Novel nonplanar and rigid fluorophores with intensive emission in water and the application in two-photon imaging of live cells

Abstract

Bright and photostable fluorophores play a key role in the imaging of specific intracellular molecules. Here we report a facile approach to improve the brightness of naphthalimide fluorophores in water. We synthesized a series of nonplanar and rigid compounds (FM1, FM2, and FM3) by fusing a seven-membered heterocycle onto a naphthalimide skeleton. Compared with classic naphthalimide (FM0), these novel fluorophores exhibited much higher quantum yield values (Φ = 0.60–0.66), and higher molar extinction coefficients in aqueous solution, which elicited superior brightness to FM0. They also showed good photostability, pH-independence, and two-photon microscope (TPM) imaging ability. This new strategy of structural nonplanarity and rigidification provides an efficient way to improve the quantum efficiencies of traditional dyes.

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During the past few decades, fluorescence techniques have been well developed in chemistry,¹ environmental science,² biology,³ material⁴ and life science,⁵ owing to their real-time, sensitive and reliable detection and labeling functions. Particularly, fluorescence imaging of critical intracellular molecules is essential to biologists for understanding the interactions and activities of biomolecules. As a fluorescence signal-emitting unit, fluorophores play a critical role in fluorescence imaging, and brightness is a key factor of a fluorophore. As is well known, resonant dyes, such as fluoresceins, rhodamines, BODIPY dyes and cyanines, exhibit good brightness, and are widely used for fluorescence imaging. However, these dyes have their own individual limitations. For instance, fluorescein is pH sensitive,⁶ BODIPY dyes exhibit small Stokes shift,⁷ and cyanines suffer from severe aggregation and photobleaching.⁸ As for naphthalimide fluorophore, it has excellent photostability and large Stokes shift.⁹ But it is still not widely used in intracellular labelling and imaging owing to its solvatochromism,^9b,10 and poor fluorophore brightness in aqueous solution. As reported before, increasing the rigidity of a known fluorophore scaffold can availably reduce the energy consumption from rotation and vibration, and has been deemed as an efficient strategy to improve the fluorescence properties and build up new skeleton.¹¹ But the concomitant poor solubility and easier aggregation impart readily quenched fluorescence in biological systems.¹² To solve this issue, we propose a general strategy to improve the fluorophore brightness. By fusing a twisted water-soluble heterocycle onto naphthalimide scaffold, we construct novel nonplanar and rigid fluorophore structures. Therein, a twisted heterocycle not only enhance the rigidity, but also confirm the nonplanarity of the dyes, mitigating the aggregation of the fluorogenic molecules.

Starting from 4-bromo-5-nitro-1,8-naphthalimide, by introducing strong electron-donating groups (i.e., –NR₂ and –OR) at 4- and 5-positions of 1,8-naphthalimide and thus fusing a seven-membered heterocycle onto naphthalimide skeleton, we first designed and synthesized a novel nonplanar but rigid naphthalimide-based fluorophore FM1. Furthermore, FM2, with 2-(2-aminoethoxy) ethanol replacing n-butylamine, was designed to improve water-solubility and biocompatibilty; FM3, with targetable alkyl morpholine group, was synthesized to image the lysosome of living cells (Fig. 1).

Fig. 1 Structures of four compounds FM0–3.

These novel heterocyclic-fused fluorophores were prepared as shown in Scheme S1.† Commercially available compound M3 was condensed with primary amine to give the known intermediate compound M4-1 according to our previous publications.¹³ M4-2 and M4-3 are also prepared via the similar procedure. Then 4-bromo and 5-nitro groups were displaced by nucleophilic nitrogen and oxygen atoms of diethanolamine simultaneously to yield target molecule FM1. Similarly, compounds FM2 and FM3 were synthesized. All new compounds were characterized by NMR spectroscopy and HRMS. In addition, FM1 was further characterized by 2D NMR and elemental analysis (Table S1, Fig. S1–S5†). ¹³C NMR (Fig. S12†) of FM1 showed carbon resonances at δ_C 163.1, 162.8, 161.6 and 153.7 ppm, indicating that FM1 possessed four carbonyl related groups. Moreover, C9 and C11 had no directly correlated hydrogen resonances in HMQC (Fig. S1, Table S1†), but demonstrated interactions with H17 and H18–H19 in HMBC (Fig. S2, Table S1†), respectively. All these data suggested that expected heterocycle was fused onto the naphthalimide core successfully.

