Design and Synthesis of ESIPT-Based Imidazole Derivatives for Cell Imaging

Excited-state intramolecular proton transfer (ESIPT)-based fluorescent molecules offer several exciting applications and are utilized most frequently as a cell imaging agent. Because of this, four distinct imidazole derivatives with ESIPT emission have been synthesized, and their fluorescence characteristics have been assessed in a variety of settings. Measurements using fluorescence spectroscopy have shown a promising candidate for cell staining, and potential candidate was specifically investigated for cell imaging uses in HT-29, MDA-MB-231, and HaCaT. Cytotoxicity of candidate molecule (1d) was analyzed using HT-29 and HaCaT cell lines, and at a dosage of 160 μM, HT-29 and HaCaT cell lines showed no signs of important cell toxicity. When spectroscopically measured, compound 1d showed no fluorescence ability in phosphate-buffered saline (PBS) solution. However, after 8 h of incubation in several cell lines, excellent fluorescence characteristics were seen in the green and red filters.


■ INTRODUCTION
Noninvasive fluorescent-based imaging agents mediated the visualization of changes at the cell and tissue level, and emerge as an important diagnostic method in medicine. 1Although tremendous advances have been made in fluorescence-based cell imaging techniques recently, efforts to develop fluorescent probes that target intracytoplasmic structures, which are less costly and more advantageous in terms of processing, enabling both in vitro and in vivo cell imaging, remain a major challenge in this area.2a Cellular homeostasis, which changes based on dynamic fluctuations in organelles and biomolecules in the cell, is particularly important in the diagnosis and treatment of many disorders.−6 ESIPT emission covers a unique mechanism that results in two distinct emission bands in most cases. 7,8−12 To enable ESIPT emission, the structure should include proton donor and acceptor functionalities that are close to each other via a 5-or 6-membered pseudo-ring system.In the literature, the most commonly structures are hydroxybenzimidazole (HBI), hydroxy-benzoxazole (HBO), and hydroxy-benzothiazole (HBT) (Figure 1B).On the other hand, in the past few years, our research group found out that the carbonyl functional group at the C-2 position of imidazole 1 and indole derivatives 2 13b can make ESIPT emission (Figure 1A).These structures are logically arranged and include significant functional groups that might be relevant in future applications. 14,15n this study, we investigated imidazole compounds with ESIPT emission for cell imaging.As a result, numerous imidazole derivatives were synthesized, and their ESIPT emission potentials were measured.A search of those molecules produced a possible molecule for further cell imaging study.The staining pattern of the fluorescent agent created based on ESIPT in both fixed and living cells was examined using several cell lines such as HT-29 (colon cancer cell line), MDA-MB-231 (breast cancer cell line), and HaCaT (keratinocyte cell line) utilizing the candidate imidazole derivative.This is the first study to utilize our previously announced ESIPT-based fluorescent probe for cell imaging.In this study, we hope to understand the probe's potential for cell imaging as well as to envision future applications such as varied analyte detection in vitro and in vivo.

