and room temperature, which is accompanied by fluorescence in the turn-on mode. The vinyl ethers do not respond to various other metal species. The extended coumarins and their precursors are versatile sensing platforms for Hg2 + ions, as well as for the development of related reaction-based sensing systems.

Keywords: coumarins fluorescent probes iminocoumarins mercury vinyl ethers

Introduction
Fluorescent probes are valuable tools for the diagnosis of disease markers, detection of hazardous compounds, and monitoring of biochemical processes.[1] Therefore, various fluorescent probes have been developed for given target analytes. Recently, reaction-based or reactive fluorescence probes came into the spotlight owing to their advantageous features in achieving very high selectivity and sensitivity for target analytes.[2] Furthermore, the reaction based approach enables us to realize the turn-on fluorescence sensing mode, even in the case of those analytes that are apt to quench the fluorescence.[3] The fluorescence of the reactive probes changes mostly through irreversible chemical transformations that are promoted or catalyzed by the analytes. Therefore, in this reaction-based approach, a creative combination of a chemical process with the target analyte is required to induce desirable changes in photophysical properties. Recently, we have introduced a unique sensing protocol for Hg2 + ions that utilizes the Hg2 + -promoted hydrolysis of aryl vinyl ethers. Fluorescein-derived vinyl ether 1 (Scheme 1) senses mercury species through hydrolysis in the turn-on fluorescent mode with excellent selectivity and sensitivity.[4] Furthermore, this new sensing protocol ena[a] I. Kim, D. Kim, Dr. S. Sambasivan, Prof. K. H. Ahn Department of Chemistry and Center for Electro-Photo Behaviors in Advanced Molecular Systems Pohang University of Science and Technology (POSTECH) Pohang, 790-784 (Republic of Korea) Fax: (+ 82) 54-279-5877 E-mail: ahn@postech.ac.kr Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/ajoc.201200034.

Scheme 1. Fluorescein-based probe for Hg2 + ions.

bled us to fluorescently image methylmercury, the typical and notoriously toxic organomercury species, for the first time. Mercury is currently used in industrial chemicals, electrical apparatus, dental amalgam, and batteries. However, mercury is a highly poisonous element and a widespread pollutant, which occurrs from natural and anthropogenic sources in the form of reduced and oxidized forms.[5] Mercury species cause damage to the central nervous system and other organs.[6] Therefore, efficient detection and imaging probes for mercury species have attracted intense research interest.[7] In spite of the tremendous efforts, it is only recently that fluorescent probes for methylmercury have been recognized as viable tools, as demonstrated by our work.[8] In our continuous study on the reaction-based approach to the Hg2 + -promoted hydrolysis of aryl vinyl ethers, we also developed a vinyl ether of a coumarin precursor (3) as a fluorescent probe for Hg2 + ions. In this case, the Hg2 + -promoted hydrolysis of the vinyl ether group results in a hydroxy-ester intermediate, which undergoes a fast cyclization reaction to afford a fluorescent coumarin compound 4 (Scheme 2).[9] This hydrolysis-cyclization approach seems to be more versatile in terms of the structural modification of the probe for further structure elaboration, relative to the fluorescein-based one.

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extended coumarins and also to develop related fluorescent probes with improved photophysical properties, we have studied new extended coumarins and their precursors that are related to 3, but with a linearly p-extended feature. We designed the extended coumarin precursors 5 and 7 (Scheme 3), which undergo Hg2 + -promoted hydrolysis to give 6 and 8,[12] respectively. These precursors have a linear p-extended feature with respect to 4 and its analogue.

Results and Discussion

Scheme 2. Reactive fluorescent probe 3 for Hg ions, which produces coumarin 4.
2+

