A Combinatorial Approach To Discover New Chelators for Optical

Anal. Chem. 2000, 72, 5250-5257

A Combinatorial Approach To Discover New Chelators for Optical Metal Ion Sensing Ferenc Szurdoki,† Dahai Ren, and David R. Walt*

Max Tishler Laboratory for Organic Chemistry, Department of Chemistry, Tufts University, 62 Talbot Avenue, Medford, Massachusetts 02155

This paper reports the synthesis and characterization of metal-binding indicators with diverse optical responses on exposure to various heavy metal ions. A combinatorial approach, based on azo coupling of diazonium salts with either phenolic compounds or aromatic amines, generated a library of azo dyes. Each reaction mixture, containing the product(s) of azo coupling, was incubated with a series of solutions, each containing a different heavy metal ion. The absorbance and, in some cases, fluorescence spectra of the resulting complexes were recorded. The metal chelates showed extensive diversity in their UV-visible absorbance spectra upon binding to selected metal ions. Of the azo dyes prepared, the terdentate dyes were particularly useful, providing distinct spectral responses to three or more metal ions in a panel of seven. Rapid analysis of metals in large numbers of samples is important for chemical and bioprocess monitoring as well as for environmental and clinical applications. On-site, real-time monitoring of hazardous heavy metal ions with the simultaneous determination of multiple metal ions is a longstanding goal. Established instrumental methods used to determine metal ions include atomic absorption1a,b and fluorescence1c spectrometry, inductively coupled plasma mass spectrometry,1c,d and neutron activation analysis.1e These analytical techniques1 are highly sensitive and specific but utilize expensive instruments housed in centralized facilities and have low sample throughput. A variety of metal analysis methods have emerged to overcome these limitations.2 Of particular note, research has focused on developing sensors for remote monitoring of toxic and carcinogenic metals that impose serious human and environmental health hazards.2c,3 A new sensing strategy is to employ an array of sensing elements, each displaying cross-reactivities for either all or some of the target analytes, to individually quantify target species in mixtures.2c,3d,4 Computational pattern analysis methods are utilized to deconvolute data generated by such arrays.2c,4 Several groups * Corresponding author: (phone) 617 627 3470; (fax) 617 627 3443; (e-mail) [email protected]. † Current address: Polestar Technologies, Inc., 220 Reservoir St., Suite 32, Needham Heights, MA 02194. (1) (a) Sakamoto, H.; Taniyama, J.; Yonehara, N. Anal. Sci. 1997, 13, 771775. (b) Jackson, K. W. Anal. Chem. 2000, 72, 159R-167R. (c) Armstrong, H. L.; Corns, W. T.; Stockwell, P. B.; O’Connor, G.; Ebdon, L.; Evans, E. H. Anal. Chim. Acta 1999, 390, 245-253. (d) Marchante-Gayo´n, J. M.; Mun ˜iz, C. S.; Alonso, J. I. G.; Sanz-Medel, A. Anal. Chim. Acta 1999, 400, 307320. (e) Sun, Y. C.; Yang, J. Y. Anal. Chim. Acta 1999, 395, 293-300.

