Design of red-shifted and environment-sensitive fluorogens based on GFP chromophore core

Journal Pre-proof Design of red-shifted and environment-sensitive fluorogens based on GFP chromophore core Alexander Yu Smirnov, Maxim M. Perfilov, Elvira R. Zaitseva, Marina B. Zagudaylova, Snizhana O. Zaitseva, Alexander S. Mishin, Mikhail S. Baranov PII:

S0143-7208(19)32324-1

DOI:

https://doi.org/10.1016/j.dyepig.2020.108258

Reference:

DYPI 108258

To appear in:

Dyes and Pigments

Received Date: 1 October 2019 Revised Date:

3 February 2020

Accepted Date: 3 February 2020

Please cite this article as: Smirnov AY, Perfilov MM, Zaitseva ER, Zagudaylova MB, Zaitseva SO, Mishin AS, Baranov MS, Design of red-shifted and environment-sensitive fluorogens based on GFP chromophore core, Dyes and Pigments (2020), doi: https://doi.org/10.1016/j.dyepig.2020.108258. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Alexander Yu. Smirnov, Investigation Maxim M. Perfilov, Visualization, Investigation Elvira R. Zaitseva, Investigation Marina B. Zagudaylova, Investigation, Writing. Snizhana O. Zaitseva, Investigation Alexander S. Mishin, Writing- Reviewing and Editing, Mikhail S. Baranov Conceptualization, Methodology, Supervision, Writing- Reviewing and Editing.

Design of red-shifted and environment-sensitive fluorogens based on GFP chromophore core Alexander Yu. Smirnov,1 Maxim M. Perfilov,1 Elvira R. Zaitseva,1,2 Marina B. Zagudaylova,1 Snizhana O. Zaitseva,1 Alexander S. Mishin,1 Mikhail S. Baranov1,3 1

Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, Moscow 117997, Russia

2

D. Mendeleev University of Chemical Technology of Russia, 9 Miusskaya Sq., Moscow 125047, Russia

3

Pirogov Russian National Research Medical University, Ostrovitianov 1, Moscow 117997, Russia

Abstract: Fluorescent staining is an indispensable method to study living systems One of the recent fundamental advances in this field is the development of fluorogenic dyes - compounds that have no pronounced fluorescence in the free state but acquire it upon binding with the target object, allowing for simple no-wash labeling. Among these dyes, it is worth to note compounds whose fluorescence is particularly sensitive to the properties of the microenvironment (e.g., solvent), which can be used, for example, for staining cell organelles. In this paper, we present a novel technique for the creation of such environment-sensitive and red-shifted fluorogenic dyes with large Stokes shifts. Novel dyes are based on the GFP chromophore core and exhibit high solvent-dependent variation of emission maxima position and fluorescence quantum yield. These compounds are cell-permeable and rapidly stain the endoplasmic reticulum in living cells.

