Fluorescent Diazapyrenium Films and Their Response to Dopamine

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Fluorescent Diazapyrenium Films and Their Response to Dopamine Mabel A. Cejas and Franç isco M. Raymo* Center for Supramolecular Science, Department of Chemistry, University of Miami, 1301 Memorial Drive, Coral Gables, Florida 33146-0431 Received January 31, 2005. In Final Form: April 5, 2005 Experimental protocols for the preparation of 2,7-diazapyrenium films on glass, quartz, and silica in one or two steps have been developed. The one-step procedures involve the adsorption of preformed 2,7diazapyrenium dications with trimethoxysilane appendages to the hydroxylated substrates. The two-step procedures consist in the formation of interfacial polysiloxanes with pendent chloromethyl groups and their subsequent coupling to monoalkylated 2,7-diazapyrene derivatives. For the modification of the glass slides, the silane building blocks have been copolymerized with Si(OEt)4. The transmission absorption spectra of the coated glass and quartz slides all reveal the characteristic bands of the 2,7-diazapyrenium chromophores. Combustion analyses confirm the adsorption of the 2,7-diazapyrenium dications on the silica particles. A comparison of the surface coverages of all films indicates that the one-step procedures are significantly more efficient than their two-step counterparts. Furthermore, the copolymerization of the silane building blocks with Si(OEt)4 translates into an increase in 2,7-diazapyrenium surface coverage of ∼1 order of magnitude. The emission and excitation spectra of all modified substrates reveal the characteristic bands of the 2,7-diazapyrenium fluorophores. The fluorescence quantum yield, however, decreases as the surface coverage increases. Presumably, interactions between adjacent fluorophores encourage nonradiative deactivation pathways. With the exception of the glass slides modified in two steps, all films respond to the presence of dopamine, in aqueous environments at neutral pH, with pronounced decreases in emission intensity. The association of the 2,7-diazapyrenium acceptors and dopamine donors at the solid/liquid interface is responsible for fluorescence quenching. The glass slides and silica particles modified in one step are the most sensitive substrates and respond to sub-millimolar concentrations of dopamine with large changes in emission intensity. Furthermore, their fluorescence is not affected by relatively large concentrations of ascorbic acid, which is the main interferent in conventional dopamine detection protocols. Thus, these results demonstrate that the supramolecular association of 2,7-diazapyrenium dications and π-electron rich substrates can be reproduced successfully at solid/liquid interfaces and suggest that the unique properties of 2,7-diazapyrenium films might lead to dopamine-sensing schemes based on fluorescence measurements.

Introduction The 2,7-diazapyrenium dication1 is a versatile building block in supramolecular chemistry.2-10 Its electrondeficient character and extended π-surface ensures the * Author to whom correspondence should be addressed (e-mail [email protected]). (1) (a) Lier, E. F.; Hu¨nig, S.; Quast, H. Angew. Chem., Int. Ed. Engl. 1968, 7, 814. (b) Hu¨nig, S.; Gross, J. Tetrahedron Lett. 1968, 21, 25992604. (c) Hu¨nig, S.; Gross, J.; Lier, E. F.; Quast, H. Liebigs Ann. Chem. 1973, 339-358. (2) (a) Blacker, A. J.; Jazwinski, J.; Lehn, J.-M.; Wihelm, F. X. J. Chem. Soc., Chem. Commun. 1986, 1035-1037. (b) Blacker, A. J.; Jazwinski, J.; Lehn, J.-M. Helv. Chim. Acta 1987, 70, 1-12. (3) (a) Brun, A. M.; Harriman, A. J. Am. Chem. Soc. 1991, 113, 81538159. (b) Brun, A. M.; Harriman, A. J. Am. Chem. Soc. 1992, 114, 36563660. (c) Coudret, C.; Harriman, A. J. Chem. Soc., Chem. Commun. 1992, 1755-1757. (d) Brun, A. M.; Harriman, A.; Hubig, S. M. J. Phys. Chem. 1992, 96, 254-257. (e) Benniston, A. C.; Harriman, A.; Yufit, D. S. Angew. Chem., Int. Ed. Engl. 1997, 36, 2356-2358. (4) (a) Ballardini, R.; Balzani, V.; Credi, A.; Gandolfi, M. T.; Langford, S. J.; Menzer, S.; Prodi, L.; Stoddart, J. F.; Venturi, M.; Williams, D. J. Angew. Chem., Int. Ed. Engl. 1996, 35, 978-981. (b) Credi, A.; Balzani, V.; Langford, S. J.; Stoddart, J. F. J. Am. Chem. Soc. 1997, 119, 26792681. (c) Ashton, P. R.; Ballardini, R.; Balzani, V.; Constable, E. C.; Credi, A.; Kocian, O.; Langford, S. J.; Preece, J. A.; Prodi, L.; Schofield, E. R.; Spencer, N.; Stoddart, J. F.; Wenger, S. Chem. Eur. J. 1998, 4, 2413-2422. (d) Credi, A.; Montalti, M.; Balzani, V.; Langford, S. J.; Raymo, F. M.; Stoddart, J. F. New J. Chem. 1998, 22, 1061-1065. (e) Balzani, V.; Credi, A.; Langford, S. J.; Raymo, F. M.; Stoddart, J. F.; Venturi, M. J. Am. Chem. Soc. 2000, 122, 3542-3543. (f) Ballardini, R.; Balzani, V.; Di Fabio, A.; Gandolfi, M. T.; Becher, J.; Lau, J.; Nielsen, M. B.; Stoddart, J. F. New J. Chem. 2001, 25, 293-298. (g) Balzani, V.; Credi, A.; Langford, S. J.; Prodi, A.; Stoddart, J. F.; Venturi, M. Supramol. Chem. 2001, 13, 303-313. (h) Balzani, V.; Credi, A.; Marchioni, F.; Stoddart, J. F. Chem. Commun. 2001, 1860-1861.