To confirm the structure of the novel fluorophores, we conducted X-ray single-crystal diffraction. The X-ray single-crystal structure of FM2 (Fig. 2 and S6, Table S2†) demonstrated that the ethanolamine moieties of reactant fused onto the zig–zag edge of naphthalimide core at the positions C-4 and C-5, forming the characteristic seven-membered ring. Besides, the water-soluble heterocycle was not on the same plane as the fluorescent aromatic core, with two hydrophilic groups branched out into bilateral space (Fig. 2b). And as shown in Fig. 2c, we can see that molecules in a crystal unit regularly dispersed in various directions, indicating that the π–π interactions between molecules were not very strong. Hence, it could be further speculated that compounds FM1 and FM3 owned the similar twisted seven-membered ring fused structure, which was in line with other structure characterization data like ¹H, ¹³C NMR, and HRMS.

Fig. 2 (a) Top view, (b) side view and (c) packing structure of compound FM2. Carbon, hydrogen, oxygen and nitrogen atoms are shown as grey, white, red and blue junctions, respectively.

Next, we measured the photophysical properties of compounds FM1, FM2, and FM3, with FM0 as control compound. As shown in Tables 1 and S3,† FM0 exhibits an absorption maximum at 442 nm with an moderate extinction coefficient (1.50 × 10⁴ M⁻¹ cm⁻¹), emission maximum at 530 nm and a high quantum yield (Φ = 0.86) in ethanol. But in aqueous solution, the quantum yield of FM0 decreased markedly (Φ = 0.082), which can be attributed to the aggregation of dye molecules in water.

Table 1 Absorption and emission properties of four representative compounds in selected solvents^a

No.	solvent	λabs (nm)	λem^c (nm)	Φ^d (%)	ε (M^−1 cm^−1)	Brightness^e (M^−1 cm^−1)
a Molar extinction coefficient at longest wavelength transition, ε; fluorescence quantum yields, Φ.b Control compound: N-butyl-4-butylamine-1,8-naphthalimide, usually used as reference (Φs = 0.81 in alcohol).c Excited at maximum absorption wavelength.d Determined relative to fluorescein (Φf = 0.79 in 0.1 M sodium hydroxide aqueous solution).e Brightness was the product of the molar extinction coefficient at λabs and the corresponding fluorescence quantum yield.
FM0^b	EtOH	442	530	86	15 000	12 900
	Water	455	542	8.2	11 100	900
FM1	EtOH	450	502	97	19 400	18 800
	Water	469	525	62	24 400	15 100
FM2	EtOH	450	505	92	16 800	15 500
	Water	467	527	66	20 500	13 500
FM3	EtOH	453	502	99	19 800	19 600
	Water	469	526	60	21 800	13 100

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By virtue of the further rigidification of naphthalimide scaffold, compounds FM1, FM2, and FM3 showed very high quantum yield (0.97, 0.92, 0.99, respectively) in ethanol, with a higher extinction coefficient than that of FM0. And most importantly, all of FM1, FM2 and FM3 exhibited very strong green fluorescence in aqueous medium, with much higher quantum yield (0.62, 0.66, 0.60, respectively) than that of FM0 (Tables 1 and S1†). As we expected, the increased rigidity of FM1, FM2, and FM3, which was caused by the fused seven-membered heterocycle, greatly reduced the nonradiative energy loss, and the structural nonplanarity of FM1, FM2, and FM3 mitigated the aggregation of the dye molecules dramatically. These two aspects of factors resulted in >15-fold increase in fluorophore brightness relative to that of the control compound FM0. It is worth mentioning that all these target compounds showed excellent pH-independence (between pH 2–12, Fig. S7†), making them much more applicable in complex clinical environment.

Moreover, when compared to fluorescein and BODIPY derivative, all the three fluorophores showed excellent photostability after exposure to the irradiation of a 150 W incandescent lamp for 200 min (Fig. S8†). Besides, cell viability experiment (MTT assay) demonstrated that FM1, FM2, and FM3 showed minor toxicity in MCF-7 cells at certain concentrations (0–10 μM) after they were added to nutrient medium for 24 h (Fig. S10†).

