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Cite this: DOI: 10.1039/c3cc47262k Received 23rd September 2013, Accepted 5th November 2013
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Aggregation-induced emission of GFP-like chromophores via exclusion of solvent–solute hydrogen bonding† Sio-Lon Tou, Guan-Jhih Huang, Po-Cheng Chen, Huan-Tsung Chang, Jun-Yun Tsai and Jye-Shane Yang*
DOI: 10.1039/c3cc47262k www.rsc.org/chemcomm
The fluorescence of GFP-like chromophores in aqueous solutions is turned on upon forming aggregates or embedment in cell membranes as a result of exclusion of solvent–solute H-bonding.
Fluorophores that display aggregation-induced emission (AIE) are fundamentally intriguing, because they are opposite to the majority of systems that undergo fluorescence quenching upon forming aggregates.1 The AIE effect generally results from an effective restriction of the internal rotations (RIR) that are responsible for the fluorescence quenching in dilute solutions. In principle, all the low emissive fluorophores are potential candidates for AIE, provided that the underlying nonradiative decay channels can be substantially blocked upon forming aggregates. However, examples differing from the RIR mechanism have not been demonstrated. We report herein the first example of AIE as a consequence of exclusion of solvent–solute hydrogen bonding (ESSHB). Because of their potential applications as biomarkers and light-emitting materials, the chromophore of green fluorescence protein (GFP), p-hydroxybenzylidenedimethylimidazolinone ( p-HBDI), and its analogues (GFPc) have been extensively investigated.2,3 The fluorescence quantum yield (Ff) of p-HBDI is extremely low (Ff o 10 3) outside the protein matrix (Ff = 0.8 for GFP) at ambient temperatures.4 The fluorescence quenching has been attributed to ultrafast internal rotations in the excited states. A variety of conformational rigidifying methods, including encapsulation in supramolecular hosts,3,5 covalent or noncovalent bridging of the two rings,6 and crystal engineering toward compact but non-stacked molecular arrangements,7,8 have been employed to restore the fluorescence. We recently demonstrated an alternative approach without structural constraint by using meta-amino substituents (i.e., the m-ABDIs, Chart 1).9,10 For example, the Ff values
Department of Chemistry, National Taiwan University, Taipei, Taiwan 10617. E-mail: [email protected] † Electronic supplementary information (ESI) available: Detailed synthetic scheme and procedure, characterization data, electronic and NMR spectra, DLS analysis, HPLC traces, and DFT-derived MOs. See DOI: 10.1039/c3cc47262k
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Chart 1 Structures of m- and p-ABDIs and p-HBDI.
of compounds m-D0A1 and m-D1A1 in hexane are 0.34 and 0.46, respectively. The origin of the m-amino effect is a large increase of the barrier for the Z - E photoisomerization, a nonradiative decay process that is responsible for the fluorescence quenching of the para-amino isomers (the p-ABDIs) and p-HBDI due to an extremely low barrier.11–14 However, the fluorescence enhancement is limited to aprotic organic solvents, because in protic solvents the solvent– solute H-bonding interactions create a new ultrafast nonradiative decay channel, which limits the application of m-ABDIs in biological systems. In this context, we have explored the possibility of AIE for m-D1A1 and its long-chain derivatives m-D8A1 and m-D1A16 in aqueous solutions. For comparison, the corresponding para systems p-D8A1 and p-D1A16 and the ionic species m-D1Aa and p-D1Aa were also investigated (Chart 1). Our results show that the phenomenon of AIE occurs only for the m-ABDIs with long alkyl chains as a result of effective ESSHB. The ESSHB-based fluorescence turn-on cell imaging is also demonstrated using m-D1Aa. In synthesis of the target GFPc the protocol15 of Bazureau and Kowalik was adopted: namely, [2+3] cyclocondensation of the corresponding arylideneimines with the imidate ylide methyl 2-(1-ethoxyethylideneamino)acetate (4). A representative example using m-D8A1 is shown in Scheme 1. The arylideneimine 3 was prepared from 3-bromoaniline via the intermediates 1 and 2 by double N-alkylation under basic conditions followed by lithiation, formylation, and imination at the bromosubstituted position. Synthetic schemes (Schemes S1–S6, ESI†) for the other compounds and the data of product
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Table 1
Compd
Solvent
labs (nm)
lfa (nm)
Ff (%)
tfa,b (ns)
m-D1A1
Hex THF MeCN MeOH H2Oc Hex THF MeCN MeOH H2Oc Hex THF MeCN MeOH H2Oc H2O
349 354 352 353 351 352 357 355 354 361 352 357 357 355 367 349
492 584 643 701 na 504 555 621 697 578 491 563 625 701 582 na
46 14 5 o0.1 o0.01 48 19 7 o0.1 14 53 21 7 o0.1 9 o0.01
22.5 15.3 8.6 na na 19.7 14.8 9.4 na 7.1d,e 17.3 12.3 5.3 na 7.0d, f na
m-D8A1 Scheme 1
Synthesis of m-D8A1.
