The Interaction of Boronic Acid-Substituted Viologens with Pyranine

Jun 7, 2005 - David B. Cordes, Soya Gamsey, Zach Sharrett, Aaron Miller, Praveen Thoniyot,. Ritchie A. Wessling, and Bakthan Singaram*. Department of ...
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The Interaction of Boronic Acid-Substituted Viologens with Pyranine: The Effects of Quencher Charge on Fluorescence Quenching and Glucose Response† David B. Cordes, Soya Gamsey, Zach Sharrett, Aaron Miller, Praveen Thoniyot, Ritchie A. Wessling, and Bakthan Singaram* Department of Chemistry and Biochemistry, University of California at Santa Cruz, 1156 High Street, Santa Cruz, California 95064 Received January 25, 2005. In Final Form: April 11, 2005 The fluorescence sensing of several monosaccharides using boronic acid-substituted viologen quenchers in combination with the fluorescent dye pyranine (HPTS) is reported. In this two-component sensing system, fluorescence quenching by the viologen is modulated by monosaccharides to provide a fluorescence signal. A series of viologen quenchers with different charges were prepared and tested for their ability both to quench the fluorescence of HPTS and to sense changes in glucose concentration in aqueous solution at pH 7.4. Both quenching efficiency and sugar sensing were found to be strongly dependent upon viologen charge. The molar ratio between HPTS and each of the viologen quenchers was varied in order to obtain an optimal ratio that provided a fairly linear fluorescence signal across a physiological glucose concentration range. Both the quenching and sugar sensing results are explained by electrostatic interaction between dye and quencher.

Introduction The design and synthesis of chemosensors for biologically important molecules have developed into a major research area over the last 15 years. Progress has been driven by advances in the analytical capabilities of biologists and chemists and from medical professionals whose practices lay increasing emphasis on accurately monitoring a patient’s biochemical balance. In particular, medical providers have long been interested in accurate and real-time measurement of blood glucose levels for diabetic patients. The shortcomings of existing glucose detection methods have driven the development of a large number of monosaccharide sensing systems. Many of these sensing systems are based on glucose oxidase, but a variety of nonenzymatic systems have also appeared over the last 10 years. Considerable success has been achieved in both enzymatic and nonenzymatic systems by utilizing potentiometric,1,2 colorimetric,3,4 and amperometric5-8 techniques. Recently, several novel methods such as the use of photoacoustics9-11 and photonic crystals 12,13 have been applied in monosaccharide sensing. Common spectroscopic * To whom correspondence may be addressed. E-mail: singaram@ chemistry.ucsc.edu. † Dedicated to the memory of Professor James Verghese. (1) Shoji, E.; Freund, M. S. J. Am. Chem. Soc. 2002, 124, 1248612493. (2) Eggert, H.; Frederiksen, J.; Morin, C.; Norrild, J. C. J. Org. Chem. 1999, 64, 3846-3852. (3) Badugu, R.; Lakowicz, J. R.; Geddes, C. D. Anal. Biochem. 2004, 327, 82-90. (4) Rusin, O.; Alpturk, O.; He, M.; Escobedo, J. O.; Jiang, S.; Dawan, F.; Lian, K.; McCarroll, M. E.; Warner, I. M.; Strongin, R. M. J. Fluoresc. 2004, 14, 611-615. (5) Yang, M. H.; Yang, Y. H.; Liu, B.; Shen, G. L.; Yu, R. Q. Sens. Actuators, B 2004, 101, 269-276. (6) Li, C. M.; Cha, C. S. Front. Biosci. 2004, 9, 3479-3485. (7) Lee, H. L.; Chen, S. C. Talanta 2004, 64, 210-216. (8) Kase, Y.; Muguruma, H. Anal. Sci. 2004, 20, 1143-1146. (9) Argaman, D.; Ashkenazi, S.; Greenberg, L.; Raz, I. Diabetologia 2003, 46, A309-A309. (10) Quan, K. M.; Christison, G. B.; Mackenzie, H. A.; Hodgson, P. Phys. Med. Biol. 1993, 38, 1911-1922. (11) Mackenzie, H. A.; Christison, G. B.; Hodgson, P.; Blanc, D. Sens. Actuators, B 1993, 11, 213-220.

technologies such NMR,2,14,15 IR,16,17 UV-vis, and both fluorescence18-22 and phosphorescence23 have also been employed to measure changes in monosaccharide concentration. Fluorescence-based methods are among the most promising of the many new approaches due to both the sensitivity of the fluorometric method and the simplicity of optical techniques. Further, an abundance of commercially available fluorophores which can be synthetically or spatially combined with the receptor makes the fluorescence-based system readily accessible. The majority of these fluorescence-based systems relies on arylboronic acids as the saccharide receptor moieties. Arylboronic acids are well-known for their ability to reversibly bind monosaccharides.24 Significant equilibria between arylboronic acid and generic diols are shown in Scheme 1. Most fluorescence-based glucose-sensing systems use a one-component design where a single sensor molecule consists of a fluorophore covalently bound to a boronic acid receptor. Upon saccharide binding, changes in the electronic and/or steric states of the molecule cause (12) Asher, S. A.; Alexeev, V. L.; Goponenko, A. V.; Sharma, A. C.; Lednev, I. K.; Wilcox, C. S.; Finegold, D. N. J. Am. Chem. Soc. 2003, 125, 3322-3329. (13) Alexeev, V. L.; Sharma, A. C.; Goponenko, A. V.; Das, S.; Lednev, I. K.; Wilcox, C. S.; Finegold, D. N.; Asher, S. A. Anal. Chem. 2003, 75, 2316-2323. (14) Uggla, R.; Sundberg, M. R.; Nevalainen, V. Tetrahedron: Asymmetry 1996, 7, 1741-1748. (15) Bielecki, M.; Eggert, H.; Norrild, J. C. J. Chem. Soc., Perkin Trans. 2 1999, 449-455. (16) Saptari, V.; Youcef-Toumi, K. Appl. Opt. 2004, 43, 2680-2688. (17) Hazen, K. H.; Arnold, M. A.; Small, G. W. Appl. Spectrosc. 1998, 52, 1597-1605. (18) Fang, H.; Kaur, G.; Wang, B. H. J. Fluoresc. 2004, 14, 481-489. (19) McNichols, R. J.; Cote, G. L. J. Biomed. Opt. 2000, 5, 5-16. (20) Chen, J. R.; Miao, Y. Q.; He, N. Y.; Wu, X. H.; Li, S. J. Biotechnol. Adv. 2004, 22, 505-518. (21) Cao, H.; McGill, T.; Heagy, M. D. J. Org. Chem. 2004, 69, 29592966. (22) James, T. D.; Shinkai, S. In Topics in Current Chemistry; Penade´s, S., Ed.; Springer-Verlag: Berlin, Heidelberg, 2002; Vol. 218. (23) Valenciagonzalez, M. J.; Liu, Y. M.; Diazgarcia, M. E.; Sanzmedel, A. Anal. Chim. Acta 1993, 283, 439-446. (24) Lorand, J. P.; Edwards, J. O. J. Org. Chem. 1959, 24, 769-774.