■ EXPERIMENTAL SECTION
General Materials and Methods for Chemistry. 1 Hand 13 C NMR spectra were recorded using a Varian NMR-400 MHz. 1 H chemical shifts were referenced to internal standard TMS (δ 0.00 ppm) or deuterated solvents such as d 6 -DMSO and CDCl 3 .All chemical shifts (δ) are indicated in ppm, and J values are indicated in Hz. 13 C NMR spectra were fully decoupled.The following patterns were designated as s, singlet; bs, broad singlet; d, doublet; dd, doublet of doublets; t, triplet; and m, multiplet.As a purification technique, column chromatography was performed on silica gel (60-mesh) and was followed with TLC that was made of Merck 0.2 mm silica gel 60 F 254 analytical aluminum plates.Absorbance measurements were performed using Shimadzu UV-3600 Plus ultraviolet−visible−near-infrared (UV−vis−NIR) spectroscopy, and fluorescence measurements were performed utilizing Agilent Cary Eclipse spectrophotometer.Melting points were obtained on an X-4 digital melting point apparatus without correction.Solvents were evaporated at reduced pressure using a rotary vacuum evaporator.
General Procedure for Synthesis of 1a−d.One mmol portion of aryl methyl ketone was dissolved in 7 mL of 1,4dioxane.The solution was then added with 2.5 mmol (0.275 g) of selenium dioxide (SeO 2 ), and the reaction mixture was refluxed overnight in an oil bath.The TLC method was used to monitor the reaction's completion.The mixture was separated from solid black Se 0 using a filter and cooled at room temperature.On the other hand, 5 mmol (0.385 g) of ammonium acetate (CH 3 COONH 4 ) was dissolved in 10 mL of ethanol and mixed at room temperature for 1 h.The solution was then mixed with 20 mL of ice water and stirred for 1 h.The solid was finally filtered and dried on phosphorus pentoxide.
The intracellular distribution of molecule 1d was investigated using a fluorescence microscope.To determine both the fluorescence potential and the optimum concentration of molecule 1d, MDA-MB-231 cells were incubated with different concentrations of molecule 1d (20, 10, 2 μM) for 30 min, and then the cells were washed with PBS and visualized using blue, green, and red channels.To reveal the compartments stained by molecule 1d in different fixed cells (HT-29, MDA-MB-231, and HaCaT), typically cells were fixed with 4% PFA for 15 min and washed with PBS, and cells were stained with molecule 1d for 30 min and observed under a fluorescence microscope.To measure the live cell imaging (without fixation) potential of molecule d and to detect the areas it has stained inside the cell, HT-29 and HaCaT cells were incubated with molecule d for 8 h, washed with PBS, and visualized in both green and red channels with a fluorescence microscope.
Cytotoxicity Test for Molecule 1d.HaCaT keratinocyte cells were cultured in DMEM medium with 10% FBS and 1% penicillin/streptomycin, and HT-29 colon cancer cells were grown in RPMI 1640 medium with 10% FBS and 1% penicillin/streptomycin. ESIPT was prepared as 1 molar amount in DMSO.Cells were grown in 96-well plates at a density of 5 × 10 3 .It was applied to cells at various conditions for 24 h.The cell toxicity using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was then quantified and assessed with a plate reader. 17tatistical Analysis.Paired Student's t test was used to compare two groups to determine cell imaging data measurements.One-way ANOVA and Dunnett's multiple comparison tests were used to compare more than two groups.P values less than 0.05 were considered to be statistically significant.