characterization by NMR, IR, mass, and elemental analysis are provided in the ESI.† In dilute organic solutions, the photophysical behaviour of the long-chain derivatives m-D8A1 and m-D1A16 resembles that of m-D1A1.10 The absorption spectra are characterized with an intense peak maximum (labs) near 350 nm and a long-wavelength shoulder in the 400–500 nm region (Fig. S1, ESI†). The shoulder is sensitive to solvent polarity, corresponding to a charge-transfer electronic transition (Table S1, ESI†). The charge-transfer character of the lowest singlet excited state is consistent with a large positive solvatofluorochromism (Fig. S2, ESI†). For example, the fluorescence maximum (lf) is red-shifted by 3700–4800 cm 1 on going from hexane to acetonitrile. The lf displays a linear relationship with the solvent polarity parameter16 ET(30) (Fig. 1a). The dependence of Ff on solvent polarity is also significant, which is 0.46–0.53 in hexane, 0.14–0.21 in THF, and 0.03–0.07 in acetonitrile, DMF, or DMSO (Table 1 and Table S2, ESI†). To the best of our
Fig. 1 (a) Linear plots of lf against ET(30) for the m-ABDIs, the AIE behaviour of (b) m-D8A1 (0.05 mM) and (c) m-D1A16 (0.05 mM) in H2O–MeOH mixed solvents (lex = 350 nm), (d) fluorescence spectra of m-D8A1 (0.05 mM) aggregates in H2O in the presence of CTAB at 0, 0.5, 1.0, 1.5, 2.0, 2.5, and 5.0 mM, (e) TEM image of the m-D8A1 aggregates, and (f) photos of the powder fluorescence of m-D1A1, m-D8A1, p-D0A1, and p-D8A1 with the corresponding Ff values shown for each sample.
Chem. Commun.
Photophysical data for the m-ABDIs
m-D1A16
m-D1Aa a
na: not available, because fluorescence is too weak and the lifetime is too short to be determined. b The tf was determined with excitation and emission wavelengths at the spectral maxima. c Containing 3% THF for pre-dissolution of the substrate. d tf is fitted with biexponential functions and reported as a mean value: tf = (A1 t12 + A2 t22)/(A1 t1 + A2 t2). e A1 = 10%, t1 = 1.9 ns, A2 = 90%, t2 = 7.3 ns. f A1 = 18%, t1 = 2.4 ns, A2 = 82%, t2 = 7.3 ns.
