Fluorescent Chemosensors of Ion and Molecule Recognition

Chapter 27

Fluorescent Chemosensors of Ion and Molecule Recognition Recent Applications to Pyrophosphate and to Dopamine Sensing

Downloaded by NORTHWESTERN UNIV on February 21, 2017 | http://pubs.acs.org Publication Date: July 16, 1994 | doi: 10.1021/bk-1994-0561.ch027

Anthony W. Czarnik Department of Chemistry, Ohio State University, Columbus, OH 43210

1,8-Bis(TRPN)methylanthracene, available from 1,8dibromomethylanthracene, binds phosphate and pyrophosphate anions in aqueous solution, pH 7, with signalling in the form of chelation enhancedfluorescence.The affinity of this convergent chemosensor for pyrophosphate (K = 2.9 μΜ) is over 2000-times that for phosphate (K = 6.3 mM); a reference nonconvergent chemosensor [1mono(TRPN)methylanthracene] displays only a 100-fold difference in binding affinities. The ion discrimination thus realized permits real -timeanalysis in the hydrolysis of pyrophosphate by inorganic pyrophosphatase. It has been known for almost 35 years that catechol complexes reversibly to boronic acids. We observe that 2-anthrylboronic acid complexes catechol in water with K 330 μΜ and concomitant 20-fold reduction influorescenceintensity. L-DOPA and dopamine behave similarly, suggesting a mechanism for the development of real-time sensing schemes. d

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Over the past several decades, chemists have devoted their collective attention to the design of molecules that bind other molecules reversibly. A primary goal of such work is the desire to engineer synthetic catalysts fromfirstprinciples. However, an equally appealing goal, and one with significant practical potential, is the design of chemosensors with selectivity for analytes such as anions (e.g., phosphate), zwitterions (e.g., amino acids), and neutral species (e.g., O2). Insufficient attention has been paid to this application of ion and molecule recognition, given the potential utility of chemosensors as essential components in the construction of real-time monitoring devices. Because many sensing applications will take place in an aqueous environment, aqueous recognition is prerequisite. Considerable recent effort has focused on fluorescence as a signal transduction mechanism because of its sensitivity, its potential application to fiber

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Mallouk and Harrison; Interfacial Design and Chemical Sensing ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

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Fluorescent Chemosensors of Ion & Molecule Recognition

optic-based remote sensing schemes, and because the multiplicity of mechanisms for modulating fluorescence provides for the incorporation of many known binding mechanisms.

Anion Chemosensing: Pyrophosphate While an ability to monitor the concentrations of simple anions in real-time is surely as desirable as that for metal ions, there is almost no literature on the chemosensing of monomelic, inorganic anions in water. Batch methods have been described for fluorimetric chemodosimetry of several halides, but sensing methods (i.e., nondestructive and reversible) reported to date all rely on the ability of the analyte (e.g., iodide) to quench collisionally the fluorescence of compounds such as rhodamine 6G. Thus, it would seem useful to apply the substantial literature on anion recognition to this purpose. As a signal type, fluorescence is both more sensitive than absorption spectroscopies and more amenable to the conception of signal transduction mechanisms on the molecular level. In 1989, we reported previously that the aqueous complexation of anions such as phosphate and sulfate to anthrylpolyamines (e.g., 1) result in chelation-enhanced fluorescence (CHEF) signalling. Compound 1 proved to be the first fluorescent chemosensor for an inorganic anion that does not rely on an inherent quenching property of the anion. Even so, it is a conception far from usable form. For example, chemosensor 1 binds phosphate at pH 6 with ^=150 mM; in other words, the midpoint of thetitrationoccurs at a phosphate concentration of 150 mM, which is very high for many applications. Likewise, selectivity between a variety of simple anions (sulfate, acetate) is nonexistent. In an effort to improve upon the conceptual design of anion sensors, we synthesized a convergent chemosensor demonstrating anion binding in a more useful concentration range (i.e., μΜ K ) and designed to discriminate between phosphate ('Ρ^) and pyrophosphate ('ΡΡ^) ions based on size. Chemosensor 3, prepared by the reaction of 1,8-dibromomethylanthracene with excess tris(3-aminopropyl)amine (TRPN), yields a pH-fluorescence titration with pK = 6.7; thus, anion binding could be measured at pH 7 even though that using 1 (p^ = 5.4) could not. Besides possessing a high charge density at neutral pH, the anion receptor groups of 3 are geometrically disposed to bind both sides of pyrophosphate simultaneously. Our previously reported rationale for CHEF signalling of phosphate applies similarly to the current situation, as shown in Scheme I. We anticipated that the binding of pyrophosphate (non-fluorescent) to chemosensor 3 (low fluorescence at pH 7 due to photoinduced-electron transfer) would yield enhanced fluorescence as a result of intracomplex amine protonation. Aniontitrationswere thus carried out in pH 7 solution containing 4 μΜ 3 and excess cyclen (1 mM), which does not itself bind to 3 but does chelate adventitious transition metal ions that would otherwise bind to TRPN ligands in 3. We were gratified to observe (Figure 1) that the binding of pyrophosphate to 3 occurs with fluorescence enhancement (λ^ 368 nm, λ^, 414 nm), and that the 1:1 complexation occurs with Kj= 2.9 μΜ under these conditions. By contrast, the association of 3 to phosphate occurs with a K of 6.3 mM, which is only 13-times tighter binding than that of phosphate with 1-monoTRPNanthracene (not shown; Kj= 82 mM). Thus, chemosensor 3 displays a pyrophosphate/phosphate discrimination of 2200-fold at pH 7.