Synthesis An efficient synthesis of compounds 58 is outlined in Scheme 4. Monosubstitution of 2,7-dihydroxynaphthalene (9) by the Bucherer reaction by using dimethylamine afforded 7-(dimethylamino)naphthalen-2-ol (10) in 60 % yield. Protection of the hydroxy group of 10 as its methoxymethyl ether, then directed lithiation with tert-butyllithium and subsequent formylation with N,N-dimethylformamide (DMF) afforded aldehyde 12 in 40 % overall yield after the two steps. Deprotection of the methoxymethyl group of 12 with aqueous HCl afforded hydroxyaldehyde 13, which is a platform compound for further structure elaboration. Vinylation of the hydroxyl group of 13 was most efficiently carried out by a conventional two-step process of bromoethylation to give 14, then b-elimination to give vinyl ether 15 in 44 % overall yield. Finally, the Knoevenagel condensation at the formyl group of 15 with dimethylmalonate or malononitrile in presence of piperidine afforded 5 or 7, respectively. The p-extended coumarin derivatives 6 and 8 can be prepared directly from the key intermediate 13 by the Knoevenagel condensation by using dimethylmalonate or malononitrile, respectively, in presence of piperidine. The detailed experimental procedures, 1H NMR and 13 C NMR spectra, as well as HRMS data are provided in the Supporting Information. The established synthetic route is versatile and thus would also be useful for the synthesis of other functionalized naphthalene derivatives. Sensing Properties With vinyl ethers 5 and 7 in hand, we have evaluated their response to Hg2 + ions by monitoring UV/vis absorption and emission changes. All the spectroscopic measurements were carried out in solutions of phosphate buffered saline (PBS) at physiological pH (pH 7.4) containing dimethylsulfoxide (DMSO, 5 %) at room temperature. Vinyl ether 5 has one major absorption band centered at 400 nm (Figure 1 a), but emits little fluorescence when excited at the absorption maximum (the bottom spectrum in Figure 1 e). When 5 was treated with an equimolar amount of Hg2 + ions, the resulting solution showed turn-on type fluorescence change (lem at 612 nm) upon excitation at 460 nm, together with a bathochromic shift in the absorption maximum (lmax : 400 nm !460 nm). The time-dependent fluorescence change is shown in Figure 1 c. Such spec-

Coumarins, including 4, constitute an important class of fluorophores that are widely used for sensing and imaging purposes.[10] In spite of their usefulness as fluorophores, pextended coumarin derivatives, which would have improved photophysical properties, remain largely unexplored. In particular, the extended coumarin derivatives with electron-donating substituents, such as amino groups, are rare. To our knowledge, only one example is known so far, which is an angularly extended coumarin with a dimethylamino substituent.[11] We attempted to synthesize a key intermediate of the extended coumarin by following the literature, but found that it required a multistep synthesis that is lowyielding. To explore an efficient synthetic route toward p-

Scheme 3. Aryl vinyl ethers 5 and 7 designed as reactive probes for Hg2 + ions, which can produce p-extended coumarin derivatives 6 and 8, respectively.

Abstract in Korean:

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tral changes suggest that 5 undergoes the Hg2 + -promoted hydrolysis and produces the extended coumarin 6, which emits orange-red fluorescence. Spectral changes were also detected in the case of 7 before and after treatment with Hg2 + ions. Vinyl ether 7 showed little change in the absorption maximum before and after treatment with Hg2 + ions (Figure 1 b), but showed turn-on fluorescence changes (lem at 585 nm) upon treatment with Hg2 + Scheme 4. Reagents and conditions: a) Na2S2O5, Me2NH, H2O, 150 8C, 8 h, 60 %; b) NaH, DMF, chloromethyl ions when excited at 446 nm. methyl ether, 15 8C, 7 h, 95 %; c) tBuLi, diethyl ether, DMF, 15 8C, 2 h, 52 %; d) HCl, iPrOH, 60 8C, 3 h, Vinyl ether 5 responds to 97 %; e) 1,2-dibromoethane, KOH, H2O, Bu4NOH, 50 8C, 6 h, 74 %; f) tBuOK, DMSO, RT, 3 h, 59 %; g) dimethylmalonate, piperidine, THF, reflux, 6 h, 38 %; h) malononitrile, piperidine, EtOH, RT, 1 h, 70 %; i) dimethylHg2 + ions faster than vinyl malonate, piperidine, THF, reflux, 1 h, 70 %; j) malononitrile, piperidine, EtOH, RT, 1 h, 90 %. MOM = meether 7 does, as the time-dethoxymethyl ether. pendent fluorescence changes show (Figure 1 c and 1 d). Both 5 and 7 showed gradual increase in the fluorescence upon titration with Hg2 + ions (Figure 1 e and 1 f). Vinyl ethers 5 and 7 emit little fluorescence in aqueous media. It is likely that the free rotation of the dimethylmalonate and the dicyanovinyl groups causes nonradiative decay. The supposed Hg2 + -promoted hydrolysis of 5 and 7 was confirmed by identifying the final products 6 and 8, respectively, after the titration experiments. Because 6 and 8 are linearly p-extended coumarin derivatives, their photophysical properties were evaluated and compared with those of coumarin 4. The results are summarized in Table 1. CouTable 1. Photophysical properties of extended coumarin analogues 6 and 8,[a] and coumarin 4. Compound 6 8 4 lmax ; lem [nm][b] 460; 612 446; 585 405; 475 FF[c] 0.61 0.67 0.35[d]

[a] The data were obtained for a solution of the fluorophore (10 mm) dissolved in the solvent. [b] Measured in aqueous media. [c] Quantum yields were determined by exciting at the lmax by using rhodamine 6G (FF = 0.6) as standard in EtOH.[13, 14] [d] Taken from ref. [14].

