Monosaccharide Detection with 4,7-Phenanthrolinium Salts: Charge

In addition, a variety of boronic acid-functional quenchers can be screened for their quenching ability and sugar response with a variety of dyes.21 T...
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Langmuir 2003, 19, 5145-5152

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Monosaccharide Detection with 4,7-Phenanthrolinium Salts: Charge-Induced Fluorescence Sensing Jeff T. Suri, David B. Cordes, Frank E. Cappuccio, Ritche A. Wessling, and Bakthan Singaram* Department of Chemistry and Biochemistry, University of California at Santa Cruz, Santa Cruz, California 95064 Received February 17, 2003. In Final Form: April 14, 2003 Herein we report the preparation of novel boronic acid-substituted 4,7-phenanthrolinium viologens and demonstrate their ability to quench the fluorescence of 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (pyranine). In an aqueous buffer solution at pH 7.4, the quenching is suppressed upon binding to monosaccharides, as was determined by Stern-Volmer analysis. Thus, the combination of the viologen and pyranine is shown to act as a saccharide sensor. A spectroscopic study reveals a ground-state complexation between the viologens and pyranine. In the presence of monosaccharide, the complex dissociates, resulting in a relative increase in the fluorescence. In comparison to the dipyridyl-derived viologen, the phenanthrolinederived viologen shows a greater fluorescence quenching as well as sensitivity to and an unusual selectivity for glucose.

Introduction The molecular recognition of biological analytes by synthetic receptors is a subject of great interest in many fields of biology and clinical chemistry.1 In particular, the design of optical chemosensing systems capable of detecting zinc,2 fluoride,3 or glucose4 have attracted much attention because of the biological significance of such substrates. One of the most successful techniques implemented for this purpose involves the fluorescence modulation of a fluorophore. In such systems, an abiotic sensing molecule comprised of a dye bound to a receptor stoichiometrically interacts with an analyte in such a way as to change the measured fluorescence intensity of the dye/ receptor molecule.5,6 Consequently, the fluorescence intensity change is dependent on the analyte concentration, so the latter can be conveniently measured and quantified. Regarding glucose sensing, one approach is to use a boronic acid group as a receptor site and to attach it to a fluorescent dye. Russell first described this type of glucose chemosensor in a patent.7 The first scientific study was reported by Yoon and Czarnik in which an anthracene (dye) was modified with a boronic acid (receptor);8 upon * Author to whom correspondence should be addressed. E-mail: [email protected]. (1) Hartley, J. H.; James, T. D.; Ward, C. J. J. Chem. Soc., Perkin Trans. 1 2000, 3155-3184. (2) (a) Jiang, P. J.; Chen, L. Z.; Lin, J.; Liu, Q.; Ding, J.; Gao, X.; Guo, Z. J. J. Chem. Soc., Chem. Commun. 2002, 1424-1425. (b) Kim, T. W.; Park, J. H.; Hong, J. I. J. Chem. Soc., Perkin Trans. 2 2002, 923-927. (c) Kimura, E.; Aoki, S. Biometals 2001, 14, 191-204. (d) Pearce, D. A.; Jotterand, N.; Carrico, I. S.; Imperiali, B. J. Am. Chem. Soc. 2001, 123, 5160-5161. (3) (a) DiCesare, N.; Lakowicz, J. R. Anal. Biochem. 2002, 301, 111116. (b) Gunnlaugsson, T.; Davis, A. P.; Glynn, M. J. Chem Soc., Chem. Commun. 2001, 2556-2557. (c) Kim, S. K.; Yoon, J. J. Chem Soc., Chem. Commun. 2002, 770-771. (d) Yamaguchi, S.; Akiyama, S.; Tamao, K. J. Am. Chem. Soc. 2001, 123, 11372-11375. (4) James, T. D.; Shinkai, S. Top. Curr. Chem. 2002, 218, 187-200. (5) de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley, A. J. M.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. Rev. 1997, 97, 1515-1566. (6) Czarnik, A. W. Fluorescent Chemosensors for Ion and Molecule Recognition; American Chemical Society: Washington, DC, 1993. (7) Russell, A. P. U.S. Patent 5,137,833, 1992. (8) Yoon, J.; Czarnik, A. W. J. Am. Chem. Soc. 1992, 114, 58745875.

