DOI: 10.1002/ajoc.201200034

Synthesis of p-Extended Coumarins and Evaluation of Their Precursors asReactive Fluorescent Probes for Mercury Ions

Inae Kim, Dokyoung Kim, Sunderraman Sambasivan, and Kyo Han Ahn*[a]

Introduction

Fluorescent probes are valuable tools for the diagnosis ofdisease markers, detection of hazardous compounds, andmonitoring of biochemical processes.[1] Therefore, variousfluorescent probes have been developed for given targetanalytes. Recently, reaction-based or “reactive” fluores-cence probes came into the spotlight owing to their advan-tageous features in achieving very high selectivity and sensi-tivity for target analytes.[2] Furthermore, the reaction basedapproach enables us to realize the “turn-on” fluorescencesensing mode, even in the case of those analytes that areapt to quench the fluorescence.[3] The fluorescence of thereactive probes changes mostly through irreversible chemi-cal transformations that are promoted or catalyzed by theanalytes. Therefore, in this reaction-based approach, a crea-tive combination of a chemical process with the target ana-lyte is required to induce desirable changes in photophysi-cal properties.

Recently, we have introduced a unique sensing protocolfor Hg2+ ions that utilizes the Hg2+-promoted hydrolysis ofaryl vinyl ethers. Fluorescein-derived vinyl ether1 (Scheme 1) senses mercury species through hydrolysis inthe turn-on fluorescent mode with excellent selectivity andsensitivity.[4] Furthermore, this new sensing protocol ena-

bled us to fluorescently image methylmercury, the typicaland notoriously toxic organomercury species, for the firsttime. Mercury is currently used in industrial chemicals, elec-trical apparatus, dental amalgam, and batteries. However,mercury is a highly poisonous element and a widespreadpollutant, which occurrs from natural and anthropogenicsources in the form of reduced and oxidized forms.[5] Mer-cury species cause damage to the central nervous systemand other organs.[6] Therefore, efficient detection and imag-ing probes for mercury species have attracted intense re-search interest.[7] In spite of the tremendous efforts, it isonly recently that fluorescent probes for methylmercuryhave been recognized as viable tools, as demonstrated byour work.[8]

In our continuous study on the reaction-based approachto the Hg2+-promoted hydrolysis of aryl vinyl ethers, wealso developed a vinyl ether of a coumarin precursor (3) asa fluorescent probe for Hg2+ ions. In this case, the Hg2+

-promoted hydrolysis of the vinyl ether group results ina hydroxy-ester intermediate, which undergoes a fast cycli-zation reaction to afford a fluorescent coumarin compound4 (Scheme 2).[9] This hydrolysis-cyclization approach seemsto be more versatile in terms of the structural modificationof the probe for further structure elaboration, relative tothe fluorescein-based one.

Abstract: An efficient synthetic routeto p-extended coumarin derivativesand their precursors that containamino substituents has been devel-oped. The synthesis involves mono-substitution of 2,7-dihydroxynaphtha-lene with dimethylamine through theBucherer reaction. The extended cou-marins emit at much longer wave-lengths and with higher quantumyields relative to a related coumarin.

Two vinyl ethers, precursors of the ex-tended coumarins, were prepared andevaluated as reactive probes for Hg2+

ions. The vinyl ethers undergo hydrol-ysis promoted by Hg2+ ions to pro-duce the extended coumarins at phys-iological pH (pH 7.4) and room tem-perature, which is accompanied byfluorescence in the “turn-on” mode.The vinyl ethers do not respond tovarious other

metal species. The extended coumar-ins and their precursors are versatilesensing platforms for Hg2+ ions, aswell as for the development of relatedreaction-based sensing systems.

Keywords: coumarins · fluorescentprobes · iminocoumarins ·mercury · vinyl ethers

[a] I. Kim, D. Kim, Dr. S. Sambasivan, Prof. K. H. AhnDepartment of Chemistry and Center for Electro-Photo Behaviorsin Advanced Molecular SystemsPohang University of Science and Technology (POSTECH)Pohang, 790-784 (Republic of Korea)Fax: (+82) 54-279-5877E-mail : ahn@postech.ac.kr

Supporting information for this article is available on the WWWunder http://dx.doi.org/10.1002/ajoc.201200034.