KeyWords GFP, Kaede, fluorogen, fluorescence, endoplasmic reticulum, staining 1. Introduction Fluorogenic dyes, compounds which are not fluorescent in a free state, but kindle upon binding with some target, have attracted considerable interest of researchers.[1] The dyes can be used in fluorescence microscopy for staining various components of living systems, including proteins,[2] nucleic acids[3] and other components.[4] However, despite the abundance of existing dyes, all of them have some drawbacks, such as low cell permeability, off-target staining, toxicity, or ability to alter the functions of living cells. In this regard, the task of creating new labels is still vital. Among the numerous fluorogenic dyes, it is worth to separately emphasize structurally modified analogues of GFP-like fluorescent proteins’ chromophore representing diverse benzylidene imidazolones (BDI, Figure 1).[5] These compounds possess intense and multifarious colors, small size, high solubility in water, and also they are easy to synthesize.[6] Even though the chromophores are highly emissive as a part of natural GFP-like fluorescent proteins, they typically have low fluorescence quantum yield (FQY) in a free state[7] that makes them very attractive as fluorogens. Their applicability for fluorogenic labeling in living systems has already been shown earlier: they were used to stain RNA[8] or proteins.[9] At the same time, their applicability for staining of the cells’ components is still limited. Intriguingly, some GFP chromophore analogues (Figure 1) show a notable increase in fluorescence upon the transition from aqueous medium to less polar solvents. For instance, derivatives containing electron-withdrawing substituents in the para-position of the benzylidene fragment were characterized by a significant solvent-dependent variation in FQY (e.g., 4-nitrile [10] or 4-pyridinyl[11] substituted). Some of meta-electron-donating substituted derivatives (e.g., 2,5-dimethoxy[12]) were also characterized by the high variation of FQY. All these compounds possess the large Stokes shift (the gap between absorption and emission maxima) that made them even more attractive for imaging. Indeed, such dyes are particularly interesting for multi-color labeling, since they can be observed separately in different channels together with conventional dyes with small Stokes shift, being excited by a single wavelength. Similarly, the use of such dyes as donors in Förster resonance energy transfer (FRET) pairs may decrease the channel crosstalk due to direct excitation of the acceptor.

Figure 1. GFP chromophore and previously proposed environment-sensitive fluorophores based on its core. The extremely short-wave absorption maxima in a phototoxic UV range was the main drawback of all previously proposed compounds, which significantly limited the area of their use in living systems. Previously, we showed that the maxima positions of pyridinium analogues of GFP chromophore could be red-shifted simply by the extension of the conjugated system while keeping the solvatochromic variations of the FQY.[11] In this paper, we systematically investigate the results of such modifications for other environment-sensitive GFP chromophore analogues. First, we made close derivatives of GFP chromophore containing various para-electron-withdrawing and meta-electron-donating substitutions in benzylidene moiety (e.g., “parent” compounds, group a, Figure 2). Next, we made a series of analogues with enlarged pi-system. For this purpose, two approaches were used - the introduction of styrene residues (to form compounds similar to chromophore of protein Kaede[13]) and the introduction of a cyclic fragment containing two double bonds (Figure 2, groups b-e).

Figure 2. The proposed technique for the creation of red-shifted and environment-sensitive fluorogenic dyes and the structures of synthesized compounds.

We showed that the proposed technique in all cases leads to a significant red-shift of emission and absorbance spectra while preserving sensitivity of the FQY to the local environment. Moreover, we found that some of the synthesized dyes were suitable for fluorogenic labeling of the endoplasmic reticulum (ER) in living cells. The endoplasmic reticulum is a reticular membranous structure bound to an external nuclear membrane. As a part of a cellular secretory system, ER takes part in protein folding and posttranslational modification[14] and also is a place, where a synthesis of phospholipids occurs.[15] ER could suffer from the so-called ER-stress – an imbalance between lumen capacity and amount of synthesized proteins, which could occur as a result of a pathological state of the cell (i.e., hypoxia, hypoglycemia).[16] As a consequence, labeling of ER is an important component in the study of cellular pathologies. Moreover, even the very structure of ER and many features of its work in the secretory pathways of eukaryotic cells may differ in various organisms and are not fully studied, and therefore are still the subject of fundamental research (see, for example, [17]). However, the most common trackers for endoplasmic reticulum - ER tracker Red and Green (Invitrogen) – are not fluorogenic microenvironment-sensitive dyes. Their structure contain a conventional fluorescent dye and an additional part that allows a selective binding to specific proteins (sulfonylurea receptors) in the ER membrane.[18] Thereby, variations in the expression of these proteins or in cell type could result in mislabeling of a target structure. In contrast, labeling with membranespecific dyes will be more versatile for staining ER due to independence from an expression of specific proteins. Still, the majority of membrane-binding ER-specific fluorescent dyes exhibit absorption maxima in the phototoxic UV region, hindering live-cell imaging applications.[11,19] The created tags are free from abovementioned disadvantages. The absence of a specific protein target makes it possible to stain the ER evenly with less dependence on the state of the cell. Their absorption and emission maximum lie in a sufficiently long-wavelength, less phototoxic region, which allows for longer experiments in living cells. Moreover, the high value of the Stokes shift and the noticeable photostability allows to use them for multi-color labeling together with fluorescent proteins or other tags.