10.1021/la050219x CCC: $30.25 © 2005 American Chemical Society Published on Web 06/07/2005

Boronic Acid-Substituted Viologen Quenchers Scheme 1. Boronic Acid Equilibria for Generic Diols

changes in the fluorescence emission of the fluorophore. In our two-component system, however, the fluorophore is not covalently attached to the boronic acid receptor unit. In the present study, we chose the anionic dye pyranine, 8-hydroxy-1,3,6-pyrenetrisulfonic acid trisodium salt (HPTS) (1), as the fluorophore and a boronic acidsubstituted viologen as the receptor. Pyranine, hereafter HPTS, is a photostable, anionic, and highly water soluble green-fluorescing dye with a quantum yield of nearly 1. Viologens, the cationic salts of 4,4′-bipyridinium, are wellknown electron acceptors that have been found to quench the fluorescence of numerous dyes25-27 and macromolecular systems.28-31 The quenching of HPTS fluorescence by viologen quenchers was first reported by Baptista who studied complexation of the dye with methyl viologen (MV2+).25 In our previous studies, we found that anionic HPTS and its derivatives are generally sensitive to quenching by boronic acid-substituted viologens and related phenanthrolinium compounds. It was also determined that by changing the concentration of glucose and other monosaccharides, the fluorescence intensity of a quenched solution of dye could be modulated to provide a sensing signal.32-35 The generality of this two-component sensing system led us to conduct further research in this area, and we now describe a systematic study of the effect of quencher charges on both quenching efficiency and sugar sensing. A general mechanism which accounts for both fluorescence quenching and saccharide sensing is depicted in Scheme 2. The quencher/receptor molecule, a boronic acidsubstituted viologen, m-BBV2+ (4), is doubly charged at a physiological pH of 7.4. In this cationic state the boron substituents are trigonal and neutral and the viologen forms a nonfluorescent ground-state complex with anionic HPTS. Upon sugar binding, the pKa of boron in its ester configuration is lowered, causing the boron to convert to its tetrahedral, anionic form in which it bears a charge of (25) de Borba, E. B.; Amaral, C. L. C.; Politi, M. J.; Villalobos, R.; Baptista, M. S. Langmuir 2000, 16, 5900-5907. (26) Nakashima, K.; Kido, N. Photochem. Photobiol. 1996, 64, 296302. (27) Zhao, Z. G.; Shen, T.; Xu, H. J. J. Photochem. Photobiol., A 1990, 52, 47-53. (28) Gaylord, B. S.; Wang, S. J.; Heeger, A. J.; Bazan, G. C. J. Am. Chem. Soc. 2001, 123, 6417-6418. (29) Wang, D. L.; Gong, X.; Heeger, P. S.; Rininsland, F.; Bazan, G. C.; Heeger, A. J. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 49-53. (30) Chen, L. H.; McBranch, D. W.; Wang, H. L.; Helgeson, R.; Wudl, F.; Whitten, D. G. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 12287-12292. (31) DiCesare, N.; Pinto, M. R.; Schanze, K. S.; Lakowicz, J. R. Langmuir 2002, 18, 7785-7787. (32) Cappuccio, F. E.; Suri, J. T.; Cordes, D. B.; Wessling, R. A.; Singaram, B. J. Fluoresc. 2004, 14, 521-533. (33) Suri, J. T.; Cordes, D. B.; Cappuccio, F. E.; Wessling, R. A.; Singaram, B. Langmuir 2003, 19, 5145-5152. (34) Camara, J. N.; Suri, J. T.; Cappuccio, F. E.; Wessling, R. A.; Singaram, B. Tetrahedron Lett. 2002, 43, 1139-1141. (35) Suri, J. T.; Cordes, D. B.; Cappuccio, F. E.; Wessling, R. A.; Singaram, B. Angew. Chem., Int. Ed. 2003, 42, 5857-5859.

