Pyrene-based D–π–A dyes that exhibit solvatochromism and high fluorescence brightness in apolar solvents and water

Abstract

A novel pyrene-based D–π–A dye consisting of piperidine (D) and a secondary N-alkyl carboxamide (A) was prepared. This dye showed solvatochromism and bright fluorescence in solvents with a wide range of polarities, including water. Such emission properties are derived from the role of the secondary N-alkyl carboxamide group as both a weak acceptor and a stabilizer of the n electrons on the carbonyl group.

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Fluorescent organic dyes have attracted considerable attention in many research fields because they can be used as imaging tools with high sensitivity, rapid response, and good selectivity.^1–6 In particular, solvatochromic fluorescent dyes have been extensively developed because of their potential applications as polarity-sensitive molecular probes.^3–11 Because the properties of a solvatochromic dye, such as the fluorescence intensity, colour, or lifetime, are sensitive to the environment in which it is located, these molecules can be used to investigate conformational changes and chemical or biological events in multimolecular systems.^4,7–11 The simplest method for developing solvatochromic dyes is to design molecules consisting of an ‘electron donor–π–acceptor’ (D–π–A) system, as seen in PRODAN,¹² which is a widely used solvatochromic dye. Dyes possessing such structures can undergo excited-state intramolecular charge transfer (ESICT), where the electronic structure of the dyes are rearranged in response to environmental polarity and therefore show solvatochromic fluorescence. It is also known that the photophysical properties of D–π–A dyes can be tuned by changing the combination of chromophore, donor, and acceptor moieties because each moiety shows inherent electron donating or withdrawing ability, electronic transitions, or interactions with the media. In fact, a variety of D–π–A dyes have been developed using this concept and improvements have been made for the practical use of these dyes as probes.^{9b,c,d,13–16} However, the types of electron donor and acceptor moieties used are still limited.^8b We consider that this structural limitation may restrict the fluorescence behaviour obtainable in the resulting solvatochromic dyes.

Herein, we focused on D–π–A dyes possessing secondary N-alkyl or tertiary N,N-dialkyl carboxamide substituents as new acceptor groups (Fig. 1). Previously, we reported the photophysical properties of polycyclic aromatic hydrocarbons (PAHs) possessing carboxamide groups.¹⁷ We demonstrated that (1) PAHs with carboxamide groups do not undergo intersystem crossing (ISC) as would generally be expected owing to the n electrons of the carbonyl group.^18,19 On the contrary, secondary N-alkyl carboxamide groups instead enhance the fluorescence intensity of PAHs. Moreover, both mono- and dialkylated amine groups possess smaller Hammett constant values than hydrogen, alkyl groups, and alkoxy groups, indicating (2) carboxamides may act as much weaker acceptors than other carbonyl groups, such as aldehydes, ketones, and esters.²⁰ Because of these two aspects, it is expected that D–π–A dyes containing carboxamide groups, especially secondary carboxamides, as an acceptor will not be quenched by ISC or large charge separation, and thus will be solvatochromic and exhibit bright fluorescence both in apolar and polar solvents. To the best of our knowledge, typical solvatochromic dyes become less fluorescent owing to internal conversion assisted by hydrogen bonding, large charge separation, or aggregation in water or other protic solvents.^8b,14a,15,21 In addition, some examples of dyes that are fluorescent in water and show solvatochromism are quenched in apolar solvents owing to ISC.¹⁸ That is, there is little known about dyes that show solvatochromism and fluoresce in both apolar solvents and water. Therefore, we believe the abovementioned fluorescence properties obtained from D–π–A dyes possessing carboxamide groups are important and will open new ways to apply these fluorescent dyes.

Fig. 1 Chemical structure of the new D–π–A dye possessing N-alkyl or N,N-dialkyl carboxamide as an acceptor.

