Optically Active Supramolecular Complexes of Water-Soluble Achiral

Oct 23, 2008 - Fax: +86-10-6277-1149. ... PMTEA, having one fewer carbon in the hydrophobic side chain relative to PMTPA, showed better selectivity to...
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Langmuir 2008, 24, 12829-12835

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Optically Active Supramolecular Complexes of Water-Soluble Achiral Polythiophenes and Folic Acid: Spectroscopic Studies and Sensing Applications Zhiyi Yao, Chun Li,* and Gaoquan Shi* Department of Chemistry, and the Key Laboratory of Bio-organic Phosphorous Chemistry and Chemical Biology, Tsinghua UniVersity, Beijing 100084, P. R. China ReceiVed July 3, 2008. ReVised Manuscript ReceiVed September 17, 2008 Optically active supramolecular complexes of water-soluble achiral polythiophene (PT) derivatives, PMTPA or PMTEA (Chart 1), and folic acid have been prepared; and the complex formation processes have been studied by absorption, emission, and circular dichroism (CD) spectroscopies. The complexes exhibited unique split-type induced CDs in the π-π* transition region of PTs, indicating that the molecular chirality of the glutamic acid moiety in folic acid was expressed in PT backbones. The influences of temperature, solvent composition, and the structures of the inducing molecules on the chirality induction to PTs were also investigated, and a possible mechanism for the formation of chiral superstructures was proposed. Furthermore, it was found that, upon addition of folic acid into aqueous solution of PTs (PMTPA or PMTEA), a dramatic color change from yellow to purple along with the emission quenching of PT derivatives was observed. PMTEA, having one fewer carbon in the hydrophobic side chain relative to PMTPA, showed better selectivity toward folic acid sensing over ATP because of its higher solubility in water and the appropriate hydrophilic/hydrophobic balance in the complex. Therefore, it can be applied as a colorimetric and fluorescent probe for detecting folic acid with high selectivity and sensitivity. Besides naked-eye detection of folic acid, the detection limit can be extended to be 10-8 M by using fluorometry and PMTEA as the probing molecule.

Introduction The design and construction of optically active macromolecular π-conjugated systems have attracted widespread interest in view of their potential applications in molecular electronics, sensing, and asymmetric catalysis.1 Optically active polythiophene (PT) derivatives are among the most investigated conjugated polymers (CPs) due to the ease of synthesis and functionalization.2-14 Hitherto, several intriguing strategies have been developed to prepare chiral PTs through covalent or noncovalent approaches. * Corresponding author. E-mail: [email protected]: gshi@ tsinghua.edu.cn. Tel: +86-10-6279-8909. Fax: +86-10-6277-1149. (1) (a) Pu, L. Acta Polym. 1997, 48, 116–141. (b) Hill, D. J.; Mio, M. J.; Prince, R. B.; Hugher, T. S.; Moore, J. S. Chem. ReV. 2001, 101, 3893–4012. (c) Yashima, E; Maeda, K; Nishimura, T Chem.-Eur. J. 2004, 10, 43–51. (d) Hoeben, F. J. M.; Jonkheijm, P.; Meijer, E. W.; Schenning, A. P. H. J. Chem. ReV. 2005, 105, 1491–1546. (e) Gottarelli, G.; Lena, S.; Masiero, S.; Pieraccini, S.; Spada, G. P. Chirality 2008, 20, 471–485. (2) (a) Bouman, M. M.; Meijer, E. W. AdV. Mater. 1995, 7, 385–387. (b) Langeveld-Voss, B. M. W.; Christiaans, M. P. T.; Janssen, R. A. J.; Meijer, E. W. Macromolecules 1998, 31, 6702–6704. (c) Langeveld-Voss, B. M. W.; Janssen, R. A. J.; Meijer, E. W. J. Mol. Struct. 2000, 521, 285–301. (d) Brustolin, F.; Goldoni, F.; Meijer, E. W.; Sommerdijk, N. A. J. M. Macromolecules 2002, 35, 1054–1059. (e) Grenier, C. R. G.; George, S. J.; Joncheray, T. J.; Meijer, E. W.; Reynolds, J. R. J. Am. Chem. Soc. 2007, 129, 10694–10699. (3) Bidan, G.; Guillerez, S.; Sorokin, V. AdV. Mater. 1996, 8, 157–160. (4) (a) Andreani, F.; Angiolini, L.; Caretta, D.; Salatelli, E. J. Mater. Chem. 1998, 8, 1109–1111. (b) Andreani, F.; Angiolini, L.; Grenci, V.; Salatelli, E. Synth. Met. 2004, 145, 221–227. (5) (a) Yashima, E.; Goto, H.; Okamoto, Y. Macromolecules 1999, 32, 7942– 7945. (b) Goto, H.; Yashima, E.; Okamoto, Y. Chirality 2000, 12, 396–399. (c) Yashima, E.; Goto, H. J. Am. Chem. Soc. 2002, 124, 7943–7949. (d) Goto, H.; Okamoto, E.; Yashima, E. Macromolecules 2002, 35, 4590–4601. (6) (a) Zhang, Z.-B.; Fujiki, M.; Motonaga, M.; Nakashima, H.; Torimitsu, K.; Tang, H.-Z. Macromolecules 2002, 35, 941–944. (b) Zhang, Z.-B.; Fujiki, M.; Motonaga, M.; McKenna, C. E. J. Am. Chem. Soc. 2003, 125, 7878–7881. (7) (a) Iarossi, D.; Mucci, A.; Parenti, F.; Schenetti, L.; Seeber, R.; Zanardi, C.; Forni, A.; Tonelli, M. Chem.-Eur. J. 2001, 7, 676–685. (b) Cagnoli, R.; Lanzi, M.; Mucci, A.; Parenti, F.; Schenetti, L. Polymer 2005, 46, 3588–3596. (c) Mucci, A.; Parenti, F.; Cagnoli, R.; Benassi, R.; Passalacqua, A.; Preti, L.; Schenetti, L. Macromolecules 2006, 39, 8293–8302. (8) (a) Koeckelberghs, G.; Vangheluwe, M.; Samyn, C.; Persoons, A.; Verbiest, T. Macromolecules 2005, 38, 5554–5559. (b) Vangheluwe, M.; Verbiest, T.; Koechelberghs, G. Macromolecules 2008, 41, 1041–1044.