knowledge, the Ff value of 0.53 for m-D1A16 in hexane reaches the maximum recorded for unconstrained and neutral GFPc. Nevertheless, its fluorescence lifetime (tf) of 17.3 ns is shorter than that of m-D1A1 in hexane (22.5 ns). The fluorescence is nearly quenched (Ff o 10 3) in methanol or in 60/40 (v/v) H2O–THF, which is attributable to solvent–solute H-bonding that leads to excited-state proton transfer.11 Our previous studies on m-D1A1 reveal that the H-bonding at the carbonyl group is of prime importance in accounting for the fluorescence quenching.10 The extremely weak fluorescence in methanol or mixed H2O– THF renders the m-ABDIs potential AIE fluorophores. Indeed, the AIE phenomenon was observed for both m-D8A1 and m-D1A16 in water (Fig. 1b and c). The lf is located at 580 2 nm, corresponding to a data point at ET(30) = 41.2 in the linear plots shown in Fig. 1a. This is indicative of aggregate formation, as the expected lf for an individual molecule in water (ET(30) = 63.1) is at much longer wavelength (765 5 nm). Other evidence for aggregate formation of m-D8A1 and m-D1A16 in water includes broadening of the absorption and 1H NMR spectra (Fig. S1 and S3, ESI†) and a more complex fluorescence decay functional (Table 1) relative to those in organic solvents, observation of fluorescent micrometer-sized particles under an optical microscope (Fig. S4, ESI†), and a decrease of the fluorescence upon adding the good solvent THF (Fig. S5, ESI†) or the surfactant cetyltrimethylammonium bromide (CTAB) as a de-aggregating agent (Fig. 1d). The dynamic light scattering (DLS) analysis indicates an averaged particle size of B370 and B200 nm for m-D8A1 and m-D1A16, respectively (Fig. S6, ESI†). This is consistent with the TEM image of a drop-cast film of m-D8A1 in water, in which the size of aggregates is generally less than 500 nm (Fig. 1e). Furthermore, the water-soluble m-D1Aa could serve as a reference model of the non-aggregating m-ABDI system in water, which is essentially nonfluorescent (Ff o 10 4). This again indicates that the fluorescence of m-D8A1 and m-D1A16 in water is not due to individual molecules but due to aggregates, and the AIE effect
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increases the fluorescence by more than 900-fold (Ff = 0.14 and 0.09 vs. o10 4). In contrast, no fluorescence enhancement could be observed for m-D1A1 in water, which could be attributed to its poor ability to form aggregates according to DLS and 1H NMR analysis (Fig. S3, ESI†). Likewise, an attempt to induce aggregate formation in m-D1Aa, despite its insolubility in THF, in mixed THF–H2O was unsuccessful (Fig. S7, ESI†). These results show that long alkyl chains facilitate aggregate formation in water and are required for m-ABDIs to be AIE fluorophores. To elucidate the AIE mechanism of m-D8A1 and m-D1A16, the corresponding behaviour of p-D8A1 and p-D1A16 was investigated. Like p-HBDI and p-D0A1,9 both p-D8A1 and p-D1A16 display very weak fluorescence (Ff o 10 3) in both protic and aprotic organic solvents (Table S3, ESI†), which is attributable to the ultrafast Z–E photoisomerization.12,13 Such a weak emission is unchanged for them in water, although aggregate formation (averaged size B200 nm) is evidenced by the DLS analysis and by the absorption and 1H NMR spectroscopy (Fig. S3 and S6, ESI†). It is also noted that even in the solid powder both p-D8A1 and p-D1A16 display much weaker fluorescence than the m-ABDIs (Fig. 1f) and the O-alkylated p-HBDIs,7 although the Z–E photoisomerization is inhibited for both the m- and p-ABDIs in solids (Fig. S9, ESI†). These results show that the RIR for p-ABDIs in aggregates or solids is ineffective in enhancing the fluorescence.17 Therefore, the observed AIE of m-D8A1 and m-D1A16 in water is more likely a consequence of ESSHB rather than RIR that is responsible for the previously reported AIE fluorophores such as silole and tetraphenylethene derivatives.1 Inspired by the ESSHB-based AIE for the long-chain m-ABDIs in water, we envisioned that a similar fluorescence turn-on behavior might also occur for m-ABDIs upon transferring from the bulk aqueous solutions to hydrophobic environments such as micelles or lipid bilayers. To prove this concept, we have carried out two experiments. One is to titrate the aqueous solution of m-D8A1 (in aggregates) using CTAB over the critical micelle concentration (0.92 mM).18 As shown in Fig. 1d, the fluorescence (578 nm) is diminished due to de-aggregation by CTAB but remained to be significant with red-shifted lf (618 nm) even at 5.0 mM CTAB. This is attributable to an embedment of m-D8A1 in a water-free environment. The second experiment is to incubate human mammary epithelial cells MCF-10A in the presence of m-D1Aa. More than 90% of the cells incubated with 1 mM of m-D1Aa at 37 1C for 24 h survived, indicating a low cytotoxicity of m-D1Aa toward the cells. In addition, green fluorescence is observed for the cells, particularly in the region of the cell membrane (Fig. 2). In contrast, no such fluorescence turn-on could be observed for
Fig. 2 (a) Bright-field and (b) fluorescent images of MCF-10A incubated with m-D1Aa.
