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Nonspecific Interactions of a Carboxylate-Substituted PPE with Proteins. A Cautionary Tale for Biosensor Applications Ik-Bum Kim, Anna Dunkhorst, and Uwe H. F. Bunz* School of Chemistry and Biochemistry, Georgia Institute of Technology, 770 State Street, Atlanta, Georgia 30332 Received April 28, 2005. In Final Form: June 4, 2005 Two carboxylate-substituted, fluorescent (Φ ) 0.08), water-soluble poly(p-phenyleneethynylene)s (PPE) and a water-soluble model compound were exposed to a series of proteins and bovine serum. While the anionic PPEs do not have any specific binding sites, they form stable complexes with histone, lysozyme, myoglobin, and hemoglobin. The complex formation was evidenced by fluorescence quenching. Bovine serum albumin does not quench the fluorescence of the PPEs but enhances it, probably due to its surfactant character. These results imply that the use of charged conjugated polymers as biosensors, while an attractive proposition, has to take into account strong nonspecific interactions between conjugated polymers and the host of proteins that is found in cells and complex biological fluids.
Introduction We report that proteins quench the fluorescence of poly(p-phenyleneethynylene) (PPE) derivatives 1 and 5. The positively charged histone is the most efficient quencher, while bovine serum albumin (BSA) enhances the fluorescence of the polymers. Heeger et al. have reported the interaction of a sulfonated poly(paraphenylenevinylene) (PPV), with cytochrome c, myoglobin, and lysozyme. They find that the fluorescence of their polyelectrolyte is strongly quenched by cytochrome c but to a lesser degree by lysozyme and by myoglobin.1 Bazan investigated the nonspecific interactions between PPV-based polyelectrolytes and avidin in combination with a biotinylated quencher, reporting complex spectral responses.2 Biosensing utilizing conjugated polymers and oligomers is attractive due to their intrinsic fluorescence and their high sensitivity to external stimuli, combining scaffolding and reporting functions into one package.3-10 Sensory recognition elements do not need to be attached covalently but self-assemble onto polyanionic fluorescent conjugated polymers. Sophisticated applications of this concept have been developed by Wudl, Heeger, Bazan, Schanze, Whitten, Leclerc, and others.3-10 * To whom correspondence should be addressed. Fax: 01 404 385 1795. Tel: 01 404 385 1795. E-mail: uwe.bunz@ chemistry.gatech.edu. (1) Fan, C.; Plaxco, K. W.; Heeger, A. J. J. Am. Chem. Soc. 2002, 124, 5642-5643. (2) (a) Dwight, S. J.; Gaylord, B. S.; Hong, J. W.; Bazan, G. C. J. Am. Chem. Soc. 2004, 126, 16850-16859. (b) Wang, D.; Gong, X.; Heeger, P. S.; Rinisland, F.; Bazan, G. C.; Heeger, A. J. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 49-53. (3) Chen, L. H.; McBranch, D. W.; Wang, H. L.; Helgeson, R.; Wudl, F.; Whitten, D. G. Proc. Natl. Acad Sci. 1999, 96, 12287-12292. (4) (a) Xu, Q. H.; Gaylord, B. S.; Wang, S.; Bazan, G. C.; Moses, D.; Heeger, A. J. Proc. Natl. Acad Sci. 2004, 101, 11634-11639. (b) Gaylord, B. S.; Heeger, A. J.; Bazan, G. C. Proc. Natl. Acad Sci. 2002, 99, 1095410957. (c) Gaylord, B. S.; Heeger, A. J.; Bazan, G. C. J. Am. Chem. Soc. 2002, 125, 896-900. (5) (a) Liu, B.; Bazan, G. C. Proc. Natl. Acad Sci. 2005, 102, 589593. (b)Wang, S.; Gaylord, B. S.; Bazan, G. C. J. Am. Chem. Soc. 2004, 126, 5446-5451. (c) Liu, B.; Baudrey, S.; Jaeger, L.; Bazan. G. C. J. Am. Chem. Soc. 2004, 126, 4076-4077. (d) Liu, B.; Bazan, G. C. J. Am. Chem. Soc. 2004, 126 1942-1943. (6) (a) DiCesare, N.; Pinto, M. R.; Schanze, K. S.; Lakowicz, J. R. Langmuir 2002, 18, 7785-7787. (b) Pinto, M. R.; Schanze K. S. Proc. Natl. Acad. Sci. 2004, 101, 7505-7510.