It has been reported that naphthalimide derivatives possess two-photon absorption (TPA) property and can be used for two-photon microscope (TPM) imaging,¹⁴ which is excited by two low-energy NIR photons. In contrast to traditional one-photon microscope imaging (OPM), TPM imaging shows various attractive advantages, including lower photodamage, deeper tissue penetration and higher spatial localization.¹⁵ Hence we primarily measured two-photon action cross section (Φδ) of compounds FM1, FM2, and FM3. As shown in Fig. S9,† the maximum Φδ values of FM1, FM2, and FM3 were 26 GM, 17 GM and 21 GM, respectively.

In view of the excellent photophysical properties in water and low biotoxicity, FM1, FM2, and FM3 were used for both OPM and TPM imaging in MCF-7 living cells (Fig. 3). When MCF-7 cells were incubated with FM0, FM1, FM2, and FM3 at 37 °C for 10–20 min and subsequently washed with PBS buffer for three times to remove free dyes, strong green fluorescence were observed in all the four groups of cells. Given the different cellular penetration abilities of compounds FM0, FM1, FM2, FM3, we adopted different laser intensities and gain settings (Table S4†), except for exposure times. Besides, the TPM images (λ_ex = 820 nm, λ_em = 520–560 nm, Fig. 3e–h) fit in well with OPM images (λ_ex = 405 nm, λ_em = 470–570 nm, Fig. 3i–l) to a great extent, which revealed that this novel fluorescent skeleton would have potential application in TPM imaging.

Fig. 3 Fluorescence imaging of compounds FM0, FM1, FM2, and FM3 in living MCF-7 cells with one-photon microscopy (OPM, λ_ex = 405 nm, λ_em = 470–570 nm) and two-photon microscopy (TPM, λ_ex = 820 nm, λ_em = 520–560 nm). Scale bar = 10 μm.

Finally, to explore the real application of the novel fluorophores, we explored the subcellular localization of FM3, and a commercial Lyso-Tracker (DND-99) was utilized for co-localization study (Fig. 4). The intensity correlation profile (Fig. 4f) and intensity profile of ROIs across cells (Fig. 4e) showed relatively high Pearson's coefficient (0.84) and overlap coefficient (0.85), indicating the specificity of FM3 towards lysosomes.

Fig. 4 Co-localization images of FM3 (2 μM, 20 min) with commercial Lyso-Tracker red (DND-99, 0.25 μM, 5 min) in living MCF-7 cells. (a) Green channel (λ_ex = 405 nm, λ_em = 470–550 nm) for FM3, (b) red channel (λ_ex = 559 nm, λ_em = 575–675 nm) for DND-99, (c) overlay of (a) and (b), (d) bright field image, (e) intensity profile of regions of interest (ROIs) across cells, (f) intensity correlation profile of FM3 and DND-99. Scale bar = 10 μm.

In summary, based on 1,8-naphthalimide skeleton, a new class of nonplanar and rigid fluorophores, FM1, FM2, and FM3, have been facilely synthesized by fusing a rigid and twisted heterocycle. Both the structural rigidification and nonplanarity elicited a large increase in fluorophore brightness in water. FM1, FM2, and FM3 exhibited prominent photophysical properties in aqueous solution: intensive emission (Φ = 0.60–0.66), improved molar extinction coefficient (ε = 13 100–15 100 M⁻¹ cm⁻¹), pH-independence, good water solubility, excellent photostability, and low cytotoxicity. Besides, all the three compounds showed efficient TPM imaging ability. Among them, compound FM3 showed good specificity towards lysosomes. In addition, this innovative type of fluorophore may be further developed and modified at positions of imide substituent or hydroxyl group to afford versatile naphthalimide derivatives. We believe that this strategy of structural nonplanarity and rigidification provides an efficient way to improve the quantum efficiencies of the traditional dyes.

Acknowledgements

We thank the financial support from National Natural Science Foundation of China (Grants 21476077, 21236002), and Shanghai Pujiang Program. W. Zhu is grateful for the support from Chinese Scholarship Council.