the binding to glucose (analyte), a measurable change in the fluorescence intensity was observed in aqueous dimethyl sulfoxide solutions. It was well-known at that time that boronic acids bind reversibly with glucose to form boronate esters under aqueous conditions,9 but Yoon and Czarnik’s work was significant in that it established the basis for an optical glucose sensor. Since that time, numerous in vitro studies have been carried out by Shinkai et al.,10 James et al.,11 Norrild et al.,12 and others13-17 to help establish the scope of the technique. We have reported a two-component system comprised of 8-hydroxypyrene-1,3,6-trisulfonic acid (pyranine) and o-BBV2+ that is capable of detecting glucose in the physiological range.18 In this system, pyranine is the reporting group and its fluorescence is modulated via interaction with the boronic acid-substituted viologen. In contrast to the unimolecular chemosensors described previously,10-17 our system is bimolecular, involving a discrete dye molecule and quencher molecule that contains the boronic acid receptor.19,20 In glucose sensor research, our two-component system offers certain advantages. For (9) Lorand, J. P.; Edwards, J. O. J. Org. Chem. 1959, 24, 769. (10) (a) James, T. D.; Harada, T.; Shinkai, S. J. Chem. Soc., Chem. Commun. 1993, 1176-1176. (b) James, T. D.; Harada, T.; Shinkai, S. J. Chem. Soc., Chem. Commun. 1993, 857-860. (c) Deng, G.; James, T. D.; Shinkai, S. J. Am. Chem. Soc. 1994, 116, 4567-4572. (d) James, T. D.; Sandanayake, K.; Shinkai, S. Angew. Chem., Int. Ed. Engl. 1994, 33, 2207-2209. (e) James, T. D.; Murata, K.; Harada, T.; Ueda, K.; Shinkai, S. Chem. Lett. 1994, 273-276. (f) James, T. D.; Sandanayake, K.; Shinkai, S. J. Chem. Soc., Chem. Commun. 1994, 477-478. (g) Ludwig, R.; Harada, T.; Ueda, K.; James, T. D.; Shinkai, S. J. Chem. Soc., Perkin Trans. 2 1994, 697-702. (h) James, T. D.; Sandanayake, K.; Iguchi, R.; Shinkai, S. J. Am. Chem. Soc. 1995, 117, 8982-8987. (i) James, T. D.; Sandanayake, K.; Shinkai, S. Supramol. Chem. 1995, 6, 141. (j) James, T. D.; Shinkai, S. J. Chem. Soc., Chem. Commun. 1995, 1483-1485. (k) Linnane, P.; James, T. D.; Shinkai, S. J. Chem. Soc., Chem. Commun. 1995, 1997-1998. (l) Sandanayake, K.; James, T. D.; Shinkai, S. Chem. Lett. 1995, 503-504. (m) Sandanayake, K.; Imazu, S.; James, T. D.; Mikami, M.; Shinkai, S. Chem. Lett. 1995, 139-140. (n) James, T. D.; Sandanayake, K.; Shinkai, S. Angew. Chem., Int. Ed. Engl. 1996, 35, 1911-1922. (o) James, T. D.; Shinmori, H.; Takeuchi, M.; Shinkai, S. J. Chem Soc., Chem. Commun. 1996, 705-706. (p) James, T. D.; Linnane, P.; Shinkai, S. J. Chem Soc., Chem. Commun. 1996, 281-288. (q) Sandanayake, K.; James, T. D.; Shinkai, S. Pure Appl. Chem. 1996, 68, 1207-1212. (r) James, T. D.; Shinmori, H.; Shinkai, S. J. Chem Soc., Chem. Commun. 1997, 71-72. (s) Kijima, H.; Takeuchi, M.; Robertson, A.; Shinkai, S.; Cooper, C.; James, T. D. J. Chem Soc., Chem. Commun. 1999, 2011-2012.

10.1021/la034270h CCC: $25.00 © 2003 American Chemical Society Published on Web 05/14/2003

