Substituent Effects on Fluorescence Properties of AIEgens Based on Coumarin-3-formylhydrazone and their Application in Cell Imaging

AIE-active compounds have lately attracted considerable attention owing to their versatile applications, especially in OLED and bioimaging. Herein, a series of coumarin-3-formylhydrazone derivatives (CFH-1, 2, 3 and 4) were developed for investigating their AIE and solid-state luminescence behaviors. All the obtained compounds emit varying degrees of solid-state fluorescence. CFH-1 and 2 show the typical AIE characteristics, while CFH-3 and 4 exhibit stronger solution fluorescence than their aggregation-induced emission. The single crystal X-ray diffraction analysis of CFH-1 and CFH-3 showed that both of them adopt planar conformation and the CH···O hydrogen bonding plays a crucial role in their crystal packing. Meanwhile, there is a notable difference between them. Successive π-π stacking interaction was observed in the crystal packing of CFH-1, while the crystals of CFH-3 contain dimeric π-π stacking interaction. Their distinct crystal packing interactions result in their different fluorescence properties. Moreover, both CFH-1 and CFH-2 displayed excellent bioimaging performances in living cells.


Introduction
Conventional luminophores often encounter a thorny problem of aggregation-caused quenching (ACQ), which greatly limits their application as aggregation/solid-state luminescent materials. In recent decades, a family of new luminogens with aggregation-induced emission (AIE) properties has emerged as a hot topic owing to their wide applications in organic light-emitting diodes [1], bioimaging [2], theranostics [3,4] and so on. A variety of explanations for the AIE working mechanisms have been proposed [5][6][7]. According to any of the mechanisms, it is indispensable to avoiding the successive π-π stacking interaction in aggregation/solid state which leads to radiationless relaxation. Hence, the AIE-active molecules usually adopt twisted conformations instead of planar conformations for hampering the intermolecular π-π stacking interaction [8,9]. Nevertheless, when the planar luminogens are stacked via hydrogen-bonding (H-bonding) or other intermolecular interactions instead of π-π stacking interaction, they should emit fluorescence in aggregation/solid state [10][11][12][13][14][15]. Therefore, the groups involved in H-bonding might dominate the fluorescence performance of luminogens in aggregation/ solid state. Based on this consideration, we developed a series of coumarin-3-formylhydrazone derivatives with planar conformation (Scheme 1) for exploring their molecular packing and AIE activity. All the obtained compounds emit bright fluorescence in solid state and exhibit varying degrees of AIE activity in MeCN/H 2 O mixtures. The single crystal X-ray diffraction analysis showed that CH···O hydrogenbonding plays a very important role in the crystal packing. It was demonstrated that the planar fluorophores can be AIE-active luminogens when stacked via H-bonding and the introduction of building blocks involved in H-bonding is an effective means to construct AIEgens.

Reagents and Apparatus
All chemicals were obtained from commercial suppliers and directly used without further purification. 7-(diethylamino)coumarin-3-carbohydrazide was prepared according to the literature method [16]. Analytical grade acetonitrile and deionized water were used as solvents for spectral measurements. 1 H NMR and 13 C NMR spectra were recorded on a Bruker Av400 NMR spectrometer. HRMS spectra were performed on a Shimadzu LCMS-IT-TOF apparatus. Fluorescence spectra were taken on a Hitachi F-7000 fluorescence spectrometer. The X-ray diffraction data was collected on an Agilent Gemini A Ultra diffractometer (MoKα, λ = 0.71073 Å). Images of cells were obtained using a Nikon A1 confocal laser scanning microscope.

