Enhanced Fluorescence of Loosely Packed Dye Aggregates

Received: October 20, 2014 | Accepted: November 8, 2014 | Web Released: February 5, 2015

CL-140962

Enhanced Fluorescence of Loosely Packed Dye Aggregates Naoya Ryu* and Hiroshi Hachisako Division of Applied Chemistry, Graduate School of Engineering, Sojo University, 4-22-1 Ikeda, Nishi-ku, Kumamoto 860-0082 (E-mail: [email protected]) The fluorescence of a hemicyanine dye was considerably enhanced by the formation of loosely packed aggregates, compared to tightly aggregated and molecularly dispersed hemicyanine dyes. Loose dye aggregates were realized by introducing a self-assembling unit to the dye molecules and by adjusting the mixture of good solvent and nonsolvent for the dye moiety.

The aggregation morphology of organic dyes can greatly affect their fluorescence intensity, wavelength, and lifetime. For example, when arranged in a parallel orientation, the so-called H-aggregation generally causes a significant decrease in fluorescence intensity accompanied by a blue shift of the absorption band with respect to the monomer band.1 Conversely, in a slipped arrangement, the so-called J-aggregation exhibits a narrow red-shifted absorption band which often results in enhanced fluorescence with a very small Stokes shift.1 Therefore, how dye molecules are designed and the way in which their aggregation morphology is controlled are very important for applications of fluorogenic materials (for example, sensors, laser sources, sensitizers, and nonlinear optical materials). Fluorescent dyes are very useful materials and are usually used in solution at low concentration so that the dye molecules do not form aggregates and self-quench. In contrast, a number of dyes have recently been developed that exhibit strongly enhanced fluorescence by the formation of rigid aggregates in the solid (nanoparticles and thin films)1a,2 or gel3 states, through a process known as aggregation-induced emission (AIE). This finding has expanded the range of fluorogenic dye applications. Their enhanced fluorescence is caused by, for example, conformational planarization, J-aggregate formation, and physical constraint. The formation of tightly packed dye aggregates has been limited to the solid state or at sufficiently high solution concentrations at which gel formation can occur. In this paper, we demonstrate the fluorescence enhancement of a hemicyanine (stilbazolium) dye based on a new concept, that is, the formation of “loosely packed aggregates” in the sol state. In general, it is difficult to form such loose aggregates in the solution state because dyes can be molecularly dispersed in good solvents, whereas tightly packed dye aggregates are formed in poor- or nonsolvents. We have achieved the formation of loose dye aggregates by functionalizing the dye molecule with an L-glutamic acid-derived peptide lipid which has two long alkyl chains that act as a self-assembling unit4,5 and by using a mixture of ethanol as a good solvent and toluene as a nonsolvent for the dye moiety. It was found that the doublechain lipid moiety was effective at improving the solubility of the whole molecule in some nonpolar solvents that are practically nonsolvents for the hemicyanine moiety. The hemicyanine derivative with the peptide lipid (abbreviated as St-Gln-2C12, Chart 1) was synthesized by the Chem. Lett. 2015, 44, 211–213 | doi:10.1246/cl.140962

Chart 1. Hemicyanine derivatives used in this study. Table 1. Solubility tests of St-Gln-2C12 and St-C22 in various solvents (1.0 mM)a

Solvent Water Methanol Ethanol Acetonitrile Dimethyl sulfoxide N,N-Dimethylformamide Chloroform Tetrahydrofuran Acetone Ethyl acetate Toluene Xylene Diethyl ether n-Hexane Cyclohexane

St-Gln-2C12 I S S P S S S S P P S S I I I

St-C22 I S S S S S S P I I P P I I I

a

S: soluble at ambient temperature after 1 h, P: precipitates at ambient temperature within 1 h, I: insoluble even at the boiling point.

quaternization of 4-[4-(dimethylamino)styryl]pyridine with N,N¤-didodecyl-N ¡-3-bromopropanoyl-L-glutamide6 (see Supporting Information, Scheme S1 and Figures S1S4). Table 1 lists the results of the solubility tests of St-Gln-2C12 and trans-4-[4-(dimethylamino)styryl]-1-docosylpyridinium bromide (abbreviated as St-C22, Chart 1) in various common solvents at 1.0 mM after they were heated to their boiling points and then slowly cooled at ambient temperature for 1 h. These hemicyanine derivatives showed good solubility in polar organic solvents such as methanol, ethanol, dimethyl sulfoxide, and N,N-dimethylformamide. However, only St-Gln-2C12 could be solubilized in nonpolar aromatic solvents such as toluene and xylene, indicating that the introduction of the double-chain peptide lipid was more effective than the single long alkyl chain at improving the solubility of the hemicyanine dye moiety in the nonpolar solvents. It was found that St-Gln-2C12 formed © 2015 The Chemical Society of Japan | 211