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example, the chemical structure of the quencher can be modified to obtain glucose selectivity over fructose without having to modify the structure of the dye. Dyes are typically complex organic molecules with optical properties sensitive to subtle changes in structure. Being able to use existing dyes with minimal structural modification is, thus, an advantage. In addition, a variety of boronic acidfunctional quenchers can be screened for their quenching ability and sugar response with a variety of dyes.21 To this end, we have screened several types of quenchers to optimize the system with pyranine. Herein, we report our findings using novel 4,7-phenanthroline-based quenchers and demonstrate their ability to detect monosaccharides. Design of the Quencher. Viologens are well-known to be good electron acceptors.22 This property has been exploited in the fluorescence quenching of simple dyes23 as well as macromolecular dyes.24 The ability of the viologens to quench fluorescence stems partly from the fact that they contain two positive charges. The charges facilitate two possible types of processes: electron transfer and Coulombic attraction. The fluorescence quenching of an anionic dye molecule by nonionic 4,4′-dipyridyl follows simple Stern-Volmer kinetics (dynamic quenching); however, quenching with a cationic viologen is dramatically enhanced as a result of electrostatic attraction (static quenching). Wang et al. have shown this to be the case in the fluorescence quenching of MBL-PPV25 with 4,4′dipyridyl and 4,4′-dipyridinium salts.26 Although methyl viologen (MV2+) is the compound that is most often utilized, other types of viologens with different redox potentials (11) (a) Arimori, S.; Ward, C. J.; James, T. D. J. Chem. Soc., Chem. Commun. 2001, 2018-2019. (b) Arimori, S.; Bosch, L. I.; Ward, C. J.; James, T. D. Tetrahedron Lett. 2001, 42, 4553-4555. (c) Arimori, S.; Bell, M. L.; Oh, C. S.; Frimat, K. A.; James, T. D. J. Chem. Soc., Chem. Commun. 2001, 1836-1837. (d) Ward, C. J.; Patel, P.; Ashton, P. R.; James, T. D. J. Chem. Soc., Chem. Commun. 2000, 229-230. (e) Cooper, C. R.; James, T. D. Chem. Lett. 1998, 883-884. (12) (a) Eggert, H.; Frederiksen, J.; Morin, C.; Norrild, J. C. J. Org. Chem. 1999, 64, 3846-3852. (b) Bielecki, M.; Eggert, H.; Norrild, J. C. J. Chem. Soc., Perkin Trans. 2 1999, 449-455. (c) Norrild, J. C.; Eggert, H. J. Am. Chem. Soc. 1995, 117, 1479-1484. (13) Yang, W.; He, H.; Drueckhammer, D. G. Angew. Chem., Int. Ed. 2001, 40, 1714-1718. (14) Tong, A. J.; Yamauchi, A.; Hayashita, T.; Zhang, Z. Y.; Smith, B. D.; Teramae, N. Anal. Chem. 2001, 73, 1530-1536. (15) Adhikiri, D. P.; Heagy, M. D. Tetrahedron Lett. 1999, 40, 78937896. (16) (a) DiCesare, N.; Lakowicz, J. R. J. Photochem. Photobiol., A 2001, 143, 39-47. (b) DiCesare, N.; Lakowicz, J. R. Tetrahedron Lett. 2001, 42, 9105-9108. (c) DiCesare, N.; Lakowicz, J. R. J. Chem. Soc., Chem. Commun. 2001, 2022-2023. (d) DiCesare, N.; Lakowicz, J. R. J. Phys. Chem. A 2001, 105, 6834-6840. (e) DiCesare, N.; Lakowicz, J. R. Anal. Biochem. 2001, 294, 154-160. (17) Springsteen, G.; Wang, B. H. J. Chem. Soc., Chem. Commun. 2001, 1608-1609. (18) Camara, J. N.; Suri, J. T.; Cappuccio, F. E.; Wessling, R. A.; Singaram, B. Tetrahedron Lett. 2002, 43, 1139-1141.

are known. In 1973, Huenig et al. reported the effect of extended conjugation in viologens where the redox potentials of 17 different N-substituted heteroaromatic salts were determined in water, DMF, and acetonitrile.27 This study showed that extended conjugation via the addition of aromatic rings improved the electron-accepting ability of the viologens. Specifically, N,N′-dimethyl-4,7-phenanthrolinium tetrafluoroborate (1b, Chart 1) gave a redox potential of -490 mV (vs AgCl) in water for a single electron transfer. This is significantly more positive than that of MV2+ (-640 mV), indicating that 4,7-phenanthrolinium salts are better electron acceptors. Despite this study, there has been only one report of a 4,7-phenanthrolinium salt being used as a fluorescence quencher. Ihara et al. used a porphyrin dye in combination with 1a to determine the base mismatching in a DNA sequence.28 In this study, 1a is thought to quench the fluorescence of the poryphyrin via a photoinduced electron transfer mechanism. The enhanced ability of 4,7-phenanthrolinium salts to accept electrons prompted us to study the quenching ability of these compounds and compare them to our previously reported dipyridyl-based quenchers in anticipation that phenanthrolinium-derived viologens might show greater quenching. We were also interested in modifying the viologens by the attachment of a boronic (19) Shinkai et al. have reported a biomolecular system where the boronic acid is attached to the dye molecule; see (a) Suenaga, H.; Arimori, S.; Shinkai, S. J. Chem. Soc., Perkin Trans. 2 1996, 607-612. (b) Takeuchi, M.; Taguchi, M.; Shinmori, H.; Shinkai, S. Bull. Chem. Soc. Jpn. 1996, 69, 2613-2618. (c) Arimori, S.; Murakami, H.; Takeuchi, M.; Shinkai, S. J. Chem Soc., Chem. Commun. 1995, 961-962. (20) Other groups have reported two-component dye-assay systems utilizing boronic acid-sensitive dyes; see (a) Springsteen, G.; Wang, B. J. Chem Soc., Chem. Commun. 2001, 17, 1608-1609. (b) Arimori, S.; Ward, C. J.; James, T. D. Tetrahedron Lett. 2002, 43, 303-305. (21) Recently another group has published their results using a twocomponent system to sense saccharides; see DiCesare, N.; Pinto, M. R.; Schanze, K. S.; Lakowicz, J. R. Langmuir 2002, 18, 7785-7787. (22) Monk, P. M. S. The viologens: physicochemical properties, synthesis, and applications of the salts of 4,4′-bipyridine; Wiley: Chichester, New York, 1998. (23) (a) de Borba, E. B.; Amaral, C. L. C.; Politi, M. J.; Villalobos, R.; Baptista, M. S. Langmuir 2000, 16, 5900-5907. (b) Nakashima, K.; Kido, N. Photochem. Photobiol. 1996, 64, 296-302. (c) Zhao, Z. G.; Shen, T.; Xu, H. J. J. Photochem. Photobiol., A 1990, 52, 47-53. (24) (a) 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, 1228712292. (b) Gaylord, B. S.; Wang, S. J.; Heeger, A. J.; Bazan, G. C. J. Am. Chem. Soc. 2001, 123, 6417-6418. (c) 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. (25) Poly[5-methoxy-2-(4-sulfobutoxy)-1,4-phenylenevinylene]. (26) Wang, D. L.; Wang, J.; Moses, D.; Bazan, G. C.; Heeger, A. J. Langmuir 2001, 17, 1262-1266. (27) Huenig, S.; Gross, J.; Lier, E. F.; Quast, H. Justus Liebigs Ann. Chem. 1973, 2, 339-358. (28) Ihara, T.; Takata, J.; Takagi, M. Anal. Chim. Acta 1998, 365, 49-54.