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have applied the sensor array technique for the simultaneous determination of multiple metal ions.2c,3d,4c,5 For instance, metal ion concentrations were measured with cross-reactive fluorescent metal chelators using full-spectrum analysis and multivariate calibration in our laboratory.5d The performance of such metal analysis methods is diminished by the lack of recognition of some of the target metal ions by the chelators and by the uniformity of (2) (a) Czarnik, A. W., Ed. Fluorescent Chemosensors for Ion and Molecular Recognition; ACS Symposium Series 538; American Chemical Society: Washington, DC, 1993. (b) Walker, B.; Kasianowicz, J.; Krishnasastry, M.; Bayley, H. Protein Eng. 1994, 7, 655-662. (c) Mu ¨ller-Ackermann, E.; Panne, U.; Niessner, R. Anal. Methods Instrum. 1995, 2, 182-189. (d) Szurdoki, F.; Kido, H.; Hammock, B. D. Bioconjugate Chem. 1995, 6, 145-149. (e) Walkup, G. K.; Imperiali, B. J. Am. Chem. Soc. 1997, 119, 3443-3450. (f) Wang, B.; Wasielewski, M. R. J. Am. Chem. Soc. 1997, 119, 12-21. (g) Yoon, J.; Ohler, N. E.; Vance, D. H.; Aumiller, W. D.; Czarnik, A. W. Tetrahedron Lett. 1997, 38, 3845-3848. (h) Ramachandram, B.; Samanta, A. J. Phys. Chem. A 1998, 102, 10579-10587. (i) Ramanathan, S.; Shi, W. P.; Rosen, B. P.; Daunert, S. Anal. Chim. Acta 1998, 369, 189-195. (j) Blake, D. A.; Blake, R. C., II; Khosraviani, M.; Pavlov, A. R. Anal. Chim. Acta 1998, 376, 13-19. (k) Thompson, R. B.; Maliwal, B. P.; Fierke, C. A. Anal. Biochem. 1999, 267, 185-195. (l) Kido, H.; Szurdoki, F.; Gustin, M. S.; Hammock, B. D. In Metals and Genetics; Sarkar, B., Ed.; Kluwer Academic/Plenum Publishers: New York, 1999; pp 101-116. (m) Hennrich, G.; Sonnenschein, H.; Resch-Genger, U. J. Am. Chem. Soc. 1999, 121, 50735074. (n) Grandini, P.; Mancin, F.; Tecilla, P.; Scrimin, P.; Tonellato, U. Angew. Chem., Int. Ed. 1999, 38, 3061-3064. (o) Fabbrizzi, L.; Licchelli, M.; Parodi, L.; Poggi, A.; Taglietti A. Eur. J. Inorg. Chem. 1999, 35-39. (p) de Silva, A. P.; Eilers, J.; Zlokarnik, G. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 8336-8337. (q) Westhoff, C. M.; Lopez, O.; Goebel. P.; Carlson, L.; Carlson, R. R.; Wagner, F. W.; Schuster, S. M.; Wylie, D. E. Proteins 1999, 37, 429-440. (r) Bandyopadhyay, P.; Bharadwaj, P. K.; Roy, M. B.; Dutta, R.; Ghosh, S. Chem. Phys. 2000, 255, 325-334. (s) Wu, X. Q.; Kim, J.; Dordick, J. S. Biotechnol. Prog. 2000, 16, 513-516. (t) Hirayama, E.; Sugiyama, T.; Hisamoto, H.; Suzuki, K. Anal. Chem. 2000, 72, 465-474. (u) Shamsipur, M.; Yousefi, M.; Ganjali, M. R. Anal. Chem. 2000, 72, 23912394. (3) (a) Lin, Z. H.; Booksh, K. S.; Burgess, L. W.; Kowalski B. R. Anal. Chem. 1994, 66, 2552-2560. (b) Schweyer, M. G.; Andle, J. C.; McAllister, D. J.; Vetelino, J. F. Sens Actuators B 1996, 35, 170-175. (c) Oehme, I.; Wolfbeis, O. S. Mikrochim. Acta 1997, 126, 177-192. (d) Vlasov, Y.; Legin, A.; Rudnitskaya, A. Sens Actuators B 1997, 44, 532-537. (e) Herdan, J.; Feeney, R.; Kounaves, S. P.; Flannery, A. F.; Storment, C. W.; Kovacs, G. T. A.; Darling, R. B. Environ. Sci. Technol. 1998, 32, 131-136. (4) (a) Cheung, P. Y. K.; Kauvar, L. M.; Engqvist-Goldstein, A. E.; Ambler, S. M.; Karu, A. E.; Ramos, L. S. Anal. Chim. Acta 1993, 282, 181-192. (b) Dickinson, T. A.; White, J.; Kauer, J. S.; Walt, D. R. A. Nature 1996, 382, 697-700. (c) Lavigne, J. J.; Savoy, S.; Clevenger, M. B.; Ritchie, J. E.; McDoniel, B.; Yoo, S.-J.; Anslyn, E. V.; McDevitt, J. T.; Shear, J. B.; Neikirk, D. J. Am. Chem. Soc. 1998, 120, 6429-6430. (d) Bessant, C.; Saini, S. Anal. Chem. 1999, 71, 2806-2813. (5) (a) Engstrom, E.; Karlberg, B. J. Chemom. 1996, 10, 509-520. (b) Wrobel, K.; Lopez de Alba, P. L.; Lopez-Martinez, L. Anal. Lett. 1997, 30, 717-737. (c) da Silva, J. C. G. E.; Oliveira, C. J. S. Talanta 1999, 49, 889-897. (d) Tabacco, M. B.; Chadha, S.; Hammond, J. D.; Walt, D. R. Unpublished results, 1999. 10.1021/ac0008542 CCC: $19.00

© 2000 American Chemical Society Published on Web 09/28/2000

Figure 1. Scheme for azo dye synthesis.

Figure 2. Putative chelate structures formed between various library members and metal ions.