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-1. Using 11B NMR, we have confirmed that these changes occur to the boronic acids in each of the viologen quenchers used in this study.36 These NMR studies have shown that in the absence of glucose, the boron exists in its neutral trigonal form at pH 7.4 giving a signal at ∼30 ppm. As the glucose ester forms under increasing glucose concentrations the boron converts to its anionic tetrahedral “-ate” form which gives a characteristic signal at ∼8 ppm. In its fully glucose bound form, the zwitterionic m-BBV quencher will have a net charge of zero. The neutral quencher displays a diminished affinity for the anionic dye, thus weakening the ground-state complex and resulting in a fluorescence increase (Scheme 2). As the mechanism indicates, the quenching process appears heavily dependent on the degree of electrostatic attraction between the cationic viologen quencher and anionic dye. The proposed mechanism is consistent with the results described in this study and with our prior observation that complex formation does not occur at pH 10 where the boron exists exclusively in its tetrahedral, negatively charged form.33 Experimental Section Materials. All reagents including HPTS dye were purchased from Aldrich Co. and used as received. DMF was dried over CaH2 prior to use. General Methods. 1H NMR spectra were recorded on a Varian 500 MHz spectrometer and are reported in ppm with respect to TMS (δ ) 0). Proton-decoupled 13C NMR spectra were recorded on a Varian at 125 MHz and are reported in ppm.37 11B NMR spectra were recorded on Bruker at 80.25 MHz and are reported in ppm with respect to BF3‚OEt2 (δ ) 0). High-resolution mass measurements were obtained on a benchtop Mariner ESITOF mass spectrometer or a JEOL HX-110 double focusing mass spectrometer for fast atom bombardment. For elemental analysis, C, H, and N were determined by combustion, Br was determined by titration, and boron was determined by PGNAA.38 Syntheses. All reactions were performed using standard syringe techniques, and carried out in oven-dried glassware under an argon atmosphere. 2-(3,5-Bis(bromomethyl)phenyl)[1,3,2]dioxaborinane (7). To a 500-mL round-bottom flask fitted with a condenser and a sidearm was added 3,5-dimethylphenylboronic acid (10.5 g, 70 mmol), calcium hydride (5.9 g, 140 mmol), and dichloroethane (300 mL). After 10 min of stirring under argon, 1,3-propanediol was added via syringe. The reaction was refluxed for 1.5 h, cooled to room temperature, and filtered. The clear filtrate was mixed with N-bromosuccinimide (27.4 g, 154 mmol) and 2,2′-azobisisobutryonitrile (2.3 g, 14 mmol) and refluxed for 3 h. The orange solution was cooled overnight, and the succinate crystals that formed were filtered off. The filtrate was evaporated to dryness, leaving an off-white chunky solid, which was recrystallized from methanol (ca. 300 mL) to give 11.0 g (46%) of pure 7: 1H NMR (CDCl3, 500 MHz) δ 2.07 (q, J ) 5.5 Hz, 2H), 4.17 (t, J ) 5.5 Hz, 4H), 4.49 (s, 4H), 7.48 (s, 1H), 7.74 (s, 2H); 13C NMR (CDCl3, 125 MHz) δ 27.51, 33.39, 62.22, 131.92, 134.52, 137.73; 11B NMR (80 MHz, CDCl3) δ 28.5. Anal. Calcd for C11H13BBrO2: C, 37.98; H, 3.77; Br, 45.94. Found: C, 38.08; H, 3.68; Br, 46.12. 1-(3-Boronic acid-5-bromomethyl-benzyl)-pyridinium Bromide (8). Pyridine (0.56 mL, 7 mmol) was added via syringe to a solution of compound 7 (9.73 g, 28 mmol) in CH2Cl2 (370 mL) and CH3OH (180 mL), and the reaction was stirred at 40 °C for 22 h. The CH2Cl2 was removed in vacuo, and the excess 10 which precipitated out of methanol was filtered off and washed with ice-cold methanol. The filtrate was concentrated down to ca. 20 mL, and then acetone (ca. 300 mL) was added, followed by the addition of ether until turbidity occurred. Storage at -4 °C for 24 h resulted in the formation of white needle-shaped crystals. (36) See Supporting Information for details. (37) Due to relaxed 13C-11B spin-spin coupling, signals for carbons directly attached to boron are not observed. (38) Boron results may be low due to high B concentration affecting neutron flux.

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Cordes et al.

Scheme 2. Two-Component Sensing System Depicting Interaction of HPTS (1) and m-BBV2+ (4)

The solid was collected by centrifugation, washed several times with acetone, and dried under argon to yield 1.3 g of pure 8 (48% yield): 1H NMR (CD3OD, 500 MHz) δ 4.57 (s, 2H), 5.88 (s, 2H), 7.64 (s, 1H), 7.75-7.90 (m, 2H), 8.13 (dd, J ) 7.0, 7.5 Hz, 2H), 8.61 (tt, J ) 8.0, 1.5 Hz, 1H), 9.10 (d, J ) 5.5 Hz, 2H); 13C NMR (CD3OD, 125 MHz) δ 31.9, 64.1, 128.4, 130.9, 133.0, 133.8, 135.6, 139.1, 144.6, 146.1; 11B NMR (80 MHz, CD3OD) δ 28.3. 1-(3-[4,4′]Bipyridinium-1-methyl-5-boronic acid-benzyl)-pyridinium Dibromide (9). To a solution of 8 (0.4 g, 1.03 mmol) in DMF (20 mL) was added 4,4′-dipyridyl (0.8 g, 5.2 mmol), and the reaction was heated in an oil bath. Once the temperature reached 80 °C (ca. 5 min), a small amount of yellow precipitate began to form. The reaction was filtered hot, and acetone (ca. 50 mL) was added to the clear yellow filtrate until a fluffy white precipitate formed. The precipitate was collected by centrifugation, washed with acetone several times, and dried under a stream of argon to yield pure 9 as an off-white solid (0.41 g, 74% yield):. 1H NMR (CD3OD, 500 MHz) δ 5.94 (s, 2H), 5.98 (s, 2H), 7.89 (br s, 1H), 7.93 (br s, 1H), 7.96 (br s, 1H) 7.99 (dd, J ) 4.5, 1.5 Hz, 2H), 8.14 (t, J ) 7.0 Hz, 2H), 8.54 (d, J ) 7.0 Hz, 2H), 8.61 (tt, J ) 7.5, 1.5 Hz, 1H), 8.80 (dd, J ) 5.0, 1.5 Hz, 2H), 9.17 (d, J ) 6.0 Hz, 2H), 9.25 (d, J ) 7.0 Hz, 2H); 13C NMR (CD3OD, 125 MHz) δ 63.4, 63.7, 122.2, 126.1, 128.4, 133.7, 133.8, 135.2, 135.3, 142.1, 144.7, 145.3, 146.0, 149.5, 150.3, 154.0; 11B NMR (80 MHz, CD3OD) δ 27.1. m-BBVMP3+ (3). Compound 9 (0.4 g, 0.8 mmol) was dissolved in DMF (35 mL), and insolubles were filtered off. m-Bromomethylphenylboronic acid32 (0.2 g, 0.93 mmol) was added, and the reaction was stirred at 70 °C for 48 h. After being cooled to room temperature, the clear yellow solution was diluted with acetone (200 mL) to precipitate the product. The resulting yellow precipitate was collected by centrifugation, washed with DMF and then acetone, and dried under a stream of argon to yield pure 3 as a bright yellow solid (0.26 g, 43% yield): 1H NMR (CD3OD, 500 MHz) δ 5.94 (s, 2H), 5.98 (s, 2H), 6.03 (s, 2H), 7.45-