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Scheme I. Reaction schemerationalizingthe observed fluorescence increase upon the binding of pyrophosphate to chemosensor 3.


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The ion selectivity, which is also readily apparent in Figure 1, allows for real-time assay of pyrophosphate hydrolysis. For example, thefluorescenceof a reaction containing 20 μΜ pyrophosphate would be expected to decrease (by 54%) on conversion at constant pH to 40 μΜ phosphate (Scheme Π). This reaction is catalyzed by inorganic pyrophosphatase, a Mg(H)-dependent hydrolase that serves to drive reactions dependent upon PPj release by further hydrolysis to P^ Previously reported assay methods for this enzyme include; phosphomolybdate analysis, paper electrophoresis, starch gel electrophoresis, isotopic labelling, pH stat analysis and enyzmic-UV coupling. To test the use of chemosensor 3 for this purpose, we prepared solutions containing 50 mM HEPES, 1 mM cyclen, 20 μΜ Na P 0 , 20 μΜ Mg(C10 ) and 4 μΜ 3. Inorganic PPase from baker's yeast was added, and after a brief mixing period (30 seconds)fluorescenceintensity (λ^ 368 nm; 414 nm) was monitored as a function of time. As shown in Figure 2, the hydrolysis of pyrophosphate generates the predictedfluorescenceresponse, with the rate of hydrolysis increasing as does the enzyme concentration. Neutral Chemosensing: Dopamine and Other Catechols There is a substantial classical literature describing the fluorimetric indication of ions in water, which has been augmented by more recent studies. We have been studying additionally the synthesis offluorescentchemosensors for uncharged organic analytes in water. The design of anyfluorescentchemosensor requires knowledge of three topics: (1) how can one bind a specie with selectivity from water, (2) how can one generate signals from such binding events that are easy to measure, and (3) what mechanisms for binding and signal transduction intersect. Mechanisms have been elucidated by which ion binding events can be transduced tofluorescentevents, but these is less literature available on how to designfluorescentchemosensors of neutral analytes. The complexation of carbohydrates with phenylboronic acid has been known for at least 40 years, and the reversibility of that interaction serves as a basis for the chromatographic separation of sugars. In 1959, Lorand and Edwards reported association constants (listed here as KJs) for aqueous associations of phenylboronic acid with many saturated polyols; binding interactions range from very weak (e.g., ethylene glycol, £^=360 mM) to moderately strong (e.g., glucose, Κ^9Λ mM). We have utilized these results to evaluate a mechanism forfluorescentchemosensing based on polyol-boronic acid association. This abiotic sensing scheme yieldsfluorescencequenching.of 40% upon polyol chelation, owing to a modulation of the boronic acid pK . Lorand and Edwards also described the binding of phenylboronic acid to catechol, with significantly stronger association (^=57 μΜ) than that exhibited by carbohydrates. As efficient intramolecular quenching has been observed previously by other electron-rich organic compounds, it seemed reasonable that the catechol complexes of anthrylboronic acids might similarly demonstrate chelation-enhanced quenching (CHEQ). Indeed, we found that anthrylboronic acids bind catechol and catecholamines from water, Kj=330 μΜ (for the 2-isomer), with concomitant 20-fold changes in fluorescence intensity. Fluorescencetitrationsof 6 and 9 with catechol (1,2-dihydroxybenzene; 7) were carried out using an 0.75 μΜ solution of chemosensor at room temperature and pH 7.4 (Scheme ΙΠ), and the results are shown in Figure 3. (Excitation was 7


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1mM Cyclen, 50mM HEPES, pH7

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Figure 1. Fluorescence titrations of chemosensor 3 with Pj and PPi.