212 | Chem. Lett. 2015, 44, 211–213 | doi:10.1246/cl.140962

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Figure 1. UVvis absorption (a), CD (b), and fluorescence (c) spectra of St-Gln-2C12 at 0.1 mM in ethanoltoluene mixtures of various composition at 20 °C; (a) path length: 0.1 cm; (b) path length: 0.1 cm; (c) path length: 1.0 cm, band width: 3.0 nm, excitation wavelengths are listed in Table S1. (d) Effect of ethanol on the fluorescence intensity of St-Gln-2C12 at 0.1 mM in toluene at 20 °C.

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transparent orange gels in toluene (Figure S5a) and xylene at higher concentrations (>10 mM), although they were not stable for very long. Transmission electron microscopy (TEM) revealed that St-Gln-2C12 self-assembled and formed ribbonlike structures in toluene (Figure S5b). Upon heating, a gelsol transition of the toluene gel was observed visually. The gelsol transition temperature (Tgel) was detected using differential scanning calorimetry (DSC) and was estimated to be 75 °C (transition enthalpy, ¦H = 15 kJ mol¹1, Figure S5c). Meanwhile, St-C22 could not be dispersed in the nonpolar solvents under the same conditions. St-Gln-2C12 in toluene gave a sol state when the toluene gel was diluted to 0.1 mM, and the resulting toluene solution was subjected to ultravioletvisible (UVvis) absorption, circular dichromism (CD), and fluorescence measurements. The St-Gln2C12 toluene solution showed an absorption peak (max) at 435 nm (Figure S6a) and split Cotton effect in the region of the absorption band at 20 °C (Figure S6b). As the temperature increased the max red-shifted to 477 nm with an isosbestic point at 453 nm and the intensity of the Cotton effect decreased. From these results, the max at 20 °C is attributed to the S-chiral (lefthanded) H-aggregates of the hemicyanine moieties, according to the exciton chirality theory.7 At the same time, these results indicate that the peptide lipid self-assembles at this concentration (0.1 mM); that is, this concentration is above the critical aggregation concentration of the peptide lipid. Conversely, in a good solvent, ethanol, the hemicyanine moieties did not form Haggregates and had a max at 484 nm (Figure S7a). This max value was the same as that of St-C22 in ethanol. Furthermore, the fluorescence spectra of these dyes in ethanol were very similar irrespective of the N-substituents (Figure S7b). The fluorescence intensity of St-Gln-2C12 was remarkably enhanced when toluene was used as the nonsolvent for the hemicyanine moieties in the presence of ethanol and is different to that of St-C22 under the same conditions. Figure 1 shows the effect of toluene on the UVvis absorption, CD, and fluorescence spectra of St-Gln-2C12 in ethanol. The fluorescence intensity of St-Gln-2C12 increased with the toluene content and reached a maximum at 95 vol %, as shown in Figures 1c and 1d. Above 95 vol % toluene the fluorescence intensity decreased because of the formation of tightly packed H-aggregates that are almost nonfluorescent. The fluorescence intensity of St-C22 in ethanol was also enhanced with increasing toluene content and showed the maximum value at 70 vol % (Figure S8). This could be ascribed to the decrease in solvent polarity. However, the fluorescence was very weak. The difference of the fluorescence intensities between St-Gln-2C12 and St-C22 was maximized at 95 vol % of toluene, and the fluorescence intensity of St-C22 was only about one-seventh that of St-Gln-2C12 as shown in Figure 2b. TEM observations revealed that St-Gln-2C12 formed fibrillar assemblies with this solvent composition as shown in Figure 3a. DSC measurements also revealed that the Tgel was located at 60 °C (¦H = 25 kJ mol¹1) in the heating process as shown in Figure 3b. These results indicate that the assemblies of St-Gln2C12 were maintained in the ethanoltoluene mixture (5:95 v/v) at 20 °C. Moreover, a weak Cotton effect was maintained in these conditions as shown in Figure 1b, indicating that the hemicyanine moiety adopted a nearly monomeric state (Figure 1a). Therefore, it was concluded that the monomeric hemicyanine