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p-D8A1, p-D1A16, and p-D1Aa in the corresponding experiments. Evidently, the ESSHB is responsible for the observed fluorescence turn-on of the m-ABDIs. This is again different from the RIR mechanism previously reported for the fluorescence turn-on of GFPc in bile salt aggregates.19 The fluorescence turn-off in aqueous solutions but turn-on in cells renders m-D1Aa and its analogues potential fluorescent probes for continuous real-time cell imaging.20 Further studies on this issue are in progress. In summary, we have demonstrated the first example of ESSHBbased AIE using the GFPc m-D8A1 and m-D1A16. Because of the AIE effect, these long-chain derivatives of m-ABDIs are fluorescent not only in aprotic solvents but also in protic media, which renders m-ABDIs potential sensory materials and novel fluorescent dyes. The concept of fluorescence turn-on by ESSHB can also be applied in cell imaging. We thank the National Science Council and the Ministry of Education, Taiwan, for financial support, Prof. Chung-Yuan Mou for DLS analysis, Ms C.-Y. Chien of Precious Instrument Center (NTU) for assistance in TEM experiments, and Mr Che-Jen Lin for DFT calculations.
Notes and references 1 (a) Y. Hong, J. W. Lam and B. Z. Tang, Chem. Commun., 2009, 4332–4353; (b) M. Wang, G. Zhang, D. Zhang, D. Zhu and B. Z. Tang, J. Mater. Chem., 2010, 20, 1858–1867. 2 (a) S. R. Meech, Chem. Soc. Rev., 2009, 38, 2922–2934; (b) L. M. Tolbert, A. Baldridge, J. Kowalik and K. M. Solntsev, Acc. Chem. Res., 2012, 45, 171–181. 3 A. Baldridge, S. Feng, Y.-T. Chang and L. M. Tolbert, ACS Comb. Sci., 2011, 13, 214–217. 4 (a) H. Niwa, S. Inouye, T. Hirano, T. Matsuno, S. Kojima, M. Kubota, M. Ohashi and F. I. Tsuji, Proc. Natl. Acad. Sci. U. S. A., 1996, 93, 13617–13622; (b) W. Weber, V. Helms, J. A. McCammon and P. W. Langhoff, Proc. Natl. Acad. Sci. U. S. A., 1999, 96, 6177–6182. 5 A. Baldridge, S. R. Samanta, N. Jayaraj, V. Ramamurthy and L. M. Tolbert, J. Am. Chem. Soc., 2011, 133, 712–715. 6 (a) L. Wu and K. Burgess, J. Am. Chem. Soc., 2008, 130, 4089–4096; (b) A. Baldridge, K. M. Solntsev, C. Song, T. Tanioka, J. Kowalik, K. Hardcastle and L. M. Tolbert, Chem. Commun., 2010, 46, 5686–5688. 7 J. Dong, K. M. Solntsev and L. M. Tolbert, J. Am. Chem. Soc., 2009, 131, 662–670. 8 G. Huang, G. Zhang, Y. Wu, Q. Liao, H. Fu and D. Zhang, Asian J. Org. Chem., 2012, 1, 352–358. 9 J.-S. Yang, G.-J. Huang, Y.-H. Liu and S.-M. Peng, Chem. Commun., 2008, 1344–1346. 10 G.-J. Huang, J.-H. Ho, C. Prabhakar, Y.-H. Liu, S.-M. Peng and J.-S. Yang, Org. Lett., 2012, 14, 5034–5037. 11 C.-W. Cheng, G.-J. Huang, H.-Y. Hsu, C. Prabhakar, Y.-P. Lee, E. W.-G. Diau and J.-S. Yang, J. Phys. Chem. B, 2013, 117, 2705–2716. 12 G.-J. Huang, C.-W. Cheng, H.-Y. Hsu, C. Prabhakar, Y.-P. Lee, E. W.-G. Diau and J.-S. Yang, J. Phys. Chem. B, 2013, 117, 2695–2704. 13 G.-J. Huang and J.-S. Yang, Chem.–Asian J., 2010, 5, 2075–2085. 14 K. M. Solntsev, O. Poizat, J. Dong, J. Rehault, Y. Lou, C. Burda and L. M. Tolbert, J. Phys. Chem. B, 2008, 112, 2700–2711. 15 (a) J. M. Lerestif, J. P. Bazureau and J. Hamelin, Tetrahedron Lett., 1993, 34, 4639–4642; (b) J. Kowalik, A. Baldridge and L. M. Tolbert, Synthesis, 2010, 2424–2436. 16 C. Reichardt, Chem. Rev., 1994, 94, 2319–2358. 