PTs bearing chiral β-substituents have been widely studied, and the highly ordered, optically active superstructures were constructed by intermolecular π-π stacking in poor solvents at low temperature or in thin films.2-8 The synthesis of main-chaintype optically active PTs without chiral centers has been confirmed to be possible by polymerization in a chiral liquid crystal matrix. In this case, chiral PTs were prepared from achiral monomers by conformational locking of their repeating units, and the chirality of polymers is preserved due to their insolubility and infusibility.9 Recently, optically active supramolecular complexes between achiral PTs and biomacromolecules with helical conformations, such as DNA,10 peptides,11 and polysaccharides,12 have been prepared. The induced chirality in the backbone of PTs is due to the complex formation through electrostatic or hydrophobic interactions. The induced conformational and aggregation mode changes of PTs in the supramolecular complexes have been used (9) (a) Goto, H.; Akagi, K. Macromol. Rapid Commun. 2004, 25, 1482–1486. (b) Goto, H.; Akagi, K. Chem. Mater. 2006, 18, 255–262. (c) Togashi, F.; Ohta, R.; Goto, H. Tetrahedron Lett. 2007, 48, 2559–2562. (d) Goto, H. J. Electrochem. Soc. 2007, 154, E63–E67. (e) Togashi, F.; Ohta, R.; Goto, H. Macromolecules 2007, 40, 5228–5230. (10) (a) Ewbank, P. C.; Nuding, G.; Suenaga, H.; McCullough, R. D.; Shinkai, S. Tetrahedron Lett. 2001, 42, 155–157. (b) Nilsson, K. P. R.; Ingana¨s, O. Nat. Mater. 2003, 2, 419–424. (c) Ho, H.-A.; Bera-Aberem, M.; Leclerc, M. Chem.Eur. J. 2005, 11, 1718–1724. (11) (a) Nilsson, K. P. R.; Rydberg, J.; Baltzer, L.; Ingana¨s, O. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 11197–11202. (b) Nilsson, K. P. R.; Herland, A.; Olsson, J. D. M.; Hammarstro¨m, P.; Ingana¨s, O. Biochemistry 2005, 44, 3718– 3724. (c) Nilsson, K. P. R.; Olsson, J. D. M.; Stabo-Eeg, F.; Lindgren, M.; Konradsson, P.; Ingana¨s, O. Macromolecules 2005, 38, 6813–6821. (12) (a) Li, C.; Numata, M.; Bae, A.-H.; Sakurai, K.; Shinkai, S. J. Am. Chem. Soc. 2005, 127, 4548–4549. (b) Li, C.; Numata, M.; Hasegawa, T.; Sakurai, K.; Shinkai, S. Chem. Lett. 2005, 34, 1354–1355. (13) A few examples of chiral polythiophenes that exhibit optical activity owing to a one-handed helix without aggregation in solution were reported: Nilsson, K. P. R.; Olsson, J. D. M.; Konradsson, P.; Ingana¨s, O. Macromolecules 2004, 37, 6316–6321. (14) Examples for optically active oligothiophenes bearing chiral R or β-substituents:(a) Sakurai, S.-i.; Goto, H.; Yashima, E. Org. Lett. 2001, 3, 2379– 2382. (b) Kawano, S.-i.; Fujita, N.; Shinkai, S. Chem.-Eur. J 2005, 11, 1718– 1724. (c) Cornelis, D.; Peeters, H.; Zrig, S.; Andrioletti, B.; Rose, E.; Verbiest, T.; Koeckelberghs, G. Chem. Mater. 2008, 20, 2133–2143.