supramolecular association of its heterocyclic core with a diversity of π-electron rich partners. Charge-transfer and electrostatic terms are mainly responsible for the resulting [π‚‚‚π] stacking interactions. These attractive forces have been exploited successfully to encourage the assembly of host-guest complexes2b,3e,4a-d,h,6a,7,9 and to template the formation of molecules with interlocked components.4e-g,6b Furthermore, these robust interactions induce the intercalation of 2,7-diazapyrenium compounds (5) (a) Ikeda, H.; Fuji, K.; Tanaka, K. Bioorg. Med. Chem. Lett. 1996, 6, 101-104. (b) Ikeda, H.; Fuji, K.; Tanaka, K.; Iso, Y.; Yoneda, F. Chem. Pharm. Bull. 1999, 47, 1455-1463. (c) Uchida, T.; Takamiya, M.; Takahashi, M.; Miyashita, H.; Ikeda, H.; Terada, T.; Matsuo, Y.; Shirouzu, M.; Yokoyama, S.; Fujimori, F.; Hunter, T. Chem. Biol. 2003, 10, 15-24. (6) (a) Ashton, P. R.; Langford, S. J.; Spencer, N.; Stoddart, J. F.; White, A. J. P.; Williams, D. J. Chem. Commun. 1996, 1387-1388. (b) Ashton, P. R.; Boyd, S. E.; Brindle, A.; Langford, S. J.; Menzer, S.; Pe´rez-Garcıá, L.; Preece, J. A.; Raymo, F. M.; Spencer, N.; Stoddart, J. F.; White, A. J. P.; Williams, D. J. New J. Chem. 1999, 23, 587-602. (c) Diehl, M. R.; Steuerman, D. W.; Tseng, H.; Vignon, S. A.; Star, A.; Celestre, P. C.; Stoddart, J. F.; Heath, J. R. ChemPhysChem 2003, 4, 1335-1339. (7) (a) Lilienthal, N. D.; Enlow, M. A.; Othman, L.; Smith, E. A. F.; Smith, D. K. J. Electroanal. Chem. 1996, 414, 107-114. (b) Lilienthal, N. D.; Alsafar, H.; Conerty, J.; Fernandez, R.; Kong, C.; Smith, D. K. Anal. Chem. 2003, 75, 3322-3328. (8) (a) Becker, H.-C.; Norden, B. J. Am. Chem. Soc. 1997, 119, 57985803. (b) Becker, H. C.; Broo, A.; Norden, B. J. Phys. Chem. A 1997, 101, 8853-8860. (9) (a) Credi, A.; Prodi, L. Spectrochim. Acta A 1998, 54, 159-170. (b) Clemente-Leoń, M.; Marchioni, F.; Silvi, S.; Credi, A. Synth. Metals 2003, 139, 773-777. (10) Raymo, F. M.; Cejas, M. A. Org. Lett. 2002, 4, 3183-3185.

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Figure 1. Synthesis of the building blocks 3 and 4.

in nucleic acids2a,3a,5a,b,8a and their association with various nucleotides.3c,8b The donor/acceptor pairs responsible for the formation of these molecular and supramolecular assemblies dissociate upon either reduction of the 2,7diazapyrenium components or complexation with competing electron donors. Thus, the unique properties of the 2,7-diazapyrenium dication not only permit the assembly of fascinating chemical systems but also offer the opportunity to regulate reversibly the relative orientation of their donor and acceptor components with external stimulations.3e,4,9b The spectroscopic properties of the 2,7-diazapyrenium dication are also particularly attractive.2b,3,4a-d,g,h,9,10 This chromophore absorbs across the ultraviolet region with a well-defined vibrational structure that extends to visible wavelengths and emits between 400 and 500 nm. Once engaged in supramolecular association with electron donors, however, the fluorescence of 2,7-diazapyrenium acceptors is quenched.2b,3,4a-d,g,h,9,10 Thus, the formation of donor/acceptor complexes, involving these particular supramolecular synthons, can be conveniently probed by emission spectroscopy. It follows that protocols for the detection of biorelevant π-electron-rich analytes could be developed on the basis of the binding and emissive behavior of the 2,7-diazapyrenium dication. For example, we have demonstrated that the catechol appendage of dopamine stacks on one of the two π-faces of a 2,7-diazapyrenium core in aqueous environment at physiological pH, suppressing its ability to emit light.10 The identification of methods to detect dopamine with fluorescence measurements10-14 might contribute to the defeat of limitations associated with conventional electrochemical protocols to sense this particular neurotransmitter in vivo.15 In particular, ascorbic acid causes problematic interferences in the quantitative determination of dopamine, because the oxidation potentials of both compounds are remarkably similar.16 Ascorbic acid, however, does not have a π-electron-rich surface able to associate with the 2,7-diazapyrenium dication and should not affect the emissive behavior of this particular fluorophore. On the basis of these considerations, we have envisaged the possibility of developing fluorescent probes for the detection of catecholamine neurotransmitters17 based on 2,7-diazapyrenium derivatives immobilized on hydroxylated surfaces. In this paper, we report experimental procedures for the preparation of 2,7-diazapyrenium films on three different substrates and their fluorescence response to dopamine. Results and Discussion Design and Synthesis of 2,7-Diazapyrenium Building Blocks. Glass, quartz, and silica can be coated with organic molecules relying on the ability of trialkoxysilanes or trihalosilanes to form polysiloxanes on their hydroxy-