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Figure 2. UVvis absorption (a) and fluorescence (b) spectra of St-C22 and St-Gln-2C12 at 0.1 mM in a tolueneethanol (95:5 v/v) mixture at 20 °C; (a) path length: 0.1 cm; (b) path length: 1.0 cm, excited at 528 nm for St-C22 and 527 nm for St-Gln-2C12, band width: 3.0 nm. Inset in (b) shows pictures of St-C22 and St-Gln-2C12 in a tolueneethanol (95:5 v/v) mixture under black light (352 nm) irradiation in the dark.

moieties are separated to such a degree that they cannot be regarded as densely packed H-aggregates. In contrast, St-C22 molecules were molecularly dispersed under the same conditions as evidenced by Figure 2a. These results indicate that the remarkably enhanced fluorescence of St-Gln-2C12 in an ethanol toluene mixture (5:95 v/v) is caused by loosely packed monomeric aggregates, which result in the restricted rotational motion of the hemicyanine moieties due to steric hindrance derived from self-assemblies of the peptide lipid moieties. Figure 4 illustrates the fluorescence enhancement in the system.

© 2015 The Chemical Society of Japan

(b) Endothermic

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Figure 3. (a) TEM image of self-assembled St-Gln-2C12 at 1.0 mM in an ethanoltoluene (5:95 v/v) mixture post-stained with 2 wt % aqueous uranyl acetate. (b) DSC thermogram of the heating process of St-Gln-2C12 at 20 mM in an ethanoltoluene (5:95 v/v) mixture; heating rate: 2 °C min¹1.

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Highly fluorescent, loosely arranged hemicyanines

Almost non-fluorescent, molecularly dispersed hemicyanines

Quenched hemicyanines under H-aggregation

Figure 4. Schematic illustration of fluorescence enhancement of StGln-2C12; (a) St-Gln-2C12 in an ethanoltoluene (5:95 v/v) mixture, (b) St-C22 in an ethanoltoluene (5:95 v/v) mixture, (c) St-Gln-2C12 in toluene. Dotted lines in green denote complementary hydrogen bonds between peptide lipids. Arrows in blue indicate rotation of the hemicyanine molecules.

Although St-Gln-2C12 self-assembled in ethanol, as evidenced by TEM observations (Figure S9a) and DSC measurements (Figure S9b), the fluorescence intensity was very weak and almost the same as that of St-C22 (Figure S7b). This is ascribed to the strong solvation of the hemicyanine moieties that undergo predominantly nonradiative deactivation. In conclusion, intense fluorescence was successfully induced in the loosely arranged hemicyanine dye. The key to such enhanced fluorescence is to allow the hemicyanine moieties to pack moderately well in mixed solvents of nonsolvent (toluene) with small amounts of good solvent (ethanol) for the hemicyanine moieties and to maintain the arrangement of the hemicyanine moieties through self-assembly of the lipid moieties into scaffolds. The mechanism of fluorescence enhancement in this work is different from that of conventional densely packed fluorescent dye aggregates that depend on hydrophobic interactions, in the sense that an “amphiphilic molecule” is used to realize and maintain a highly arranged dye moiety in nonaqueous mixed organic media. The present findings provide a new molecular design strategy for fluorogenic materials. This work was supported by The Ministry of Education, Culture, Sports, Science and Technology, Japan (No. S0801085). We are grateful to Prof. Dr. H. Ihara and Assoc. Prof. Dr. M. Takafuji of the Department of Applied Chemistry and Bio-