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

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Coumarins, including 4, constitute an important class offluorophores that are widely used for sensing and imagingpurposes.[10] In spite of their usefulness as fluorophores, p-extended coumarin derivatives, which would have improvedphotophysical properties, remain largely unexplored. In par-ticular, the extended coumarin derivatives with electron-do-nating substituents, such as amino groups, are rare. To ourknowledge, only one example is known so far, which is anangularly extended coumarin with a dimethylamino sub-stituent.[11] We attempted to synthesize a key intermediateof the extended coumarin by following the literature, butfound that it required a multistep synthesis that is low-yielding. To explore an efficient synthetic route toward p-

extended coumarins and also to develop related fluorescentprobes with improved photophysical properties, we havestudied new extended coumarins and their precursors thatare related to 3, but with a linearly p-extended feature. Wedesigned the extended coumarin precursors 5 and 7(Scheme 3), which undergo Hg2+-promoted hydrolysis togive 6 and 8,[12] respectively. These precursors have a linearp-extended feature with respect to 4 and its analogue.

Results and Discussion

Synthesis

An efficient synthesis of compounds 5–8 is outlined inScheme 4. Monosubstitution of 2,7-dihydroxynaphthalene(9) by the Bucherer reaction by using dimethylamine af-forded 7-(dimethylamino)naphthalen-2-ol (10) in 60 %yield. Protection of the hydroxy group of 10 as its methoxy-methyl ether, then directed lithiation with tert-butyllithiumand subsequent formylation with N,N-dimethylformamide(DMF) afforded aldehyde 12 in 40 % overall yield after thetwo steps. Deprotection of the methoxymethyl group of 12with aqueous HCl afforded hydroxyaldehyde 13, which isa platform compound for further structure elaboration. Vi-nylation of the hydroxyl group of 13 was most efficientlycarried out by a conventional two-step process of bromoe-thylation to give 14, then b-elimination to give vinyl ether15 in 44 % overall yield. Finally, the Knoevenagel conden-sation at the formyl group of 15 with dimethylmalonate ormalononitrile in presence of piperidine afforded 5 or 7, re-spectively. The p-extended coumarin derivatives 6 and 8can be prepared directly from the key intermediate 13 bythe Knoevenagel condensation by using dimethylmalonateor malononitrile, respectively, in presence of piperidine.The detailed experimental procedures, 1H NMR and13C NMR spectra, as well as HRMS data are provided inthe Supporting Information. The established synthetic routeis versatile and thus would also be useful for the synthesisof other functionalized naphthalene derivatives.

Sensing Properties

With vinyl ethers 5 and 7 in hand, we have evaluated theirresponse to Hg2+ ions by monitoring UV/vis absorptionand emission changes. All the spectroscopic measurementswere carried out in solutions of phosphate buffered saline(PBS) at physiological pH (pH 7.4) containing dimethylsulf-oxide (DMSO, 5 %) at room temperature.

Vinyl ether 5 has one major absorption band centered at400 nm (Figure 1 a), but emits little fluorescence when ex-cited at the absorption maximum (the bottom spectrum inFigure 1 e). When 5 was treated with an equimolar amountof Hg2+ ions, the resulting solution showed turn-on typefluorescence change (lem at 612 nm) upon excitation at460 nm, together with a bathochromic shift in the absorp-tion maximum (lmax: 400 nm!460 nm). The time-depen-dent fluorescence change is shown in Figure 1 c. Such spec-

Abstract in Korean:

Scheme 3. Aryl vinyl ethers 5 and 7 designed as reactive probes forHg2+ions, which can produce p-extended coumarin derivatives 6 and 8,respectively.

Scheme 2. Reactive fluorescent probe 3 for Hg2+ions, which producescoumarin 4.

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tral changes suggest that 5undergoes the Hg2+-promot-ed hydrolysis and producesthe extended coumarin 6,which emits orange-red fluo-rescence.