lated surfaces.18 Following these procedures, fluorescent compounds can be attached to all three substrates in either one or two steps.19-24 For example, chromophores with trialkoxysilane appendages can be condensed to the hydroxylated surfaces of these substrates in a single step. Alternatively, a polysiloxane with reactive termini protruding away from the underlying surface can be formed first, and then chromophores can be coupled to the functional groups on the modified surface in a second step. To explore both strategies for the preparation of 2,7diazapyrenium films, we have designed the dication 3 and the monocation 4 (Figure 1). The trimethoxysilane group at one end of 3 is expected to ensure the attachment of this compound to a hydroxylated surface in a single step. Instead, an N-alkylation at one end of 4 can be exploited to couple this species to a preformed polysiloxane with pendent alkylating sites. We have synthesized both compounds in either one or two steps starting from 2,7diazapyrene 1. In particular, the N-alkylation of a single nitrogen atom of 1 with 3-iodopropyltrimethoxysilane gave the monocationic species 2. A second N-alkylation step with benzyl bromide afforded the target dication 3. (11) Czarnik, A. W. In Interfacial Design and Chemical Sensing; Mallouk, T. E., Harrison, D. J., Eds.; American Chemical Society: Washington, DC, 1994; pp 314-323. (12) (a) Lin, V. S.-Y.; Lai, C. Y.; Huang, J. G.; Song, S. A.; Xu, S. J. Am. Chem. Soc. 2001, 123, 11510-11511. (b) Radu, D. R.; Lai, C.-Y.; Wiench, J. W.; Pruski, M.; Lin, V. S.-Y. J. Am. Chem. Soc. 2004, 126, 1640-1641. (13) Coskun, A.; Akkaya, E. U. Org. Lett. 2004, 6, 3107-3109. (14) Secor, K. E.; Glass, T. E. Org. Lett. 2004, 6, 3727-3730. (15) Colliver, T. L.; Ewing, A. G. In Encyclopedia of Analytical Chemistry: Applications, Theory and Instrumentation; Meyers, R. A., Ed.; Wiley: Chichester, U.K., 2000; Vol. 11, pp 9959-9983. (16) Coury, L. A., Jr.; Huber, E. W.; Heineman, W. R. In Applied Biosensors; Davies, J. E., Ed.; Butterworth: Stoneham, MA, 1989; pp 1-37. (17) McIwan, H.; Bachelard, H. S. Biochemistry and the Central Nervous System; Churchill Livingstone: Melbourne, Australia, 1985. (18) Ulman, A. An Introduction to Ultrathin Organic Films: from Langmuir-Blodgett to Self-Assembly; Academic Press: Boston, MA, 1991. (19) Ayadim, M.; Habib Jiwan, J. L.; de Silva, A. P.; Soumillon, J. Ph. Tetrahedron Lett. 1996, 37, 7039-7042. (20) (a) Flink, S.; van Veggel, F. C. J. M.; Reinhoudt, D. N. Chem. Commun. 1999, 2229-2230. (b) van der Veen, N. J.; Flink, S.; Deij, M. A.; Egberink, R. J. M.; van Veggel, F. C. J. M.; Reinhoudt, D. N. J. Am. Chem. Soc. 2000, 122, 6112-6113. (c) Crego-Calama, M.; Reinhoudt, D. N. Adv. Mater. 2001, 13, 1171-1174. (d) Basabe-Desmonts, L.; Beld, J.; Zimmerman, R. S.; Hernando, J.; Mela, P.; Parajo, M. F. G.; van Hulst, N. F.; van den Berg, A.; Reinhoudt, D. N.; Crego-Calama, M. J. Am. Chem. Soc. 2004, 126, 7293-7299. (21) Sekar, M. M. A.; Hampton, P. D.; Buranda, T.; Lo´pez, G. P. J. Am. Chem. Soc. 1999, 121, 5135-5141. (22) (a) Montalti, M.; Prodi, L.; Zaccheroni, N.; Falini, G. J. Am. Chem. Soc. 2002, 124, 13540-13546. (b) Montalti, M.; Prodi, L.; Zaccheroni, N.; Zattoni, A.; Reschiglian, P.; Falini, G. Langmuir 2004, 20, 2989-2991. (23) van der Boom, T.; Evmenenko, G.; Dutta, P.; Wasielewski, M. R. Chem. Mater. 2003, 15, 4068-4074. (24) Gao, L. N.; Fang, Y.; Wen, X. P.; Li, Y. G.; Hu, D. D. J. Phys. Chem. B 2004, 108, 1207-1213.

Fluorescent Diazapyrenium Films

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Figure 2. Modification of hydroxylated substrates with 2,7-diazapyrenium building blocks in one (a) or two (b, c) steps.

Similarly, the N-alkylation of only one of the two nitrogen atoms of 1, again, with benzyl bromide yielded the target monocation 4. Preparation and Characterization of 2,7-Diazapyrenium Films. We have modified the hydroxylated surfaces of quartz slides in a single step with the dicationic trimethoxysilane 3 (pathway a in Figure 2). Alternatively, we have first treated the substrates with 4-chloromethylphenyltrichlorosilane (pathway b in Figure 2) and then coupled the pendent chloromethyl groups of the resulting polysiloxane to the monocation 4 (pathway c in Figure 2). In both instances, the final result was the immobilization of multiple 2,7-diazapyrenium dications on the surface of the quartz substrates. Consistently, the absorption spectra (a and b in Figure 3) of the modified slides reveal a band at ∼250 nm, which resembles the main absorption (spectrum c in Figure 3) of the model 2,7-diazapyrenium dication 5 in MeCN. From the absorbances of these bands, the surface coverages (Γ in Table 1) of the films prepared in one and two steps can be estimated to be 0.2 and 0.1 nmol cm-2, respectively.25 Both values are slightly lower than the limiting surface coverage expected for a closepacked monolayer of 2,7-diazapyrenium dications, which is ∼0.4 nmol cm-2.26 The absorption spectrum of 5 (c in Figure 3) has additional bands at wavelengths longer than 300 nm. These weak absorptions can hardly be distinguished in the spectra of the films (a and b in Figure 3). However, they are well-defined in the corresponding excitation