Molecular design and synthesis

We designed new pyrene-based D–π–A dyes containing piperidine (D) and either a secondary N-alkyl (PSA) or tertiary N,N-dialkyl (PTA) carboxamide group (A). We chose pyrene as a chromophore because of its superior fluorescence properties, including good molar absorption coefficients (ε) and fluorescence quantum yields (Φ_FL) compared with other PAHs (e.g. naphthalene and anthracene).^17b In addition, it can be deduced that pyrene-based D–π–A structures do not have large dipole moments in the excited state when compared with fluorene- and oligophenylene-based D–π–A structures with similar donor and acceptor moieties.^14a,22 Furthermore, to enable us to evaluate the fluorescence properties of these dyes in water, we also synthesized water soluble PSAC, which possesses a carboxylic acid in the secondary N-alkyl carboxamide group. For our purpose, the secondary carboxamide group is a more preferable acceptor than the tertiary group, as demonstrated later. The synthetic protocol for PSA, PTA, and PSAC is outlined in Scheme 1. All the final compounds were characterized by ¹H and ¹³C NMR, FT-IR, and high-resolution mass spectrometry. The water solubility of PSAC was checked using the fluorescence spectra to confirm that PSAC possesses enough solubility for photophysical measurements (see Fig. S5 in the ESI†).

Scheme 1 Synthetic routes to PSA, PTA, and PSAC.

Initially, we studied the photophysical properties of both PSA and PTA to determine whether secondary N-alkyl or tertiary N,N-dialkyl carboxamides are preferable for the design of D–π–A dyes that exhibit both solvatochromism and bright fluorescence in a wide range of solvents with different polarities, including water.

Spectroscopic properties of PSA and PTA

The absorption and fluorescence spectra of PSA and PTA were measured in several solvents of different polarities (Fig. 2); the spectroscopic properties are listed in Table 1 (detailed data are summarised in Fig. S6–S10 and Table S1†). For all solvents examined, the absorption maxima for PSA and PTA were observed around 375 and 365 nm, respectively. The ε values of both PSA and PTA in EtOH were calculated as 21000 M⁻¹ cm⁻¹, and pronounced changes of these values were not observed in other solvents. The fluorescence spectra revealed that PSA exhibits bright and solvatochromic fluorescence, with an 80 nm (3410 cm⁻¹) red shift observed from hexane (446 nm, Φ_FL = 97%) to trifluoroethanol (TFE) (526 nm, Φ_FL = 98%). Similarly, PTA showed solvatochromism comparable to PSA; i.e. an 88 nm (4059 cm⁻¹) red shift was observed from hexane (428 nm, Φ_FL = 66%) to TFE (518 nm, Φ_FL = 97%). Thus, it was revealed that both PSA and PTA show high brightness with solvatochromism in a wide polarity region: from hexane to TFE. Interestingly, the pyrene-based D–π–A dye PA,^14b which we have reported previously and which possesses an aldehyde group as an acceptor instead of a carboxamide, exhibits a decrease in Φ_FL in protic solvents, even though PA possesses a similar structure to PSA and PTA. These results indicate that the high brightness of PSA and PTA over a wide polarity range is derived from the characteristics of the secondary N-alkyl and tertiary N,N-dialkyl carboxamides. The extent of solvatochromism exhibited by PSA and PTA might seem small; however, the magnitude of the red shift observed here is similar to that of Nile red (583 nm (Φ_FL = 91%) and 657 nm (Φ_FL = 5%) in dioxane and phosphate buffer, respectively),²³ a well-known and commonly used solvatochromic dye, indicating that PSA and PTA might also be utilizable as polarity probes.

Fig. 2 The absorption and fluorescence spectra of (a) PSA and (b) PTA in several solvents of different polarities (λ_ex = λ_{abs, max}, optical density (O.D.) = 0.1, room temperature).