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Figure 1. 11B NMR spectrum of (a) o-BBV2+ and (b) o-PBBV2+ as a function of pH (initial [boronic acid] ) 30 mM in 0.15 M KCl; titrated with 0.1 M KOH). Scheme 1. Equilibria between Boronic Acids and Generic Diols

acid group and determining how their quenching ability is modulated in the presence of monosaccharides. Thus, we prepared N,N′-bisbenzyl-4,7-phenanthrolinium dibromide (PV2+), N,N′-4,4′-bis(benzyl-2-boronic acid)-bipyridinium dibromide (o-BBV2+), N-(benzyl-2-boronic acid)4,7-phenanthrolinium bromide (o-PBV+), and N,N′bis(benzyl-2-boronic acid)-4,7-phenanthrolinium dibromide (o-PBBV2+, Chart 1) and compared their ability to quench the fluorescence of pyranine and act as sugar sensors. Results and Discussion Boronic Acid pKa’s. Arylboronic acids are known for their ability to complex monosaccharides, as is indicated in Scheme 1.9 Under aqueous conditions, the boronic acid forms reversible covalent bonds with the diol moiety, creating a boronate ester. When the boron is tetrahedral, the rate of interconversion is much faster. Thus, from the standpoint of sensor design, it is thought advantageous to utilize a more acidic boron to facilitate a greater saccharide binding at a neutral pH.29 Many groups have accomplished this by incorporating a tertiary amine adjacent to the boronic acid functional group.4 The amine donates its electrons to the empty p orbital of boron, converting it from trigonal to tetrahedral at or near a neutral pH.30 Recently, Anslyn et al. has further demonstrated that secondary amines are also just as effective.31 Another approach places a boronic acid group near a positively charged nitrogen. When boron is directly (29) Recently Springsteen and Wang have challenged this notion; see Springsteen, G.; Wang, B. H. Tetrahedron 2002, 58, 5291-5300. (30) Wulff, G. Pure Appl. Chem. 1982, 54, 2093-2102. (31) Wiskur, S. L.; Lavigne, J. J.; Ait-Haddou, H.; Lynch, V.; Chiu, Y. H.; Canary, J. W.; Anslyn, E. V. Org. Lett. 2001, 3, 1311-1314.

Figure 2. UV-visible absorbance spectra of pyranine (4 × 10-6 M) at pH 3 (light solid line), pH 7.4 (bold solid line), and pH 10 (dashed line).

connected to a pyridinium ring, the pKa is lowered to 3.7.12a The viologens used in our study contain a positive charge in close proximity to the boronic acid group. We, therefore, anticipated a drop in the pKa relative to that of simple phenylboronic acid (pKa ) 8.8). Using 11B NMR to monitor the geometry of the boronic acid,31,32 we obtained titration curves for o-BBV2+ and o-PBBV2+. As is indicated in Figure 1, the boronic acids show a resonance of ∼30 ppm at a low pH. As the pH increases, the trigonal sp2 boron is converted to the tetrahedral sp3 boron, resulting in an upfield shift. From these plots, we found the apparent pKa(o-PBBV2+) ) 7.6 and pKa(o-BBV2+) ) 8.8.33 The 4,7-phenanthrolinium salt does show a slight drop in the pKa relative to that of simple phenylboronic acid; however, the bipyridinium salt pKa is identical to that of phenylboronic acid. These results indicate that boron is only slightly influenced by the positive charge.34 The pKa’s of the boronic acid adducts were also determined in the presence of glucose. Titrations carried out with 100 mM glucose present result in a relative decrease in the pKa’s, giving pKa(o-BBV2+) ) 6.7 and pKa(o-PBBV2+) ) 5.5. The decrease in the pKa is in line with what has been observed previously.9,19b Absorption Studies. Figure 2 shows the absorbance of pure pyranine at pH 3, 7.4, and 10. At pH 7.4, two (32) Nagai, Y.; Kobayashi, K.; Toi, H.; Aoyama, Y. Bull. Chem. Soc. Jpn. 1993, 66, 2965-2971. (33) Only one resonance was observed in the 11B NMR spectrum and was taken as the apparent pKa. (34) Shinkai et al. also observed similar pKa’s for a boronic acidsubstituted pyridinium salt; see ref 19b.