17 Both the m- and p-ABDIs have low Z–E photoisomerization quantum yields in aggregates (o0.05) and solids (B0). A possible explanation is that new fluorescence quenching channels such as nonfluorescent excimers are induced in aggregates and solids and offset the gain from diminished Z–E photoisomerization reactivity. 18 I. Garcia-Mateos, M. Mercedes Velazquez and L. J. Rodriguez, Langmuir, 1990, 6, 1078–1083. 19 A. Baldridge, A. Amador and L. M. Tolbert, Langmuir, 2011, 27, 3271–3274. 20 A. Palma, L. A. Alvarez, D. Scholz, D. O. Frimannsson, M. Grossi, S. J. Quinn and D. F. O’Shea, J. Am. Chem. Soc., 2011, 133, 19618–19621.
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Cite this: DOI: 10.1039/c3cc47262k Received 23rd September 2013, Accepted 5th November 2013
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Aggregation-induced emission of GFP-like chromophores via exclusion of solvent–solute hydrogen bonding† Sio-Lon Tou, Guan-Jhih Huang, Po-Cheng Chen, Huan-Tsung Chang, Jun-Yun Tsai and Jye-Shane Yang*
DOI: 10.1039/c3cc47262k www.rsc.org/chemcomm
The fluorescence of GFP-like chromophores in aqueous solutions is turned on upon forming aggregates or embedment in cell membranes as a result of exclusion of solvent–solute H-bonding.
Fluorophores that display aggregation-induced emission (AIE) are fundamentally intriguing, because they are opposite to the majority of systems that undergo fluorescence quenching upon forming aggregates.1 The AIE effect generally results from an effective restriction of the internal rotations (RIR) that are responsible for the fluorescence quenching in dilute solutions. In principle, all the low emissive fluorophores are potential candidates for AIE, provided that the underlying nonradiative decay channels can be substantially blocked upon forming aggregates. However, examples differing from the RIR mechanism have not been demonstrated. We report herein the first example of AIE as a consequence of exclusion of solvent–solute hydrogen bonding (ESSHB). Because of their potential applications as biomarkers and light-emitting materials, the chromophore of green fluorescence protein (GFP), p-hydroxybenzylidenedimethylimidazolinone ( p-HBDI), and its analogues (GFPc) have been extensively investigated.2,3 The fluorescence quantum yield (Ff) of p-HBDI is extremely low (Ff o 10 3) outside the protein matrix (Ff = 0.8 for GFP) at ambient temperatures.4 The fluorescence quenching has been attributed to ultrafast internal rotations in the excited states. A variety of conformational rigidifying methods, including encapsulation in supramolecular hosts,3,5 covalent or noncovalent bridging of the two rings,6 and crystal engineering toward compact but non-stacked molecular arrangements,7,8 have been employed to restore the fluorescence. We recently demonstrated an alternative approach without structural constraint by using meta-amino substituents (i.e., the m-ABDIs, Chart 1).9,10 For example, the Ff values
Department of Chemistry, National Taiwan University, Taipei, Taiwan 10617. E-mail: [email protected] † Electronic supplementary information (ESI) available: Detailed synthetic scheme and procedure, characterization data, electronic and NMR spectra, DLS analysis, HPLC traces, and DFT-derived MOs. See DOI: 10.1039/c3cc47262k
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Chart 1 Structures of m- and p-ABDIs and p-HBDI.