■ RESULTS AND DISCUSSION
Design and Synthesis.Figure 1A,B demonstrates that compounds 1 and 2 have several benefits over the popular skeletons in Figure 1B.Compounds 1 and 2 include a carbonyl group, and compound 2 has additional ester functionality.Furthermore, substituents on the benzene ring and potential replacements on the free nitrogen atom in compounds 1 and 2 may give substantial advantages when researchers strive to develop alternative functionality for ESIPT-based emitting compounds.These benefits are confined to compounds 1 and 2 and do not apply to the compounds mentioned in Figure 1B.Customizing the functional groups of compounds 1 and 2 may not influence the ESIPT mechanism, as seen in our previous study. 15or the imidazole ring, we have used our previous synthetic method. 13In this method, acetyl functionality on the benzene ring was oxidized using SeO 2 following cyclization with NH 4 OAc in ethanol via two-step and one-pot reaction protocol that makes this protocol highly applicable (Scheme 1).
Four different molecules were synthesized to identify the best candidates for the study.Derivatives containing unsubstituted benzene 1a, para-iodo benzene 1b, para methoxybenzene 1c, and para piperidine benzene 1d groups were obtained with good yields.We have stated in our previous studies that electrondonating groups in the benzene ring of molecules are important for fluorescence.In this study, fluorescence effects were investigated using both OMe 1c and piperidine group 1d.In addition, a para-iodo benzene derivative 1b was synthesized to examine the heavy atom effect.
UV−Vis and Fluorescence Spectra.According to the spectroscopic investigation, the imidazole derivative with an unsubstituted benzene ring (1a) displays an emission band of 459 nm while having no absorption in the visible area.The presence of an iodine atom in the para position of the benzene ring 1b produced an emission band at 480 nm with a red shift of around 21 nm (1b) (Table 1).The emission band of an imidazole derivative with an electron-donating group such as OMe at the para position (1c) was measured to be 494 nm with a red shift of up to 40 nm.Finally, compared with other derivatives, the derivative containing a piperidine ring at the para position of benzene (1d) exhibited the most significant   red shift, with an emission band at 590 nm (in DMSO solvent).
Our group was the first to publish on the ESIPT emission theory of this skeleton.As a result of these investigations, our group discovered the optimal emission value for the corresponding skeleton.As a result, we believe that it might be an ideal candidate for cell imaging research.
The selected chemical's behavior in various solvents was also studied.Different solvents were used for this purpose (Figure 2A−B, and Table 2), and the absorbance value did not vary significantly even when employing cell media PBS.Furthermore, none of the absorbances in the different solutions revealed the value of the visible area, demonstrating that the molecule does not interact differently with different solvents in the ground state.However, the solvents used have an influence on the fluorescence spectrum of 1d.When compared with the other solvents, MeOH and EtOH, like many ESIPT-based luminescent compounds, did not display significant fluorescence properties.The fluorescence intensity was highest in EtOAc, THF, and CHCl 3 solutions, which had 0.710, 0.767, and 0.557 quantum yields, respectively, with maximum values of 535, 533, and 553 nm.In DMSO and DMF solvents, two distinct bands with modest fluorescence intensity were identified, which might be because of solvent interactions due to the nature of solvent and nonbonding electrons.The fact that these two solvents give two bands, unlike the others, is due to high-energy excitation because it was observed that the fluorescence emission decreased to a single band as the excitation energy decreased up to 440 nm (see Figures S1 and  S2).The fluorescence intensity of the solution generated in toluene, an aromatic molecule, was equally modest, with a max value of 507 nm with 0.132 quantum yield.It is worth noting   that 1d in PBS solution did not show any fluorescence intensity, as predicted because of the nature of ESIPT emission.However, when the molecule of 1d was introduced into the cell, we found considerable green and red fluorescence images on the microscopy, even at 2 μM solution (Figure 3), which means that 1d can penetrate the cell and have some interactions with cell organelles.
Cell Imaging Studies.To evaluate the potential biological application of molecule 1d in cell imaging, fluorescent microscopy experiments were performed using fixed MDA-MB-231 cells.In the first step, (i) in which channels molecule d generates a detectable fluorescent signal and (ii) optimum concentrations of 1d were investigated.Three different channels (blue, green, and red) and concentrations (20, 10, and 2 μM) were tested.MDA-MB-231 cells were incubated with molecule d for 30 min, and cells were visualized using the blue, green, and red channels.As seen in Figure 3A, a weak fluorescent signal was obtained in the blue channel, but a strong fluorescent signal was detected in the green and red channels (p < 0.0001).
When the staining pattern of the cells is evaluated specifically, it cannot be distinguished exactly which compartments are stained in the cell due to the intense fluorescent signal.To gain a deeper insight into the structures with which molecule 1d specifically interacts in cells, MDA-MB-231 cells were stained using different concentrations of molecule 1d (20, 10, and 2 μM).Measurements with ImageJ showed that the mean fluorescent intensity (MFI) values were statistically significant due to the increasing concentration of molecule d (p < 0.0001).