Table 1 Spectroscopic parameter of PSA and PTA in organic solvents with different polarities

Solvent	Δf	λabs, max [nm]		λem, max [nm]		ΦFL [%]
		PSA	PTA	PSA	PTA	PSA	PTA
Hexane	0.000	371	359	446	428	97	66
Toluene	0.013	376	363	469	446	95	85
Dioxane	0.020	373	362	471	454	95	92
THF	0.210	372	360	474	455	94	81
Chloroform	0.148	376	373	485	465	91	52
DCM	0.217	378	372	488	456	99	83
Acetone	0.284	372	361	480	471	93	94
DMF	0.274	375	371	495	474	93	83
DMSO	0.263	376	374	500	482	88	85
Acetonitrile	0.305	373	363	498	472	95	89
Ethanol	0.289	372	362	500	485	99	91
Methanol	0.309	372	362	510	487	93	83
TFE	0.280	363	357	528	516	98	97

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Fluorescence mechanism of PSA and PTA

To understand why PSA and PTA showed the above mentioned high Φ_FL values, we investigated the dependence of the Stokes shifts of PSA and PTA on the orientation polarizability function, Δf, and calculated the change in the dipole moments between the ground and excited states (Δμ = μ_e − μ_g) (Fig. S15†).²⁴ As expected, the Δμ values of PSA and PTA are not large (7.3 D and 7.1 D, respectively), unlike the values for the above mentioned PA and PRODAN (8.3 D and 7.9 D, respectively), meaning that the secondary N-alkyl and tertiary N,N-dialkyl carboxamides act as weaker acceptors than aldehydes or ketones, and therefore the extent of ESICT characteristic in PSA and PTA is not large. As a result, unlike with typical D–π–A dyes, PSA and PTA are not quenched through internal conversion assisted by hydrogen bonding or large charge separation in highly polar solvents, including strong hydrogen bond donating solvents such as methanol and TFE. Conversely, from the high brightness of PSA and PTA in apolar solvents like hexane, it can be deduced that PSA and PTA are not quenched by ISC based on ‘El-Sayed's rule’.¹⁹

We have demonstrated that pyrene-based dyes possessing secondary N-alkyl or tertiary N,N-dialkyl carboxamide substituents do not have n–π* first singlet excited states (S₁) that would promote ISC to the triplet state and subsequent internal conversion to the ground state. In contrast to other carbonyl compounds possessing formyl or ketone groups, amide conjugation (i.e. the resonance effect within (C=O)-NHR) stabilizes the energy of the n electrons on the carbonyl group; therefore, the energy of the n–π* state becomes higher than that of the first singlet π–π* state. We believe that this characteristic of pyrene-based dyes functionalized with secondary N-alkyl or tertiary N,N-dialkyl carboxamides, which stabilize the n electrons on the carbonyl group, is expressed for PSA and PTA in apolar solvents. In fact, TD-DFT calculations supported this explanation, i.e. the n–π* transitions of PSA and PTA were not observed for S₁ and adjacent triplet states (Fig. S17 and S18, and Table S3†).

Differences between PSA and PTA

Briefly, the spectroscopic measurements demonstrated that PSA showed stronger absorption and fluorescence at slightly longer wavelengths than PTA. As seen in the optimized structures of PSA and PTA (Fig. 3) obtained from DFT calculations, this difference is most likely a result of the larger dihedral angle between the carbonyl group and the pyrene for the tertiary N,N-dialkyl carboxamide than for the secondary N-alkyl carboxamide, as we reported previously. Therefore, there is less perturbation of pyrene by the tertiary N,N-dialkyl carboxamide. As a result, both the absorption and fluorescence spectra of PTA were located at shorter wavelengths than those of PSA. In addition, the oscillator strength of PTA was not as strongly enhanced as that of PSA, which is supported by the TD-DFT calculations (Table S3†).

Fig. 3 Optimized structures of PSA and PTA obtained from DFT calculations (ωB97X-D/6-31 G (d.p)). The dihedral angles in PSA and PTA between the pyrene moiety and the carbonyl group (ϕ (C_αC_βCO)) were calculated as 43.2° and 105.8°, respectively.