7.96 (m, 7H), 8.13 (t, J ) 7.0 Hz, 2H) 8.60 (t, J ) 7.5 Hz, 1H), 8.67 (d, J ) 4.0 Hz, 2H), 8.69 (d, J ) 4.5 Hz, 2H), 9.17 (d, J ) 6.0 Hz, 2H), 9.33 (d, J ) 6.0 Hz, 2H), 9.37 (d, J ) 7.0 Hz, 2H); 13C NMR (CD OD, 125 MHz) δ 63.6, 63.9, 64.7 127.16, 127.22, 3 127.3, 128.4, 128.5, 130.7, 131.4, 132.0, 133.5, 133.9, 134.3, 135.1, 135.3, 135.5, 144.7, 145.5, 145.8, 145.9, 150.2; 11B NMR (80 MHz, CD3OD) δ 28.4. Anal. Calcd for C30H30B2Br3N3O4: C, 47.54; H, 3.99; B, 2.85; Br, 31.63; N, 5.54; O, 8.94. Found: C, 46.18; H, 3.94; B, 2.10; Br, 34.88; N, 5.22. m-BBV2+ (4). To a solution of m-bromomethylphenylboronic acid (2.68 g, 12.5 mmol) in DMF (25 mL) was added 4,4′-dipyridyl (0.78 g, 5 mmol), and the reaction was stirred at 65 °C for 24 h. The yellow precipitate was collected by filtration under argon, washed with DMF and then acetone, and dried under a stream of argon. Recrystallization from methanol yielded pure 4 as light yellow crystals (2.11 g, 73% yield): 1H NMR (D2O, 500 MHz) δ 6.08 (s, 4H), 7.67 (t, J ) 7.5 Hz, 2H), 7.76 (d, J ) 7.0 Hz, 2H), 7.96 (d, J ) 7.5 Hz, 2H), 7.97 (s, 2H), 8.67 (d, J ) 6.5 Hz, 4H), 9.29 (d, J ) 6.5 Hz, 4H); 13C NMR (DMSO-d6, 125 MHz) δ 63.5, 127.2, 128.3, 130.6, 133.2, 134.6, 135.0, 145.6, 149.1; 11B NMR (80 MHz, CD3OD) δ 28.5. Anal. Calcd for C24H24B2Br2N2O4: C, 49.20; H, 4.13; B, 3.69; Br, 27.28; N, 4.78; O, 10.90. Found: C, 49.27; H, 4.29; B, 2.40; Br, 30.80; N, 4.47. 1-(3-(Bromomethyl)benzyl)pyridinium Bromide (10). Intermediate 10 was prepared analogously to 8, except R,R′-dibromom-xylene (7.4 g, 28 mmol) and pyridine (0.56 mL, 7 mmol) were used. The product was isolated as a white solid (0.685 g, 29% yield): 1H NMR (CD3OD, 500 MHz) δ 4.57 (s, 2H), 5.88 (s, 2H), 7.42-7.52 (m, 3H), 7.61 (s, 1H), 8.13 (t, J ) 7.0 Hz, 2H), 8.62 (tt, J ) 8.0, 1.5 Hz, 1H), 9.10 (dd, J ) 6.5, 1.5 Hz, 2H); 13C NMR (CD3OD, 125 MHz) δ 31.5, 63.9, 128.4, 138.5, 129.3, 129.7, 130.3, 133.7, 140.0, 144.6, 146.0. m-BBVBP4+ (2). To a solution of 8 (0.49 g, 1.26 mmol) in DMF (20 mL) was added 4,4′-dipyridyl (0.094 g, 0.6 mmol), and the reaction was stirred at 70 °C for 72 h. The bright yellow precipitate