Considering its distribution in the cell and the appropriate fluorescent signal, it has been determined that the 10 μM concentration of 1d was optimum (Figure 3B), and this concentration was used in both fixed and living cells.
To explore the fluorescent staining pattern of 1d in the fixed cells, HT-29, MDA-MB-231, and HaCaT cells were incubated for 30 min and viewed under an Olympus BX53 light microscope with an attached DP74 camera.Furthermore, the cells were examined under both an inverted microscope and a light microscope to confirm the stained areas inside the cell (Figure 4).In particular, H&E staining clearly reveals the shape of the cell mediated the differentiation of cytoplasm and nucleus in the cell.
As seen in Figure 4, green and red fluorescent signals were obtained in HT-29, MDA-MB-231, and HaCaT when cells were stained with molecule 1d.Considering the localizations in the cell, it was determined that molecule d fluorescence was intense in the cell membrane (white arrow) and nucleolus (white arrowhead) structure of HT-29 cells.In MDA-MB-231 cells, a strong fluorescence was obtained in the nuclear membrane.In the image presented in Figure 4, a different staining pattern was displayed in HaCaT cells compared with the others.It was determined that molecule 1d intensely fluoresced in the subnuclear region (black arrow).Interestingly, in HaCaT cells, it was noted that dividing cells (white asterisks) were stained extensively with 1d.These results revealed that 1d specifically stains cell membranes, nuclei, and subnuclear areas in cells.
Fluorescent-based live cell imaging allows real-time observation of the interaction processes between cellular structures and molecules, providing more insight into cellular processes than a snapshot provided by imaging fixed cells.
A chemical cannot be used for live imaging if it produces any harmful side effects.For this purpose, we first tested whether compound 1d we used in our study had a toxic effect on the cell.The dose range of 10−160 μm has no cytotoxic effect on HT-29 and HaCaT cells during 24 h (Figure 5).
In light of these data, encouraged by the fluorescent images obtained from fixed cells, we evaluated the potential for the use of 1d in live cell imaging.HT-29 and HaCaT cells were incubated with 1d for 8 h, and then the cells were washed with PBS and viewed under a fluorescent microscope.Similar staining patterns were obtained in live imaging, confirming the results in fixed cells (Figure 6).In HT-29 cells, the fluorescence intensity was quite prominent in the cell membranes and nuclei (Figure 6A).Mean fluorescence intensity (MFI) values of molecule 1d were measured for both fixed and live cells using ImageJ.MFI values were higher in fixed cells compared with live cells in HT-29 green channel (p < 0.0001) and red channel (p < 0.001) (Figure 6C).However, the staining pattern in living cells was observed to be more specific.The MFI value was higher in the fixed cells, probably due to some nonspecific weak staining in the cytoplasm.Three-dimensional (3D) surface plot analyses of HT-29 cells showed that the intensity of fluorescent staining was in the nuclear membrane and nucleolus regions (Figure 6A).
Live imaging of HaCaT cells is both more intense and highly specific with fluorescence intensity in the green and red channels compared with fixed cells (Figure 6).Furthermore, MFI values were obtained higher in live cells than in fixed cells (p < 0.0001) (Figure 6C).In addition to a prominent nuclear membrane and nucleolus fluorescence staining, the intensity of the staining in the subnucleus structure is quite remarkable.Condensed fluorescence areas were also observed in certain regions in the HaCaT cytoplasm (double-headed white arrow).This staining pattern in the cytoplasm indicates that a specific organelle or molecule is stained in the cell.As seen in Figure 6B, surface plot analyses of HaCaT cells showed that the fluorescence intensity was particularly prominent in the subnucleus region.
To summarize, the absorbance and fluorescence properties of four distinct imidazole derivatives, the ESIPT properties of which were recently discovered by our research group, were studied.The imaging capabilities of 1d, which was shown to have the greatest shift to the red area, were studied in both healthy (HacaT) and malignant cell lines (HT-29 and MDA-MB-231).Furthermore, 1d's cytotoxicity was tested, and the cell viability of HT-29 and HaCaT at 160 μM concentration was determined to be at least 80%.Compound 1d emitted at a high level and fluoresced in both green and red filters.Furthermore, in terms of organelle staining, it was discovered that it stained distinct locations in healthy and malignant cells, which is the most relevant concern.The chosen ESIPT-based skeleton was employed for the first time in the literature in cellstaining investigations, and it was discovered that it had significant potential.In future experiments, we will study the mechanistic explanations of the organelles dyed by compound 1d and report on more specific findings.

Figure 5 .
Figure 5. Cytotoxicity effect of compound 1d on two different cell lines.

Figure 6 .
Figure 6.Live imaging of molecule d in cells.HT-29 (A) and HaCaT (B) cells were incubated with molecule d for 8 h and visualized in the green channel and red channel, and a 3D surface plot was obtained.(C) Both fixed and live imaging graphs of HT-29 and HaCaT cells.The white arrow, white arrowhead, and double-headed white arrow point to the cell membrane, nucleolus, and subnucleus, respectively (scale bar: 100 and 50 μm).

Table 1 .
Scheme 1. Synthesis of Imidazole Derivatives That Have ESIPT Emission Absorbance and Fluorescence Max Values for Compounds 1a−d a a 30 μM in DMSO.