spectra (d and e in Figure 3). In fact, the excitation of both films in this range of wavelengths results in the characteristic 2,7-diazapyrenium fluorescence (spectra g and h in Figure 3) consistently with the emissive behavior of 5 in solution (spectra f and i in Figure 3). The sub-monolayer surface coverages of the modified quartz slides translate into a relatively poor spectral response (a and b in Figure 3). To increase the signalto-noise ratio and facilitate the spectroscopic investigation of the interfacial polysiloxanes, we have copolymerized the 2,7-diazapyrenium building block 3 and Si(OEt)4 on the surface of glass slides. Alternatively, we have copolymerized 4-chloromethylphenyltrichlorosilane and Si(OEt)4 first and then coupled the pendent chloromethyl (25) The surface coverage (Γ) was calculated from the absorbance (A) of the film and the molar extinction coefficient () of the surface-confined 2,7-diazapyrenium dications using eq 1. The A at ∼250 nm of the modified quartz substrates was determined from spectra a and b in Figure 3, considering that both faces of each slide were covered by a 2,7diazapyrenium film. In the case of the glass slides, only one of the two faces was modified with a 2,7-diazapyrenium film, and the A at ∼340 nm was determined from spectra a and b in Figure 4. In all instances, the at the appropriate wavelength of the surface-confined 2,7diazapyrenium dications was approximated to that of the model compound 5 in MeCN, which was determined from the spectra c in Figures 3 and 4. Γ ) A/ (1) (26) The limiting surface coverage was calculated from the cross section (∼41 Å2) of the 2,7-diazapyrenium core paired to two hexafluorophosphate counterions. The cross section was estimated using GaussView 2.1 (Gaussian, Inc., Pittsburgh, PA).

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Figure 4. Absorption spectra of glass slides modified in one (a) and two (b) steps and of 5 (c, scaled by 0.1) in MeCN (5 × 10-5 M, 25 °C): excitation spectra (λem ) 432 nm) of glass slides modified in one (d) and two (e) steps and of 5 (f, scaled by 0.1) in MeCN (1 × 10-5 M, 25 °C); emission spectra (λex ) 342 nm) of quartz slides modified in one (g) and two (h) steps and of 5 (i, scaled by 0.1) in MeCN (1 × 10-5 M, 25 °C). Figure 3. Absorption spectra of quartz slides modified in one (a) and two (b) steps and of 5 (c, scaled by 0.02) in MeCN (5 × 10-5 M, 25 °C): excitation spectra (λem ) 432 nm) of quartz slides modified in one (d) and two (e) steps and of 5 (f) in MeCN (5 × 10-7 M, 25 °C); emission spectra (λex ) 342 nm) of quartz slides modified with in one (g) and two (h) steps and of 5 (i) in MeCN (5 × 10-7 M, 25 °C). Table 1. Surface Coverage (Γ), Fluorescence Quantum Yield (O), Response (c50) to 6, and Emission Intensity (IS) at Saturation of the 2,7-Diazapyrenium Films Prepared in One and Two Stepsa substrate

parameter

one step

two steps

cm-2)

φc

0.2 0.4

0.1 0.6

glass

Γb (nmol cm-2) φc c50d (mM) ISd (%)

2.0 0.1 0.6 34

1.8 0.02 -e -e

silica

Γf (nmol cm-2) φc c50d (mM) ISd (%)

4.4 0.02 0.6 1

0.2 0.1 4.6 11

quartz

Γb

(nmol

a The one- and two-step procedures are illustrated in Figure 2. The value of Γ was determined by absorption spectroscopy (ref 25). c The value of φ was determined as described in ref 27. d The values of c50 and IS were determined as described in ref 30. e The concentration of 6 has a negligible effect on the fluorescence of glass slides modified in two steps. f The value of Γ was determined by combustion analysis from the content of carbon atoms in the modified particles.

b

groups of the resulting polysiloxane to the monocation 4. In both instances, the absorption spectra of the films (a

and b in Figure 4) show the characteristic bands of the 2,7-diazapyrenium dications between 300 and 450 nm and resemble closely the spectrum of 5 in MeCN (c in Figure 4). From the absorbance of the band at ∼340 nm, the surface coverages of the films prepared in one and two steps can be estimated to be 2.0 and 1.8 nmol cm-2, respectively (Table 1).25 These values are ∼1 order of magnitude greater than those of the 2,7-diazapyrenium films prepared on quartz in the absence of Si(OEt)4. The absorption bands of the surface-confined 2,7diazapyrenium dications between 300 and 400 nm can also be observed in the excitation spectrum of the film prepared in one step (d in Figure 4), which is in good agreement with the one of 5 in MeCN (f in Figure 4). Instead, these bands are poorly defined in the excitation spectrum of the film prepared in two steps (e in Figure 4). Nonetheless, the excitation of both films in this range wavelengths results in the characteristic diazapyrenium fluorescence between 400 and 500 nm (g and h in Figure 4). Once again, the emission spectra of the films resemble closely the spectrum of 5 in MeCN (i in Figure 4). Following the one- and two-step procedures of Figure 2, we have also coated the hydroxylated surfaces of nanoscaled silica particles. The remarkably high surface densities (∼20 cm2 mg-1) of these substrates facilitate the interfacial polysiloxane formation. Indeed, the combustion analysis of the particles treated with the dicationic trimethoxysilane 3 indicates the loading of the 2,7diazapyrenium dications to be 87 nmol mg-1. This value corresponds to a surface coverage of 4.4 nmol cm-2 (Table 1). Similarly, the combustion analysis of the particles