individual sensor responses.2c,5d This approach requires optical chemosensor materials that respond to multiple heavy metal ions. Few such chelators are known,2f,g,m,3c,6 which is undoubtedly a consequence of the bias toward specificity. The objective of this work was to develop a panel of azo dye chelators capable of binding to a variety of heavy metal ions and producing diverse optical responses upon chelation (Figures 1 and 2). Such chelators could be used for the optical detection of multiple metal ions in a sensor array format.2c,s,5a,b We sought to distinguish between various metal chelates using optical changes in the absorbance or fluorescence maximum wavelengths, signal intensities, and/or spectral shapes. Both diversity between response patterns of multiple chelators and heterogeneity among signals produced by an individual chelator upon exposure to various metal ions are of value for sensor array applications. Azo dyes and their metal chelates are used primarily in the textile industry and in photoelectronic devices.7 Metal complexes of azo compounds also have recently been employed in optical recording media.7b The metal chelating properties of azo dyes have been utilized in a number of analytical methods,5a-c,8 including sensors,3c,9 to determine metal ions. EXPERIMENTAL SECTION General Information. All chemicals were obtained from commercial suppliers and used without purification. Metal salts, common reagents, and solvents were purchased from Aldrich (Milwaukee, WI) and Lancaster (Windham, NH). Metal salts of (6) (a) Blanco, M.; Coello, J.; Gonza´lez, F.; Iturriaga, H.; Maspoch, S.; Puigdome`nech, A. R. Talanta 1996, 43, 1489-1496. (b) Giraudi, G.; Baggiani, C.; Giovannoli, C.; Marletto, C.; Vanni A. Anal. Chim. Acta 1999, 378, 225-233. (c) Bodenant, B.; Weil, T.; Businelli-Pourcel, M.; Fages, F.; Barbe, B.; Pianet, I.; Laguerre, M. J. Org. Chem. 1999, 64, 7034-7039. (7) (a) Gordon, P. F.; Gregory, P. Organic Chemistry in Color; Springer-Verlag: Berlin, Heidelberg, New York, 1983. (b) Wang, S.; Shen, S.; Xu, H.; Gu, D.; Yin, J.; Tang, X. Dyes Pigm. 1999, 42, 173-177. (8) (a) Farias, P. A. M.; Ohara, A. K.; Takase, I.; Ferreira, S. L. C.; Gold, J. S. Anal. Chim. Acta 1993, 271, 209-215. (b) Ding, X.-J.; Mou, S.-F.; Liu, K.N.; Siriraks, A.; Riviello, J. Anal. Chim. Acta 2000, 407, 319-326. (9) (a) Wang, K. M.; Seiler. K.; Rusterholz, B.; Simon, W. Analyst 1992, 117, 57-60. (b) Malcik, N.; Oktar, O.; Ozser, M. E.; Caglar, P.; Bushby, L.; Vaughan, A.; Kuswandi, B.; Narayanaswamy, R. Sens Actuators B 1998, 53, 211-221. (c) Ayora-Can ˜ada, M. J.; Pascual-Reguera, M. I.; Molina-Dı´az, A. Anal. Chim. Acta 1998, 375, 71-80.

analytical grade or equivalent used to form chelates were as follows: cadmium chloride, copper(II) sulfate, iron(II) sulfate, lead(II) chloride, mercury(II) acetate, nickel(II) sulfate, zinc chloride. 3-(N-Morpholino)propanesulfonic acid (MOPS) hemisodium salt from Sigma (St. Louis, MO) was used to prepare MOPS buffer. Preparative TLC purifications were carried out on PLK5F 150A (F254, 20 cm × 20 cm × 1000 µm) silica gel plates (Whatman Inc., Clifton, NJ). Uncorrected melting points were taken with a capillary apparatus. UV-visible absorbance values were recorded on a U-2000 spectrometer (Hitachi Ltd., Tokyo, Japan). Infrared spectra were measured on a Mattson 1000 FT-IR spectrophotometer (Unicam Ltd., Cambridge, U.K.) in KBr pellets; wavenumber (cm-1) values are given. 1H NMR spectra were obtained with a 300-MHz Bruker AM300 NMR spectrometer (Bruker NMR, Billerica, MA). Chemical shifts (δ) are reported in ppm relative to tetramethylsilane as internal reference. Electron impact (EI) and chemical ionization (CI) mass spectrometry (MS) data were recorded on a 5988A mass spectrometer (HewlettPackard, Palo Alto, CA) at 70 eV and reported as m/z (relative intensity). Matrix-assisted laser desorption/ionization (MALDI) mass spectra were obtained on a Voyager RP (PerSeptive Biosystems, Framingham, MA) time-of-flight MALDI mass spectrometer. High-resolution MS (HR-MS) was performed on an SX-102A (JEOL) spectrometer operating in fast atom bombardment (FAB) mode using nitrobenzyl alcohol (NBA) plus NaI matrix. Chelates were screened using 96-well microtiter plates. A SPECTRAmax 250 microplate spectrophotometer (Molecular Devices, Sunnyvale, CA) was employed to record absorbance spectra. Fluorescence excitation and emission spectra were measured by a SPECTRAmax Gemini dual-scanning microplate spectrofluorometer (Molecular Devices). Data, recorded using the software Softmax PRO (Molecular Devices), were imported into Microsoft Excel 97 spreadsheets and sorted by a self-designed macro for data analysis. Spectra were plotted using KaleidaGraph version 3.0 software (Synergy Software, Reading, PA). Synthesis. The reactions were carried out in glass vials with magnetic stirring. 1. General Procedures for Diazotation and Azo Coupling. Most reactions were carried out in aqueous solutions (vide infra). However, in a few cases, aqueous organic solvent (e.g., ethanol) was used to enhance the solubility of a reactant. 1.1. Azo Coupling with a Phenolic Compound. In a typical reaction, a primary aromatic amine (1a-1h, 0.1 mmol) was dissolved in 0.25 M hydrochloric acid (1.0 mL), cooled to 0-4 °C; 0.12 M sodium nitrite solution (1.0 mL) was then added to the solution dropwise at 4 °C. If the amine had other basic group(s), an additional equivalent of acid was used per basic group (see section 2.1.). The reaction mixture was stirred at 0-4 °C for 15 min to yield a solution of the corresponding diazonium salt (2a-2h). Excess nitrous acid was then quenched by adding a small amount of urea to the solution at 0-4 °C. A commercially available diazonium salt (2i-2k, 0.1 mmol) was dissolved in water (3 mL). Meanwhile, the phenolic compound (3aa-3bl, 0.1 mmol) was dissolved in 80 mM sodium hydroxide solution (5 mL). The solution was then cooled to 4 °C. If the phenol had other acidic group(s), an additional equivalent of base was used per acidic group. The cold solution of the diazonium salt (2a-2k) was added dropwise to this solution at 4 °C without delay. The mixture was Analytical Chemistry, Vol. 72, No. 21, November 1, 2000