Chem. Lett. 2015, 44, 211–213 | doi:10.1246/cl.140962

chemistry, Kumamoto University for CD spectral measurements and TEM observations using a JEOL JEM-1400Plus transmission electron microscope and Prof. Dr. R. Tomoshige of the Faculty of Engineering, Department of Nanoscience, Sojo University for taking TEM images using a Philips TECNAI F20 S-TWIN transmission electron microscope. We also thank Dr. S. Nagaoka of Kumamoto Industrial Research Institute for his generous support. Supporting Information is available electronically on J-STAGE. References and Notes 1 a) B.-K. An, S.-K. Kwon, S.-D. Jung, S. Y. Park, J. Am. Chem. Soc. 2002, 124, 14410. b) S. Gadde, E. K. Batchelor, J. P. Weiss, Y. Ling, A. E. Kaifer, J. Am. Chem. Soc. 2008, 130, 17114. c) Y. Xu, P. Xue, D. Xu, X. Zhang, X. Liu, H. Zhou, J. Jia, X. Yang, F. Wang, R. Lu, Org. Biomol. Chem. 2010, 8, 4289. d) F. Würthner, T. E. Kaiser, C. R. Saha-Möller, Angew. Chem., Int. Ed. 2011, 50, 3376. e) A. N. Jordan, S. Das, N. Siraj, S. L. de Rooy, M. Li, B. El-Zahab, L. Chandler, G. A. Baker, I. M. Warner, Nanoscale 2012, 4, 5031. f) M. Şinoforoğlu, B. Gür, M. Arık, Y. Onganer, K. Meral, RSC Adv. 2013, 3, 11832. 2 J. Luo, Z. Xie, J. W. Y. Lam, L. Cheng, H. Chen, C. Qiu, H. S. Kwok, X. Zhan, Y. Liu, D. Zhu, B. Z. Tang, Chem. Commun. 2001, 1740; H. Tong, Y. Hong, Y. Dong, M. Häußler, J. W. Y. Lam, Z. Li, Z. Guo, Z. Guo, B. Z. Tang, Chem. Commun. 2006, 3705; Y. Hong, J. W. Y. Lam, B. Z. Tang, Chem. Commun. 2009, 4332; A. Asefa, A. K. Singh, J. Lumin. 2010, 130, 24; N. Zhao, Z. Yang, J. W. Y. Lam, H. H. Y. Sung, N. Xie, S. Chen, H. Su, M. Gao, I. D. Williams, K. S. Wong, B. Z. Tang, Chem. Commun. 2012, 48, 8637. 3 S. Y. Ryu, S. Kim, J. Seo, Y.-W. Kim, O.-H. Kwon, D.-J. Jang, S. Y. Park, Chem. Commun. 2004, 70; C. Bao, R. Lu, M. Jin, P. Xue, C. Tan, T. Xu, G. Liu, Y. Zhao, Chem.®Eur. J. 2006, 12, 3287; G. Palui, A. Banerjee, J. Phys. Chem. B 2008, 112, 10107; Y. Chen, Y. Lv, Y. Han, B. Zhu, F. Zhang, Z. Bo, C.-Y. Liu, Langmuir 2009, 25, 8548; J.-H. Wan, L.-Y. Mao, Y.-B. Li, Z.-F. Li, H.-Y. Qiu, C. Wang, G.-Q. Lai, Soft Matter 2010, 6, 3195. 4 H. Hachisako, H. Ihara, T. Kamiya, C. Hirayama, K. Yamada, Chem. Commun. 1997, 19; H. Hachisako, H. Nakayama, H. Ihara, Chem. Lett. 1999, 1165; H. Hachisako, Y. Murata, H. Ihara, J. Chem. Soc., Perkin Trans. 2 1999, 2569; M. Takafuji, T. Sakurai, T. Yamada, T. Hashimoto, N. Kido, T. Sagawa, H. Hachisako, H. Ihara, Chem. Lett. 2002, 548; H. Hachisako, R. Murakami, Chem. Commun. 2006, 1073; H. Hachisako, N. Ryu, H. Hashimoto, R. Murakami, Org. Biomol. Chem. 2009, 7, 2338; N. Ryu, H. Hachisako, Org. Biomol. Chem. 2011, 9, 2000; Y. Okazaki, H. Jintoku, M. Takafuji, R. Oda, H. Ihara, RSC Adv. 2014, 4, 33194; H. Jintoku, M. Yamaguchi, M. Takafuji, H. Ihara, Adv. Funct. Mater. 2014, 24, 4105. 5 H. Hachisako, N. Ryu, R. Murakami, Org. Biomol. Chem. 2009, 7, 2327. 6 V. Gopal, T. K. Prasad, N. M. Rao, M. Takafuji, M. M. Rahman, H. Ihara, Bioconjugate Chem. 2006, 17, 1530. 7 R. V. Person, K. Monde, H. Humpf, N. Berova, K. Nakanishi, Chirality 1995, 7, 128; N. Berova, Chirality 1997, 9, 395; N. Berova, K. Nakanishi, in Circular Dichroism: Principles and Applications, 2nd ed., ed. by N. Berova, K. Nakanishi, R. W. Woody, Wiley-VCH, New York, 2000, Chap. 12, pp. 337382; L.-C. Lo, J.-Y. Chen, C.-T. Yang, D.-S. Gu, Chirality 2001, 13, 266; L.-C. Lo, C.-T. Yang, C.-S. Tsai, J. Org. Chem. 2002, 67, 1368; M. Simonyi, Z. Bikádi, F. Zsila, J. Deli, Chirality 2003, 15, 680.

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