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Figure 5. Excitation spectra (λem ) 432 nm, MeCN, 25 °C) of suspensions of silica particles (0.5 mg mL-1) modified in one (a) and two (b) steps and of a solution of 5 (c, scaled by 0.3, 1 × 10-5 M); emission spectra (λex ) 342 nm, MeCN, 25 °C) of suspensions of silica particles (0.5 mg mL-1) modified in one (d) and two (e) steps and of a solution of 5 (f, scaled by 0.3, 1 × 10-5 M).

treated with 4-chlorophenyltrichlorosilane shows the loading of the 4-chloromethylphenyl groups to be 1.6 µmol mg-1. However, most of these groups can hardly be accessed by the monocation 4. After the N-alkylation step, the loading of the diazapyrenium dications is only 4 nmol mg-1, which translates into a surface coverage of just 0.2 nmol cm-2 (Table 1). The excitation spectra (a and b in Figure 5) of MeCN suspensions of the modified particles confirm the incorporation of the 2,7-diazapyrenium dications. Their characteristic bands between 300 and 400 nm can be clearly observed and are essentially coincident with those of the model compound 5 in MeCN (spectrum c in Figure 5). The excitation of the modified particles in this range of wavelengths results in the typical 2,7-diazapyrenium fluorescence between 400 and 500 nm (d and e in Figure 5). Once again, the emission bands of the surface-confined 2,7-diazapyrenium dications are very similar to those of the model compound 5 in MeCN (spectrum f in Figure 5). The fluorescence quantum yields (φ in Table 1) for the surface-confined 2,7-diazapyrenium dications can be estimated by comparing the emission spectra of the films (d and e in Figures 3-5) to those of the model compound 5 (f in Figures 3-5) recorded under identical excitation conditions.27 The resulting values demonstrate that an increase in surface coverage translates into a decrease in quantum yield. This trend is particularly evident from the data listed in Table 1 for the films prepared in a single (27) The fluorescence quantum yield (φ) of the 2,7-diazapyrenium films prepared on quartz and glass was calculated with eq 2. The fluorescence intensities of the film (IF) and of the model compound 5 (IM) were measured at ∼430 nm in air and MeCN, respectively, under identical excitation conditions. The fluorescence detected for the modified quartz substrates was corrected to account for the fact that both faces of each slide were coated by a 2,7-diazapyrenium film. The surface coverage (Γ) of the film was determined by absorption spectroscopy (ref 25). The fluorescence quantum yield (φΜ) of 5 is 0.28 in MeCN (ref 4a). The terms cM and d are the concentration of 5 and the path length of the cell, respectively. The value of φ for the modified silica particles was calculated with eq 3. In this instance, IF was measured by suspending the particles in MeCN. The pseudomolarity (cF) is the number of moles of surface-confined 2,7-diazapyrenium dications per liter of suspension. This term was calculated from the 2,7-diazapyrenium loading determined by combustion analysis. φ ) (IFφMcMd)/(IMΓ)

(2)

φ ) (IFφMcM)/(IMcF)

(3)

Figure 6. Emission spectra (λex ) 342 nm, sodium phosphate buffer, pH 7.0, 32 °C) of a glass slide modified in one step (a) and of silica particles (0.33 mg mL-1) modified in one (b) and two (c) steps in the presence of increasing amounts of 6 (0-0.1 M).

step on the three different substrates. The quantum yield is 0.4 at the sub-monolayer surface coverages of the quartz slides, but drops to only 0.02 with the 1 order of magnitude increase in surface coverage associated with the silica particles. Thus, the surface coverage has a pronounced influence on the radiative deactivation of the 2,7-diazapyrenium singlet excited state. Presumably, the close proximity of the 2,7-diazapyrenium dications at high surface coverages opens nonradiative deactivation pathways, which lead to partial fluorescence quenching. Influence of Dopamine on the Fluorescence of 2,7Diazapyrenium Films. We have assessed the fluorescence response of the modified glass slides and silica particles to dopamine (6 in Figure 6) in aqueous environment at neutral pH.28 Under these conditions, the primary amino group of 6 is almost exclusively in a protonated state.29 Despite the cationic character of the resulting ammonium group, the electron-rich catechol appendage of 6 associates with the electron-deficient core of 2,7-

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Figure 7. Relative emission intensities (λex ) 342 nm, sodium phosphate buffer, pH 7.0, 32 °C) of a glass slide modified in one step (a) and of silica particles (0.33 mg mL-1) modified in one (b) and two (c) steps against the concentration of 6 and the corresponding hyperbolic fittings (coefficients of determination > 0.98).