Quenching processes are utilized to monitor binding events, either directly or by displacement assays that remove a prebound quencher and turn on the fluorescence of a conjugated polymer.7d Quenching processes of conjugated polymers are an effective means to transmit information. Why? An exciton in a conjugated polymer is delocalized over 15-20 repeating units and can move along the polymer chain via fluorescence resonance energy transfer (FRET).11 If each repeating unit carries a recognition element, then one exciton can “service” up to 70 recognition elements. If one of the recognition elements binds to a quencher, the fluorescence of the whole chain is shut down. Swager has elaborated this principle in his classic 1995 paper.11 The static quenching processes that are at the core of the sensing principle for most conjugated polymers can be described best by the Stern-Volmer equation12
F0/F ) 1 + KSV[Q] with F0 being the fluorescence intensity of the conjugated (7) (a) Rininsland, F.; Xia, W. S.; Wittenburg, S.; Shi, X. B.; Stankewicz, C.; Achyuthan, K.; McBranch, D.; Whitten, D. Proc. Natl. Acad. Sci. 2004, 101, 15295-15300. (b) Kumaraswamy, S.; Bergstedt, T.; Shi, X. B.; Rininsland, F.; Kushon, S.; Xia, W. S.; Ley, K.; Achyuthan, K.; McBranch, D.; Whitten, D. Proc. Natl. Acad. Sci. 2004, 101, 75117515. (c) Kushon, S. A.; Bradford, K.; Marin, V.; Suhrada, C.; Armitage, B. A.; McBranch, D.; Whitten, D. Langmuir 2003, 19, 6456-6464. (d) Kushon, S. A.; Ley, K. D.; Bradford, K.; Jones, R. M.; McBranch, D.; Whitten, D. Langmuir 2002, 18, 7245-7249. (8) (a) Ho, H. A.; Boissinot, M.; Bergeron, M. G.; Corbeil, G.; Dore, K.; Boudreau, D.; Leclerc, M. Angew. Chem. 2002, 41, 1548-1551. (b) Bera-Aberem, M.; Ho, H. A.; Leclerc, M. Tetrahdron 2004, 60, 1116911173. (c) Dore, K.; Dubus, S.; Ho, H. A.; Levesque, I.; Brunette, M.; Corbeil, G.; Boissinot, M.; Boivin, G.; Bergeron, M. G.; Boudreau, D.; Leclerc, M. J. Am. Chem. Soc. 2004, 126, 4240-4244. (d) Ho, H. A.; Leclerc, M. J. Am. Chem. Soc. 2004, 126, 1384-1387. (e) Bernier, S.; Garreau, S.; Bera-Aberem, M.; Gravel, C.; Leclerc, M. J. Am. Chem. Soc. 2002, 124, 12463-12468. (9) Wosnick, J. H.; Mello, C. M.; Swager, T. M. J. Am. Chem. Soc. 2005, 127, 3400-3405. (10) (a) Kim, I. B.; Wilson, J. N.; Bunz, U. H. F. Chem. Commun. 2005, 1273-1275. (b) Kim, I. B.; Erdogan, B.; Wilson, J. N.; Bunz, U. H. F. Chem.-Eur. J. 2004, 10, 6247-6254. (c) Wilson, J. N.; Wang, Y. Q.; Lavigne, J. J.; Bunz, U. H. F. Chem. Commun. 2003, 1626-1627. (11) Zhou, Q.; Swager, T. M. J. Am. Chem. Soc. 1995, 117, 1259312602. (12) Turro, N. J.; Modern molecular photochemistry; Benjamin Cummings: Menlo Park, 1978; pp 246-248.