10.1021/la802086d CCC: $40.75  2008 American Chemical Society Published on Web 10/24/2008

12830 Langmuir, Vol. 24, No. 22, 2008 Chart 1. Chemical Structures of PMTEA, PMTPA, and Folic Acid

for the colorimetric and fluorescent DNA sensing10 and the detection of conformation transition of peptides and polysaccharides.11,12 Sensory technology based on water-soluble CPs has attracted considerable interest due to the signal amplification effects of the polymers, and thus is sensitive to very minor perturbations.15 In particular, aqueous systems provide a unique platform for the development of chemosensors for biologically relevant targets. Most CP sensors are based on the direct quenching of the inherent fluorescence intensity of the polymer through electron transfer and energy transfer between the CP and an analyte (quenching species);15c whereas scarce CP-based biosensors are reported to detect nonquenching analytes.16-18 Shinkai and co-workers confirmed that adenosine 5′-triphosphate (ATP), a small chiral bioanion, is a versatile building block for the self-assembly of optically active PT superstructures,18 in which the chirality of the inducing molecules is expressed in the main chain of PTs through noncovalent interactions. Meanwhile, exposure of cationic water-soluble PT derivative (PMTPA, Chart 1) to aqueous ATP solution leads to a distinct red shift in the absorption spectrum, along with a color change from yellow to purple and a pronounced fluorescence quenching of PMTPA. These spectral changes are due to the induced conformation change and aggregation of PMTPA through electrostatic and hydrophobic cooperative interactions, making PMTPA a sensitive colorimetric and fluorescent probe for the detection of ATP.18a Cationic water-soluble PMTPA is characteristic of amphiphilic molecules, in which the backbone and alkyl chains are hydrophobic moieties, whereas the cationic charged quaternary ammonium groups are hydrophilic ones. It has been confirmed that, upon addition of nucleotides with different structures into aqueous PMTPA solution, the interplay of hydrophilic and hydrophobic interactions accounts for the formation of PMTPA aggregates with different molecular ordering, and thus results in the formation of optically active PMTPA complexes with different circular dichroism (CD) patterns and intensities.18b To further address this proposed mechanism for the optically active PT complex formation, herein we adjust the length of hydrophobic alkyl moieties to control the amphiphilic nature of PT derivatives (PMTPA and PMTEA; Chart 1) and investigate the optical and (15) (a) Swager, T. M. Acc. Chem. Res. 1998, 31, 201–207. (b) McQuade, D. T.; Pullen, A. E.; Swager, T. M. Chem. ReV. 2000, 100, 2537–2574. (c) Thomas, S. W.; Joly, G. D.; Swager, T. M. Chem. ReV. 2007, 107, 1339–1386. (16) An analyte that cannot participate in directly quenching the photoexcited conjugated polymer due to incompatible redox and spectral properties is descried as “nonquenching” analyte. (17) (a) Liu, B.; Bazan, G. C. J. Am. Chem. Soc. 2004, 126, 1942–1943. (b) Hong, J. W.; Hemme, W. L.; Keller, G. E.; Rinke, M. T.; Bazan, G. C. AdV. Mater. 2006, 18, 878–882. (c) Satrijo, A.; Swager, T. M. J. Am. Chem. Soc. 2007, 129, 16020–16028. (18) (a) Li, C.; Numata, M.; Takeuchi, M.; Shinkai, S. Angew. Chem., Int. Ed. 2005, 44, 6371–6374. (b) Li, C.; Numata, M.; Takeuchi, M.; Shinkai, S. Chem. Asian J. 2006, 1-2, 95–101.

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aggregation properties of these PT derivatives by the introduction of identical guest molecules. On the other hand, folic acid, a B vitamin, acts as a coenzyme substrate in many reactions of the metabolism of amino acids and nucleotides, and also an important nutrient for health.19 It plays a key role in the stability of DNA in human cells.20 A low level of folic acid in the body may increase the risk of many diseases, such as coronary heart disease,21 Alzheimer’s disease,22 and cancers.23 Especially, it is an essential diet for pregnant women to reduce the risk of neural tube birth defects.24 Thus, it is important to monitor easily the concentration of folic acid in aqueous solution. Folic acid content in food is currently detected using microbiological assays,25 high-performance liquid chromatography,26 electrochemical assays,27 biospecific methods,28 and microfluidic devices,29 which are relatively time-consuming and require expensive equipment. Therefore, the rapid visual detection of folic acid using a colorimetric and fluorescent CP probe may be applicable for facile monitoring of folic acid content in food. In the present work, we have used folic acid as a building block for constructing optically active PT supramolecular aggregates,30 and further elucidating the mechanism behind this strategy. Upon the introduction of folic acid into aqueous PMTPA or PMTEA solution, both PTs can form optically active aggregates, along with a distinct solution color change from yellow to purple and a pronounced fluorescence quenching of PTs. Furthermore, PMTEA exhibits better selectivity relative to PMTPA for sensing folic acid and can be used as a sensitive and selective colorimetric and fluorescent probe for the detection of folic acid through supramolecular complex formation.