of compounds m-D0A1 and m-D1A1 in hexane are 0.34 and 0.46, respectively. The origin of the m-amino effect is a large increase of the barrier for the Z - E photoisomerization, a nonradiative decay process that is responsible for the fluorescence quenching of the para-amino isomers (the p-ABDIs) and p-HBDI due to an extremely low barrier.11–14 However, the fluorescence enhancement is limited to aprotic organic solvents, because in protic solvents the solvent– solute H-bonding interactions create a new ultrafast nonradiative decay channel, which limits the application of m-ABDIs in biological systems. In this context, we have explored the possibility of AIE for m-D1A1 and its long-chain derivatives m-D8A1 and m-D1A16 in aqueous solutions. For comparison, the corresponding para systems p-D8A1 and p-D1A16 and the ionic species m-D1Aa and p-D1Aa were also investigated (Chart 1). Our results show that the phenomenon of AIE occurs only for the m-ABDIs with long alkyl chains as a result of effective ESSHB. The ESSHB-based fluorescence turn-on cell imaging is also demonstrated using m-D1Aa. In synthesis of the target GFPc the protocol15 of Bazureau and Kowalik was adopted: namely, [2+3] cyclocondensation of the corresponding arylideneimines with the imidate ylide methyl 2-(1-ethoxyethylideneamino)acetate (4). A representative example using m-D8A1 is shown in Scheme 1. The arylideneimine 3 was prepared from 3-bromoaniline via the intermediates 1 and 2 by double N-alkylation under basic conditions followed by lithiation, formylation, and imination at the bromosubstituted position. Synthetic schemes (Schemes S1–S6, ESI†) for the other compounds and the data of product
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Table 1
Compd
Solvent
labs (nm)
lfa (nm)
Ff (%)
tfa,b (ns)
m-D1A1
Hex THF MeCN MeOH H2Oc Hex THF MeCN MeOH H2Oc Hex THF MeCN MeOH H2Oc H2O
349 354 352 353 351 352 357 355 354 361 352 357 357 355 367 349
492 584 643 701 na 504 555 621 697 578 491 563 625 701 582 na
46 14 5 o0.1 o0.01 48 19 7 o0.1 14 53 21 7 o0.1 9 o0.01
22.5 15.3 8.6 na na 19.7 14.8 9.4 na 7.1d,e 17.3 12.3 5.3 na 7.0d, f na
m-D8A1 Scheme 1
Synthesis of m-D8A1.
characterization by NMR, IR, mass, and elemental analysis are provided in the ESI.† In dilute organic solutions, the photophysical behaviour of the long-chain derivatives m-D8A1 and m-D1A16 resembles that of m-D1A1.10 The absorption spectra are characterized with an intense peak maximum (labs) near 350 nm and a long-wavelength shoulder in the 400–500 nm region (Fig. S1, ESI†). The shoulder is sensitive to solvent polarity, corresponding to a charge-transfer electronic transition (Table S1, ESI†). The charge-transfer character of the lowest singlet excited state is consistent with a large positive solvatofluorochromism (Fig. S2, ESI†). For example, the fluorescence maximum (lf) is red-shifted by 3700–4800 cm 1 on going from hexane to acetonitrile. The lf displays a linear relationship with the solvent polarity parameter16 ET(30) (Fig. 1a). The dependence of Ff on solvent polarity is also significant, which is 0.46–0.53 in hexane, 0.14–0.21 in THF, and 0.03–0.07 in acetonitrile, DMF, or DMSO (Table 1 and Table S2, ESI†). To the best of our
Fig. 1 (a) Linear plots of lf against ET(30) for the m-ABDIs, the AIE behaviour of (b) m-D8A1 (0.05 mM) and (c) m-D1A16 (0.05 mM) in H2O–MeOH mixed solvents (lex = 350 nm), (d) fluorescence spectra of m-D8A1 (0.05 mM) aggregates in H2O in the presence of CTAB at 0, 0.5, 1.0, 1.5, 2.0, 2.5, and 5.0 mM, (e) TEM image of the m-D8A1 aggregates, and (f) photos of the powder fluorescence of m-D1A1, m-D8A1, p-D0A1, and p-D8A1 with the corresponding Ff values shown for each sample.