Notes and references

1. (a) J. A. Kitchen, P. N. Martinho, G. G. Morgan and T. Gunnlaugsson, Dalton Trans., 2014, 43, 6468; (b) C. Lincheneau, B. Jean-Denis and T. Gunnlaugsson, Chem. Commun., 2014, 50, 2857; (c) T. D. James, P. Linnane and S. Shinkai, Chem. Commun., 1996, 281; (d) T. D. James, K. R. A. S. Sandanayake and S. Shinkai, Angew. Chem., Int. Ed. Engl., 1996, 35, 1910; (e) Z. Xu, J. Yoon and D. R. Spring, Chem. Commun., 2010, 46, 2563; (f) A. P. de Silva, H. Q. N. Gunaratne, J.-L. Habib-Jiwan, C. P. McCoy, T. E. Rice and J.-P. Soumillion, Angew. Chem., Int. Ed. Engl., 1995, 34, 1728; (g) A. H. Younes, L. Zhang, R. J. Clark and L. Zhu, J. Org. Chem., 2009, 74, 8761.
2. (a) H. N. Kim, W. X. Ren, J. S. Kim and J. Yoon, Chem. Soc. Rev., 2012, 41, 3210; (b) H. Zhu, Y. Ding, A. Wang, X. Sun, X.-C. Wu and J.-J. Zhu, J. Mater. Chem. B, 2015, 3, 458.
3. (a) Z. Xu, X. Liu, J. Pan and D. R. Spring, Chem. Commun., 2012, 48, 4764; (b) Z. Xu, X. Chen, H. N. Kim and J. Yoon, Chem. Soc. Rev., 2010, 39, 127; (c) Y. Yuan, S. Sun, S. Liu, X. Song and X. Peng, J. Mater. Chem. B, 2015, 3, 5261; (d) K. Sreenath, J. R. Allen, M. W. Davidson and L. Zhu, Chem. Commun., 2011, 47, 11730; (e) D. Bumann and R. H. Valdivia, Nat. Protoc., 2007, 2, 770; (f) C. J. Chang, T. Gunnlaugsson and T. D. James, Chem. Soc. Rev., 2015, 44, 4484.
4. (a) L. Cui, Y. Baek, S. Lee, N. Kwon and J. Yoon, J. Mater. Chem. C, 2016, 4, 2909; (b) R. Gong, H. Mu, Y. Sun, X. Fang, P. Xue and E. Fu, J. Mater. Chem. B, 2013, 1, 2038; (c) C.-P. Liu, T.-H. Wu, C.-Y. Liu, H.-J. Cheng and S.-Y. Lin, J. Mater. Chem. B, 2015, 3, 191; (d) Y. Zhang and J. Yang, J. Mater. Chem. B, 2013, 1, 132.
5. (a) H. M. Burke, T. Gunnlaugsson and E. M. Scanlan, Chem. Commun., 2015, 51, 10576; (b) Z. Xu, N. J. Singh, J. Lim, J. Pan, H. N. Kim, S. Park, K. S. Kim and J. Yoon, J. Am. Chem. Soc., 2009, 131, 15528; (c) Z. Xu, J. Yoon and D. R. Spring, Chem. Soc. Rev., 2010, 39, 1996; (d) J. Yin, Y. Hu and J. Yoon, Chem. Soc. Rev., 2015, 44, 4619; (e) S. Banerjee, E. B. Veale, C. M. Phelan, S. A. Murphy, G. M. Tocci, L. J. Gillespie, D. O. Frimannsson, J. M. Kelly and T. Gunnlaugsson, Chem. Soc. Rev., 2013, 42, 1601; (f) P. Chandra, H. B. Noh and Y. B. Shim, Chem. Commun., 2013, 49, 1900; (g) X. Zhang, Y. Xiao and X. Qian, Angew. Chem., Int. Ed., 2008, 47, 8025.
6. R. Sjöback, J. Nygren and M. Kubista, Spectrochim. Acta, Part A, 1995, 51, L7.
7. A. Loudet and K. Burgess, Chem. Rev., 2007, 107, 4891.
8. A. Mishra, R. K. Behera, P. K. Behera, B. K. Mishra and G. B. Behera, Chem. Rev., 2000, 100, 1973.