Spectral changes were alsodetected in the case of 7before and after treatmentwith Hg2+ ions. Vinyl ether 7showed little change in theabsorption maximum beforeand after treatment withHg2+ ions (Figure 1 b), butshowed turn-on fluorescencechanges (lem at 585 nm)upon treatment with Hg2+

ions when excited at 446 nm.Vinyl ether 5 responds toHg2+ ions faster than vinylether 7 does, as the time-de-pendent fluorescencechanges show (Figure 1 c and1 d). Both 5 and 7 showed

gradual increase in the fluorescence upon titration withHg2+ ions (Figure 1 e and 1 f). Vinyl ethers 5 and 7 emitlittle fluorescence in aqueous media. It is likely that thefree rotation of the dimethylmalonate and the dicyanovinylgroups causes nonradiative decay.

The supposed Hg2+-promoted hydrolysis of 5 and 7 wasconfirmed by identifying the final products 6 and 8, respec-tively, after the titration experiments. Because 6 and 8 arelinearly p-extended coumarin derivatives, their photophysi-cal properties were evaluated and compared with those ofcoumarin 4. The results are summarized in Table 1. Cou-

marin 6 and iminocoumarin 8 emitted at much longerwavelengths (Dlem: 110–137 nm) with larger quantum yields(FF: 0.61 and 0.67) relative to 4, probably owing to their p-extended nature. The extended coumarin compounds them-selves can thus be used together with the conventional cou-marins for the purpose of multiwavelength imaging.

We evaluated whether other metal species also promotethe hydrolysis of 5 and 7. Only Hg2+ ions promotes the hy-drolysis, whereas other metal species (Mg2+ , Ba2+ , Mn2+ ,

Scheme 4. Reagents and conditions: a) Na2S2O5, Me2NH, H2O, 150 8C, 8 h, 60 %; b) NaH, DMF, chloromethylmethyl ether, �15 8C, 7 h, 95 %; c) tBuLi, diethyl ether, DMF, �15 8C, 2 h, 52 %; d) HCl, iPrOH, 60 8C, 3 h,97%; e) 1,2-dibromoethane, KOH, H2O, Bu4NOH, 50 8C, 6 h, 74 %; f) tBuOK, DMSO, RT, 3 h, 59 %; g) dime-thylmalonate, piperidine, THF, reflux, 6 h, 38 %; h) malononitrile, piperidine, EtOH, RT, 1 h, 70%; i) dimethyl-malonate, piperidine, THF, reflux, 1 h, 70%; j) malononitrile, piperidine, EtOH, RT, 1 h, 90%. MOM =me-thoxymethyl ether.

Figure 1. UV/vis absorption spectra of a) 5 (b) and its equimolar mix-ture with Hg2+ ions measured after 1 h (c), and b) 7 (b) and itsequimolar mixture with Hg2+ ions measured after 3 h (c). Time-de-pendent fluorescence changes measured for c) an equimolar mixture ofHg2+ ions and 5, and d) an equimolar mixture of Hg2+ ions and 7. Fluo-rescence changes upon treatment of e) 5 and f) 7 with varying amountof 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 tem-perature with the vinyl ethers (3 mm) in a solution of PBS (pH 7.4) con-taining DMSO (5 %).

Table 1. Photophysical properties of extended coumarin analogues 6and 8,[a] and coumarin 4.

Compound lmax; lem [nm][b] FF[c]

6 460; 612 0.618 446; 585 0.674 405; 475 0.35[d]

[a] The data were obtained for a solution of the fluorophore (10 mm) dis-solved in the solvent. [b] Measured in aqueous media. [c] Quantumyields 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].

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Co2+ , Fe3+ , Ni2+ , Ag+ , Cr2+ , Ca2+ , Cd2+ , Cu2+ , Zn2+ , andPb2+ as chloride salts) examined do not exhibit any reactiv-ity (Figure 2). These results demonstrate again that thevinyl ether approach to sensing mercury species can secureexcellent selectivity over other metal species. A good linearrelationship between the fluorescence intensity and Hg2+

ions was obtained for a wide concentration range of Hg2+

ions (0–200 ppb, Figure 3). On the basis of the criterion ofsignal-to-noise ratio�3, the detection limits of 5 and 7 areestimated to be 18 and 50 ppb, respectively (Figure S1 inthe Supporting Information).