Results and Discussion 1. Absorption, Emission, and CD Spectroscopic Studies on Complexation of PMTPA with Folic Acid. PMTEA and PMTPA have a similar structure and their complexation behaviors with folic acid are almost the same. Herein, we take PMTPA as an example, and the data of PMTEA are provided as Supporting Information. PMTPA has good solubility in water, and its aqueous solution exhibits an absorption maximum around 400 nm (Figure 1A), which indicates that PMTPA backbones are in a randomcoiled conformation. Upon addition of folic acid, the absorption maximum of the aqueous PMTPA solution was red-shifted, featuring two major peaks at 546 and 588 nm and a broad shoulder around 510 nm (Figure 1A), along with a color change from (19) Acrot, J.; Shrestha, A. Trends Food Sci. Technol. 2005, 16, 253–266. (20) Fenech, M. Mutat. Res.-Fundam. Mol. Mech. Mutagen. 2001, 475, 57–

67. (21) Malinow, M. R.; Duell, P. B.; Hess, D. L.; erson, P. H.; Kruger, W. D.; Phillipson, B. E.; Gluckman, R. A.; Block, P. C.; Upson, B. M. New Engl. J. Med. 1998, 338, 1009–1015. (22) Kruman, I. I.; Kumaravel, T. S.; Lohani, A.; Pedersen, W. A.; Cutler, R. G.; Kruman, Y.; Haughey, N.; Lee, J.; Evans, M.; Mattson, M. P. J. Neurosci. 2002, 22, 1752–1762. (23) Bayston, R.; Russell, A.; Wald, N. J.; Hoffbrand, A. V. Lancet 2007, 370, 2004. (24) Berry, R. J.; Li, Z.; Erickson, J. D.; Li, S.; Moore, C. A.; Wang, H.; Mulinare, J.; Zhao, P.; Wong, L. Y. C.; Gindler, J.; Hong, S. X.; Correa, A. New Engl. J. Med. 1999, 341, 1485–1490. (25) Tamura, T. J. Nutr. Biochem. 1998, 9, 285–293. (26) Ginting, E.; Acrot, J. J. Agric. Food Chem. 2004, 52, 7752–7758. (27) Wang, C. H.; Li, C. Y.; Ting, L.; Xu, X. L.; Wang, C. F. Microchim. Acta 2006, 152, 233–238. (28) Finglas, P. M.; Morgan, M. R. A. Food Chem. 1994, 49, 191–201. (29) Crevillen, A. G.; Avila, M.; Pumera, M.; Gonzalez, M. C.; Escarpa, A. Anal. Chem. 2007, 79, 7408–7415. (30) Folic acid moieties have been used as building blocks in covalent approach for the construction of chiral supramolecular aggregate:(a) Kato, T.; Matsuoka, T.; Nishii, M.; Kamikawa, Y.; Kanie, K.; Nishimura, T.; Yashima, E.; Ujiie, S. Angew. Chem., Int. Ed. 2004, 43, 1969–1972. (b) Kamikawa, Y.; Nishii, M.; Kato, T. Chem.-Eur. J. 2004, 10, 5942–5951. (c) Sakai, N.; Kamikawa, Y.; Nishii, M.; Matsuoka, T.; Kato, T.; Matile, S. J. Am. Chem. Soc. 2006, 128, 2218–2219.

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Figure 2. CD spectra of folic acid (blue) and PMTPA (1.0 × 10-4 M) in water (pH ) 9.0) in the absence (black) and the presence (red) of folic acid. [PMTPA] ) [folic acid] ) 1.0 × 10-4 M.

Figure 1. Absorption (A) and emission (B) spectra of PMTPA (1.0 × 10-4 M) in water (pH ) 9.0) in the absence (black) and the presence (red) of an equimolar amount of folic acid. (λex ) 450 nm).

yellow to purple. The appearance of well-resolved vibronic transitions is assigned to the changes in the conformation and the aggregation mode of PT backbones, and the formation of a highly ordered phase of PTs. In the complex, PMTPA takes a more planar conformation, and a stronger intermolecular π-π stacking interaction is induced upon noncovalent binding with folic acid. This ordered phase of substituted PTs is usually associated with the formation of small aggregates or microcrystallites with interchain interactions,2c which is confirmed by the results of dynamic light scattering (DLS) and microscopic observations.31,32 Moreover, the yellow, random-coiled form of PMTPA is fluorescent and exhibits an emission band around 525 nm (λex ) 450 nm) as shown in Figure 1B. The excitation spectrum (emission recorded at λem ) 525 nm) closely follows the absorption spectrum of free PMTPA chains with random-coiled conformation (Supporting Information Figure S2). However, in the presence of an equimolar amount of folic acid, this emission is strongly quenched, indicating the formation of π-stacked aggregates of PMTPA.2 PMTPA has no chiral center; thus, it is intrinsically optically inactive, and no CD pattern in the π-π* transition region can be detected. Interestingly, the introduction of folic acid into an aqueous PMTPA solution resulted in an intense induced CD (ICD) signal in the π-π* transition region of PMTPA (Figure 2). The contribution of linear dichroism under the same measurement conditions was negligibly small, being characteristic (31) Dynamic light scattering (DLS) analysis provides direct information about the size of chiral superstructures in solution. In the absence of folic acid, DLS experiments hardly showed scattering, indicating the absence of aggregate in PMTPA solution. However, the PMTPA/folic acid complexes have an average hydrodynamic diameter of 154 nm, being consistent with those observed by microscopic methods. (32) TEM images of PMTPA and its complex with folic acid were recorded. It can be seen that PMTPA itself gives small particles, whereas the interactions between PMTPA and folic acid lead to the formation of bigger aggregates (Supporting Information Figure S1). Combining DLS and TEM results, one can conclude that the introduction of folic acid induces the formation of chiral aggregates and the superstructures observed by microscopic methods are already formed in solution, not during the sample preparation processes.