2. Experiment 2.1 Materials and instruments Commercially available reagents were used without additional purification. Merck Kieselgel 60 was used for column chromatography. Thin-layer chromatography (TLC) was performed on silica gel 60 F254 glass-backed plates (MERCK). Visualization was performed using UV light (254 or 312 nm) and staining with KMnO4. NMR spectra were recorded on a 700 MHz Bruker Avance III NMR at 303 K, Avance III 800 (with a 5-mm CPTXI cryoprobe) and Bruker Fourier 300. Chemical shifts are reported relative to residue peaks of CDCl3 (7.27 ppm for 1H and 77.0 ppm for 13C) or DMSO-d6 (2.51 ppm for 1H and 39.5 ppm for 13 C). Melting points were measured on a SMP 30 apparatus without correction. High-resolution mass spectra (HRMS) spectra were recorded on a Bruker micrOTOF II instrument using electrospray ionization (ESI). The measurements were done in a positive ion mode (interface capillary voltage – 4500 V) or in a negative ion mode (3200 V); mass range from m/z 50 to m/z 3000; external or internal calibration was done with ESI Tuning Mix, Agilent. A syringe injection was used for solutions in acetonitrile, methanol, or water (flow rate 3 mL/min). Nitrogen was applied as a dry gas; interface temperature was set at 180°C. UV-VIS spectra were recorded on a Varian Cary 100 spectrophotometer. Fluorescence excitation and emission spectra were recorded on an Agilent Cary Eclipse fluorescence spectrophotometer. 2.2 Synthesis General procedure for the synthesis of compounds 1a-6a

Scheme 1. Synthesis of compounds 1a-6a. The corresponding aromatic aldehyde (5 mmol) was dissolved in CHCl3 (25 mL) and mixed with 0.87 ml of 40% aq. methylamine solution (10 mmol) and Na2SO4 (5 g). The mixture was stirred for 48 h at room temperature and filtered. The solvent was evaporated, 1.13 g (7 mmol) of ethyl((1methoxy)amino)acetate was added and the mixture was stirred for 24 h at room temperature. The solution was evaporated and the residue was purified with column chromatography (eluent – mixture of CHCl3 and EtOH, v/v 100:1). Compounds 2a, 3a, and 4a were synthesized according to the literature procedures. [20] Synthesis of 3,7-dimethoxy-2-naphthaldehyde is described in Supporting information. Methyl (Z)-4-((1,2-dimethyl-5-oxo-1,5-dihydro-4H-imidazol-4-ylidene)methyl)benzoate (1a). Yield 619 mg (48%), yellow solid, m.p. 162-164°C; 1H NMR (DMSO-d6), δ (ppm): 2.39 (s, 3 H), 3.11 (s, 3 H), 3.87 (s, 3 H), 7.01 (s, 1 H), 8.00 (d, J=8.4 Hz, 2 H), 8.32 (d, J=8.2 Hz, 2 H); 13C NMR (DMSO-d6), δ (ppm): 15.5, 26.3, 52.2, 122.7, 129.2, 129.8, 131.8, 138.6, 140.6, 165.8, 166.1, 169.7; HRMS found, m/z: 259.1075 [M-H]-. C14H14N2O3. Calculated, m/z: 259.1077. (Z)-5-((3,7-dimethoxynaphthalen-2-yl)methylene)-2,3-dimethyl-3,5-dihydro-4H-imidazol-4one (5a). Yield 1.13 g (73%), yellow solid, m.p. 242-244°C; 1H NMR (700 MHz, DMSO-d6), δ (ppm): 2.42 (s, 3 H), 3.13 (s, 3 H), 3.87 (s, 3 H), 3.95 (s, 3 H), 7.16 (dd, J=8.8, 2.5 Hz, 1 H), 7.24 (d, J=2.2 Hz, 1 H), 7.34 (s, 1 H), 7.41 (s, 1 H), 7.73 (d, J=8.8 Hz, 1 H), 9.15 (s, 1 H) ;13C NMR (75 MHz, DMSO-d6), δ (ppm): 15.4, 26.2, 55.2, 55.7, 105.8, 106.5, 117.5, 120.3, 123.8, 128.0, 128.9, 130.2, 131.5, 139.2, 154.0, 156.0, 164.9, 169.8; Found, m/z: 311.1388 [M+H]+. C18H18N2O3. Calculated, m/z: 311.1390. (Z)-2,3-dimethyl-5-(quinolin-4-ylmethylene)-3,5-dihydro-4H-imidazol-4-one (6a). Yield 1.03 g (82%), yellow solid, m.p. 218-220°C; 1H NMR (700 MHz, DMSO-d6), δ (ppm): 2.42 (s, 3 H), 3.15 (s, 3