General Procedure for the Synthesis of Target Compounds
As shown in Scheme 1, 7-(diethylamino)coumarin-3-carbohydrazide (550.6 mg, 2.0 mmol) and one of the aromatic aldehydes (2.0 mmol) were dissolved in absolute ethanol (6 mL). the mixture was stirred at 70 °C for 5 h. In the meanwhile, massive amounts of solid product precipitated out, which was collected by filtration and then washed several times with a small amount of ethanol to afford the target compounds (CFH-1, 2, 3 and 4) respectively. All the structures were characterized by NMR and HRMS (Fig. S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11 and S12).  13

Solid-state Luminescence and AIE Behaviors
In the course of exploring AIE-active molecules, it was accidentally found that some the condensation products of coumarin-3-formylhydrazine and aromatic aldehydes emitted superbright solid-state fluorescence. Herein, a series of 7-(diethylamino)coumarin-3-formylhydrazones (CFHs) were synthesized for investigating the effects of substituent groups and molecular packing on their luminescent properties. All the obtained CFHs emit varying degrees of solid-state fluorescence. The fluorescence intensities and emission wavelengths of them vary greatly depending on the arylidene groups. In general, the electron-donating arylidene group leads to weaker emission intensity and longer emission wavelength. On the contrary, the electronwithdrawing arylidene group results in stronger emission intensity and shorter emission wavelength (Fig. 1). The aggregation behaviors of them were studied by means of dynamic light scattering (DLS) and scanning electron microscope (SEM). The DLS data manifested that all the CFHs can form submicron particles. The average diameters of their particles increase from 150 to 520 nm (CFH-1: 190 nm, CHF-2: 470 nm, CHF-3: 520 nm, CHF-4: 150 nm). But the SEM images revealed that the particles are smaller. The particle diameters of CFH-1 and CHF-2 are obviously less than 100 nm, and that of CFH-3 and CHF-4 are less than 200 nm (Fig. S13). The AIE behaviors of them in MeCN/H 2 O with varied volume ratios were showed in Fig. 2. CFH-1 showed weak emission at 480 nm in pure MeCN. When f w increased to over 60%, the AIE phenomenon began to occur. Ultimately, its emission intensity increased by over 5 times and the emission peak was red shifted by about 70 nm ( Fig. 2A). CFH-2 had similar AIE characteristics as CFH-1, but its emissions both in pure MeCN and in aggregation state were stronger than CFH-1 (Fig. 2B). CFH-3 and CFH-4 emitted very strong fluorescence in MeCN. When f w was up to 90%, both of them exhibited a degree of AIE activity, but their fluorescence intensities of AIE were much weaker than their solution fluorescence. On the whole, the electron-donating arylidene groups on CFHs would result in AIE activity and relatively weak solid-state fluorescence (CFH-1 and CFH-2), while the electron-withdrawing arylidene groups would lead to strong solution fluorescence and solid-state fluorescence, but were of no benefit to AIE (CFH-4). It should be noted that CFH-3, being an exception, emitted superbright green solid-state fluorescence, strong cyan solution fluorescence and yellow aggregation-state fluorescence, implying its potential application as a versatile luminescent material.