Conclusions

We have established an efficient synthetic route to novel p-extended coumarin derivatives that contain amino substitu-ents and their vinyl ether precursors. Upon hydrolysis pro-moted by Hg2+ ions, the vinyl ethers generate the linearlyp-extended coumarin derivatives that emit at much longerwavelengths with higher quantum yields relative to the con-ventional coumarin dye. The vinyl ethers selectively senseHg2+ ions over various metal species in the fluorescenceturn-on mode. Further applications of this sensing platformto develop reactive fluorescent probes for other analytesare under active investigation and will be reported else-where.

Experimental Section

Only essential experimental information is given here; comprehensiveexperimental 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) wasadded 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 sealedtube. The reaction mixture was stirred at 150 8C for 8 h. After cooling toroom temperature, dichloromethane (100 mL) was added, and then theorganic layer was washed with brine, dried over anhydrous Na2SO4, andconcentrated. The residue was purified by chromatography on a silicagel 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=7.66–7.59 (m,2H), 7.05–7.02 (m, 1H), 6.96–6.95 (d, 1 H), 6.85–6.82 (m, 1H), 6.78–6.77(d, 1 H), 5.10 (s, 1 H), 3.05 ppm (s, 6H); 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)

1H NMR (CDCl3, 300 MHz, 25 8C): d =10.54 (s, 1 H), 9.89 (s, 1 H), 7.90(s, 1 H), 7.70–7.67 (d, 1 H), 7.02–6.98 (m, 2H), 6.66–6.65 (d, 1 H),3.13 ppm (s, 6H); 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-2-yl]methylene}dimethylmalonate (5)

1H NMR (CDCl3, 500 MHz, 25 8C): d =8.17 (s, 1 H), 7.76 (s, 1 H), 7.64–7.61 (d, 1H), 7.10–7.03 (m, 2H), 6.76–6.68 (m, 2H), 4.91–4.86 (dd, 1H),4.59–4.56 (dd, 1 H), 3.88 (s, 3H), 3.82 (s, 3H), 3.07 ppm (s, 6H);13C 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 forC20H21NO5 [M+ H]+ : 355.1420; found: 355.1421.

8-Dimethylamino-2-oxo-2H-benzo[g]chromene-3-methylcarboxylate (6)

1H NMR (CDCl3, 300 MHz, 25 8C): d =8.65 (s, 1 H), 7.95 (s, 1 H), 7.80–7.77 (d, 1H), 7.42 (s, 1 H), 7.18–7.14 (dd, 1 H), 6.82–6.81 (d, 1H), 3.98 (s,3H), 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 forC16H13N3O [M+H]+ : 297.1001; found: 297.0997.

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

1H NMR (CDCl3, 500 MHz, 25 8C): d =8.75 (s, 1 H), 8.28 (s, 1 H), 7.77–7.73 (d, 1H), 7.09–7.04 (m, 2H), 6.74–6.67 (m, 2H), 4.99–4.94 (dd, 1H),4.68–4.66 (dd, 1 H), 3.17 ppm (s, 6H); 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)

1H NMR (DMSO, 300 MHz, 25 8C): d =8.75 (s, 1 H), 8.30 (s, 1H), 7.90(s, 1 H), 7.79–7.76 (d, 1H), 7.24–7.15 (m, 2 H), 6.86 (s, 1H), 3.08 ppm (s,6H); 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.

Figure 2. Fluorescence emission spectra obtained for a 1:1 mixture of 5or 7 (3 mm) and Hg2+ (b), Mg2+, Ba2+ , Mn2+ , Co2+ , Fe3+ , Ni2+, Ag+ ,Cr2+ , Ca2+ , Cd2+ , Cu2+ , Zn2+ , or Pb2+ ions (all overlapping spectra atthe bottom on each plot), or a mixture of Hg2+ and all of the othermetal 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.

Figure 3. Fluorescence intensity changes of 5 and 7 (3 mm) depending onthe concentration of Hg2+ ions (0–200 ppb) in a solution of PBS(pH 7.4) containing DMSO (5 %). a) Data for 5 ; (b) Data for 7. The rel-ative emission intensity was approximated as the peak height.

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Probes for Mercury Ions

Acknowledgements

This work was supported by grants from the EPB Center (R11–2008–052–01001) through the National Research Foundation, Korea.

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Received: May 16, 2012Published online: July 2, 2012

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