10.1021/la051152g CCC: $30.25 © 2005 American Chemical Society Published on Web 07/12/2005
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polymer by itself and F being the fluorescence intensity of the conjugated polymer after addition of a given concentration of quencher [Q], with the square brackets defining the quencher’s concentration. KSV denotes the Stern-Volmer constant and can be extracted as the slope from a graph that plots quencher concentration vs F0/F. Conjugated polymers (unless they contain organometallic fragments)13 feature short emissive lifetimes, on the order of 0.2-0.5 ns;14 therefore, only static quenching is of importance. In static quenching, the quencher forms a ground-state complex with the fluorophore that is then quenched after excitation: KSV in static quenching equals the apparent complex formation constant of quencher to fluorophore. Using the Stern-Volmer relationship is a facile way to extract binding constants. Conjugated polyelectrolytes are generally water soluble and impart a significant electrostatic attraction between the fluorophore and a given oppositely charged (receptor) group. An example is the complexes formed from positively charged conjugated polymers and DNA.5,8 These complexes are used as sensors and work successfully in aqueous solution containing only DNA (as in most DNA chips) but no other biomolecules. If conjugated polymers, however, (13) (a) Haskins-Glusac, K.; Pinto, M. R.; Tan, C. Y.; Schanze, K. S. J. Am. Chem. Soc. 2004, 126, 14964-14971. (b) Tan, C. Y.; Alas, E.; Muller, J. G.; Pinto, M. R.; Kleiman, V. D.; Schanze, K. S. J. Am. Chem. Soc. 2004, 126, 13685-13694. (14) (a) Sluch, M. I.; Godt, A.; Bunz, U. H. F.; Berg, M. A. J. Am. Chem. Soc. 2001, 123, 6447-6448. (b) Bunz, U. H. F.; Imhof, J. M.; Bly, R. K.; Bangcuyo, C. G.; Rozanski, L.; Vanden Bout, D. A. Macromolecules 2005, 38, 5892-5896.
are to be used to detect specific components in blood serum, urine, saliva, or tear fluid, proteins, salts, and other species will be present and may interfere with the desired biosensory response. To explore the influence of proteins on the fluorescence of conjugated polymers, we exposed PPEs 1 and 5 and a model compound 2 to several proteins and examined their response.15,16 Results and Discussion Schanze reported the synthesis and the amplified fluorescence sensing of protease activity by 1 and investigated the photophysics of this fluorescent polyelectrolyte.6b,13,17 We have independently developed a synthesis for 1 and model compound 2 (Scheme 1).16 We are interested in the synthesis of water-soluble and gluco-substituted PPEs such as 5. Its precursor, 5prot, was synthesized by the Pd-catalyzed reaction of the monomer 3 with the diyne 4 utilizing triethylamine as a base.10a,16 The polymer 5prot is soluble in DMF, DMSO, and chloroform but insoluble in ethyl ether. The structure assignment was performed by 1H and 13C NMR spectros(15) (a) Bunz, U. H. F. Chem. Rev. 2000, 100, 1605-1644. (b) Bunz U. H. F. Acc. Chem. Res. 1991, 24, 998-1010. (c) Kloppenburg, L.; Jones, D.; Bunz U. H. F. Macromolecules 1999, 32, 4194-4203. (16) Kim I. B.; Dunkhorst, A.; Gilbert, J.; Bunz, U. H. F. Macromolecules 2005, 38, 4560-4562. (17) (a) Halkyard, C. E.; Rampey, M. E.; Kloppenburg, L.; StuderMartinez, S. L.; Bunz, U. H. F. Macromolecules 1998, 31, 8655-8659. (b) Miteva, T.; Palmer, L.; Kloppenburg, L.; Neher, D.; Bunz, U. H. F. Macromolecules 2001, 33, 652-654. (c) Bunz U. H. F.; Wilson J. N.; Bangcuyo, C. G. ACS Symp. Ser. 2005, 888, 147-160.