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stirred at 4 °C for 30 min and at room temperature overnight. The pH value of the solution was maintained between 9 and 10 by addition of either diluted sodium hydroxide or hydrochloric acid solution. The resulting reaction mixture containing the azo compound 4 was stored at 4 °C in the dark. 1.2. Azo Coupling with an Aromatic Amino Compound. An aromatic amino compound (3bm-3bx, 0.1 mmol) was dissolved in 0.1 M sodium acetate buffer solution (pH 5.5, 5 mL). The cold solution of the diazonium salt, prepared as described above, was added dropwise to this solution at 4 °C. The mixture was stirred at 4 °C for 30 min and at room temperature overnight. The resulting reaction mixture containing the azo compound 4 was stored at 4 °C in the dark. 2. Preparation of Specific Examples. 2.1. 5-Hydroxy-6(8-quinolinylazo)-1,3-benzodioxole (4aah). 8-Aminoquinoline (1a) was diazotized and coupled with 5-hydroxy-1,3-benzodioxole (3ah) by essentially the same procedure described in section 1.1. In brief, a solution of 8-aminoquinoline (1a, 144 mg, 1.0 mmol) in 0.5 M hydrochloric acid (7 mL) was treated with 1.2 M sodium nitrite solution (1.0 mL). Excess nitrous acid was then quenched by adding a small amount of urea to the solution at 0-4 °C. Meanwhile, 5-hydroxy-1,3-benzodioxole (3ah, 166 mg, 1.2 mmol) was dissolved in 0.2 M sodium hydroxide solution (20 mL). After completion of the azo coupling reaction, the pH of the azo compound solution was adjusted to 5 with diluted acetic acid. The resulting precipitate was collected by filtration and washed by cold water. The crude product was dried in a vacuum desiccator and then recrystallyzed from ethyl acetate/hexane to yield 174 mg (59%) of red powder (4aah), mp 212-215 °C: IR 3440, 1512, 1441, 1412, 1242, 1194, 1020, 859, 833, 786 cm-1; 1H NMR (CDCl3) δ 17.1 (s, 1H), 9.0 (dd, J ) 4.2 Hz, 1.6 Hz, 1H), 8.18 (dd, J ) 8.3 Hz, 1.6 Hz, 1H), 8.16 (m, 1H), 7.59 (m, 2H), 7.48 (dd, J ) 8.3 Hz, 4.2 Hz, 1H), 6.56 (s, 1H), 6.21 (s, 1H), 6.0 (s, 2H); MS (CI/CH4) [C16H11N3O3] 294 [M + H]+; HR-MS (FAB/NBA, NaI) 316.0698 [M + Na]+ (theoretical mass 316.0698). 2.2. 8-Hydroxy-7-(2-methoxy-4-nitrophenylazo)quinoline5-sulfonic Acid Sodium Salt Hydrate (4jaf). 2-Methoxy-4nitrobenzenediazonium tetrafluoroborate (Aldrich’s Fast Red B tetrafluoroborate salt, 2j, 95%, 281 mg, 1.0 mmol) was dissolved in water (25 mL) and cooled to 0-4 °C. 8-Hydroxyquinoline-5sulfonic acid hydrate (Aldrich, 98%, 3af, 241 mg, 1.05 mmol) was dissolved in 2 M sodium hydroxide solution (2.5 mL). The azo coupling reaction was carried out as described in section 1.1; the pH of the solution was then adjusted to 5 by diluted acetic acid. Sodium chloride was added to enhance precipitation of the product. The precipitate was filtered and washed with cold water. The crude product was dissolved in diluted sodium hydroxide solution, and the pH of the resulting solution was adjusted to 5 by diluted acetic acid again. The solid product was isolated using the same method as above and then dried in a vacuum desiccator to yield 205 mg of dark red powder (4jaf), mp > 300 °C: IR 36003140, 1623,1587, 1502, 1397, 1338, 1222, 1044, 648 cm-1; 1H NMR (DMSO-d6) δ 8.93 (dd, J ) 8.3 Hz, 1.6 Hz, 1H), 8.83 (dd, J ) 4.3 Hz, 1.5 Hz, 1 H), 7.97-8.07 (m, 3H), 7.75 (dd, J ) 8.3 Hz, 4.3 Hz, 1 H), 7.69 (s, 1 H) 4.16 (s, 3H); MS (MALDI) [C16H12N4O7S] 427 [M + Na]+, 449 [M - H + 2Na]+; Anal. (C16H11N4O7SNa)Na: calcd, 5.39, found, 4.03. The sodium content of the sample indicated that the monosodium salt (sulfonate) was the major 5252