of the presence of strong exciton coupling between PT backbones in the chiral π-stacked PMTPA/folic acid complex. Moreover, the complex also displays a positive Cotton effect in the absorption range of folic acid (Supporting Information Figure S3), which is due to folic acid in the complexes. Chirality induction in an optically inactive CP through noncovalent binding interaction with small chiral molecules is well-known for polyacetylene derivatives,33 polyaniline,34 and polypyrrole;35 few examples were reported for the construction of an optically active PT supramolecular complex by this strategy.18b 2. Effects of the Structures of Inducing Molecules on the Optically Active Complex Formation. With a view to understanding the mechanism of chiral supramolecular complex formation, effects of inducing molecules with different structures on the aggregation structure and chirality induction in achiral PMTPA were examined. Figure 3 compares the absorption and CD spectra of PMTPA in the absence and the presence of an equimolar amount of glutamic acid (Glu), N-(4-aminobenzoyl)L-glutamic acid, and folic acid. It is clear that the introduction of Glu can scarcely induce the change of absorption maximum of PMTPA; whereas upon the addition of N-(4-aminobenzoyl)L-glutamic acid and folic acid, the absorption maxima are redshifted from 400 (PMTPA only in water) to 410 and 546 nm, respectively. These results indicate that a more planar conformation and a strong intermolecular π-π stacking are induced upon nocovalent binding with folic acid. CD spectroscopic results indicate that chiral aggregates of PMTPA can only be induced by noncovalent complexation with folic acid, whereas no chiral superstructure is formed from Glu and N-(4-aminobenzoyl)-Lglutamic acid. Both the absorption spectral change and the ICD pattern observed in the presence of folic acid suggest that the aromatic pterin moiety in the folic acid molecule is indispensable for the formation of π-stacked optically active supramolecular aggregates. In other words, the π-stacking interaction between pterin moieties is one of the important factors for the formation of chiral superstructures of PMTPA.36 3. Temperature-Dependent Absorption and CD spectra of the Chiral Supramolecular Complexes in Water. To obtain more insight into the formation mechanism of the optically active (33) (a) Yashima, E.; Maeda, K.; Okamoto, Y. Nature 1999, 399, 499–451. (b) Maeda, K.; Morino, K.; Okamoto, Y.; Sato, T.; Yashima, E. J. Am. Chem. Soc. 2004, 126, 4329–4342. (c) Nagai, K.; Maeda, K.; Takeyama, Y.; Sakajiri, K.; Yashima, E. Macromolecules 2005, 38, 5444–5451. (34) Majidi, M. R.; Kane-Maguire, L. A. P.; Wallace, G. G. Polymer 1995, 36, 3597–3599. (35) Zhou, Y.; Yu, B.; Zhu, G. Polymer 1997, 38, 5493–5495. (36) The differences between the 1H NMR spectra of the pterin moiety in folic acid and the PMTPA/folic acid complex further support this conclusion (Supporting Information Figure S4). In the complex, the protons in the pterin moiety are broadened and vanished relative to those for “free” folic acid in solution, indicating the significant contribution of π stacking between pterin moieties.

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Figure 5. Solvent composition dependence of the ICD intensity of PMTPA in the water/methanol mixed solvents. [PMTPA] ) 1.0 × 10-4 M; [folic acid] ) 2.5 × 10-5 M.

Figure 3. Absorption (A) and CD (B) spectra of PMTPA (1.0 × 10-4 M) in water in the absence (black) and the presence of equimolar amount of glutamic acid (Glu) (green), N-(4-aminobenzoyl)-L-glutamic acid (blue), and folic acid (red).

