INTRODUCTION

Fluorescent proteins are the most common type of genetically encoded fluorescent labels. These proteins can autocatalytically generate internal aromatic structures from their amino acid residues, thus leading to various arylidene-imidazolone chromophores. To date, many multicolored fluorescent proteins that contain chromophores of different structures are used in fluorescence microscopy [1]. A separate place among these proteins is occupied by those that contain chromophores with the acyl group in the second position of the imidazolone cycle, e.g., the AsFP protein [2]. The presence of this group leads to a significant bathochromic shift in the maxima of absorption and emission spectra of these proteins.

Arylidene-imidazolones in a protein-free form are known to fluoresce extremely weakly [3], which is explained by the possibility of nonradiative release of the excitation energy because of the mobility of the arylidene fragment [4]. However, due to these properties, arylidene-imidazolones can be used as fluorogenic dyes, e.g., for staining proteins, nucleic acids, and individual cellular organelles [5–9].

It has been previously shown that some arylidene-imidazolones are characterized by a noticeable variation in the fluorescence intensity in different media [10–13]. This property made it possible to use these compounds as a kind of fluorescent “polarity sensors” for staining the endoplasmic reticulum and other organelles. It has been found that arylidene-imidazolones belong to a similar group and have simultaneously two electron-donor substituents in the meta- and ortho-positions of the arylidene fragment (Scheme 1, compounds (I)) [11, 14, 15]. We showed later that the introduction of styrene substituents into the second position of the imidazolone cycle of these compounds preserves this variation [14, 15]. The introduction of theY styrene groups is an important modification in the chemistry of dyes, which makes it possible to increase the size of the π-system, thus leading to a bathochromic shift of spectral maxima. Similar dyes are particularly in demand in fluorescence microscopy because long-wave radiation is the least toxic to living tissues.

Scheme 1 . Structure of compounds (I).

In this paper, we studied the effect of another modification, which can lead to a shift of the absorption and emission maxima to the long-wavelength region. Our previous studies have shown that the introduction of the keto group into the second position of the imidazolone ring of different arylidene-imidazolones often leads to a bathochromic shift of spectral maxima by 50–100 nm [16, 17]. Therefore, the goal of this work was to synthesize keto-derivatives of arylidene-imidazolones (I) and study the effect of the keto group in the structure on the optical properties of these dyes.

RESULTS AND DISCUSSION

At the first stage of the synthesis, the corresponding arylimines were obtained from various aromatic aldehydes (II), which, without additional purification, were used for the synthesis of arylidene-imidazolones (III) by [3+2]cycloaddition (Scheme 2). Then arylidene-imidazolones (III) was oxidized to the corresponding keto derivatives (IV) under the action of selenium dioxide (Scheme 2).

Scheme 2 . Synthesis of compounds (III) and (IV).

In the next stage, we studied the optical properties of ketones (IV). We found that the absorption maxima were in the region of 410–450 nm, and the emission maxima were in the region of 560–630 nm (Table 1). A comparison of these results with the data known for compounds (I) shows that the introduction of the keto group leads to a bathochromic shift of the maxima by 50–100 nm (marked in Fig. 1 with arrows) and a noticeable increase in the Stokes shift (the difference between the emission and absorption maxima) (Fig. 1). The chosen modification also leads to a slight decrease in the fluorescence intensity. Thus, the quantum yield of fluorescence of ketones (IV) in dioxane is 1.2–4.2%, while compounds (I) fluoresce in this solvent with a quantum yield of 6–40% [14, 15]. However, new derivatives (IV), as well as compounds (I), are characterized by a pronounced variation in the quantum yield (Table 1), which indicates the prospects of their use as polarity sensors for living systems.

Table 1. Optical properties of compounds (IV) in various solvents
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Fig. 1.
figure 1

Absorption (green) and emission (red) spectra of compounds (I) (dotted) and keto derivatives (IV) (solid) in dioxane. Compounds (Ia) and (IVa) (a), compounds (Ib) and (IVb) (b), and compounds (Ic) and (IVc) (c).

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Due to the variation of the quantum yield of compounds (IV), we decided to study the possibility of their use for staining cell cultures at the final stage of this work. It was found that the addition of the ketone (IV) solutions to the HeLa Kyoto cells at a final concentration of 10 µM led to a pronounced fluorescence in the structures of the endoplasmic reticulum (Fig. 2). The brightest fluorescence was observed in the case of derivative (IVc). However, the staining, in this case, was the least selective because, in addition to the fluorescence of the structures of the endoplasmic reticulum, bright fluorescent droplets were formed, which is caused by probable aggregation of the dye molecules. Similar properties of derivative (IVb) were observed, although the formation of aggregates, in this case, was less pronounced. Probably, the presence of the naphthalene fragment in the structure of these two compounds reduces their solubility in water and facilitates the formation of aggregates. The best result was achieved for derivative (IVa) because of its smallest size.