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component in agreement with literature data for structurally related compounds.10,11 The IR spectrum showed the presence of water; similar compounds were reported to crystallize with various amounts of water.10,11 The water content contributed to the difference between the sodium content of the sample and the calculated amount for the anhydrous monosodium salt. Thus, the general formula C16H12-xN4O7SNax‚(H2O)y was proposed for the components of this product. No further effort was made to determine the exact composition and ratio of these forms because they should have no considerable influence on metal chelation under conditions used in our experiments. 2.3. 2-(8-Quinolinylazo)-3,4,5-trimethylphenol (4aan). 8-Aminoquinoline (1a, 144 mg, 1.0 mmol) was diazotized as described in section 2.1. Meanwhile, 3,4,5-trimethylphenol (3an, 136 mg, 1.0 mmol) was dissolved in 0.2 M sodium hydroxide solution (20 mL). The azo coupling reaction was carried out as described in section 1.1. Saturated sodium chloride solution was added; the product was then extracted with ethyl acetate (5 × 10 mL). The combined organic extracts were washed successively with water (10 mL), 1 M hydrochloric acid (3 × 5 mL), saturated NaCl (2 × 5 mL), 5% Na2CO3 (3 × 5 mL), and saturated NaCl solutions (4 × 5 mL). The organic solution was dried over Na2SO4 and evaporated in vacuo. One-fourth of the crude product was purified by preparative thin-layer chromatography (ethyl acetate/ hexane/acetic acid, 375:125:1) to yield 34 mg (47%) of dark red, amorphous material (4aan): IR 3456, 1609, 1476, 1384, 1088, 824, 788 cm-1; 1H NMR (CDCl3) δ 15.95 (s, 1H), 9.08 (dd, J ) 4.1 Hz, 1.4 Hz, 1H), 8.22 (dd, J ) 8.3 Hz, 1.4 Hz, 1H), 8.20 (d, J ) 7.4 Hz, 1H), 7.85 (d, J ) 8.0 Hz, 1H), 7.65 (m, 1H), 7.51 (dd, J ) 8.3 Hz, 4.1 Hz, 1H), 6.70 (s, 1 H), 2.68 (s, 3H), 2.30 (s, 3H), 2.20 (s, 3H); MS (EI) [C18H17N3O] 129 (100), 291 (9.5, [M]+); MS (CI/CH4) 292 [M + H]+; HR-MS (FAB/NBA, NaI) 314.1273 [M + Na]+ (theoretical mass, 314.1270). High-Throughput Screening. 1. General Screening Procedure. Either 0.1 M MOPS (pH 7.0, 294 µL/well) or 0.1 M sodium acetate buffer solution (pH 5.5, 294 µL/well) and the reaction mixture containing the azo compound (4, 3 µL/well, obtained by the General Procedures for Diazotation and Azo Coupling and diluted to 10 mM concentration) were dispensed into the wells of a 96-well microplate. If the azo compound 4 was not completely soluble in any of the above buffers, ethanol (100 µL/well), one of the above buffer solutions (194 µL), and the reaction mixture containing the azo dye (4, 3 µL/well) were mixed in the wells to form a clear solution. Metal complex solutions (0.1 mM) were prepared by adding the appropriate 10 mM metal ion solution in 0.1 M sodium acetate buffer (pH 5.5, 3 µL/well) to the wells containing the solution of the azo compound. The contents of the wells were mixed, and the plate was read with either an absorbance or a fluorescence microplate reader after 20 min. The wells of the first column of the microplate contained the buffer solution (blank) and the seven individual metal ion solutions in the same solvent. The wells of the other microplate columns contained the solution of an azo dye and the solutions of seven individual metal ions. Chelation experiments for only visual inspection of color changes were carried out in plastic (10) Huang, H.; Chikushi, H.; Nakamura, M.; Kai, F. Bull. Chem. Soc. Jpn. 1990, 63, 1985-1993. (11) Noritake, M.; Okamoto, K.; Hidaka, J.; Einaga, H. Bull. Chem. Soc. Jpn. 1990, 63, 353-358.

Figure 3. Structures of aromatic amino compounds 1a-1h and commercially available diazonium salts 2i-2k.