Molecular Stacking Effects on AIE and Solid-state Luminescence
The single crystals of CFH-1 and CFH-3 suitable for the crystallographic analysis were obtained by slow evaporation of their solutions in THF/CH 2 Cl 2 (20:1) at ambient temperature. The crystallographic data are summarized in supplementary material (Table S1). For more information, see the crystallographic information file that has been deposited in the Cambridge Crystallographic Data Centre (CCDC 2,178,542 and 2,178,543). CFH-1 crystallizes in the triclinic space group P-1. All the atoms in CFH-1 molecule, except for the ethyl groups, are coplanar. An intramolecular hydrogen bond N(3)-H(3)···O(2) was observed. There is a dichloromethane molecule coexisting with one CFH-1 molecule in the asymmetric unit (Fig. 3A). CFH-1 molecules are liked via CH···O hydrogen bonds to form planar molecular dimers, which are further linked by dichloromethane via CH···O hydrogen bonding to form molecular chains. The molecular chains are parallelly arranged affording a molecular sheet (Fig. 3B). The CFH-1 molecules in different sheets are arranged in brickwork-like pattern. Multiple weak H-bonding and π-π stacking interactions were observed between the adjacent molecular sheets (Fig. 3C). It is the π-π stacking interaction with interplanar distance of 3.48 Å that should be responsible for its relatively weak solid-state fluorescence intensity (Fig. 1A). All the hydrogen bonds were summarized in Table 1.
CFH-3 also crystallizes in the triclinic space group P-1. The asymmetric unit of the crystal contains one CFH-3 molecule adopting planar conformation (Fig. 4A). Two molecules form an antiparallel dimer liked via CH···O hydrogen bonds. In addition to the H-bonding, two identical π-π stacking interactions with interplanar distance of 3.50 Å were observed. The molecular dimers adopting a brickworklike arrangement were linked via CH···O hydrogen bonds to form a planar molecular sheet (( Fig. 4B and Table 2). The dimeric π-π stacking would bring about excimer fluorescence that was described as excimer-induced enhanced emission (EIEE) [17][18][19][20]. The superbright solid-state fluorescence of CFH-3 should be based on the EIEE mechanism. When f w is 90%, CFH-3 show dual-wavelength emission (Fig. 1C, inset). The short-wavelength emission band should be assigned to the single molecule fluorescence, while the long-wavelength emission band should be assigned to the excimer fluorescence.

Cell Imaging
The excellent AIE performance of CFH-1, CFH-2 motivated us to examine their utilization in live cell imaging. Cultured HeLa cells were seeded in a glass bottom cell culture dish. After overnight culture, the cells were stained with 10 μmol/L CFH-1 and CFH-2 in MeCN/DMEM mixture (5/95) for 30 min respectively. After washed by using PBS buffer to remove extracellular fluorescent dyes, the cell samples were imaged using confocal laser scanning fluorescence microscopy in blue, green and red channels. As shown in Fig. 5, the self-aggregates of CFH-1 can efficiently permeate cells and emit bright green fluorescence and bright red emission, though the image in blue channel was less than satisfactory. The image performance of CFH-2 was of inferior quality compared to CFH-1. It was

Conclusion
A series of 7-diethylaminocoumarin-3-formylhydrazone derivatives (CFH-1, 2, 3 and 4) have be synthesized through amine-aldehyde condensation between 7-(diethylamino)coumarin-3-carbohydrazide and different aromatic aldehydes. All of them exhibit varying degrees of solid-state emission and AIE activities depending on the electronic effects of the arylidene groups. On the whole, the electron-donating arylidene groups result in AIE activity and relatively weak solid-state fluorescence (CFH-1 and CFH-2), while the electron-withdrawing arylidene groups would lead to strong solution fluorescence and relatively weak aggregation fluorescence (CFH-4). CFH-3 stands out as an exception: it emits the strongest solid-state fluorescence and superbright solution fluorescence, meanwhile, it exhibits distinct visible aggregation-state fluorescence which should be related to its dimeric π-π stacked packing. Furthermore, both CFH-1 and CFH-2 displayed fast cell staining and multi-channel bioimaging performances.
Author Contributions EW contributed to the study conception and design, and was the major contributor in writing the manuscript. The synthesis, spectrum test, data analysis and so on were performed by PC with the assistance of YZ. The X-ray diffraction data collection and structure determination was performed by ZN. The crystallographic information files that have been deposited in the Cambridge Crystallographic Data Centre (CCDC 2,178,542 and 2,178,543) which are freely available for all. The link for CCDC: https:// www. ccdc. cam. ac. uk/.
Funding This work is financially supported by Hainan Provincial Natural Science Foundation of China (222MS059) and the National Natural Science Foundation of China (22061016).

Availability of Data and Material
The data supporting the findings of this study are included in this published article and its supplementary information files. The raw data are available from the corresponding author on reasonable request.
Code Availability Not applicable.

Declarations
Ethical Approval NoT applicable.

Conflicts of Interest/Competing Interests
The authors have no conflicts of interest to declare that are relevant to the content of this article.