From these results, we concluded that the use of secondary N-alkyl carboxamide is preferable for the development of fluorescent materials in terms of high Φ_FL values and longer absorption wavelengths.

Finally, to demonstrate the unique fluorescence properties of the D–π–A dyes possessing secondary carboxamide groups as weak acceptors, i.e. strong solvatochromic fluorescence in apolar to polar solvents, including water, we synthesized PSAC and investigated its fluorescence properties. PSAC contains a secondary carboxamide group with a carboxylic acid, as described in Scheme 1.

Spectroscopic properties of PSAC

The absorption and fluorescence spectra of PSAC were measured in the same manner as those of PSA and PTA (Fig. 4; details are shown in Fig. S12–S14 and Table S2†). As was expected from the structure of PSAC, the observed fluorescence properties of PSAC were similar to those of PSA. PSAC was found to have Φ_FL > 90% in all solvents, even water. As mentioned above, D–π–A dyes possessing such properties, i.e. solvatochromism and high fluorescence quantum yields, even in water, are not commonly known. In fact, our previous D–π–A dye, PA, exhibits a drastic decrease in Φ_FL in the presence of water, even though PA has high Φ_FL values in polar solvents compared with existing solvatochromic dyes (Fig. S16†).^3–16 Moreover, considering that the spectral shifts of solvatochromic dyes that fluoresce strongly in water are also similar to that of PSAC, e.g. 1-formylpyrene (391 nm (Φ_FL = 0.3%) and 470 nm (Φ_FL > 90%) in toluene and water, respectively),¹⁸ the obtained solvatochromic properties of PSAC are significant. Importantly, although it might be possible to predict that weak CT characteristics can realize high brightness in water, as seen for 1-formylpyrene, solvatochromism and the high brightness in both hexane and water cannot be achieved without the intentional use of an acceptor such as N-alkyl carboxamide that acts both as a weak acceptor and as a stabilizer of the n electrons.

Fig. 4 The absorption and fluorescence spectra of PSAC in several solvents of different polarities (λ_ex = λ_{abs, max}, optical density (O.D.) = 0.1, room temperature).

The region in which the absorption and fluorescence of PSAC are located, from ultraviolet to yellow, might be inappropriate for biological applications.¹³ However, the absorption of PSAC is fully compatible with the 405 nm diode laser commonly used for fluorescence microscopy. In addition, given that synthetic methodologies using pyrene^8b,14b,25 and several advanced optical techniques, such as single-molecule^13,26 and two-photon excited^9,14,27 fluorescence microscopies employing solvatochromic dyes including PRODAN, have progressed, it can be expected that further modification of PSAC or strategic use of the carboxamide groups as an acceptor will be extended to practical applications of fluorescent probes for investigating multimolecular systems.

In summary, we have developed a new D–π–A dye, PSAC, which fluoresces with high Φ_FL and shows solvatochromism in solvents with a wide range of polarities, including water, and we have shown the fundamental role of the secondary N-alkyl and tertiary N,N-dialkyl carboxamide as electron acceptors. The excellent fluorescence properties of PSAC are derived from the intentional use of the N-alkyl carboxamide substituent, which acts not only as a ‘weak’ acceptor, but also stabilizes the n electrons on the carbonyl group. These characteristics suppress the nonfluorescent deactivation pathways, both by reducing the large charge separation and by minimizing ISC, while maintaining the underlying property of D–π–A dyes, i.e. solvatochromism. Because dyes possessing the properties of PSAC are not commonly known, the fluorescence properties obtained here are significant. Therefore, we believe that the strategic use of carboxamides as acceptors will allow the development of novel fluorescent probes and lead to new applications for fluorescent molecules.