Eppendorf tubes in a similar manner. When a starting material (e.g., 3bj) used in the synthesis of an azo dye itself displayed chelating properties, control experiments were performed to evaluate the spectral responses of this reactant upon exposure to the panel of metal ions. Only the spectral responses of the corresponding dye 4, which were considerably different from those observed in the control experiments, were taken into account in azo dye categorization. 2. Comparison of Spectra of Chelates 4hay‚M2+ Based on Dye 4hay Obtained by the General Synthetic Procedure and from a Commercial Source. A solution of dye 4hay was prepared by the usual method from diazotized 4-nitroaniline and 4,5-dihydroxynaphthalene-2,7-disulfonic acid disodium salt. Aldrich’s Chromotrope 2B azo dye, 4,5-dihydroxynaphthalene-3-(4nitrophenylazo)-2,7-disulfonic acid disodium salt (4hay), served as control. The two sets of spectra of the chelates 4hay‚M2+ (Figures 1, 3, and 4), which were obtained by the general screening procedure using MOPS buffer, were very similar (see Supporting Information for spectra). Only incubation with Cu(II) resulted in a significant shift of the wavelength of the absorbance maximums. RESULTS AND DISCUSSION Aromatic amines (1a-1h, Figures 1 and 3) were diazotized by a standard method.7a Azo coupling of the resulting (2a-2h) diazonium ions, as well as several commercially available diazonium salts (2i-2k, Figure 3), with phenolic compounds (3aa3bl, Figures 1 and 4) and aromatic amines (3bm-3bx, Figures 1 and 4) were carried out following established procedures.7a,12 This simple combinatorial approach produced a diverse library (12) Lang-Fugmann, S. In Methoden der organischen Chemie (Houben-Weyl), Vol. E16d, Organische Stickstoff-Verbindungen IV, Part I; Klamann, D., Ed.; G. Thieme: Stuttgart, New York, 1992; pp 1-93.

of chelating azo dyes 4 with many new structures. We assumed that the structures of the azo compounds 4 were determined by the known empirical orientation rules for azo couplings.7,12,13 Mixtures of the likely products (e.g., regioisomers, compounds with multiple azo substitution) were obtained when reactant structures permitted the formation of such compounds. However, it was not the objective of this pilot study to identify and isolate all the products. Instead, each reaction mixture, containing the azo dye chelator(s) 4, was treated with a series of solutions, with each solution containing one of seven heavy metal ions. The absorbance and, in some cases, fluorescence spectra of the resulting chelate solutions were measured. For compounds (4aah, 4jaf, 4aan) that were investigated in more detail, the main product of each azo coupling was isolated (vide infra). Solvent molecules can be incorporated in azo dye-metal complexes as ligands.10,14,15 The character of the solvent and the pH of the chelate solution often influence the structure and stability of the dissolved metal chelate.10,14-16 In our experiments, buffers and pH values were selected in order to minimize solvent interferences with metal ion chelation. Thus, metal complexes were formed in either a neutral MOPS17 or a slightly acidic acetate buffer.2d The former was preferred because it often gave better results (e.g., faster chelation). Nevertheless, both buffers were employed in a number of experiments to further study the diversity of spectral responses and because some metal chelates are best formed in acidic media.11 The UV-visible absorbance spectra displayed diverse responses when some of the azo compounds bound to heavy metal ions. Azo ligands 4 were classified according to the number of bivalent metal ions they responded to upon screening with a panel of seven ions (Table 1). Azo dye categories 1, 2, and 3 gave significant spectral responses (e.g., shift of the wavelength of the absorbance maximum) upon exposure to one or more metal ions (Table 1; see Supporting Information for further details). Figures 5 (4aak‚M2+), 6 (4iab‚M2+), and 7 (4aah‚M2+) present illustrative examples of spectra, recorded in MOPS buffer, displayed by chelates in categories 1, 1, and 2, respectively. In Table 1, all the compounds, except 4aah, listed as examples in categories 1-3 have not been previously reported. Terdentate azo dyes (e.g., 4aaj) can form two chelate rings per dye molecule (Figure 2) and thus bring about highly stable complexes with a number of metal ions.7a,13b In our studies, most azo dyes in categories 1 and 2 can form terdentate structures. These dyes gave color changes with three or more metal ions in a panel of seven. Bidentate ligands form only one chelate ring per molecule, which is usually less stable than a similar chelate structure with two rings.7a,13 All bidentate azo dyes we studied can be included in categories 3 (13) (a) Hirohata, M.; Kai, F.; Kurosawa, K.; Huang, H.; Ishii, K.; Matsuoka, Y.; Komori, K.; Nakamura, M. J. Coord. Chem. 1993, 30, 379-391. (b) Hirohata, M.; Kai, F.; He, P. L.; Komori, K.; Matsuoka, Y.; Huang, H. J. Coord. Chem. 1994, 31, 237-247. (14) Huang, H.; Kai, F.; Asai, Y.; Hirohata, M.; Nakamura, M. Bull. Chem. Soc. Jpn. 1991, 64, 2464-2469. (15) Colacio, E.; Ruiz, J.; Moreno, J. M.; Kiveka¨s, R.; Sundberg, M. R.; DominguezVera, J. M.; Laurent, J. P. J. Chem. Soc., Dalton Trans. 1993, 157-163. (16) Cingi, M. B.; Bigoli, F.; Leporati, E.; Pellinghelli, M. A. J. Chem. Soc., Dalton Trans. 1982, 1411-1416. (17) (a) Delnomdedieu, M.; Boudou, A.; Georgescauld, D.; Dufourc, E. J. Chem.Biol. Interact. 1992, 81, 243-269. (b) Soares, H. M. V. M.; Conde, P. C. F. L.; Almeida, A. A. N.; Vasconcelos, M. T. S. D. Anal. Chim. Acta 1999, 394, 325-335.