H), 7.65 (s, 1 H), 7.70 (t, J=7.6 Hz, 1 H), 7.82 (t, J=7.5 Hz, 1 H), 8.08 (d, J=8.4 Hz, 1 H), 8.35 (d, J=8.2 Hz, 1 H), 8.61 (d, J=4.6 Hz, 1 H), 9.00 (d, J=4.4 Hz, 1 H); 13C NMR (75 MHz, CDCl3), δ (ppm): 15.8, 26.7, 119.6, 123.1, 123.4, 126.7, 127.1, 129.3, 130.3, 137.7, 142.4, 148.7, 150.3, 165.7, 170.1; Found, m/z: 252.1130 [M+H]+. C15H13N3O. Calculated, m/z: 252.1131. General procedure for the synthesis of the compounds 1b-1d, 2b-2d, 3b-3d, 4b-4d, 5b-5d, 6b-6d

Scheme 2. Synthesis of compounds 1a-6a. The corresponding 2-methylimidazolone (1 mmol), aldehyde (5 mmol) and a catalytic amount of piperidine (10 mg) were dissolved in 5 ml of dry pyridine and the resulting mixture was refluxed for 4-24 h (controlled with TLC until no 2-methylimidazolone remaining). The solution was evaporated and the residue was purified with column chromatography (eluent – mixture of CHCl3 and EtOH, v/v 100:1). Methyl 4-((Z)-(1-methyl-5-oxo-2-((E)-styryl)-1,5-dihydro-4H-imidazol-4-ylidene)methyl) benzoate (1b). Yield 138 mg (40%), yellow solid, m.p. 115-118°C; 1H NMR (700 MHz, DMSO-d6), δ (ppm): 3.88 (s, 3 H), 7.08 (s, 1 H), 7.29 (d, J=15.8 Hz, 1 H), 7.45 - 7.52 (m, 3 H), 7.90 (d, J=7.2 Hz, 2 H), 8.03 (d, J=8.2 Hz, 2 H), 8.13 (d, J=15.8 Hz, 1 H), 8.44 (d, J=8.2 Hz, 2 H); 13C NMR (75 MHz, DMSOd6), δ (ppm): 26.6, 52.3, 113.8, 122.7, 128.6, 129.0, 129.3, 129.7, 130.5, 132.1, 135.0, 139.1, 141.3, 141.6, 162.2, 165.9, 170.1; Found, m/z: 347.1387 [M+H]+. C21H18N2O3. Calculated, m/z: 347.1390. Methyl 4-((Z)-(2-(4-methoxystyryl)-1-methyl-5-oxo-1,5-dihydro-4H-imidazol-4ylidene)methyl)benzoate (1c). Yield 145 mg (39%), yellow solid, m.p. 173-175°C; Mixture of E and Zstyryl isomers (5:4); E-isomer: 1H NMR (700 MHz, DMSO-d6), δ (ppm): 3.12 (s, 3 H), 3.85 (s, 3 H), 3.88 (s, 3 H), 7.01 (s, 1 H), 7.06 (d, J=8.7 Hz, 2 H), 7.12 (d, J=15.7 Hz, 1 H), 7.87 (m, J=8.7 Hz, 2 H), 7.97 8.06 (m, 2 H), 8.10 (d, J=15.7 Hz, 1 H), 8.43 (d, J=8.4 