Chem. Commun.
Photophysical data for the m-ABDIs
m-D1A16
m-D1Aa a
na: not available, because fluorescence is too weak and the lifetime is too short to be determined. b The tf was determined with excitation and emission wavelengths at the spectral maxima. c Containing 3% THF for pre-dissolution of the substrate. d tf is fitted with biexponential functions and reported as a mean value: tf = (A1 t12 + A2 t22)/(A1 t1 + A2 t2). e A1 = 10%, t1 = 1.9 ns, A2 = 90%, t2 = 7.3 ns. f A1 = 18%, t1 = 2.4 ns, A2 = 82%, t2 = 7.3 ns.
knowledge, the Ff value of 0.53 for m-D1A16 in hexane reaches the maximum recorded for unconstrained and neutral GFPc. Nevertheless, its fluorescence lifetime (tf) of 17.3 ns is shorter than that of m-D1A1 in hexane (22.5 ns). The fluorescence is nearly quenched (Ff o 10 3) in methanol or in 60/40 (v/v) H2O–THF, which is attributable to solvent–solute H-bonding that leads to excited-state proton transfer.11 Our previous studies on m-D1A1 reveal that the H-bonding at the carbonyl group is of prime importance in accounting for the fluorescence quenching.10 The extremely weak fluorescence in methanol or mixed H2O– THF renders the m-ABDIs potential AIE fluorophores. Indeed, the AIE phenomenon was observed for both m-D8A1 and m-D1A16 in water (Fig. 1b and c). The lf is located at 580 2 nm, corresponding to a data point at ET(30) = 41.2 in the linear plots shown in Fig. 1a. This is indicative of aggregate formation, as the expected lf for an individual molecule in water (ET(30) = 63.1) is at much longer wavelength (765 5 nm). Other evidence for aggregate formation of m-D8A1 and m-D1A16 in water includes broadening of the absorption and 1H NMR spectra (Fig. S1 and S3, ESI†) and a more complex fluorescence decay functional (Table 1) relative to those in organic solvents, observation of fluorescent micrometer-sized particles under an optical microscope (Fig. S4, ESI†), and a decrease of the fluorescence upon adding the good solvent THF (Fig. S5, ESI†) or the surfactant cetyltrimethylammonium bromide (CTAB) as a de-aggregating agent (Fig. 1d). The dynamic light scattering (DLS) analysis indicates an averaged particle size of B370 and B200 nm for m-D8A1 and m-D1A16, respectively (Fig. S6, ESI†). This is consistent with the TEM image of a drop-cast film of m-D8A1 in water, in which the size of aggregates is generally less than 500 nm (Fig. 1e). Furthermore, the water-soluble m-D1Aa could serve as a reference model of the non-aggregating m-ABDI system in water, which is essentially nonfluorescent (Ff o 10 4). This again indicates that the fluorescence of m-D8A1 and m-D1A16 in water is not due to individual molecules but due to aggregates, and the AIE effect
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increases the fluorescence by more than 900-fold (Ff = 0.14 and 0.09 vs. o10 4). In contrast, no fluorescence enhancement could be observed for m-D1A1 in water, which could be attributed to its poor ability to form aggregates according to DLS and 1H NMR analysis (Fig. S3, ESI†). Likewise, an attempt to induce aggregate formation in m-D1Aa, despite its insolubility in THF, in mixed THF–H2O was unsuccessful (Fig. S7, ESI†). These results show that long alkyl chains facilitate aggregate formation in water and are required for m-ABDIs to be AIE fluorophores. To elucidate the AIE mechanism of m-D8A1 and m-D1A16, the corresponding behaviour of p-D8A1 and p-D1A16 was investigated. Like p-HBDI and p-D0A1,9 both p-D8A1 and p-D1A16 