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Figure 4. Structures of phenolic compounds 3aa-3bl and aromatic amines 3bm-3bx.

and 4. Consistent with this observation, Hirohata et al. found that a multidentate azo dye formed chelates with more metal ions than a structurally related bidentate azo compound.13b The structure and stability of the chelate(s) formed between an azo dye and a 5254

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metal ion depend on a number of factors including the size of the chelate ring(s), the number of chelate rings per molecule, steric restrictions, the basicity of the ligand, and the nature of the metal.7a,13a The hard and soft acid and base (HSAB) properties of

Figure 5. Absorbance spectra of chelates of dye 4aak with selected metal ions.

Table 1. Spectral Responses of Azo Ligands to Metal Ionsa category

no. of metal ions responded to

1

five-six

2

three-four

3 4

one-two none

examplesb 4aai*, 4aaj*, 4aak*, 4iaa*, 4iab*, 4iac*, 4jaf* 4aaa*, 4aab*, 4aac*, 4aah*, 4aam*, 4aan* 4jae*, 4jag*, 4jbm* 4aas*, 4bac, 4daa, 4fah, 4jav, 4jbf

a The experiments were carried out in MOPS buffer. b Examples with an asterisk were categorized on the basis of spectra recorded by an absorbance microplate reader. The rest of the examples were classified on the basis of visual inspection of color changes in test tube-experiments.

the chelating sites and metal ion strongly influence the coordination mode of a multidentate ligand toward a metal ion, when multiple types of chelates can be formed.10,14,18 Multidentate azo dyes usually form complexes with a variety of heavy metal ions.10,14,19 The UV-visible absorbance spectra of such complexes often show spectral diversity among the metal ions bound to the same chelator.19 Therefore, most of our lead structures were designed to be multidentate dyes (4). The structures were chosen following literature data about similar azo chelators;7a,15 in particular, o-hydroxy-azo and 8-quinolyl-azo derivatives are known multidentate ligands.10,11,14,16 For instance, 7-[(3,5dihalo-2-pyridyl)azo]-8-hydroxyquinoline-5-sulfonic acids (halo: chloro or bromo) were reported to form highly stable complexes with soft or borderline Lewis acids such as Ni(II), Zn(II), and Cd(II).10,14 In these chelates, the metal ions were coordinated selectively to the N-N-O terdentate moiety, containing the azo group.10,14 On the other hand, VIVO, a hard Lewis acid, bonded selectively to (18) Huang, H.; Kai, F.; Shoda, T.; Nakamura, M. J. Coord. Chem. 1993, 28, 155-166. (19) (a) Furukawa, M. Anal. Chim. Acta 1982, 140, 281-289. (b) Furukawa, M.; Shibata, S. Anal. Chim. Acta 1982, 140, 301-307.

the N-O skeleton in the 8-hydroxyquinoline moiety of the same ligand.14 This example illustrates that multidentate azo dyes can form various types of chelates depending on the nature of the metal ion. Chelate structural diversity among different types of metal ions can broaden the spectral diversity of such complexes. Extensive studies on metal chelates of azo dyes elucidated some common structural patterns among subclasses of these complexes.7a,14,16,20 The scope of these rules is limited. A chelator and several similar metal ions often form complexes with different compositions.10 The complex influence of various factors (vide supra) on the structures of azo dye-metal complexes is not entirely understood. Therefore, we did not speculate on the exact structures of the numerous new chelates (4‚M2+, Figure 2) prepared in this work. Although most chelates in this study were not fluorescent, the excitation and emission spectra of some complexes were measured. For example, the fluorescence of azo dye 4aaa, synthesized from the fluorogenic coumarin derivative 3aa, was entirely quenched upon binding to Hg(II) (data not shown). This quenching property was specific for Hg(II) among the studied metal ions and can serve as the basis for detecting Hg(II) using a number of optical sensor formats. Unfortunately, fluorescence excitation and emission spectra of most other studied fluorescent chelates were similar to those of the parent chelators (data not shown). In a control experiment, we determined that reaction mixtures containing the azo dyes 4, prepared with this approach, gave results similar to those obtained with the corresponding purified azo derivative(s). A purified azo dye (4hay), obtained from a commercial source, was compared with a reaction mixture, containing the same dye prepared by our method. The spectra of the resulting two sets of chelates were virtually identical (see Supporting Information for spectra). Furthermore, in control runs, starting materials, utilized in syntheses of the azo compounds, (20) Chakraborty, I.; Bhattacharyya, S.; Banerjee, S.; Dirghangi, B. K.; Chakravorty, A. J. Chem. Soc., Dalton Trans. 1999, 3747-3753.