Notes and references

1. R. W. Sinkeldam, N. J. Greco and Y. Tor, Chem. Rev., 2010, 110, 2579.
2. A. P. de Silva, H. Q. N. Gunaratne, T. Gunnlaugsson, A. J. M. Huxley, C. P. McCoy, J. T. Rademacher and T. E. Rice, Chem. Rev., 1997, 97, 1515.
3. A. R. Katritzky and T. Narindoshvili, Org. Biomol. Chem., 2009, 7, 627.
4. A. S. Klymchenko and R. Kreder, Chem. Biol., 2014, 21, 97.
5. Z. Yang, J. Cao, Y. He, J. H. Yang, T. Kim, X. Peng and J. S. Kim, Chem. Soc. Rev., 2014, 43, 4563.
6. (a) K. Li, W. Qin, D. Ding, N. Tomczak, J. Geng, R. Liu, J. Liu, X. Zhang, H. Liu, B. Liu and B. Z. Tang, Sci. Rep., 2013, 3, 1150; (b) A. S. Klymchenko, E. Roger, N. Anton, H. Anton, I. Shulov, J. Vermot, Y. Mély and T. F. Vandamme, RSC Adv., 2012, 2, 11876; (c) J. H. Kim, S. Lee, K. Park, H. Y. Nam, S. Y. Jang, I. Youn, K. Kim, H. Jeon, R. W. Park, I. S. Kim, K. Choi and I. C. Kwon, Angew. Chem., Int. Ed., 2007, 46, 5779.
7. A. M. Fakhari and S. E. Rokita, Chem. Commun., 2011, 47, 4222.
8. (a) R. F. Landis, M. Yazdani, B. Creran, X. Yu, V. Nandwana, G. Cooke and V. M. Rotello, Chem. Commun., 2014, 50, 4579; (b) Y. Niko, S. Sasaki, S. Kawauchi, K. Tokumaru and G. Konishi, Chem.–Asian J., 2014, 9, 1797.
9. (a) K. P. Divya, S. Savithri and A. Ajayaghosh, Chem. Commun., 2014, 50, 6020; (b) H. M. Kim and B. R. Cho, Acc. Chem. Res., 2009, 42, 863; (c) H. M. Kim and B. R. Cho, Chem.–Asian J., 2011, 6, 58; (d) X. Dong, J. H. Han, C. H. Heo, H. M. Kim, Z. Liu and B. R. Cho, Anal. Chem., 2012, 84, 8110.
10. L. Ding, Z. Zhang, X. Li and J. Su, Chem. Commun., 2013, 49, 7319.
11. (a) M. Nitz, A. R. Mezo, M. H. Ali and B. Imperiali, Chem. Commun., 2002, 1912; (b) B. E. Cohen, T. B. MacAnaney, E. S. Park, Y. N. Jan, S. G. Boxer and L. Y. Jan, Science, 2002, 296, 1700; (c) A. V. Strizhak, V. Y. Postupalenko, V. V. Shvadchak, N. Morellet, E. Guittet, V. G. Pivovarenko, A. S. Klymchenko and Y. Mely, Bioconjugate Chem., 2012, 23, 2434.
12. G. Weber and F. J. Farris, Biochemistry, 1979, 18, 3075.
13. (a) Z. Lu, S. J. Lord, H. Wang, W. E. Moerner and R. J. Twieg, J. Org. Chem., 2006, 71, 9651; (b) S. J. Lord, Z. Lu, H. Wang, K. A. Willets, P. J. Schuck, H. D. Lee, S. Y. Nishimura, R. J. Twieg and W. E. Moerner, J. Phys. Chem. A, 2007, 111, 8934.
14. (a) O. A. Kucherak, P. Didier, Y. Mely and A. S. Klymchenko, J. Phys. Chem. Lett., 2010, 1, 616; (b) Y. Niko, S. Kawauchi and G. Konishi, Chem.–Eur. J., 2013, 19, 9760; (c) P. D. Zoon and A. M. Brouwer, ChemPhysChem, 2005, 6, 1574; (d) S. Sasaki, Y. Niko, K. Igawa and G. Konishi, RSC Adv., 2014, 4, 33474; (e) S. Sasaki, Y. Niko, A. S. Klymchenko and G. Konishi, Tetrahedron DOI:10.1016/j.tet.2014.08.002 , in press.