Nonspecific Interactions of PPE with Proteins
Figure 1. Bar graph of the Stern-Volmer constants for the quenching of the fluorescence of 1, 2, and 5 by different proteins. Top, histone included; bottom, histone excluded.
copy. Gel permeation chromatography (GPC) shows that the molecular weight (Mn) of 5prot is 1.1 × 104 with a polydispersity Mw/Mn of 2.8. Upon treatment of 5prot with NaOH in methanol, the polyelectrolyte 5 forms in high yield and is purified by careful dialysis to remove lowmolecular-weight materials. The polymer 5 can be precipitated from water by addition of acetone or by careful acidification. Further molecular weight determination is difficult because an attempt to perform GPC on 5 was not successful due to its ionic character. The polymer 5 could not be eluted from the column. The PPEs 5prot and 5 show absorption and emission properties that are expected for a dialkoxy-PPE.15,17 The quantum yields of 1 and 5 in water are both around Φ ) 0.08, in good accord with Schanze’s determination of the quantum yield of 1. The low quantum yield of 1 and 5 in water is not attributed to aggregation effects but due to faster internal conversion in single chains. It is not clear which specific pathways are followed, but intramolecular electron transfer of the carboxylate anions is one possibility. Of course, at the same time, we cannot exclude that well-solvated and charged main-chain defects are responsible for the observed low quantum yield. We exposed the polyelectrolytes 1 and 5 and the model compound 2 to a series of commercially available proteins, viz. histone, cytochrome c, myoglobin, lysozyme, bovine serum albumin, and hemoglobin in phosphate buffer (10 mM). Figure 1 and Table 1 show the obtained SternVolmer constants (KSV) and the isoelectric points of the proteins. Histone shows for both polymers, 1 (KSV ) 3 × 107) and 5 (KSV ) 107), the largest Stern-Volmer constants. For 1,
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the quenching is approximately 3 times as effective as for 5. The herein employed histone is isolated from calf thymus and consists of a mixture of histones H1-H4. Histones are basic proteins that are used in the cell to wrap the negatively charged DNA into a solenoid. Histones bear a positive charge at pH 7.2, and the increased interaction of histones with 1 compared to 2 and 5 is simply due to increased electrostatic attraction. When comparing fluorescence quenching of 1 and its model compound 2 by histone and cytochrome c, large quenching enhancement effects are observed, suggesting multivalency effects in the binding of the polyelectrolyte to the protein.18 Myoglobin, hemoglobin, and lysozyme on the other hand show only moderate molecular wire effects. The absorption spectrum of hemoglobin and myoglobin overlaps well with the emission spectrum of the model 2, explaining their relatively increased KSV by electron transfer quenching through particularly efficient FRET. The five investigated proteins have different properties. Hemoglobin, myoglobin, and cytochrome c are iron metalloproteins in which quenching can occur by an energy transfer mechanism. While cytochrome is positively charged at physiological pH, both myoglobin and hemoglobin are neutral. Histone and lysozyme are highly positively charged proteins, while bovine serum albumin (BSA) is negatively charged and responsible for the trafficking of lipophilic materials in vivo. The proteins in this study can be grouped in a matrix according to their isoelectric point and their redox activity (Table 2). From the known properties of the proteins and from the spectral changes, we infer that myoglobin and hemoglobin must quench by electron transfer. The heme portion would be the electron acceptor because the proteins were obtained in their Fe(III) forms from commercial sources. In the case of cytochrome c, both energy and charge transfer can play a role. For histone and lysozyme, energy transfer does not play a role but either charge transfer or induced aggregation is repsonsible for the quenching processes. Emission spectra of protein-quenched samples of 1 all look identical to the emission spectrum of 1 by itself. The lysozyme-quenched sample of 1 is an exception. In lysozyme, the emission peak of 1 is shifted from 464 to 474 nm upon quenching. This suggests that not only charge transfer but also self-quenching by aggregation might play a role in this case. In the polymer 5, aggregative effect is more pronounced for lysozyme and histone due to the lower negative charge of 5. It seems that the gluco-moieties are imparting less water solubility than the carboxylates, facilitating the aggregation of the chains under addition of the positively charged proteins. The polymer 1 has the largest overall negative charge of the three investigated fluorophores, 2 is a model compound, and 5 has half of the negative charge per repeat unit when compared to 1. Figures 2-4 display their respective Stern-Volmer plots. In Figure 2, the fluorescence quenching of 1 is shown; both myoglobin and cytochrome c are similarly effective as quencher for 1 and for 5 (Figure 4). The Stern-Volmer plot is linear for these proteins, suggesting static quenching without any other effects. The similarly high quenching efficiency of myoglobin compared to that of cytochrome c was surprising, as Heeger reports for a sulfonated PPV that quenching is highly effective for cytochrome c but not for myoglobin.1 Lysozyme quenches 1 in a fashion similar to that reported by Heeger for its quenching of PPV-sulfonate, i.e., effective quenching is observed at low concentrations but less effective quenching occurs at higher concentrations.1 (18) Mammen, M.; Choi, S. K.; Whitesides, G. M. Angew. Chem. 1998, 37, 2755-2794.