Hz, 2 H); Z-isomer: 1H NMR (700 MHz, DMSOd6), δ (ppm): 3.33 (s, 3 H), 3.85 (s, 3 H), 3.88 (s, 3 H), 6.37 (d, J=12.9 Hz, 1 H), 6.99 (d, J=8.7 Hz, 2 H), 7.10 (s, 1 H), 7.20 (d, J=12.9 Hz, 1 H), 7.94 (d, J=8.4 Hz, 2 H), 7.97 - 8.06 (m, 2 H), 8.25 (d, J=8.4 Hz, 2 H); 13C NMR (75 MHz, DMSO-d6), δ (ppm): 26.5, 52.3, 55.4, 110.9, 114.5, 121.6, 127.7, 129.3, 129.5, 130.5, 131.9, 139.2, 141.4, 141.6, 161.3, 162.4, 165.9, 170.1; HRMS found, m/z: 377.1493 [M+H]+. C22H20N2O4. Calculated, m/z: 377.1496. Methyl 4-((Z)-(1-methyl-5-oxo-2-((E)-2-(pyridin-4-yl)vinyl)-1,5-dihydro-4H-imidazol-4ylidene)methyl)benzoate (1d). Yield 107 mg (31%), yellow solid, m.p. 162-165°C; 1H NMR (700 MHz, DMSO-d6), δ (ppm): 3.88 (s, 3 H), 7.15 (s, 1 H), 7.54 (d, J=15.8 Hz, 1 H), 7.85 (d, J=5.9 Hz, 2 H), 8.03 (d, J=8.4 Hz, 2 H), 8.06 (d, J=15.8 Hz, 1 H), 8.44 (d, J=8.4 Hz, 2 H), 8.70 (d, J=5.9 Hz, 2 H); 13C NMR (75 MHz, DMSO-d6), δ (ppm): 26.6, 52.3, 118.5, 122.2, 124.1, 129.3, 130.0, 132.2, 138.5, 138.8, 141.0, 142.0, 150.3, 161.5, 165.8, 169.9; Found, m/z: 348.1341 [M+H]+. C20H17N3O3. Calculated, m/z: 348.1343. 4-((Z)-(1-methyl-5-oxo-2-((E)-styryl)-1,5-dihydro-4H-imidazol-4-ylidene)methyl) benzonitrile (2b). Yield 94 mg (30%), yellow solid, m.p. 228-230°C; 1H NMR (700 MHz, DMSO-d6), δ (ppm): 3.30 (s, 3 H), 7.09 (s, 1 H), 7.29 (d, J=15.8 Hz, 1 H), 7.45 - 7.52 (m, 3 H), 7.90 (t, J=8.4 Hz, 4 H), 8.12 (d, J=15.8 Hz, 1 H), 8.48 (d, J=8.4 Hz, 2 H); 13C NMR (75 MHz, DMSO-d6), δ (ppm): 26.6, 111.2, 113.8, 118.9, 121.9, 128.7, 129.0, 130.6, 132.3, 132.4, 134.9, 139.1, 141.8, 162.7, 170.0; HRMS found, m/z: 314.1286 [M+H]+. C20H15N3O. Calculated, m/z: 314.1288. 4-((Z)-(2-(4-methoxystyryl)-1-methyl-5-oxo-1,5-dihydro-4H-imidazol-4-ylidene)methyl) benzonitrile (2c). Yield 216 mg (63%), yellow solid, m.p. 218-220°C; Major form (e-isomer, 75%): 1H NMR (700 MHz, DMSO-d6), δ (ppm): 3.29 (s, 3 H), 3.84 (s, 3 H), 7.02 (s, 1 H), 7.06 (d, J=8.8 Hz, 2 H),