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Figure 3. UV-visible absorbance spectra of pyranine (4 × 10-6 M) with increasing concentrations of (a) PV2+, (b) o-PBBV2+, and (c) o-BBV2+ in a pH 7.4 phosphate buffer.

Figure 4. UV-visible absorbance spectra of pyranine (4 × 10-6 M) with increasing concentrations of (a) PV2+, (b) o-PBBV2+, and (c) o-BBV2+ in a pH 10 carbonate buffer. Scheme 2. Pyranine in Equilibrium in Its Acidic Form POH and Its Basic Form PO- at pH 7.4

Table 1. Association Constants (Ks) Determined for Pyranine (4 × 10-6 M) Complexed with PV2+, o-PBBV2+, and o-BBV2+ at Different pHs o-BBV2+ o-PBBV2+ PV2+

distinct absorption bands are apparent in the blue region, one at 404 nm and the other at 454 nm. The ground-state pKa of the phenolic group in pyranine is 7.2.35 At pH 3, pyranine is in its protonated form POH, and at pH 10, it is in its unprotonated form PO(Scheme 2). Thus, at pH 7.4 there exists a mixture of two different species, POH and PO-, giving rise to the 404- and 454-nm bands, respectively. It has been reported that POH and PO- reversibly react with MV2+ to form a ground-state complex that can be observed by UV-visible spectroscopy.22a It has also been shown that pyranine forms a groundstate complex with o-BBV2+.18 This suggests that a similar interaction between the viologens used in this study and the different forms of pyranine is possible at pH 7.4. Figure 3 shows the pyranine absorbance at pH 7.4 in the presence of the viologens PV2+, o-PBBV2+, and o-BBV2+. As was anticipated, a significant change occurs in the POH and PO- bands of the absorption spectra. For PV2+, o-PBBV2+, and o-BBV2+, a decrease in the 404-nm band is apparent. For PV2+ an isosbestic point is observed at 434 nm, for o-PBBV2+ one is observed at 416, and for o-BBV2+ isosbestic points occur at 412, 446, and 470 nm. To gain a better understanding of the perturbed spectra, experiments were carried out at pH 3 and 10. At pH 3, the band at 404 nm, due to POH, decreases upon the addition (35) (a) Politi, J. J.; Fernandez, A. M. C. J. Photochem. Photobiol., A 1997, 104, 165-172. (b) Wolfbeis, O. S.; Fuerlinger, E.; Kroneis, H.; Marsoner, H. Fresenius’ J. Anal. Chem. 1983, 314, 119-124. (c) Chulman, S. G.; Chen, S.; Bai, F.; Leiner, M. J. P.; Weis, L.; Wolfbeis, O. S. Anal. Chim. Acta 1995, 304, 165-170.

pH 3 (104 M-1)

pH 7.4 (104 M-1)

pH 10 (104 M-1)

4.9 ( 0.3 7.7 ( 0.5 7.7 ( 0.6

1.6 ( 0.2 3.1 ( 0.5 6.0 ( 0.7

0 0 4.6 ( 0.8

of PV2+ (see the Supporting Information). A clear isosbestic point is revealed at 416 nm. At pH 10, the UV-visible spectra also reveal an isosbestic point at 470 nm upon the addition of PV2+ (Figure 4).36 This suggests complex formation between the viologen and pyranine at pH 3 and 10. Therefore, at pH 7.4, the overall spectra are due to the presence of multiple complexes between the different forms of pyranine and PV2+. For the boronic acid-substituted viologens, similar spectra are obtained at pH 3. However, at pH 10, o-PBBV2+ and o-BBV2+ show no significant change in the absorption spectra of pyranine (360-500-nm range). This can be explained by comparison of the association constants calculated using Benesi-Hildebrand37 plots for each derivative at the different pHs and assuming 1:1 binding stoichiometry (Table 1). It is clear that the boronic acid functional group affects complex formation, especially at a high pH. The pKa of o-PBBV2+ is 7.6. In a pH 7.4 buffer, it exists as a mixture of boronic acid and an “ate” complex (Figure 1), but at pH 10, the molecule is purely zwitterionic. Consequently, at a high pH, o-PBBV2+ is completely neutralized to o-PBBV. The neutralization of o-PBBV2+ results in the loss of the electrostatic attraction to pyranine; with the loss of the electrostatic attraction, there is no ground-state complex formation and no perturbation in the UV-visible spectra. The same argument also holds for o-BBV2+. Quenching. Data Analysis. The fluorescence quenching of dyes can be quantitatively monitored by Stern-Volmer (36) At pH 10 the phenanthrolinium derivatives show some absorbance in the range 400-500 nm. To determine the degree of complexation with pyranine, the absorbance due to phenanthrolinium was subtracted from the overall absorbance due to pyranine and phenanthrolinium salt. See the Supporting Information for details. (37) Connors, K. A. Binding ConstantssThe measurement of Molecular Complex Stability; John Wiley: New York, 1987.