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Table 1. Stern-Volmer Constants KSV for the Quenching of 1, 2, and 5 by Different Proteins histone isoelectric point of protein charges/molecule size of protein 1 2 MW effect KSV1/ KSV2 5
pI )
11.7a
calf thymus, mix of H1-H4 2.8 × 107 3.1 × 103 9 × 102 9.4 × 106
cytochrome c pI )
10.2-10.7b
+9 3.4 nm spherical, 6.5 × 105 0 > 105 2.6 × 105
myoglobin pI )
hemoglobin pI )
7.0-7.2b
0 4.5 × 3.5 × 2.5 nm3 6.9 × 105 1.9 × 105 3.5 3.8 × 105
7.0-7.4d
0 5.5 nm spherical 1.3 × 106 6.6 × 105 2 1.0 × 105
lysozyme pI )
BSA pI ) 4.8-4.9
11c
+7 4.5 × 3 × 3 nm3 2.2 × 105 1.4 × 104 16 1.3 × 106
-14 14 × 4 × 4 nm3 -4.3 × 106 7.6 × 105 na -0.8 × 105
a Luck, J. M.; Rasmussen, P. S.; Satake, K.; Tsvetikov, A. N. J. Biol. Chem. 1958, 233, 1407-1414. b Browning, M.; Vanable, J. Am. Biol. Teacher 2002, 64, 536-537. See also: http://www.bioone.org/bioone/?request)get-document&issn)00027685&volume)064|issue)07&page)0535. c http://biochem4.okstate.edu/srford/2344_Lab/ 2344%20Exp%2010%20Protein%20Purification.pdf. d Mohammad, A. A.; Okorodudu, A. O.; Bissell, M. G.; Dow, P.; Reger, G.; Meier, A.; Guodagno, P.; Petersen, J. R. Clin. Chem. 1997, 43, 1798-1799. e Voet, D.; Voet, J. G. Biochemistry, 2nd ed.; Wiley: New York, 1995; p 133.
Figure 2. (a) Fo/F plots for polymer 1 with different proteins. (b) Low-concentration inset for quenching of 1 by histone. Note there is a region of superquenching for hemoglobin and histone where (Fo/F) - 1 ≈ (a[Q]2 + b[Q]). Table 2. Classification of the Herein-Investigated Proteins and Possible Mode of Action
electron transfer active not electron transfer active
positively charged at pH 7.2
neutral at pH 7.2
cytochrome c, energy transfer, charge transfer histone, mixture of proteins, lysozyme, charge transfer, and small influence on aggregation (lysozyme)
myoglobin, hemoglobin, energy transfer
Hemoglobin and histone show a superquenching effect for 1,7 i.e., KSV increases with added quencher concentration. The Stern-Volmer plot can be fitted by a parabolic instead of a linear function, i.e., F/Fo - 1 ≈ [Q]2. We have obtained the Stern-Volmer constant for the quenching of 1 by histone (KSV ) 2.8 × 107) from the lowest concentrations by linear extrapolation. Superquenching of 1 and 5 is observed up to a 100 nM concentration of histone. Between 100 and 150 nM concentration of histone, the fluorescence of 1 and 5 is still further quenched but less dramatic. Above that concentration, additional histone reconstitutes 1’s fluorescence. This was unexpected, and the experiment was repeated four times at this specific polymer concentration. A large excess of histone added to 1 or 5 seems to enhance the solubilization of polymer chains, and deaggregation of both polyelectrolytes occurs. We presume (Figure 5) that the isolation of the polymer chains from each other by the histones leads to an increase in the observed fluorescence. BSA is known to be a fatty acid transporter and has surfactant qualities.19 Addition of BSA to 1 leads to a 3-fold (19) Lavigne, J. J.; Broughton, D. L.; Wilson, J. N.; Erdogan, B.; Bunz, U. H. F. Macromolecules 2003, 35, 7409-7412.