9. (a) X. Qian, Y. Xiao, Y. Xu, X. Guo, J. Qian and W. Zhu, Chem. Commun., 2010, 46, 6418; (b) R. M. Duke, E. B. Veale, F. M. Pfeffer, P. E. Kruger and T. Gunnlaugsson, Chem. Soc. Rev., 2010, 39, 3936; (c) S. Banerjee, E. B. Veale, C. M. Phelan, S. A. Murphy, G. M. Tocci, L. J. Gillespie, D. O. Frimannsson, J. M. Kelly and T. Gunnlaugsson, Chem. Soc. Rev., 2013, 42, 1601.
10. (a) D. Yuan and R. G. Brown, J. Phys. Chem. A, 1997, 101, 3461; (b) S. Saha and A. Samanta, J. Phys. Chem. A, 2002, 106, 4763.
11. (a) J. B. Grimm, B. P. English, J. Chen, J. P. Slaughter, Z. Zhang, A. Revyakin, R. Patel, J. J. Macklin, D. Normanno, R. H. Singer, T. Lionnet and L. D. Lavis, Nat. Methods, 2015, 12, 244; (b) W. Zhao and E. M. Carreira, Angew. Chem., Int. Ed., 2005, 44, 1677; (c) Y. Xiao, F. Liu, X. Qian and J. Cui, Chem. Commun., 2005, 239; (d) Y. Mei, P. A. Bentley and W. Wang, Tetrahedron Lett., 2006, 47, 2447; (e) M. Y. Berezin and S. Achilefu, Chem. Rev., 2010, 110, 2641.
12. (a) J. Wu, W. Liu, J. Ge, H. Zhang and P. Wang, Chem. Soc. Rev., 2011, 40, 3483; (b) Y. Hong, J. W. Y. Lam and B. Z. Tang, Chem. Commun., 2009, 4332; (c) U. Resch-Genger, M. Grabolle, S. Cavaliere-Jaricot, R. Nitschke and T. Nann, Nat. Methods, 2008, 5, 763.
13. (a) Z. Xu, X. Qian and J. Cui, Org. Lett., 2005, 7, 3029; (b) L. Duan, Y. Xu and X. Qian, Chem. Commun., 2008, 6339; (c) J. Huang, Y. Xu and X. Qian, Org. Biomol. Chem., 2009, 7, 1299; (d) C. Satriano, G. T. Sfrazzetto, M. E. Amato, F. P. Ballistreri, A. Copani, M. L. Giuffrida, G. Grasso, A. Pappalardo, E. Rizzarelli, G. A. Tomaselli and R. M. Toscano, Chem. Commun., 2013, 49, 5565; (e) Y. Xu, F. Lu, Z. Xu, T. Cheng and X. Qian, Sci. China, Ser. B: Chem., 2009, 52, 771.
14. (a) Q. Jin, L. Feng, D.-D. Wang, Z.-R. Dai, P. Wang, L.-W. Zou, Z.-H. Liu, J.-Y. Wang, Y. Yu, G.-B. Ge, J.-N. Cui and L. Yang, ACS Appl. Mater. Interfaces, 2015, 7, 28474; (b) Z.-R. Dai, G.-B. Ge, L. Feng, J. Ning, L.-H. Hu, Q. Jin, D.-D. Wang, X. Lv, T.-Y. Dou, J.-N. Cui and L. Yang, J. Am. Chem. Soc., 2015, 137, 14488; (c) J. Fan, Z. Han, Y. Kang and X. Peng, Sci. Rep., 2016, 6, 19562.
15. (a) M. Pawlicki, H. A. Collins, R. G. Denning and H. L. Anderson, Angew. Chem., Int. Ed., 2009, 48, 3244; (b) W. Sun, J. Fan, C. Hu, J. Cao, H. Zhang, X. Xiong, J. Wang, S. Cui, S. Sun and X. Peng, Chem. Commun., 2013, 49, 3890; (c) H. M. Kim and B. R. Cho, Chem. Rev., 2015, 115, 5014.

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Footnote

† Electronic supplementary information (ESI) available: Details of synthesis, X-ray data of FM2, photophysical properties and cell imaging studies. CCDC 1478601. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6ra13226j

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