Boronic Acid-Substituted Viologen Quenchers

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was collected by centrifugation, washed with DMF and then acetone, and dried under a stream of argon to yield pure 2 (0.28 g, 51% yield): 1H NMR (CD3OD, 500 MHz) δ 5.97 (s, 4H), 6.07 (s, 4H), 7.93-7.99 (m, 6H), 8.15 (t, J ) 7.0 Hz, 4H), 8.62 (tt, J ) 7.5, 1.5 Hz, 2H), 8.71 (d, J ) 7.0 Hz, 4H), 9.19 (d, J ) 5.5 Hz, 4H), 9.40 (d, J ) 7.0 Hz, 4H); 13C NMR (CD3OD, 125 MHz) δ 63.7, 63.9, 127.4, 128.4, 131.5, 133.5, 133.8, 135.4, 135.5, 144.8, 145.8, 146.0, 150.2; 11B NMR (80 MHz, CD3OD) δ 25.9. Anal. Calcd for C36H36B2Br4N4O4: C, 46.50; H, 3.90; B, 2.33; Br, 34.37; N, 6.02; O, 6.88. Found: C, 46.34; H, 3.82; B, 1.70; Br, 34.21; N, 5.95. NBABP4+ (5). To a solution of 10 (0.67 g, 1.9 mmol) in DMF (20 mL) was added 4,4′-dipyridyl (0.14 g, 0.89 mmol), and the reaction was stirred at 70 °C for 48 h. The bright yellow precipitate was collected by centrifugation, washed with DMF and then acetone, and dried under a stream of argon to yield pure 5 (0.71 g, 95% yield): 1H NMR (CD3OD, 500 MHz) δ 5.93 (s, 4H), 6.02 (s, 4H), 7.56-7.69 (m, 6H), 7.88 (s, 2H), 8.14 (t, J ) 7.0 Hz, 4H), 8.61 (tt, J ) 8.0, 1.5 Hz, 2H), 8.70 (d, J ) 7.0 Hz, 4H), 9.15 (d, J ) 5.5 Hz, 4H), 9.36 (d, J ) 7.0 Hz, 4H); 13C NMR (D2O, 125 MHz) δ 65.3, 65.6, 128.7, 129.9, 131.2, 132.0, 132.2, 134.8, 135.5, 145.8, 147.0, 147.6, 151.7; HRMS (FAB): m/z calcd for C36H34Br4N4 [M - Br]+ 759.0333, found 759.0334. 1,3-Bis(pyridiniomethyl)benzene Dibromide (6). Pyridine (2.3 mL, 28.4 mmol) was added to a solution of R,R′-dibromo-m-xylene (3.0 g, 11.4 mmol) in DMF (25 mL), and the reaction was stirred at 80 °C for 16 h. The white precipitate was filtered, washed with DMF, and then washed with acetone several times. After airdrying, 4.43 g (92% yield) of product was obtained: 1H NMR (CD3OD, 500 MHz) δ 5.99 (s, 4H), 7.56 (t, J ) 8.0 Hz, 1H), 7.66 (dd, J ) 7.5, 1.5 Hz, 2H), 7.92 (s, 1H), 8.16 (t, J ) 7.5 4H), 8.64 (tt, J ) 8.0, 1.5 Hz, 2H), 9.21 (dd, J ) 6.5, 1.0 Hz, 4H); 13C NMR (CD3OD, 125 MHz) δ 63.50, 128.44, 129.96, 130.26, 130.48, 134.66, 144.82, 146.04; HRMS (ESI) m/z calcd for C18H18Br2N2 [M Br]+ 341.06479, found 341.05966. 62.22, 131.92, 134.52, 137.73; 11B NMR (80 MHz, CDCl ) δ 28.5. Anal. Calcd for C H BBrO : 3 11 13 2 C, 37.98; H, 3.77; Br, 45.94. Found: C, 38.08; H, 3.68; Br, 46.12. Fluorescence Emission and UV-vis Absorption Studies (General). All studies were carried out in pH 7.4 buffer solution prepared with water purified via a Nanopure Ultrafiltration system. Buffer solution (pH 7.4, 0.1 ionic strength) was freshly prepared using KH2PO4 and Na2HPO4. Fluorescence spectra were taken on a Perkin-Elmer LS50-B luminescence spectrometer, except for temperature studies which were conducted on a Varian Cary Eclipse fluorescence spectrophotometer. All other studies were carried out at 20 °C without exclusion of air. Absorption spectra were taken on a Hewlett-Packard 8452A diode array spectrophotometer. For fluorescence titration experiments, the added volume did not exceed 3% of the total volume and the absorbance for all fluorescent measurements was below 0.1.39 All experiments except the quencher/dye optimization were carried out in triplicate, and the error is reported as the standard deviation. All data were analyzed using the Solver (nonlinear least-squares curve fitting) in Microsoft Excel. Absorbance Studies. Measurements were done in situ by taking the absorbance spectra of HPTS with each of the quenchers. The emission of HPTS dye (2 mL of 1 × 10-5 M in buffer) was first obtained, then aliquots of quencher (0.005 M) were added, the solution was shaken for 60 s, and the new absorbance was measured. Association constants (KUV) were calculated by means of Benesi-Hildebrand plots and eq 1

b/(∆A) ) 1/(StKUV∆[L]) + 1/(St∆)

(1)

with association constants evaluated by eq 2

KUV ) (y-intercept)/(slope) ) -(x-intercept)

(2)

where b is the y-intercept, ∆A is the change in absorbance at the monitored wavelength, St is the substrate (dye) concentration, ∆ is the change in the molar absorptivity, and [L] is the ligand (quencher) concentration.40 Representative absorbance data and (39) Credi, A.; Prodi, L. Spectrochim. Acta, Part A 1998, 54, 159170. (40) Connors, K. A. Binding Constants: The Measurement of Complex Stability; John Wiley & Sons: New York, 1987.