Fig. 2.
figure 2

Staining of the endoplasmic reticulum with keto derivatives (IV) in living HeLa Kyoto cells. Compound (IVa) (a), compound (IVb) (b), and compound (IVc) (c). Final concentration of the compounds was 10 µM. The bar is 10 µm.

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Thus, we have found that ketones (IV), as well as arylidene-imidazolones (I), can stain the endoplasmic reticulum and can find their application in fluorescence microscopy.

EXPERIMENTAL

Equipment. The NMR spectra (δ, ppm; J, Hz) were recorded on Bruker Fourier 300 (300 MHz; Bruker, United States), Bruker Avance III NMR (700 MHz; Bruker, United States), and Bruker Avance III NMR (800 MHz; Bruker, United States) spectrometers equipped with a 5-mm TXI cryodetector in DMSO-d6 and CDCl3 (the internal standard, Me4Si). The absorption spectra in the UV and visible ranges were recorded on a Varian Cary 100 Bio spectrophotometer (Varian, United States); the fluorescence spectra were recorded on a Varian Cary Eclipse spectrofluorimeter (Varian, United States). Melting temperatures were measured on an SMP30 device (Stuart Scientific, United Kingdom) and were not corrected. The high-resolution mass spectra were recorded on a TripleTOF 5600+ spectrometer (AB Sciex, United States) with electrospray ionization (ESI). The voltage on the capillary was 5.5 kV in the positive ion registration mode and 4.5 kV in the negative ion registration mode. The flows of the carrier gas and spray gas were 15 Arb and 25 Arb, respectively. The samples were injected using a syringe pump with a flow rate of 20 mL/min.

Synthesis of 2-ethyl-1-methyl-1H-imidazol-5(4H)-ones (II). A 40% aqueous solution of methylamine (2.2 mL, 25 mmol, 5 equiv.) was added to the aromatic aldehyde solution (5 mmol, 1 equiv.) in chloroform (50 mL), followed by the addition of anhydrous sodium sulfate until the water layer in the reaction mixture disappeared. The flask was closed with a stopper, and the reaction mixture was kept at room temperature for 96 h. The suspension was filtered and dried over anhydrous sodium sulfate. The desiccant was filtered out and the solution was evaporated at reduced pressure. Carboxyimidate (1.13 g, 6.5 mmol, 1.3 equiv.) was added to the residue after evaporation. The resulting mixture was stirred at room temperature for 96 h. The target product was isolated and purified by flash chromatography (eluent, chloroform–ethanol, 100 : 3).

Synthesis of 2-ethyl-1-methyl-1H-imidazol-5(4H)-ones (III). Selenium dioxide (IV) (88 mg, 0.8 mmol, 2 equiv.) was added to a solution of 2-ethyl-1-methyl-1H-imidazol-5(4H)-one (0.4 mmol, 1 equiv.) in dioxane (8 mL). The resulting mixture was kept in an oil bath at 100°C for 30 min. The mixture was cooled to room temperature, diluted with ethyl acetate (80 mL), transferred to a separating funnel, and washed successively with a saturated solution of potassium carbonate (100 mL) and a saturated solution of potassium chloride (3 × 100 mL). The organic phase was dried over anhydrous sodium sulfate. The desiccant was filtered out and the solution was evaporated at reduced pressure. The target compounds were isolated after evaporation by flash chromatography (eluent, ethyl acetate–hexane, 1 : 2).

The reaction yields, melting temperatures, and spectral characteristics of synthesized compounds (II) and (III) are given in the supplementary materials.

Fluorescence microscopy. Screening of compounds was performed on the HeLa Kyoto living cell cultures (ATCC). The compounds were added to the HeLa Kyoto cells at concentrations of 1–10 µM (the compounds were diluted from 1-mM stock DMSO solution). The images were observed on an inverted wide-field BZ-9000 fluorescence microscope (Keyence, Japan) with a Nikon Plan Apo 60 × 1.40 Oil lens (Nikon, United States) and a set of Keyence GFP-B EX 470/40 DM 495 BA 535/50 light filters.

CONCLUSIONS

Four keto-derivatives of arylidene-imidazolones were synthesized. It was found that the introduction of the keto group does not lead to significant changes in optical properties. All new compounds, as well as the original arylidene-imidazolones, are characterized by a variation in the magnitude of the fluorescence quantum yield when replacing the solvent. It has been demonstrated that the synthesized ketones are promising compounds as dyes in fluorescence microscopy because they can stain the endoplasmic reticulum.