Figure 4. Absorbance at 546 nm (left axis, •) and ICD intensity at 600 nm (right axis, 0) of the chiral PMTPA/folic acid supramolecular complexes in water vs temperature. [PMTPA] ) 1.0 × 10-4 M; [folic acid] ) 2.5 × 10-5 M.

supramolecular PMTPA/folic acid complex in water, temperaturedependent absorption and CD spectra of the PMTPA/folic acid complex were recorded (Supporting Information Figure S5). It is clear that, with increasing temperature, the absorbance of the bands originating from the chiral superstructures decreased gradually, whereas the intensity of ICD bands increased initially and then decreased (Figure 4). These results indicated that aging at appropriate temperatures may result in a rearrangement of the PMTPA chains, and the most ordered chiral superstructure of PMTPA was achieved around 40 °C. By further increasing temperature, the chiral aggregated structure is dissociated gradually. It is noted here that, even at 90 °C, the optically active PMTPA/folic acid complex exhibits only 40% decrease in ICD intensity with respect to that at 20 °C, indicating that the complex has a good thermal stability. However, for the chiral PMTPA/ ATP complex reported previously,18b the ICD signal almost totally disappears at 80 °C and the vibronic bands from the aggregated phase are displaced by a broad peak around 410 nm, signifying that the chiral superstructures formed at room temperature are totally dissociated. Considering the structural difference between

the inducing molecules, one can conclude that the stronger hydrophobic interaction between the pterin moieties in the PMTPA/folic acid complex would be responsible for the improved thermal stability with respect to the PMTPA/ATP complex. 4. CD Spectra of the Chiral Supramolecular Complexes in Water/Methanol Mixed Solvents. To further elucidate the nonlinear temperature-dependent ICD signals observed for the chiral PMTPA/folic acid complexes in water and the formation mechanism of the chiral complexes, CD spectra of the PMTPA/ folic acid complexes in the water/methanol mixed solvents were recorded (Figures 5 and Supporting Information S6). It was found that the introduction of folic acid can induce an ICD pattern in the π-π* transition region of PMTPA, and the strongest ICD of PMTPA was achieved around Vw ) 0.5 (Vw: the volume fraction of water in the mixed solvent). Although both water and methanol are good solvents for PMTPA, methanol favors dissolving the hydrophobic moieties (PT backbones and alkyl moieties) with respect to water, and water favors accommodating the quaternary ammonium groups. As a result, water tends to induce fast aggregation and stronger solvophobic association among PT backbones, leading to the formation of tightly packed aggregates with lower molecular ordering and improved thermal stability, whereas adding an appropriate amount of methanol into the aqueous solution (Vw > 0.5) results in a weakening of the π-π stacking of the PT backbones because of the solubility improvement of the hydrophobic moieties. Therefore, more ordered (optically active) assemblies of PTs were formed in the mixed solvents. By further increasing the content of methanol and moving to a more methanol-rich region, the solubility of PMTPA becomes too good to induce the formation of aggregates effectively, and thus ICD intensity was decreased gradually. The appearance of a maximum value in the solvent compositiondependent ICD spectra is similar to that observed in the temperature-dependent spectra of the PMTPA/folic acid complexes in water, being related to the hydrophilic/hydrophobic balance controlled by the solvent composition and temperature, respectively. To confirm the hypothesis described above, temperaturedependent absorption and CD spectra of the PMTPA/folic acid complexes in the water/methanol (1:1 by volume) mixed solvent were recorded (Supporting Information Figure S7). It can be seen that, with increasing temperature, the magnitudes of the absorption and ICD bands from the chiral complexes are decreased monotonously. At 70 °C,37 the complex exhibits 90% decrease in ICD intensity with respect to that at 20 °C (Figure 6), exhibiting poor thermal stability relative to that in water (only 40% decrease (37) The highest temperature examined here is restricted by the low boiling point of methanol in the mixed solvent.

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Figure 6. Absorbance at 546 nm (left axis, •) and ICD intensity at 600 nm (right axis, 0) of the chiral PMTPA/folic acid supramolecular complexes in water/methanol (1:1 by volume) mixed solvent against temperature. [PMTPA] ) 1.0 × 10-4 M; [folic acid] ) 2.5 × 10-5 M.

Figure 7. Job plot for the formation of the chiral PMTPA/folic acid complex in water at 20 °C. [PMTPA] + [folic acid] ) 0.2 mM; XPMTPA ) [PMTPA]/([PMTPA] + [folic acid]).

in the ICD intensity at 90 °C with respect to the one at 20 °C). On the basis of these observations, one may conclude that hydrophilic/hydrophobic balance accounts for the appearance of the maximum values in the temperature and solvent compositiondependent CD spectra. For the tightly packed PMTPA/folic acid complexes in water, appropriately elevating temperature (below 40 °C), to some extent, weakens the π-π interaction between PT backbones, and thus leads to a rearrangement of the PMTPA chains and the formation of a more ordered chiral PMTPA superstructure. Further increasing temperature makes the complex dissociate gradually, and therefore, a maximum value was observed in the temperature-dependent ICD curve. 5. Stoichiometry of the Chiral Supramolecular Complex Formation. The stoichiometry of the complex formation was evaluated by means of continuous-variation plots from CD spectroscopic studies. It can be seen from Figure 7 that the ICD intensity increases gradually with the increase in the molar fraction of PMTPA (repeating unit) and attains a maximal value around 0.80, which corresponds to the molar ratio of 4/1 (PMTPA/folic acid). Obviously, the stoichiometric ratio obtained here deviates from that simply expected from the complementary electrostatic interaction and gives the overall positive net charge to the supramolecular complexes. The finding allows us to conclude that stable homogeneous dispersion in water is maintained by these excess cationic charges. The cooperative effects of electrostatic, hydrophobic, and aromatic stacking interactions would be responsible for this ratio, like those reported for supramolecular complexes formed between functional dyes and