Low Fluorescence

Scheme IL Reaction scheme rationalizing the observed fluorescence assay of inorganic pyrophosphatase activity using chemosensor 3.


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Figure 2. Fluorescence intensity as a function of time during the pyrophosphatase-catalyzed hydrolysis of PPi to 2Pi, as monitored using chemosensor 3.

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[catechol! (mM) Figure 3. Fluorescence titrations of 2- and 9-anthrylboronic acids with catechol (-Δ- 6; - • - 9). (Reproduced with permissionfromreference 16. Copyright 1993 Pergamon Press.)


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#+4 ionization,pΚ obsdfor 3 likely is an aggregate of two +3-->+4 ionizations, one at each TRPN group. 7) (a) Josse, J. and Wong, S. C. K., "Enzymes," 3rd ed., Vol. 4, pp 499, Boyer, P. D., Ed., Academic Press, New York, New York, 1971; (b) Butler, L. G. ibid, 529. 8) (a) Josse, J. J. Biol. Chem. 1966, 241, 1938; (b) Moe, Ο. Α.; Butter, L. G. J. Biol. Chem. 1972, 247, 7308; (c) Cooperman, B. S.; Chiu, Ν. Y.; Bruckman, R. H.; Bunick, G. J.; McKenna, G. P. Biochem. 1973, 12, 1665; (d) Ryan, L. M.; Koxin, F.; McCarty, D. J.; ArthritisRheum.1979, 22, 892; (e) Baltscheffsky, M.; Nyrén, P. Methods Enzymol. 1988, 126, 538. Only the pH a

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stat-method is non-destructive with real-time signalling, but it is not useful in the μΜ concentration range. (a) Schwarzenbach, G. & Flaschka, H. Complexometric Titrations, translation by H. Irving (Metheun, London, 1969); (b) West, T. S. Complexometry with EDTA and Related Reagents (BDH, London, 1969); (c) Bishop, E. ed.,\ Indicators (Pergamon, NY, 1972); (d) Guilbault, G. G. Practical Fluorescence (Marcel Dekker, NY, 1973), chapter 6. For lead references to recent work by several authors, see: Fluorescent Chemosensors for Ion and Molecule Recognition, Czarnik, A. W., Ed., ACS Books, Washington, 1993. A notable exception is found in recent work by Ueno: Ueno, Α., Kuwabara, T., Nakamura, Α.; Toda, F. Nature 356, 136 (1992). Kuivila, H. G. , Keough, A. H.; Soboczenki, E. J. J. Org. Chem. 19, 780 (1954). (a) Weith, H. L., Wiebers, J. L.; Gilham, P. T. Biochemistry 9, 4396 (1970); (b) Schott, H. Angew.Chem.Int. Ed. Eng. 11, 824 (1972); (c) Barker, S. Α., Hatt, B. W., Somers, P. J.; Woodbury, R. R. Carhohyd. Res. 26, 55 (1973); (d). Yurkevich, A. M , Kolodkina, I. I., Ivanova, Ε. Α.; Pichuzhkina, Ε. I Carbohyd. Res. 43, 215 (1975). Lorand, J. P.; Edwards, J. O. J. Org. Chem. 24, 769 (1959). Yoon, J.-Y.; Czarnik, A. W. J. Am. Chem. Soc. 114, 5874 (1992). Yoon, J.-Y.; Czarnik, A. W. Bioorg. Med. Chem. 1, 267 (1993). The current method is to be distinguished clearly from the well-known fluorimetric trihydroxyindol assay for catecholamines, which consumes the analyte (i.e., it is a batch analysis) and is not intended to afford real-time response: von Euler, U. S.; Lishajko, F. Acta Physiol. Scand. 51, 348 (1961).

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Fluorescent Chemosensors of Ion and Molecule Recognition

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