Figure 1. UV/vis absorption spectra of a) 5 (b) and its equimolar mixture with Hg2 + ions measured after 1 h (c), and b) 7 (b) and its equimolar mixture with Hg2 + ions measured after 3 h (c). Time-dependent fluorescence changes measured for c) an equimolar mixture of Hg2 + ions and 5, and d) an equimolar mixture of Hg2 + ions and 7. Fluorescence changes upon treatment of e) 5 and f) 7 with varying amount of Hg2 + ions (from bottom to top spectrum: 0, 0.1, 0.2, 0.3, 0.5, 1.0, 1.5, 2.0, and 3.0 equiv). All the measurements were carried out at room temperature with the vinyl ethers (3 mm) in a solution of PBS (pH 7.4) containing DMSO (5 %).

marin 6 and iminocoumarin 8 emitted at much longer wavelengths (Dlem : 110137 nm) with larger quantum yields (FF : 0.61 and 0.67) relative to 4, probably owing to their pextended nature. The extended coumarin compounds themselves can thus be used together with the conventional coumarins for the purpose of multiwavelength imaging. We evaluated whether other metal species also promote the hydrolysis of 5 and 7. Only Hg2 + ions promotes the hydrolysis, whereas other metal species (Mg2 + , Ba2 + , Mn2 + ,

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Probes for Mercury Ions

Co2 + , Fe3 + , Ni2 + , Ag + , Cr2 + , Ca2 + , Cd2 + , Cu2 + , Zn2 + , and Pb2 + as chloride salts) examined do not exhibit any reactivity (Figure 2). These results demonstrate again that the vinyl ether approach to sensing mercury species can secure excellent selectivity over other metal species. A good linear relationship between the fluorescence intensity and Hg2 + ions was obtained for a wide concentration range of Hg2 + ions (0200 ppb, Figure 3). On the basis of the criterion of signal-to-noise ratio ! 3, the detection limits of 5 and 7 are estimated to be 18 and 50 ppb, respectively (Figure S1 in the Supporting Information).

Experimental Section
Only essential experimental information is given here; comprehensive experimental details are provided in the Supporting Information. 7-(Dimethylamino)naphthalen-2-ol (10) A solution of dimethylamine (40 % in H2O, 10.5 mL, 93.5 mmol) was added to a mixture of 2,7-dihydroxynaphthalene (9, 3 g, 18.7 mmol), sodium metabisulfite (7.11 g, 37.4 mmol), and H2O (8 mL) in a sealed tube. The reaction mixture was stirred at 150 8C for 8 h. After cooling to room temperature, dichloromethane (100 mL) was added, and then the organic layer was washed with brine, dried over anhydrous Na2SO4, and concentrated. The residue was purified by chromatography on a silica gel column (eluent: 20 % EtOAc in hexane) to afford 10 as a white solid (2.10 g, 60 %). 1H NMR (CDCl3, 300 MHz, 20 8C): d = 7.667.59 (m, 2 H), 7.057.02 (m, 1 H), 6.966.95 (d, 1 H), 6.856.82 (m, 1 H), 6.786.77 (d, 1 H), 5.10 (s, 1 H), 3.05 ppm (s, 6 H); 13C NMR (CDCl3, 75 MHz, 25 8C): d = 153.88, 149.17, 136.24, 129.39, 128.61, 122.44, 114.22, 113.80, 108.02, 105.32, 40.91 ppm; HRMS: (m/z): calcd for C12H13NO [M+H] + : 187.0997; found: 187.0999. 3-Hydroxy-6-dimethylaminonaphthalene-2-carbaldehyde (13)
1

Figure 2. Fluorescence emission spectra obtained for a 1:1 mixture of 5 or 7 (3 mm) and Hg2 + (b), Mg2 + , Ba2 + , Mn2 + , Co2 + , Fe3 + , Ni2 + , Ag + , Cr2 + , Ca2 + , Cd2 + , Cu2 + , Zn2 + , or Pb2 + ions (all overlapping spectra at the bottom on each plot), or a mixture of Hg2 + and all of the other metal ions (c) in a solution of PBS (pH 7.4) containing DMSO (5 %). a) Data for 5 ; acquired after 1 h with excitation wavelength of 460 nm; b) Data for 7; acquired after 3 h with excitation wavelength of 446 nm.