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Figure 6. Absorbance spectra of chelates of dye 4iab with selected metal ions.

Figure 7. Absorbance spectra of chelates of dye 4aah with selected metal ions.

with chelating properties were tested with the same panel of metal ions used to screen the azo dyes. The results of these control runs were taken into account in evaluating the spectral responses of the dyes (see Experimental Section). Finally, syntheses of three azo dyes (4aah, 4jaf, 4aan) were repeated on a preparative scale using essentially the same method that was employed to produce the small-volume reaction mixtures for the screening experiments. In each case, the main product was isolated and its structure confirmed by spectral methods including NMR and MS. All three coupling reactions resulted in the expected azo dyes (4aah, 4jaf, 4aan). The structure of 4aah was previously outlined in a patent,21 but the physical and spectral characteristics and the detailed synthesis of this compound were not described. For 4aah and 4aan, the unusually high chemical shifts of the OH protons in the 1H NMR spectra and the weak

νOH bands in the IR spectra indicated intramolecular hydrogen bonds between the hydroxyl and azo groups. The sulfonate (monosodium) salt appears to be the major component of product 4jaf. In conclusion, we have shown that a combinatorial library synthesis approach is an efficient means to discover new indicators. Our intention was to find a rapid method to identify promising candidate materials.22 In this proof of concept, we did not deconvolute the spectra of the main components of the reaction mixtures and isolated only a few selected compounds in pure form. On the basis of our experience with these selected compounds and on literature data,7a,12 we assumed that the yields of the azo dyes, generated by established methods,7a,12 were moderate to good across the examples studied. Also, only the reaction mixtures displaying deep color due to the presence of the azo dye(s) were

(21) Nakayama, N.; Komamura, T. Japanese Patent JP 04,267,194, 1992; Chem. Abstr. 1993, 118, 202176c.

(22) Blair, S. M.; Brodbelt, J. S.; Marchand, A. P.; Kumar, K. A.; Chong, H.-S. Anal. Chem. 2000, 72, 2433-2445.

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included in further investigations with metal ions. In a control experiment, we compared the spectral properties of a commercially available purified azo dye 4hay with a reaction mixture containing the same dye and showed that the mixture behaved similarly to the known material. UV-visible absorbance spectra of the azo dyes 4, obtained in this work, showed diverse responses upon exposure to selected heavy metal ions. These and similar azo dyes could be used for metal ion determination in various optical sensors.3c,9,23,24a,b Optical analyzers employing azo dyes usually can detect metal ions in the concentration range of about 10-6-10-3 M.9a,24,25 However, novel optical sensors, based on electrochemically assisted solvent extraction, hold promise for achieving higher sensitivities.25 The dyes that cross-reacted with multiple metal ions could be utilized in a sensor array format to quantify these ions in mixtures by means of a computational pattern analysis method. Combinatorial libraries are limited by the screen for particular properties. In the present work, we have prepared previously (23) Puyol, M.; del Valle, M.; Garce´s, I.; Villuendas, F.; Domı´nguez, C.; Alonso, J. Anal. Chem. 1999, 71, 5037-5044. (24) (a) Madden, J. E.; Cardwell, T. J.; Cattrall, R.; Deady, L. W. Anal. Chim. Acta 1996, 319, 129-134. (b) Sanchez-Pedren ˜o, C.; Ortun ˜o, J. A.; Albero, M. I.; Garcia, M. S.; Valero, M. V. Anal. Chim. Acta 2000, 414, 195-203. (c) Saldanha, T. C. B.; de Arau´jo, M. C. U.; Neto, B. B.; Chame, H. C. Anal. Lett. 2000, 33, 1187-1202. (25) Wilson, R.; Schiffrin, D. J.; Luff, B. J.; Wilkinson, J. S. Sens Actuators B 2000, 63, 115-121.

unreported azo dyes and screened them for an optical change upon metal binding. The same library may also be screened for other functions such as dye fastness, pH sensitivity, and photoresponsiveness. ACKNOWLEDGMENT The authors thank J. Lynch (Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA) and R. Smith (Department of Chemistry, Tufts University) for MS studies and Professor D. Kaplan (Department of Chemical Engineering, Tufts University) for providing access to a microtiter plate reader. Financial support by Department of Energy and Office of Naval Research is gratefully acknowledged. SUPPORTING INFORMATION AVAILABLE A table depicting all studied azo ligands 4, categorized as described in Table 1, and absorbance spectra of chelates of azo dye 4hay obtained from a commercial source as well as prepared by our general procedure without purification. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review July 27, 2000. Accepted August 24, 2000. AC0008542

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