negatively charged at pH 7.2
bovine serum albumin, fluorescence enhancement
increase of its fluorescence at BSA concentrations of 4 g L-1. PPE’s optical properties are sensitive to the addition of surfactants19 to their aqueous solutions. The increased fluorescence of 1 upon addition of BSA suggests that the PPE’s backbone is efficiently complexed by the hydrophobic patches on the surface of BSA; its negative charge does not seem to play a significant role under these conditions. The model compound 2 shows quenching for all added proteins, with the exception of cytochrome c, which increases 2’s fluorescence slightly. The Stern-Volmer plots (Figure 3) show a linear increase in Fo/F up to micromolar concentration of the other proteins, but above that, the quenching levels off. The reported KSV values were obtained from the linear part of the graph. When we exposed a solution of 1 or of 5 to native bovine serum, we find that the serum induces aggregation of either polymer but reduced the fluorescence intensity only marginally, strongly underscoring that cells or cell extracts will affect the photophysical behavior of conjugated polyelectrolytes such as 1. We predict therefore that most conjugated polyelectrolytes’ optical and fluorescent properties will be modulated by complex biological fluids.
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Experimental Section
Figure 3. Fo/F plots for model compound 2.
Figure 4. Fo/F plots for polymer 5.
Figure 5. Complex formation between 1 and histone.
Conclusions Proteins quench the fluorescence of the polymers 1 and 5 and the model compound 2. Most of the proteins show large KSV values in quenching the PPEs, with histone being 50 times more effective than any of the other investigated proteins. BSA enhances the fluorescence of the PPEs 1 and 5, probably due to its surfactant-like character. The polymer 5 with its lower charge density per repeat unit, as compared to 1, generally shows lower KSV values when quenched by proteins. Overall, the net negative charge of the PPE plays a significant role but is not the only factor in the interaction of proteins with these polyelectrolytes. Somewhat in contrast to Heeger’s result, we find that cytochrome c is not much different from myoglobin and hemoglobin in its efficiency to quench ionic PPEs fluorescence. We attribute the distinctions to the results of Heeger’s study1 to the different backbone, countercation (sodium instead of lithium), and a different ionic group (carboxylate vs sulfonate) used in this study. The sensitive responses of conjugated polyelectrolytes to proteins are fascinating, but they will make their use as sensors and fluorescent tags in living cells or biological fluids challenging. In further studies, we will evaluate concentration and aggregation effects of 1 and 5 upon their fluorescence quenching by proteins to find a regimen where quenching of these PPEs is most sensitive. We will further investigate the quenching of PPEs fluorescence by specific proteins in bovine serum and other complex biological fluids.