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Figure 8. Absorption spectra of PMTEA (1.0 × 10-4 M) in water (pH ) 9.0) in the presence of increasing concentrations of folic acid as indicated.

nucleotides.38,39 On the basis of the facts described above, one can draw a conclusion that not only electrostatic interactions but also some other secondary forces such as hydrophobic interactions and aromatic stacking play important roles in the formation of the PMTPA/folic acid supramolecular complex. In the complex, the chiral information is induced by intermolecular excitonic interaction between helically stacked PT backbones.2 6. Colorimetric Folic Acid Sensing Based on the SupramolecularComplexFormationwithCationicPTDerivative. As discussed above, upon addition of folic acid into aqueous PMTPA solution, emission of PMTPA is quenched along with a dramatic color change from yellow to purple. Thus, PMTPA exhibits the potential to be a colorimetric and fluorescent probe for the detection of folic acid. However, it remains one vital issue to be dealt with because of the lack of selectivity with respect to ATP, and both ATP and folic acid induce similar color changes of PMTPA solution from yellow to purple (Supporting Information Figure S8).40 To resolve this issue, a thiophene monomer was rationally designed and synthesized by shortening the spacer (alkyl chains) length between the hydrophilic ammonium group and the aromatic moiety. The corresponding polymer, PMTEA (Chart 1), was prepared by an oxidation polymerization in chloroform by using FeCl3 as the oxidizing agent.12 As expected, PMTEA exhibits enhanced solubility in water, and the absorption maximum (λ ) 358 nm) shows a distinct blue shift (by 42 nm) in comparison with that of PMTPA. These results indicate that shortening the spacer between the ammonium group and the PT backbone can increase the hydrophilicity efficiently, and the PT backbone takes a random-coiled conformation with more twisting structure. Systematic investigations confirmed that PMTEA can form an optically active supramolecular complex by noncovalent binding with folic acid (see Supporting Information Figures S9-12). To check the performances of PMTEA toward sensing folic acid, titration of PMTEA with folic acid in water (pH ) 9.0) was monitored by absorption spectroscopy. As shown in Figure 8, upon increasing the amount of folic acid, the absorption maximum is gradually red-shifted from 358 to 533 nm with a dramatic color change from yellow to purple. This distinct shift (by 175 nm) and the appearance of characteristic vibronic bands are attributed to the aggregation of PT backbones. These results indicated that water-soluble PMTEA could be used as a (38) (a) Morikawa, M.; Yoshihara, M.; Endo, T.; Kimizuka, N. J. Am. Chem. Soc. 2005, 127, 1358–1359. (b) Shiraki, T.; Morikawa, M.; Kimizuka, N. Angew. Chem., Int. Ed. 2008, 47, 106–108. (39) Ma, T.; Li, C.; Shi, G. Langmuir 2008, 24, 43–48. (40) More complicated technique (CD spectroscopy) can be used to differentiate each other since different ICD pattern of the complexes can be detected.

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Figure 9. Changes in the color of PMTEA solutions (1.0 × 10-4 M) in water (pH ) 9.0) induced by the addition of equimolar amounts of various bioanions.

Figure 11. Absorption spectra of PMTEA (1.0 × 10-4 M) in the presence of various amounts of folic acid in 10 mM HEPES buffer (pH ) 7.4).

Figure 10. Dependence of the absorbance of PMTEA at 533 nm on the different anions at various concentrations in water. Cl- (9); HCO3- (•); CH3COO- (2); H2PO4- (1); P2O74- (+); Phe (0); Thr (O); Gly (∆); His (3); Ser (×); ATP (]); folic acid ([).

colorimetric probe for detecting folic acid. More importantly, PMTEA exhibited specificity for sensing folic acid. It was observed that, upon the addition of the other biologically important anions such as phenylalanine (Phe), threonine (Thr), glycine (Gly), histidine (His), serine (Ser), glutamic acid (Glu), and adenosine triphosphate (ATP), as well as Cl-, HCO3-, CH3COO-, H2PO4-, and P2O74- into PMTEA solution, most of the solutions remained yellow with λmax < 370 nm (Supporting Information Figure S13) except for that containing ATP, which gave an orange solution with a shift of the absorption maximun to 395 nm along with the appearance of two shoulders at longer wavelength (Supporting Information Figure S14). However, the most remarkable effect was observed for folic acid, which gave a purple solution (Figure 9). Figure 10 shows the dependence of A533 on the addition of different amount of various anions. It is clear from these results that the most dramatic effects are observed for folic acid, and hence they confirm that the water-soluble PMTEA is selectively responsive to folic acid. Considering the facts that PMTEA and ATP are more hydrophilic in comparison with PMTPA and folic acid, respectively, the specificity of PMTEA toward folic acid sensing can be explained as follows: (i) the supramolecular complex formation is related to the hydrophilic/hydrophobic balance between the cationic PTs and bioanions; (ii) PMTPA is more hydrophobic and easy to aggregate via induction by both folic acid and ATP, and thus lacking selectivity toward folic acid and ATP; whereas the more hydrophilic ATP does not efficiently induce the aggregation of PMTEA to form highly ordered aggregates, and thus PMTEA exhibits satisfactory selectivity toward folic acid. To emphasize the biological importance and access the viability of this approach for the detection of folic acid at physiological pH value, the sensing ability of PMTEA toward folic acid in HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) buffer (10 mM, pH 7.4) was examined. It was confirmed that the buffer solution has very little influence on the solution behavior of PMTEA and the supramolecular complex formation between PMTEA and folic acid, indicating that PMTEA can be applied as a satisfactory folic acid sensor in aqueous solution at physiological pH value (Figure 11).