H NMR (CDCl3, 300 MHz, 25 8C): d = 10.54 (s, 1 H), 9.89 (s, 1 H), 7.90 (s, 1 H), 7.707.67 (d, 1 H), 7.026.98 (m, 2 H), 6.666.65 (d, 1 H), 3.13 ppm (s, 6 H); 13C NMR (CDCl3, 75 MHz, 25 8C): d = 195.26, 156.83, 151.39, 140.62, 137.75, 130.87, 120.63, 119.03, 114.18, 108.73, 103.22, 40.26 ppm; HRMS: (m/z): calcd for C13H13NO2 [M+H] + : 215.0946; found: 215.0946. {[6-(dimethylamino)-3-(vinyloxy)naphthalen-2yl]methylene}dimethylmalonate (5)

H NMR (CDCl3, 500 MHz, 25 8C): d = 8.17 (s, 1 H), 7.76 (s, 1 H), 7.64 7.61 (d, 1 H), 7.107.03 (m, 2 H), 6.766.68 (m, 2 H), 4.914.86 (dd, 1 H), 4.594.56 (dd, 1 H), 3.88 (s, 3 H), 3.82 (s, 3 H), 3.07 ppm (s, 6 H); 13 C NMR (CDCl3, 75 MHz, 25 8C): d = 167.59, 164.93, 153.41, 149.99, 147.76, 138.92, 137.26, 129.97, 129.72, 124.20, 122.48, 119.44, 115.01, 109.82, 104.44, 96.70, 52.48, 40.42 ppm; HRMS: (m/z): calcd for C20H21NO5 [M + H] + : 355.1420; found: 355.1421. 8-Dimethylamino-2-oxo-2 H-benzo[g]chromene-3-methylcarboxylate (6)
1

Figure 3. Fluorescence intensity changes of 5 and 7 (3 mm) depending on the concentration of Hg2 + ions (0200 ppb) in a solution of PBS (pH 7.4) containing DMSO (5 %). a) Data for 5 ; (b) Data for 7. The relative emission intensity was approximated as the peak height.

H NMR (CDCl3, 300 MHz, 25 8C): d = 8.65 (s, 1 H), 7.95 (s, 1 H), 7.80 7.77 (d, 1 H), 7.42 (s, 1 H), 7.187.14 (dd, 1 H), 6.826.81 (d, 1 H), 3.98 (s, 3 H), 3.18 ppm (s, 6 H); 13C NMR (CDCl3, 75 MHz, 25 8C): d = 164.36, 157.68, 151.80, 150.74, 149.95, 138.73, 130.95, 130.41, 123.51, 116.09, 114.38, 114.19, 109.53, 103.89, 52.67, 40.29 ppm; HRMS: (m/z): calcd for C16H13N3O [M+H] + : 297.1001; found: 297.0997.

{[6-(dimethylamino)-3-(vinyloxy)naphthalen-2yl]methylene}malononitrile (7)
1

Conclusions
We have established an efficient synthetic route to novel pextended coumarin derivatives that contain amino substituents and their vinyl ether precursors. Upon hydrolysis promoted by Hg2 + ions, the vinyl ethers generate the linearly p-extended coumarin derivatives that emit at much longer wavelengths with higher quantum yields relative to the conventional coumarin dye. The vinyl ethers selectively sense Hg2 + ions over various metal species in the fluorescence turn-on mode. Further applications of this sensing platform to develop reactive fluorescent probes for other analytes are under active investigation and will be reported elsewhere.

H NMR (CDCl3, 500 MHz, 25 8C): d = 8.75 (s, 1 H), 8.28 (s, 1 H), 7.77 7.73 (d, 1 H), 7.097.04 (m, 2 H), 6.746.67 (m, 2 H), 4.994.94 (dd, 1 H), 4.684.66 (dd, 1 H), 3.17 ppm (s, 6 H); 13C NMR (CDCl3, 75 MHz, 25 8C): d = 153.40, 153.30, 151.70, 147.32, 139.88, 131.73, 131.45, 122.19, 117.18, 115.55, 115.46, 114.40, 109.34, 104.18, 98.19, 40.49 ppm; HRMS: (m/z): calcd for C18H15N3O [M+H] + : 289.1215; found: 289.1213.

8-Dimethylamino-2-imino-2 H-benzo[g]chromene-3-carbonitrile (8)

1

H NMR (DMSO, 300 MHz, 25 8C): d = 8.75 (s, 1 H), 8.30 (s, 1 H), 7.90 (s, 1 H), 7.797.76 (d, 1 H), 7.247.15 (m, 2 H), 6.86 (s, 1 H), 3.08 ppm (s, 6 H); 13C NMR (DMSO, 75 MHz, 25 8C): d = 152.11, 150.45, 150.21, 146.82, 137.81, 130.38, 130.20, 122.28, 115.91, 115.68, 113.37, 108.20, 103.83, 100.56 ppm; HRMS: (m/z): calcd for C16H13N3O [M+H] + : 263.1059; found: 263.1058.

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Acknowledgements
This work was supported by grants from the EPB Center (R112008 05201001) through the National Research Foundation, Korea. [8]

Kyo Han Ahn et al.

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