Instrumentation and Materials. The 1H and 13C NMR spectra were taken on a Varian 300 MHz or a Bruker 400 MHz spectrometer using a broadband probe. The 1H chemical shifts are referenced to the residual proton peaks of CDCl3 at δ 7.24 (vs TMS). The 13C resonances are referenced to the central peak of CDCl3 at δ 77.0 and CDCl2CDCl2 at 74.0 δ (vs TMS). UV-vis measurements were made with a Shimadzu UV-2401PC spectrophotometer. Fluorescence data were obtained with a Shimadzu RF-5301PC spectrofluorophotometer. All chemicals and solvents were used without further purification as received unless otherwise noted. Histone (from calf thymus, Type II-A), hemoglobin (from bovine blood), myoglobin (from horse heart), lysozyme (from chicken egg white), cytochrome c (from bovine heart), and albumin (from bovine serum, BSA) were purchased from Sigma Chemical Co. Fluorescence quenching experiments were performed by microtitration in quartz cuvettes. All stock solutions were prepared in 0.01 M sodium phosphate buffer (pH ) 7.2). Concentrations of polymer 1 and model compound 2 solutions were adjusted to 2 × 10-7 M on the basis of the molecular weight of two repeating units of polymer 1 and the molecular weight of model compound 2. Polymer 5 is not completely soluble in 0.01 M sodium phosphate buffer, and only 25% of 5 is dissolved in phosphate buffer. The concentration of polymer 5 was adjusted to 8 × 10-7 M on the basis of the molecular weight of repeating unit of polymer 5. After filtering the solution using a 0.2 µm filter, its final concentration is to be 2 × 10-7 M (8 × 10-7 M/4 ) 2 × 10-7 M). The fluorescence and UV-vis absorption spectra were recorded at room temperature. In each quenching experiment, a small aliquot of the concentrated quencher solution was added to 5 mL of diluted fluorophore solution by using a calibrated microliter pipet. The fluorescence intensities were adjusted upon the volume of the added quencher solution. Synthesis of Polymer 5prot. Monomer 4 (0.111 g, 0.336 mmol) and monomer 3 (0.256 g, 0.286 mmol) were dissolved in dichloromethane (4 mL) and triethylamine (1 mL) in an ovendried Schlenk flask. (Ph3P)2PdCl2 (1.4 mg, 0.002 mmol) and CuI (0.4 mg, 0.002 mmol) were added to the flask. The reaction mixture was stirred under nitrogen at 40 °C for 72 h. The solution was slowly added to ether (100 mL). The precipitate was washed with ethanol. An orange solid was obtained in 66% yield (260 mg). The number-average molecular weight (Mn) was estimated to be 11 × 103 with a polydispersity (Mw/Mn) of 2.81 according to GPC in chloroform as solvent. 1H NMR (400 MHz, CDCl3): δ 7.11 (s, 2H), 7.03 (s, 2H), 5.32-5.25 (m, 6H), 4.84 (s, 2H), 4.76 (s, 4H), 4.26 (br, 8H), 4.08-4.06 (br, 4H), 3.92 (s, 4H), 3.77 (s, 6H), 3.65 (s, 10H), 2.13 (s, 6H), 2.08 (s, 6H), 2.02 (s, 6H), 1.97 (s, 6H), 1.30 (t, 6H). 13C NMR (400 MHz, CDCl3): δ 170.62, 169.96, 169.82, 169.71, 168.62, 153.52, 118.97, 117.65, 97.68, 91.56, 70.97, 70.73, 69.92, 69.60, 69.50, 69.33, 69.05, 68.34, 67.34, 66.08, 6 × 2.33, 61.27, 20.86, 20.72, 20.67, 20.65, 14.14. IR: v. 2979, 2873, 2204, 1751, 1629, 1508, 1448, 1276, 1220, 1139, 1076, 979, 732. Synthesis of Polymer 5. A solution of 5prot (150 mg, 0.110 mmol) and sodium hydroxide (0.500 g) in methanol (25 mL) and water (1 mL) was stirred at 50 °C for 24 h and cooled in a refrigerator overnight. The precipitate of 5 was filtered and washed with methanol to yield 5 (90 mg, 81%) as a water-soluble yellow powder. The polymer was purified by extended dialysis against deionized water using a 1 kD MWCO cellulose membrane and recovered by a freeze-dryer. 1H NMR (400 MHz, D2O): δ 7.58 (s, 2H), 7.37 (s, 2H), 5.08 (s, 4H), 4.80, 4.18, 4.14, 4.10, 4.02, 3.96, 3.94, 3.86, 3.79, 3.57. IR: v. 3422, 2922, 2875, 2202, 1604, 1460, 1326, 1278, 1213, 1137, 1056, 977, 881 cm-1.
Acknowledgment. We thank the Department of Energy (DE-FG02-04ER46141) for generous funding. U.B. is a Camille Dreyfus Teacher-Scholar (2000-2005). LA051152G