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Figure 5. Stern-Volmer plots of pyranine (4 × 10-6 M) with increasing concentrations of PV2+, o-PBBV2+, and o-BBV2+ at (a) pH 3, (b) pH 7.4, and (c) pH 10. λex ) 416 nm for pH 3 and 470 nm for pH 7.4 and pH 10; λem ) 510 nm.

analysis.38 Two types of quenching exist: static quenching, due to complex formation, and dynamic quenching, due to collisional encounters between the dye and the quencher. Dynamic quenching can be determined via steadystate (F0/F) or time-resolved measurements (τ0/τ) using

F0/F ) τ0/τ ) 1 + Kd[Q]

(1)

where F0 and τ0 are the fluorescence intensity and fluorescence lifetime, respectively, of a dye in the absence of the quencher, F and τ are the fluorescence intensity and fluorescence lifetime of a dye in the presence of the quencher, [Q] is the concentration of the quencher, and Kd is the dynamic Stern-Volmer quenching constant. Static quenching can only be determined through steady-state measurements. This is due to the fact that a static quenching mechanism is a very rapid process and cannot be observed through time-resolved measurements. Consequently, the static Stern-Volmer quenching constant, Ks, is obtained through use of eq 2.

F0/F ) 1 + Ks[Q]

(2)

Both expressions provide a way to determine the extent of dynamic or static quenching from the steady-state data; when a straight line is obtained, Kd or Ks is equal to the slope. However, when multiple quenching processes occur, the steady-state quenching data deviates from linearity. There are many examples of this behavior in the literature, especially in the cases of complex biological systems.39 A systematic treatment of such data has been reported.40 To account for positive deviations from linearity, Frank and Vavilov modified eq 1 and introduced a “quenching sphere of action” model.41 In this model, the quencher is always in close proximity to the dye, that is, within the sphere of action, making it very probable that the quenching will occur before the molecules diffuse apart. Recently, this model has been modified to account for charged molecules that statically quench through complex formation and dynamically quench within the sphere of action.26 This is given by

F0/F ) (1 + Ks[Q])eV[Q]

(3)

where V is the dynamic quenching constant, Ks is the static quenching constant, [Q] is the concentration of the quencher, and eV[Q] is derived from the Poisson distribution e-V[Q], the probability that a random distribution of quenchers in a solution will statically quench a dye. (38) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum Press: New York, 1983. (39) Castanho, M. A. R. B.; Prieto, M. J. E. Biochim. Biophys. Acta 1998, 1373, 1-16. (40) Laws, W. R.; Contino, P. B. Methods Enzymol. 1992, 210, 448463. (41) Frank, I. M.; Vavilov, S. I. Z. Phys. Chem. (Munich) 1931, 69, 100.

The percent fluorescence recovery (%FR) determined in the quencher sensitivity test is defined as

%FR )

Fglu - FP+Q FP

(4)

where FP is the fluorescence intensity of pyranine alone, FP+Q is the fluorescence intensity of pyranine after the addition of the quencher, and Fglu is the fluorescence intensity of the pyranine/glucose solutions after the addition of the quencher. In a similar manner, the binding studies were carried out using eq 4, where FP is the fluorescence intensity of pyranine alone, FP+Q is the fluorescence intensity of pyranine after the addition of the quencher, and Fglu is the fluorescence intensity of the pyranine/quencher solutions after the addition of the sugar. Emission Studies and Stern-Volmer Analysis. We investigated the ability of PV2+, o-BBV2+, and o-PBBV2+ to quench the fluorescence of pyranine under steady-state conditions. At pH 3 and 7.4, significant fluorescence quenching occurred for all the viologens. Data analysis using eq 1 gives nonlinear plots (Figure 5), suggesting both static and dynamic quenching. Excellent fits were obtained using eq 3, and the static and dynamic constants are summarized in Table 2. At pH 10, a significant amount of quenching is apparent with PV2+. However, this is not the case for the boronic acid-substituted derivatives, which show a large decrease in the quenching. Because Ks is an association constant for the dye/quencher ground-state complex,26 the degree of association between PV2+ and PO- would be greatest at a high pH because in the ground-state the Coulombic attraction between the cationic quencher and the anionic dye is maximized. With an increasing pH, the borons on o-BBV2+ and o-PBBV2+ become tetrahedral and negatively charged. The resultant zwitterion is electrostatically neutral. Without the full positive charges, the degree of association between PO- and o-BBV or PO- and o-PBBV is expected to be much lower because there is a loss of electrostatic attraction. This situation results in no static quenching and corresponds to what is observed in the UV-visible data, that is, the lack of complex formation. Because no shift in λmax was observed in the excitation spectra of pyranine upon the addition of the quencher (data not shown), the fluorescence is solely due to the population of uncomplexed fluorophores.38 Sugar Sensing. Quencher Sensitivity. Pyranine fluorescence is quenched by dipyridyl-derived viologens;23a by connecting a boronic acid group to the viologen, it has been shown that the fluorescence can be modulated upon interaction with glucose.18 We had envisioned a similar interaction with our 4,7-phenanthroline-derived viologen, o-PBBV2+. To determine the effect of glucose on the quenching ability of viologens, titrations of pyranine were carried out with o-PBBV2+ and o-BBV2+ at pH 7.4 in the