Benesi-Hildebrand plots for each HPTS-quencher combination and the Benesi-Hildebrand plots used to calculate the association constant for complex formation between dye and quencher are provided in the Supporting Information. Three trials were performed for each HPTS-quencher combination. Fluorescence Emission Studies. The HPTS dye excited at 460 nm and peak emission was observed at 510 nm. Fluorescence intensity was taken as the area under the curve between 480 and 650 nm for all studies. Stern-Volmer quenching constants were calculated by fitting the data with the eq 3

(Fo/F) ) (1 + Ks[Q])eV[Q]

(3)

where V is the dynamic quenching constant, Ks is the static quenching constant, and [Q] is the quencher concentration. Apparent glucose binding constants were calculated by fitting the data with the eq 4

Fcalc ) (Fmin + FmaxK[glucose])/(1+ K[glucose])

(4)

where Fcalc is the calculated fluorescence intensity, Fmin is the fluorescence intensity of the quenched dye, and Fmax is the calculated intensity at which the fluorescence increase reaches its maximum. K is the apparent binding constant, and [glucose] is the concentration of glucose.41 For quencher/dye ratio optimization for glucose sensing, the fluorescence measurements were done in situ by taking the emission spectra of a 1:1 quencher/ dye solution then adding an aliquot of buffered 1 M glucose solution and measuring the new fluorescence emission after shaking for 60 s. Additional aliquots were added, and measurements were taken until a glucose concentration of 30 mM was obtained. The overall process was then repeated at successively higher ratios until an optimal quencher/dye ratio was obtained. After determination of the optimal ratio, the glucose response procedure was repeated at the optimized ratio two additional times. Representative raw fluorescence emission data for quenching and glucose sensing studies are provided in the Supporting Information.

Results and Discussion In the present study, boronic acid-substituted viologens (2-4) were used to quench the fluorescence of HPTS (1). As in earlier studies of related bipyridinium and phenanthrolinium quenchers,33,34 saccharide binding to the boronic acids was found to diminish the efficiency of the quenching interaction. This produces an increase in fluorescence as saccharide concentration is increased. As indicated in the proposed mechanism, we hypothesize that this is the result of decreasing the net charge of the quencher and, thereby, the electrostatic attraction between quencher and dye molecules. Quenchers lacking boronic acids (5 and 6) were included in this study as reference compounds. Synthesis of Viologen Quenchers. Quenchers 2-5 were successfully prepared in high purity with relative ease (Scheme 3). In each synthesis, use of DMF as a solvent for quaternization of bipyridyl facilitated the precipitation of analytically pure product from the reaction mixture. Protection of 3,5-dimethylphenylboronic acid with propanediol, followed by bromination using NBS and AIBN, afforded compound 7. Under dilute conditions, reaction of pyridine with an excess of 7 gave the monosubstituted product 8, which was then reacted with an excess of 4,4′dipyridyl to give intermediate 9. Reaction of 9 with m-bromomethylphenylboronic acid in DMF provided mBBVMP3+ (3) as a yellow solid. Similarly, reaction of 2 equiv of 8 or 10 with 4,4′-dipyridyl in DMF produced m-BBVBP4+ (2) and NBABP4+ (5), respectively. In an (41) Cooper, C.; James, T. D. J. Chem. Soc., Perkin Trans. 1 2000, 963-969.

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Scheme 3. Synthesis of Viologen Quenchersa

a Key: (a) pyridine, CH2Cl2/CH3OH (3:1), 40 °C, 22 h, 48%; (b) (1) 4,4′-dipyridyl, DMF, 80 °C, 5 min; (2) acetone, 74%; (c) m-bromomethylphenylboronic acid, DMF, 70 °C, 48 h, 43%.

Figure 1. Structures of HPTS (1) and the cationic quenchers (2-6).

analogous manner, the doubly charged quencher, mBBV2+ (4), was readily prepared from m-bromomethylphenylboronic acid and 4,4′-dipyridyl in DMF. Fluorescence Quenching Studies. Early studies demonstrating that HPTS fluorescence could be efficiently quenched by MV2+ indicated that the quenching relied mainly on an electron-transfer reaction between the phenolate oxygen of excited-state HPTS and the viologen electron acceptor.25 It was also noted that the SternVolmer analysis of this quenching provided superlinear plots that were best explained by two independent quenching mechanisms. In the viologen-HPTS interaction, the primary contribution to fluorescence quenching is from a static quenching process in which a nonfluorescent ground-state complex is formed between dye and quencher. A second process, known as dynamic quenching, occurs due to collisions between dye and quencher in which the fluorophore is deactivated through interaction with the colliding quencher molecule.42 In this study we use eq 3 which has been developed based on a “sphere of action”

quenching model25,43 and which provides quenching constants for both static (Ks) and dynamic (V) processes where Fo is the initial fluorescence and F is the fluorescence after addition of quencher. Quenching experiments were carried out on HPTS using each of the five quenchers in this study in order to determine the effect of quencher charge. We observed a general charge dependence for quenching among the structurally similar boronic acidsubstituted viologens, m-BBVBP4+, m-BBVMP3+, and m-BBV2+, where a higher charge correlated with increased quenching efficiency (Figure 2). It appears that more positively charged viologens have either greater electron affinity or a stronger electrostatic attraction through which they can bind HPTS in an essentially nonfluorescent quencher/dye complex, or both. All of the viologens studied demonstrated extremely (42) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2 ed.; Kluwer Academic/Plenum Publishers: New York, 1999. (43) Frank, I. M.; Vavilov, S. I. Z. Phys. Chem. (Munich) 1931, 69, 100.

Boronic Acid-Substituted Viologen Quenchers

Figure 2. Stern-Volmer plot of fluorescence quenching of HPTS (4 × 10-6 M) by quenchers 2-6 at pH 7.4 with charges indicated. Studies conducted at 20 °C, λex ) 460 nm, λem ) 510 nm; Fo ) original fluorescence; F ) fluorescence after addition of quencher. Table 1. Stern-Volmer Quenching Constants for Quenching of HPTS (4 × 10-6 M) at pH 7.4 NBABP4+ m-BBVBP4+ m-BBVMP3+ m-BBV2+ PDP2+

Ks(static) (M-1)

V(dynamic) (M-1)