15. L. Giordano, V. Shvadchak, J. A. Fauerbach, E. A. Jares-Erijman and T. M. Jovin, J. Phys. Chem. Lett., 2012, 3, 1011.
16. O. A. Kucherak, L. Richert, Y. Mely and A. S. Klymchenko, Phys. Chem. Chem. Phys., 2012, 14, 2292.
17. (a) Y. Niko, S. Kawauchi and G. Konishi, Tetrahedron Lett., 2011, 52, 4843; (b) Y. Niko, Y. Hiroshige, S. Kawauchi and G. Konishi, J. Org. Chem., 2012, 77, 3986; (c) Y. Niko and G. Konishi, J. Synth. Org. Chem., Jpn., 2012, 70, 918.
18. (a) K. Kalyanasundaram and J. K. Thomas, J. Phys. Chem., 1977, 81, 2176; (b) Y. Niko, Y. Hiroshige, S. Kawauchi and G. Konishi, Tetrahedron, 2012, 68, 6177.
19. N. J. Turro, V. Ramamurthy and J. C. Scaiano, Modern Molecular Photochemistry of Organic Molecules, University Science Books, Sausalito, California, 2010, p.238.
20. M. Montalti, A. Credi, L. Prodi and M. T. Gandolfi, HANDBOOK OF PHOTOCHEMISTRY, Taylor & Francis, 3rd edn, 2006, pp.624–627.
21. (a) A. Marini, A. Muñoz-Losa, A. Biancardi and B. Mennucci, J. Phys. Chem. B, 2010, 114, 17128; (b) F. Moyano, M. A. Biasutti, J. J. Silber and N. M. Correa, J. Phys. Chem. B, 2006, 110, 11838; (c) F. Moyano, P. G. Molina, J. J. Silber, L. Sereno and N. M. Correa, ChemPhysChem, 2010, 11, 236; (d) L. Stryer, J. Am. Chem. Soc., 1966, 88, 5708; (e) H. Inoue, M. Hida, N. Nakashima and K. Yoshihara, J. Phys. Chem., 1982, 86, 3184; (f) K. Klehs, C. Spahn, U. Endesfelder, S. F. Lee, A. Fürstenberg and M. Heilemann, ChemPhysChem, 2014, 15, 637.
22. P. T. Chou, C. P. Chang, J. H. Clements and K. Meng-Shin, J. Fluoresc., 1995, 5, 369.
23. (a) P. Greenspan, E. P. Mayer and S. D. Fowler, J. Cell Biol., 1985, 100, 965; (b) O. A. Kucherak, S. Oncul, Z. Darwich, D. A. Yushchenko, Y. Arntz, P. Didier, Y. Mely and A. S. Klymchenko, J. Am. Chem. Soc., 2010, 132, 4907.
24. (a) E. L. Lippert, Organic Molecular Photophysics; Wiley-Interscience, New York, 1975; (b) N. Mataga, T. Kubota, Molecular Interactions and Electronic Spctra, Dekker, M. New York, 1970.
25. (a) T. M. Figueira-Duarte and K. Müllen, Chem. Rev., 2011, 111, 7260; (b) Y. Niko, S. Kawauchi, S. Otsu, K. Tokumaru and G. Konishi, J. Org. Chem., 2013, 78, 3196.
26. (a) T. Oba and M. Vacha, ACS Macro Lett., 2012, 1, 784; (b) K. Suzuki, S. Habuchi and M. Vacha, Chem. Phys. Lett., 2011, 505, 157.
27. S. Yao and K. D. Belfield, Eur. J. Org. Chem., 2012, 17, 3199.

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Footnote

† Electronic supplementary information (ESI) available: Experimental section: synthetic procedures, characterizations (¹H NMR, ¹³C NMR, FT-IR spectra), additional spectroscopic data, and DFT/TDDFT calculation results. See DOI: 10.1039/c4ra06282e

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