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Table 2. Static (Ks) and Dynamic (V) Quenching Constants of the Viologens PV2+, o-BBV2+, and o-PBBV2+ with Pyranine (4 × 10-6 M) in Buffered 0.1 Ionic Strength Solutions at Different pHs pH 3 o-BBV2+ o-PBBV2+ PV2+

pH 7.4

pH 10

Ks (104 M-1)

V (104 M-1)

Ks (104 M-1)

V (104 M-1)

Ks (104 M-1)

V (104 M-1)

1.3 ( 0.1 6.3 ( 0.1 6.1 ( 0

0 0.04 ( 0.01 0.3 ( 0

1.6 ( 0 4.9 ( 0 8.6 ( 0.5

2.1 ( 0 0.5 ( 0 1.1 ( 0

0 0 11 ( 0

0 0.4 ( 0.1 1.0 ( 0.1

Table 3. Apparent Binding Constants Determined for o-Pbbv2+, o-BBV2+, and o-PBV+ via Fluorescence (Kf) and UV-Visible Measurements (KUV) fructose o-BBV2+ o-PBBV2+ o-PBV2+

glucose

galactose

Kf (M-1)

KUV (M-1)

Kf (M-1)

KUV (M-1)

Kf (M-1)

KUV (M-1)

2600 ( 100 3300 ( 200 2500 ( 200

1800 ( 300 4300 ( 400 2000 ( 1000

43 ( 3 1800 ( 200 1000 ( 200

140 ( 12 1500 ( 400 900 ( 100

167 ( 4 1600 ( 100 600 ( 90

220 ( 20 1340 ( 70 450 ( 60

Figure 6. Stern-Volmer plot of pyranine (4 × 10-6 M) with o-PBBV2+ or o-BBV2+ with and without 5 mM glucose in a pH 7.4 phosphate buffer. λex ) 470 nm and λem ) 510 nm.

presence of glucose. The concentration of glucose was chosen to be 5 mM to mimic physiological conditions. Stern-Volmer analysis using eq 1 reveals data that remain linear for o-PBBV2+, giving a dynamic quenching constant of Kd ) 8.7 × 103 M-1. For o-BBV2+, the data is nonlinear, giving a dynamic quenching constant of V ) 1.2 × 103 M-1 and a static quenching constant of Ks ) 1.5 × 104 M-1. In the presence of sugar, the quenching has decreased giving a Stern-Volmer plot that is very similar to that obtained at pH 10, where the static quenching component has been removed (for o-PBBV2+) and only dynamic quenching occurs to varying degrees (Figure 6). When a Job-type plot was used,37 the optimum quencher concentration was determined for each viologen according to eq 4. As is indicated in Figure 7, the maximum fluorescence recoveries for o-BBV2+ and o-PBBV2+ occur at a quencher/dye ratio of 18:1 and 22:1, respectively. The signal for o-BBV2+ is ∼4% of the original emission (that of pyranine alone); o-PBBV2+ is dramatically different, giving ∼45% recovery. The optimum concentrations of the quencher determined from these plots were used in the binding studies. Binding Studies. The addition of monosaccharides to solutions of pyranine/quencher resulted in a relative increase in the fluorescence as well as a perturbation in the absorption spectra. The percent fluorescence recovery of pyranine was plotted as a function of the monosaccharide concentration. The apparent binding constants (Kf was determined using fluorescence data and KUV was determined using UV data) were determined from the binding isotherms37,42 (Figure 8) and are given in Table 3. For o-BBV2+, the binding order is fructose . galactose > glucose. For o-PBBV2+, the binding order is fructose > glucose > galactose. The selectivity of o-BBV2+ follows (42) Cooper, C.; James, T. D. J. Chem. Soc., Perkin Trans. 1 2000, 963-969.