Figure 12. Emission spectra of PMTEA (1.0 × 10-5 M) in water (pH ) 9.0) in the presence of increasing concentrations of folic acid as indicated. Excitated wavelength λex ) 415 nm.

Figure 13. Fluorescence quenching of PMTEA (1.0 × 10-5 M) by folic acid and Glu at various concentrations in water (pH ) 9.0). The fluorescence quenching QI ) [(I0 - I)/I0] × 100%; I0 is the fluorescence intensity at 524 nm of a solution of PMTEA (1.0 × 10-5 M); I is the fluorescence intensity at 524 nm of a solution of PMTEA (1.0 × 10-5 M) in the presence of different amounts of analytes folic acid and Glu. Excitated wavelength λex ) 415 nm.

7. Fluorescent Sensing Folic Acid Based on the Supramolecular Complex Formation. Given the fact that the fluorometric method has higher sensitivity relative to the absorption spectroscopy, one can expect that fluorometry will extend the detection limit of folic acid. PMTEA with random-coiled conformation is fluorescent in aqueous solution and exhibits an emission band at 524 nm upon excitation at 415 nm (Figure 12). Upon addition of an increasing amount of folic acid, the emission of PMTEA was quenched gradually until the emission intensity decreased to 95%; whereas only 18% quenching of the fluorescence was observed for Glu (Figure 13). Similar results were obtained in HEPES buffer (Supporting Information Figures S15 and S16). These spectral results indicate that fluorometric detection of folic acid is possible and the detection limit can be extended to the order of 10-8 M.

Optically ActiVe Supramolecular Complex

Conclusions In conclusion, we have developed an efficient strategy to the preparation of optically active supramolecular complexes from a bioanion, folic acid, and water-soluble cationic PT derivatives. The primary electrostatic interaction between folic acid and cationic PT derivatives produces the simple salt association initially, and then the synergistic effect such as π-π interaction and hydrophobic interaction would be responsible for promoting the formation of optically active PT superstructures, in which the molecular chirality of Glu moieties is expressed in the PT backbones through the cooperative interactions. Moreover, PMTEA, rationally designed and synthesized by shortening the hydrophobic alkyl chain of PMTPA, can be used as a sensitive and selective colorimetric and fluorescent probe for the detection of folic acid over ATP in aqueous solution at physiological pH value. Considering the fact that folic acid is a widely used targeting agent whose corresponding receptor is overpressed on many types of cancer cells, we expect that the present system will be applicable to the diagnosis of tumor cells by colorimetric analysis, and further research along this line is currently in progress.

Langmuir, Vol. 24, No. 22, 2008 12835 prepared following the similar procedure (see the Supporting Information). Aqueous stock solutions of folic acid and ATP were prepared in pure water, and the concentrations were determined by using ε368(folic acid) ) 9.12 × 104 M-1 cm-1 in 0.1 M NaOH aqueous solution and ε259(ATP) ) 15.4 × 104 M-1 cm-1 in phosphate buffer (100 mM, pH 7.0), respectively. 2. Sample Preparation. As a typical procedure, the supramolecular complexes were prepared by adding PMTPA or PMTEA aqueous stock solution (5 mM based on the repeating unit) into a dilute aqueous solution of folic acid with the given concentration. 3. Measurements. Absorption and emission spectra were collected by using a Hitachi 3010 UV-visible spectrometer and a LS 55 fluorescence spectrometer (PerkinElmer), respectively. Circular dichroism spectra were acquired on a Jasco J-715-150 L spectropolarimeter. TEMs were performed on a JEM 2010 (200 kV) transmission electron microscope (JEOL). DLS studies were conducted on Zetasizer 3000HS (Malvern, U.K.) instrument at 25 °C. 1H NMR spectra were carried out on a JNM-ECA600 spectrometer (JEOL).

Acknowledgment. This work has been financially supported by National Natural Science Foundation of China (20604013, 20774056, 50533030).

Experimental Section 1. Materials. All chemicals were purchased from Aldrich and Beijing Chem. Reagents Co. (Beijing, China) and were used as received. Water-soluble polythiophene derivative, PMTPA, was synthesized and purified as reported previously,12a PMTEA was

Supporting Information Available: Detail of the synthesis of polymer and additional figures. This material is available free of charge via the Internet at http://pubs.acs.org. LA802086D