diazapyrenium dications via [π‚‚‚π] stacking interactions.10 Consistently, the emission spectra of glass slides modified in one step (a in Figure 6) and of silica particles modified in one and two steps (b and c in Figure 6) show a pronounced decrease in emission intensity with an increase in the concentration of 6. Thus, the supramolecular association of the complementary electron-deficient and -rich components at the solid/liquid interface suppresses the ability of the immobilized 2,7-diazapyrenium dications to emit light. The response of the 2,7-diazapyrenium films prepared in two steps on glass to the electron-rich substrate 6, however, is in apparent contrast with these observations. Under identical experimental conditions, the concentration of 6 has only a modest influence on the fluorescence of these films (a and b in Figure S2 in the Supporting Information). Presumably, most of the 2,7diazapyrenium fluorophores are embedded in the polysiloxane matrix in this particular instance, and only a relatively small fraction of them can be accessed by the electron-rich catechol appendage of 6. The relative emission intensity of the glass slides modified in one step (a in Figure 7) and of the silica particles modified in one and two steps (b and c in Figure 7) decays hyperbolically with the concentration of 6. The three trends fit rectangular hyperbolas with coefficients of determination >0.98.30 From the fitted parameters, the concentration (c50 in Table 1) of 6 required to suppress the relative emission intensity to the mean of the original and saturation (IS in Table 1) values can be estimated for the three films. The resulting c50 for the glass slides and silica particles modified in one step is 0.6 mM. Instead, the c50 for the silica particles modified in two steps is 4.6 (28) We have also tested the fluorescence response of the modified quartz slides to various electron-rich substrates. In all instances, we have observed the expected decrease in emission intensity caused by the supramolecular association of the surface-confined 2,7-diazapyrenium dications with the electron-rich analytes. Although qualitatively reproducible, the correlations between the emission intensity and the concentration of the electron-rich species were quantitatively inconsistent for sets of experiments run under apparently identical conditions. This erratic behavior is, presumably, a result of the relatively low signalto-noise ratios arising from the sub-monolayer surface coverages of these particular films. (29) At a pH of 7.0, the ratio between the ammonium and amine forms is ∼80:1, and the concentration of the phenolate anion is negligible. The pKa values for the dissociation of the ammonium cation and the first hydroxy group of 6 are 8.9 and 10.6, respectively (Martin, R. B. J. Phys. Chem. 1971, 75, 2657-2661).

Cejas and Raymo

mM. Thus, the 2,7-diazapyrenium films prepared in one step are significantly more sensitive than those prepared in two. Presumably, this result is a consequence of the relatively high loading of 2,7-diazapyrenium dications on the substrates modified in one step. It is possible that pairs of proximal dications cooperate in binding the catechol appendage of 6 to form acceptor-donor-acceptor sandwiches on the surface of the glass or silica substrate. Indeed, similar binding motifs have been exploited in solution to reinforce the supramolecular association of 2,7-diazapyrenium acceptors with a variety of dioxyarene donors.2,3c,4e-h,6b Furthermore, it is interesting to note that the IS of the modified glass substrates is greater than those of the silica particles. Presumably, a relatively large fraction of 2,7-diazapyrenium dications is buried in the polysiloxane matrix, formed on the glass surface as a result of copolymerization with Si(OEt)4, and cannot be accessed by 6. The c50 values in Table 1 indicate that the silica particles modified in one step are particularly sensitive to 6. Therefore, we have also assessed their response to ascorbic acid, which is the main interferent in the electrochemical detection of 6 in vivo.15,16 Under identical experimental conditions and an analyte concentration of 3 mM, the 2,7diazapyrenium fluorescence drops to 20% for 6 and remains virtually unaffected for ascorbic acid (Figure S3, Supporting Information). Only when the concentration of ascorbic acid is raised to ∼10 mM can a small decrease in emission intensity be appreciated. At this high concentration, the 2,7-diazapyrenium fluorescence is completely quenched in the case of 6. Conclusions Preformed 2,7-diazapyrenium dications with trimethoxysilane appendages adsorb spontaneously on the hydroxylated surfaces of quartz slides to produce fluorescent films. Similar emissive coatings can be prepared in two steps by exposing the hydroxylated supports to 4-chloromethylphenyltrichlorosilane first and then coupling the pendent chloromethyl groups to a monoalkylated 2,7diazapyrene core. The transmission absorption spectra of the resulting films confirm the presence of 2,7-diazapyrenium chromophores, but indicate sub-monolayer surface coverages. The amount of immobilized 2,7-diazapyrenim dications, however, increases significantly when the polysiloxane formation steps of both procedures are performed either on the surface of silica nanoparticles or in the presence of Si(OEt)4 on glass slides. In all instances, the immobilized 2,7-diazapyrenium dications maintain their ability to emit light, but their quantum yield decreases with the surface coverage. Presumably, interactions between adjacent fluorophores encourage nonradiative deactivation pathways of the singlet excited state. The emission intensity of the 2,7-diazapyrenium dications decreases upon association of the surface-confined fluorophores with dopamine. The films prepared by (30) The experimental plots (Figure 7) of the relative emission intensity (IR) of the surface-confined 2,7-diazapyrenium dications against the concentration (c) of 6 were fitted to eq 4. The fitted value of the relative emission intensity (IS) at saturation was employed to calculate the mean relative emission intensity (I50) with eq 5. The fitted value of the pseudo-association constant (K) and I50 were used to determine the response parameter (c50) with eq 6. Thus, the term c50 is the concentration of 6 required to suppress the relative emission intensity to I50. IR ) (1 + IsKc)/(1 + Kc)

(4)

I50 ) (1 + Is)/2

(5)

c50 ) (I50 - 1)/(IsK - I50K)