260000 ( 10000 47000 ( 4000 25000 ( 2000 6700 ( 500 2400 ( 100

1800 ( 500 4500 ( 700 2400 ( 100 2700 ( 300 490 ( 40

efficient quenching. The observed quenching was determined by graphical methods using eq 3 to be due to a predominantly static quenching mechanism (Table 1). The non-boronic acid functionalized quencher NBABP4+ showed the most robust quenching activity with an extremely large static quenching constant. Evidently, the addition of boronic acids diminishes the quenching efficiency as indicated by the considerable difference in quenching between NBABP4+ and the boronic acidsubstituted quencher m-BBVBP4+. We have observed a similar decrease in the quenching efficiency when phenanthrolinium quenchers are substituted with boronic acid receptors.33 The presence of the boronic acids appears to limit complex formation either by sterics or due to electronic factors. The effects of boronic acid substitution on viologen properties are being further investigated. On comparison of m-BBV2+ to m-BBVBP4+, the presence of the pendant pyridinium groups was found to greatly enhance the quenching. Although simple pyridinium compounds have been known to cause fluorescence quenching,44 the degree of the quenching observed was quite small relative to that observed for bipyridinium compounds. In a control study we confirmed that the simple pyridinium compound PDP2+ (6), provided relatively weak quenching (Table 1) suggesting that the combination of pyridinium subunits with the bipyridinium core is not simply an additive quenching effect. Rather, the addition of the pyridinium groups appears to enhance the quenching by providing greater electrostatic attraction between dye and quencher due to the addition of two extra positive charges. To verify the dominance of the static quenching mechanism over dynamic quenching, we undertook a series of quenching experiments while varying temperature. We expected to observe a diminished quenching efficiency as temperature was increased because of the decreased stability of the quencher/dye complex. Conversely, lower temperatures were expected to provide an enhanced (44) Miola, L.; Abakerli, R. B.; Ginani, M. F.; Filho, P. B.; Toscano, V. G.; Quina, F. H. J. Phys. Chem. 1983, 87, 4417-4425.

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Figure 3. Stern-Volmer plot of NBABP4+ showing temperature-dependent quenching of HPTS (4 × 10-6 M) at pH 7.4.

degree of quenching due to the improved stability of the complex.42 By contrast, a dynamic process would be expected to produce the opposite effects since the collisional quenching process is directly proportional to the temperature. All of our viologen quenchers demonstrated the expected behavior thus verifying the primary role of the static mechanism in our system. A representative SternVolmer plot for the temperature-dependent quenching experiments is shown in Figure 3. UV-visible absorbance spectroscopy was used to investigate the interaction between HPTS and the viologen quenchers. Upon addition of a quencher, the absorbance peak at 460 nm grew as the absorbance maximum shifted to a longer wavelength. Plotting the difference spectra revealed a new band arising at ∼480 nm suggesting involvement of a charge transfer complex. At the same time, the peak at 404 nm diminished while an isosbestic point is visible at 418 nm. The isosbestic point strongly suggests a 1:1 binding stoichiometry for the dye/quencher complex (Figure 4). Similar UV-vis data were collected for each HPTSviologen combination and the data indicate 1:1 complexes being formed between dye and quencher. The stability constants obtained for complex formation from BenesiHildebrand plots of the absorbance data follow the same trend as with the Stern-Volmer data, indicating stronger binding for the more highly charged viologens (Table 2). Sugar-Sensing Studies. A considerable benefit of the two-component system is the ability to vary the ratio of quencher/dye in order to optimize the magnitude of the sensing response. Initially, we arrived at an optimal quencher/dye ratio by comparing fluorescence quenching in the absence of glucose with that observed in the presence of 5 mM glucose.33 The optimal ratio was the one that provided the largest difference between the two. While this provided the best results in terms of fluorescence response of the system for glucose concentrations of 5 mM, it did not give optimal results with regards to the magnitude of the signal at other glucose concentrations, nor did it provide information with respect to the shape of the binding isotherm. In the present study, a more thorough method for optimization of quencher/dye ratios has been developed. When the ratio between quencher and dye is steadily increased for each in a series of glucose titrations, a pattern emerges in which the point at which the system saturates steadily shifts to higher glucose concentrations, thus making the binding isotherm more linear in the low concentration range (Figure 5). As is evident in Figure 5, the low quencher/dye ratios such as 1:1 and 2:1 give weak signals and relatively saturated responses in the physiological range of glucose concentration (2.5-20 mM). Higher quencher/dye ratios,

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Table 2. Association Constants (Kuv) from UV-vis Data Determined for HPTS (4 × 10-6 M) Complexed with m-BBV2+, m-BBVMP3+, m-BBVBP4+, NBABP4+, and PDP2+ in pH 7.4 Phosphate Buffer NBABP4+ Kuv a

(M-1)

156000 ( 4000

m-BBVBP4+ 37000 (

2000a

m-BBVMP3+

m-BBV2+

PDP2+

22000 ( 2400

2900 ( 100

2000 ( 300

Obtained using 1 × 10-5 M HPTS.

Figure 6. Glucose response of viologens 2, 3, and 4 with HPTS (1) (4 × 10-6 M) at pH 7.4. Optimized quencher/dye ratios for m-BBV2+, m-BBVMP3+, and m-BBVBP4+ with HPTS were, respectively, 31:1, 125:1, and 125:1.

Figure 4. (a) UV-visible absorbance spectra of HPTS (1 × 10-5 M) at pH 7.4 with increasing concentrations of m-BBVBP4+ and (b) difference spectra for the same titration showing a putative CT band at ∼475 nm.

Figure 5. Binding isotherms for different quencher/dye ratios from fluorescence data for addition of glucose to a sample of HPTS (4 × 10-6 M) quenched by m-BBVBP4+ at pH 7.4. Physiological glucose range is boxed.

however, provide an isotherm that is increasingly linear. At 25:1 and 30:1, the signal response in the physiological range drops from a maximum reached at a 13:1 quencher/ dye ratio, but the linearity is improved across this same region. All of the quenchers studied displayed similar behavior with respect to quencher/dye ratios.