display very weak fluorescence (Ff o 10 3) in both protic and aprotic organic solvents (Table S3, ESI†), which is attributable to the ultrafast Z–E photoisomerization.12,13 Such a weak emission is unchanged for them in water, although aggregate formation (averaged size B200 nm) is evidenced by the DLS analysis and by the absorption and 1H NMR spectroscopy (Fig. S3 and S6, ESI†). It is also noted that even in the solid powder both p-D8A1 and p-D1A16 display much weaker fluorescence than the m-ABDIs (Fig. 1f) and the O-alkylated p-HBDIs,7 although the Z–E photoisomerization is inhibited for both the m- and p-ABDIs in solids (Fig. S9, ESI†). These results show that the RIR for p-ABDIs in aggregates or solids is ineffective in enhancing the fluorescence.17 Therefore, the observed AIE of m-D8A1 and m-D1A16 in water is more likely a consequence of ESSHB rather than RIR that is responsible for the previously reported AIE fluorophores such as silole and tetraphenylethene derivatives.1 Inspired by the ESSHB-based AIE for the long-chain m-ABDIs in water, we envisioned that a similar fluorescence turn-on behavior might also occur for m-ABDIs upon transferring from the bulk aqueous solutions to hydrophobic environments such as micelles or lipid bilayers. To prove this concept, we have carried out two experiments. One is to titrate the aqueous solution of m-D8A1 (in aggregates) using CTAB over the critical micelle concentration (0.92 mM).18 As shown in Fig. 1d, the fluorescence (578 nm) is diminished due to de-aggregation by CTAB but remained to be significant with red-shifted lf (618 nm) even at 5.0 mM CTAB. This is attributable to an embedment of m-D8A1 in a water-free environment. The second experiment is to incubate human mammary epithelial cells MCF-10A in the presence of m-D1Aa. More than 90% of the cells incubated with 1 mM of m-D1Aa at 37 1C for 24 h survived, indicating a low cytotoxicity of m-D1Aa toward the cells. In addition, green fluorescence is observed for the cells, particularly in the region of the cell membrane (Fig. 2). In contrast, no such fluorescence turn-on could be observed for
Fig. 2 (a) Bright-field and (b) fluorescent images of MCF-10A incubated with m-D1Aa.
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p-D8A1, p-D1A16, and p-D1Aa in the corresponding experiments. Evidently, the ESSHB is responsible for the observed fluorescence turn-on of the m-ABDIs. This is again different from the RIR mechanism previously reported for the fluorescence turn-on of GFPc in bile salt aggregates.19 The fluorescence turn-off in aqueous solutions but turn-on in cells renders m-D1Aa and its analogues potential fluorescent probes for continuous real-time cell imaging.20 Further studies on this issue are in progress. In summary, we have demonstrated the first example of ESSHBbased AIE using the GFPc m-D8A1 and m-D1A16. Because of the AIE effect, these long-chain derivatives of m-ABDIs are fluorescent not only in aprotic solvents but also in protic media, which renders m-ABDIs potential sensory materials and novel fluorescent dyes. The concept of fluorescence turn-on by ESSHB can also be applied in cell imaging. We thank the National Science Council and the Ministry of Education, Taiwan, for financial support, Prof. Chung-Yuan Mou for DLS analysis, Ms C.-Y. Chien of Precious Instrument Center (NTU) for assistance in TEM experiments, and Mr Che-Jen Lin for DFT calculations.
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