(6)


copolymerization on glass in two steps are the only exception to this trend. Presumably, only a relatively small fraction of electron-deficient dications can be accessed by the electron-rich substrates in this particular instance. The analysis of the fluorescence decay with the concentration of dopamine for all films indicates that the glass slides and silica particles modified in one step are the most sensitive to this catecholamine neurotransmitter. They respond to sub-millimolar concentrations of dopamine with changes in emission intensity >50%. Furthermore, they are essentially unaffected by relatively large concentrations of ascorbic acid. Thus, the unique properties of this family of 2,7-diazapyrenium films might eventually contribute to the identification of experimental protocols for dopamine detection in the presence of this common interferent. Experimental Procedures Synthesis of 2,7-Diazapyrenium Building Blocks. Chemicals were purchased from commercial sources and were used as received. MeCN was distilled over CaH2. Toluene was distilled over Na. Compounds 1 and 5 were prepared according to literature procedures, and their syntheses are reported in the Supporting Information (Figure S1).1c,6b All reactions were monitored by thinlayer chromatography, using aluminum sheets coated with silica gel 60 F254. Melting points (mp) were determined with an Electrothermal 9100 apparatus and are uncorrected. Fast atom bombardment mass spectra (FABMS) were recorded with a VG Mass Lab Trio-2 spectrometer, using 3-nitrobenzyl alcohol as matrix. Nuclear magnetic resonance (NMR) spectra were recorded with a Bruker Avance 500 spectrometer. 2-(3-Trimethoxysilylpropyl)-2,7-diazapyrenium Iodide (2). 3-Iodopropyltrimethoxysilane (0.43 g, 1.5 mmol) was added to a solution of 1 (0.15 g, 0.7 mmol) in toluene (10 mL). The mixture was heated for 16 h under reflux and N2. After cooling to ambient temperature, the precipitate was filtered, washed with heptane (5 mL), and dried to give 2 (217 mg, 60%) as a yellowish solid: mp 208-210 °C; FABMS, m/z 367 [M - I]+; 1H NMR (CD3CN) δ 9.82 (2H, s), 9.64 (2H, s), 8.67 (2H, d, 9 Hz), 8.53 (2H, d, 9 Hz), 4.99 (2H, t, 7 Hz), 3.52 (9H, s), 2.31-2.28 (2H, m), 0.75 (2H, t, 8 Hz). 2-(3-Trimethoxysilylpropyl)-7-benzyldiazapyrenium Iodide Bromide (3). Benzyl bromide (43 µL, 0.4 mmol) was added to a solution of 2 (0.15 g, 0.3 mmol) in MeCN (10 mL). The mixture was heated for 16 h under reflux and N2. After cooling to ambient temperature, the precipitate was filtered, washed with heptane (5 mL), and dried to give 3 (161 mg, 79%) as a yellowish solid: mp 256 °C (decomposition); FABMS, m/z 458 [M - I - Br]+; 1H NMR (D2O) δ 10.11 (2H, d, 3 Hz), 10.08 (2H, d, 3 Hz), 8.82 (2H, dd, 3 and 9 Hz), 8.81 (2H, dd, 3 and 9 Hz), 7.63-7.62 (2H, m), 7.627.51 (3H, m), 7.37 (2H, s), 5.17 (2H, t, 7 Hz), 2.40-2.22 (2H, m), 0.75 (2H, t, 8 Hz). 2-Benzyl-2,7-diazapyrenium Hexafluorophosphate (4). Benzyl bromide (500 µL, 4 mmol) was added over 10 min to a solution of 1 (245 mg, 1 mmol) in toluene (20 mL) heated under reflux. The resulting mixture was maintained at reflux for a further 1 h. After cooling to ambient temperature, the precipitate was filtered, washed with Et2O (40 mL), and dissolved in a mixture of H2O and acetone (1:1 v/v, 40 mL). After the addition of NH4PF6 (3 g), the aqueous solution was stirred for 30 min at ambient temperature, and then it was concentrated to a volume of 15 mL under reduced pressure. The resulting precipitate was filtered and washed with H2O (20 mL) to give 4 (240 mg, 83%) as a yellowish solid: mp 216-218 °C (decomposition); FABMS, m/z 295 [M - PF6]+; 1H NMR (CD3CN) δ 9.80 (2H, s), 9.65 (2H, s), 8.64 (2H, d, 9 Hz), 8.47 (2H, d, 9 Hz), 7.63-7.60 (2H, m), 7.52-7.50 (3H, m), 6.17 (2H, s); 13C NMR (CD3CN) δ 149.34, 139.36, 134.58, 132.35, 131.11, 130.68, 130.46, 130.37, 129.97, 127.09, 126.96, 125.25, 67.02. Preparation of 2,7-Diazapyrenium Films. Chemicals were purchased from commercial sources and were used as received. MeCN was distilled over CaH2. Heptane, toluene, and o-xylene were distilled over Na. H2O (18.2 MΩ cm) was purified with a Barnstead International NANOpure DIamond Analytical system.