the usual binding order, where the fructose binding is much greater than that of glucose.9 On the other hand, o-PBBV2+ shows an enhanced glucose selectivity, giving a binding constant for fructose that is approximately only twice that of glucose. The difference in the binding affinities between the boronic acid-derived quenchers can be partially explained by the difference in the pKa’s. For example, o-BBV2+ has an apparent pKa of 8.8, making the formation of the boronate complex with glucose unfavorable at pH 7.4. By comparison, the pKa of o-PBBV2+ is 7.6, giving rise to a more favorable binding at pH 7.4 and, as a result, a larger binding constant. The structural differences between o-BBV2+ and oPBBV2+ also play a part in the ability to bind glucose. For example, the spacing between the bisboronic acids in o-PBBV2+ may lead to cooperative binding, where two boronic acids bind to one glucose molecule.10h,12a,13,43 To probe the binding mode of o-PBBV2+, we carried out studies with the monoboronic acid derivative (o-PBV+).44 The binding constants of o-PBV+ were approximately half those of o-PBBV2+ for glucose and galactose, corresponding to half the number of binding sites. The affinity for fructose was similar for all the viologens. The relative fluorescence intensity change resulting from the addition of each sugar to o-PBV+ is in the order fructose > galactose > glucose, whereas for o-PBBV2+, the order is glucose > fructose > galactose. These results are unusual in that the relative fluorescence intensity change is not proportional to the binding constant45 and may indicate that the bisboronic acid in o-PBBV2+ binds cooperatively with glucose. Recognition Event. The UV-visible spectra of o-PBBV2+/pyranine in the presence of glucose are given in Figure 9. It is apparent that a ground-state complex forms between the viologen and pyranine at pH 7.4. The fluorescence spectra and Stern-Volmer analysis also reveal the formation of a “dark” complex that is nonfluorescent. Upon the addition of glucose, the absorption spectrum of pyranine is regenerated along with the partial recovery of the emission spectrum, indicating the dissociation of the o-PBBV2+/pyranine complex. Because the fluorescence (43) In o-BBV2+, this seems unlikely because the intramolecular distance between the boronic acids is much greater than what is needed for bis binding. (44) Stern-Volmer analysis of o-PBV+ with pyranine in the same concentration range gives Ks ) (6 ( 1) × 103 M-1 and V ) (4.1 ( 0.8) × 103 M-1; monocationic viologens are known to quench less efficiently than dicationic viologens, and that is what we observe with o-PBV+ (see ref 26). (45) Heagy et al. have also reported a similar finding; see Cao, H. S.; Diaz, D. I.; DiCesare, N.; Lakowicz, J. R.; Heagy, M. D. Org. Lett. 2002, 4, 1503-1505.

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Figure 7. Percent fluorescence recovery of pyranine (4 × 10-6 M) in the presence of glucose (5 mM) upon the addition of different concentrations of o-PBBV2+ or o-BBV2+ in a pH 7.4 buffer. λex ) 470 nm and λem ) 510 nm.

Figure 8. Percent fluorescence recovery of pyranine (4 × 10-6 M) in the presence of the quencher and upon the addition of different monosaccharides: (a) pyranine/o-BBV2+, (b) pyranine/o-PBBV2+, and (c) pyranine/o-PBV+. λex ) 470 nm and λem ) 510 nm. Scheme 3. Mechanistic Cycle Describing the Equilibria between Pyranine, o-PBBV2+, and Monosaccharides

emission is not completely recovered, the dynamic component is not removed from the quenching mechanism; however, the static quenching is essentially shut down. The pKa of o-PBBV2+ changes from 7.6 to 5.5 in the presence of 100 mM glucose, effectively giving the boron a negative charge at pH 7.4. This result is similar to that obtained at pH 10, where the lack of complex formation is apparent when the boron is negatively charged and the molecule is zwitterionic. Thus, it appears that the main signaling mechanism responsible for the sugar sensing

involves the modulation of the charge on boron, as is indicated in Scheme 3. Conclusion We have demonstrated that the combination of a fluorescent dye and a boronic acid-substituted phenanthrolinium viologen can be used to detect monosaccharides such as glucose. The system functions in a buffer solution at pH 7.4 over the physiologically important concentration range of 0-20 mM. In this study, the cationic viologens

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fluorescence recovery compared to those of the previously reported dipyridyl-derived viologen. Further, the N,N′benzylboronic acid phenanthrolinium salts show an unexpected enhanced selectivity for glucose compared to that of typical boronic acids. This suggests cooperative binding. Further studies are underway to evaluate the performance of these quenchers with other dyes to determine the optimum dye/quencher combination for sensing glucose. Figure 9. UV-visible absorbance spectra of pyranine (4 × 10-6 M; bold solid line), pyranine (4 × 10-6 M) with o-PBBV2+ (7.2 × 10-5 M; light solid line), and pyranine (4 × 10-6 M) with o-PBBV2+ (7.2 × 10-5 M) and glucose (14 mM; dashed line).

were combined with the anionic, water-soluble dye, trisodium 8-hydroxypyrene-1,3,6-trisulfonate (pyranine). In the absence of saccharide, a ground-state complex forms by ionic association between pyranine and the viologen boronic acid, resulting in a greatly reduced fluorescence. Upon the binding of the viologen to saccharide to form the negatively charged boronate ester, the electrostatic attraction between the dye and the quencher is lost, resulting in a recovery of the fluorescence that is dependent on the saccharide concentration. The phenanthroline-derived viologen reported here shows greater quenching and

Acknowledgment. We thank the BioSTAR Project and the Industry-University Cooperative Research Program with Glumetric for their continued financial support. We also thank Professor Jin Zhang of UC Santa Cruz for his helpful discussions and the reviewers for their insightful comments. Supporting Information Available: Detailed experimental section describing the synthesis, characterization, and spectroscopic studies of o-PBBV2+, PV2+, and o-PBV+; mathematical expressions used for the determination of the association constants. This material is available free of charge via the Internet at http://pubs.acs.org. LA034270H