For glucose sensor applications, a linear response across the physiological glucose range is highly desirable so that a fluorescence change can be easily correlated with the correct change in glucose concentration. The ability to tune the signal response for a particular concentration range circumvents a common problem of many onecomponent fluorescence-based systems in which there may be excellent sensitivity, but the signal response is too rapidly saturated before physiological glucose concentrations are reached. For this reason, we adjusted quencher/ dye ratios for each of the quencher and dye combinations in order to obtain a linear response across the physiological range. We found that m-BBVBP4+ gave optimal results at a quencher/dye ratio of 31:1, while m-BBVMP3+ and m-BBV2+ worked best at a ratio of 125:1. The apparent glucose binding constants for m-BBV2+, m-BBVMP3+, and m-BBVBP4+ determined from the fluorescence data at the optimized ratios were, respectively, 11 ( 3, 12 ( 2, and 27 ( 6 M-1. The largely optimized apparent glucose binding isotherms for these viologens are shown in Figure 6. When used in combination with HPTS, the relative glucose-sensing ability of these viologens was found to be inversely proportional to the magnitude of their charge. As the viologens bind saccharides, their boronic acids are converted to boronate esters (Scheme 1) leading to a negative charge on the boron atom. As described earlier, the change in boron from neutral to anionic upon addition of glucose was confirmed using 11B NMR.36 Assuming 1:1 binding stoichiometry between boronic acid and glucose, each viologen will experience a net decrease of 2 in its overall charge when converted to its boronate ester after glucose binding. This will leave viologens m-BBVBP, m-BBVMP, and m-BBV with residual charges of 2+, 1+, and 0, respectively. The changes in charge and observed relative quenching and sugar-sensing characteristics are summarized in Table 3. Evidently, after sugar binding the compound with no remaining positive charge has little attraction for the anionic dye. Thus the quencher/dye complex will dissociate to a large extent resulting in a large increase in the fluorescence signal. The viologens which retain a net positive charge after binding also experience a diminished

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Table 3. Summary of Charge Changes and Associated Relative Quenching and Glucose-Sensing Characteristics for Viologens viologen and net charge in absence of glucose

HPTS quenching

viologen net charge after glucose binding

m-BBVBP 4+ m-BBVMP 3+ m-BBV 2+

strong moderate weak

2+ 1+ 0

glucose sensing weak moderate strong

quenching interaction, but the effects are less pronounced as is the observed fluorescence signal for these compounds. Fluorescence recovery is observed as the complex becomes less stable and an increasing proportion of the HPTS dye exists in its uncomplexed fluorescent state. These results indicate that a 1:1 ratio between boronic acids and positive charges, as in m-BBV2+, is optimal for sugar sensing in this system. We also used our system to detect the monosaccharides fructose and galactose. The monosaccharide selectivity observed for each of the quenchers in our study roughly corresponded with the 33:3:1 ratio of binding affinities for fructose, galactose, and glucose determined for simple phenylboronic acid.45 This indicates that cooperative binding of monosaccharide by the two boronic acids is not occurring. With UV-vis absorbance spectroscopy, the decreasing stability of the quencher/dye complex and the return to the uncomplexed HPTS absorption spectra were evident upon addition of the monosaccharides glucose, galactose, and fructose to quenched solutions of HPTS. Because sugar binding disrupts the ability of the quencher to form a ground-state complex with the HPTS, a complex with a lower stability results. The diminished complexation between boronic acid-substituted viologens and HPTS was readily observed on addition of each of the monosaccharides but was most clearly seen with addition of fructose, which we found to bind most strongly to the viologen quencher (Figure 7). It is clear that on addition of saccharides such as fructose (shown here) that there is a considerable recovery of the original HPTS absorption spectra indicating that the complex is less stable and has been largely dissociated. Conclusion In this two-component sensing system, we have demonstrated a clear dependence on viologen charge both for quenching the fluorescence of the anionic HPTS dye and for producing a fluorescence increase upon addition of monosaccharides. The quenching interaction was studied by both fluorescence and UV-visible spectroscopy. The quenching mechanism was determined by graphical means and temperature studies to be primarily static in (45) Springsteen, G.; Wang, B. H. Tetrahedron 2002, 58, 5291-5300.

Figure 7. Absorption changes upon addition of m-BBV2+ to HPTS (1 × 10-5 M) and upon addition of fructose to the quenched solution at pH 7.4.

nature and reliant on formation of a nonfluorescent ground state complex. We determined that greater positive charges on the viologen quencher facilitated more efficient fluorescence quenching. The two-component design of our system allowed for an optimal quencher/dye ratio to be chosen so that a linear increase in fluorescence intensity could be achieved across the physiological glucose range. With regard to monosaccharide sensing, we found that the viologen with the fewest positive charges relative to the number boronic acids provided the highest glucose response signal due to greater loss of electrostatic attraction between dye and quencher. The generality of this two-component sensing system and the ease with which different viologen quenchers can be introduced allow for a wide range of quencher designs to be considered. We are actively investigating new quencher and dye combinations for use in an immobilized glucose-sensing system aimed toward medical applications. Acknowledgment. We thank GluMetrics, Inc., operating through the BioStar Industry-University Cooperative Research program (BioStar Grant Isi-bio01-10091), for continuing financial support. Thanks to Trevor Swartz for his assistance in performing temperature studies and to the referees for their helpful comments. Supporting Information Available: Experimental procedures, 1H and 13C NMR spectra of 2-6, 11B NMR data to confirm that changes occur to the boronic acids in each of the viologen quenchers, representative UV-vis absorbance difference spectra and Benesi-Hildebrand plots for each HPTS-quencher combination and the Benesi-Hildebrand plots used to calculate the association constant for complex formation between dye and quencher, and representative raw fluorescence emission data for quenching and glucose-sensing studies. This material is available free of charge via the Internet at http://pubs.acs.org. LA050219X