Langmuir, Vol. 21, No. 13, 2005 5801 All glassware was maintained for 30 min in a boiling mixture (1:2, v/v) of aqueous H2O2 (30%, v/v) and concentrated H2SO4, rinsed with H2O, dried for 5 h at 120 °C, and stored in a desiccator. Glass and quartz slides were purchased from commercial sources. Prior to modification, they were maintained for 10 min in a boiling mixture (1:2, v/v) of H2O2 (30%, v/v) and concentrated H2SO4, rinsed with H2O, and immersed for 10 min in a mixture (5:1:1, v/v/v) of H2O, aqueous NH4OH (30, v/v) and aqueous H2O2 (30%, v/v) maintained at 70 °C. After rinsing with H2O, the substrates were flushed with Ar, dried for 2 h at 100 °C, and stored in a desiccator. The silica particles (Aldrich S5505, particle size ) 14 nm, surface density ) 20 cm2 mg-1) were used as received. The combustion analyses of the modified silica particles were performed by Atlantic Microlab Inc. (Norcross, GA). Modification of Quartz Slides in One Step. Quartz slides were maintained in a solution of 3 (10 mg) in MeCN (15 mL) for 7 days at ambient temperature under Ar. The modified substrates were rinsed with MeCN, flushed with Ar, and dried for 30 min at 50 °C. Modification of Quartz Slides in Two Steps. Quartz slides were maintained in a solution of 4-chloromethylphenyltrichlorosilane (1%, v/v) in o-xylene (15 mL) for 45 min at ambient temperature under argon. The modified substrates were rinsed with o-xylene and sonicated in o-xylene, pentane, and acetone for 5 min each. Then, they were flushed with argon, dried for 1 h at 100 °C, and stored in a desiccator. After cooling to ambient temperature, they were maintained in a MeCN solution of 4 (5 mM, 15 mL) for 24 h at 50 °C under Ar. At this point, the modified slides were rinsed with MeCN, sonicated three times in MeCN and once in acetone for 5 min each, and flushed with Ar. Modification of Glass Slides in One Step. A mixture of Si(OEt)4 (1.5 mL), EtOH (2 mL), and aqueous HCl (0.1 M, 0.5 mL) was stirred for 5 h at ambient temperature. An aliquot (100 µL) of the mixture was diluted with a solution of 3 (3.5 mM) in DMSO/H2O (1:1, v/v, 100 µL) and stirred for a further 45 min at ambient temperature. A portion of the resulting solution (20 µL) was spread manually on a glass slide (1 cm2). The modified substrate was stored in a sealed container for 5 days at ambient temperature, rinsed with EtOH, and flushed with Ar. Modification of Glass Slides in Two Steps. A mixture of Si(OEt)4 (1.5 mL), EtOH (2 mL), and aqueous HCl (0.1 M, 0.5 mL) was stirred for 5 h at ambient temperature. An aliquot (100 µL) of the mixture was diluted with a heptane solution of 4-chloromethylphenyltrichlorosilane (3.5 mM, 100 µL) and stirred for a further 5 min at ambient temperature under Ar. A portion of the resulting solution (20 µL) was spread manually on a glass slide (1 cm2). The modified substrate was stored in a sealed container for 3 days at ambient temperature, rinsed with EtOH, sonicated in EtOH three times for 5 min each, flushed with Ar, and dried for 20 min at 30 °C. Then, the modified slide was maintained in a MeCN solution of 4 (5 mM, 4 mL) for 3 days at ambient temperature, rinsed with MeCN, and flushed with Ar. Modification of Silica Particles in One Step. Silica particles (227 mg) were suspended in a solution of 3 (27 mg) in MeCN (8 mL). The mixture was stirred for 16 h at ambient temperature. After filtration, the solid residue was washed with MeCN for 16 h in a continuous solid/liquid extraction apparatus and dried under reduced pressure. Combustion analysis of the modified particles revealed the content of carbon atoms to be 2.5% (w/w). Modification of Silica Particles in Two Steps. 4-Chloromethylphenyltrichlorosilane (4.5 mL) was added to a suspension of silica particles (500 mg) in toluene (25 mL). The mixture was stirred for 12 h at 50 °C under N2. After filtration, the solid residue was washed with toluene for 12 h in a continuous solid/ liquid extraction apparatus and dried under reduced pressure. Combustion analysis of the resulting particles revealed the content of carbon atoms to be 13.0% (w/w). The modified particles (250 mg) were suspended in a solution of 4 (22 mg) in MeCN (5 mL). The mixture was stirred for 1 h at 50 °C. After filtration, the solid residue was washed with MeCN for 12 h in a continuous solid/liquid extraction apparatus. Combustion analysis of the modified particles revealed the content of carbon atoms to be 13.1% (w/w). Absorption Spectroscopy. The spectra of the modified glass and quartz slides were recorded with a Varian Cary 100 Bio spectrometer, operating in transmission mode with a double-

5802


beam configuration. All spectra were determined in air, using bare glass or quartz slides as reference. The spectra of the 4-chloromethylphenyltrichlorosilane-coated substrates were subtracted from those of the 2,7-diazapyrenium films prepared in two steps. The spectra of 5 were recorded with the same instrument in and against MeCN, using cells with a path length of 0.5 cm. Emission Spectroscopy. The emission and excitation spectra of the modified quartz slides were recorded in air with a SPEX Fluoromax spectrometer. Those of the modified glass slides were recorded in air with a Varian Cary Eclipse spectrometer. The spectra of 5 were recorded with both instruments in MeCN, using cells with a path length of 1.0 cm and excitation conditions identical to those employed for the modified slides. The emission and excitation spectra of the modified particles were recorded in MeCN under constant stirring with a Varian Cary Eclipse spectrometer, using cylindrical glass cells. For comparison, spectra of 5 were also recorded under identical experimental conditions. Binding Studies. A glass slide modified in either one or two steps was inserted in a quartz cell (path length ) 1.0 cm) containing sodium phosphate buffer (2.5 mL, pH 7, 32 °C). Aliquots of a solution of 6 (1 M) in the same buffer were added to the cell to vary the concentration of 6 from 0.0001 to 0.1 M

Cejas and Raymo in 10 consecutive steps. The emission spectrum was recorded at each step, using a Varian Cary Eclipse spectrometer. The modified silica particles (5 mg) were suspended in sodium phosphate buffer (15 mL, pH 7, 32 °C). The suspension was diluted with aliquots of a solution of 6 (1 M) in the same buffer to vary the concentration of 6 from 0.0001 to 0.1 M in 10 consecutive steps. The emission spectrum was recorded at each step under constant stirring, using a Varian Cary Eclipse spectrometer and cylindrical glass cells.

Acknowledgment. We thank the National Institute of Environmental Health Sciences (ES-05705), the National Science Foundation (CAREER Award CHE0237578), and the University of Miami for financial support. Supporting Information Available: Experimental procedures for the synthesis of 1 and 5; influence of 6 on the fluorescence of glass slides modified in two steps; and influence of ascorbic acid on the fluorescence of silica particles modified in one step. This material is available free of charge via the Internet at http://pubs.acs.org. LA0502793

Fluorescent Diazapyrenium Films and Their Response to Dopamine

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