7.13 (d, J=15.8 Hz, 1 H), 7.84 - 7.88 (m, 2 H), 7.90 (d, J=8.4 Hz, 2 H), 8.09 (d, J=15.6 Hz, 1 H), 8.47 (d, J=8.4 Hz, 2 H); Minor form (z-isomer, 25%): 1H NMR (700 MHz, DMSO-d6), δ (ppm): 3.12 (s, 3 H), 3.84 (s, 3 H), 6.36 (d, J=13.0 Hz, 1 H), 7.01 (d, J=8.8 Hz, 2 H), 7.12 (s, 1 H), 7.20 (d, J=13.0 Hz, 1 H), 7.84 - 7.88 (m, 2 H), 8.04 (d, J=8.6 Hz, 2 H), 8.32 (d, J=8.4 Hz, 2 H); 13C NMR (75 MHz, DMSO-d6), δ (ppm): 26.5, 55.4, 110.8, 110.9, 114.5, 118.9, 120.8, 127.6, 130.6, 132.1, 132.3, 139.2, 141.8, 141.9, 161.4, 162.9, 170.0; Found, m/z: 344.1390 [M+H]+. C21H17N3O2. Calculated, m/z: 344.1394. 4-((Z)-(1-methyl-5-oxo-2-((E)-2-(pyridin-4-yl)vinyl)-1,5-dihydro-4H-imidazol-4-ylidene) methyl)benzonitrile (2d). Yield 91 mg (29%), yellow solid, m.p. 250-252°C; 1H NMR (700 MHz, DMSO-d6), δ (ppm): 3.31 (s, 3 H), 7.16 (s, 1 H), 7.54 (d, J=15.8 Hz, 1 H), 7.84 (d, J=5.9 Hz, 2 H), 7.92 (m, J=8.4 Hz, 2 H), 8.05 (d, J=15.8 Hz, 1 H), 8.49 (m, J=8.2 Hz, 2 H), 8.69 (d, J=5.9 Hz, 2 H); 13C NMR (75 MHz, DMSO-d6), δ (ppm): 26.7, 111.5, 118.6, 118.8, 122.2, 123.3, 132.4, 132.5, 138.8, 141.6, 141.9, 150.4, 162.1, 169.9; Found, m/z: 315.1239 [M+H]+. C19H14N4O. Calculated, m/z: 315.1240. 3-methyl-5-((Z)-4-nitrobenzylidene)-2-((E)-styryl)-3,5-dihydro-4H-imidazol-4-one (3b). 1 Yield 230 mg (69%), yellow solid, m.p. 240-242°C with decomp; H NMR (700 MHz, DMSO-d6), δ (ppm): 3.31 (s, 3 H), 7.12 (s, 1 H), 7.30 (d, J=15.9 Hz, 1 H), 7.46 - 7.53 (m, 3 H), 7.90 (d, J=6.7 Hz, 2 H), 8.16 (d, J=15.7 Hz, 1 H), 8.29 (d, J=9.0 Hz, 2 H), 8.56 (d, J=8.7 Hz, 2 H); 13C NMR (75 MHz, DMSOd6), δ (ppm): 26.4, 113.7, 121.3, 123.7, 128.7, 129.0, 132.5, 132.7, 140.6, 141.6, 142.3, 147.1, 167.4, 169.7; Found, m/z: 334.1185 [M+H]+. C19H15N3O3. Calculated, m/z: 334.1186. 2-((E)-4-methoxystyryl)-3-methyl-5-((Z)-4-nitrobenzylidene)-3,5-dihydro-4H-imidazol-4one (3c). Yield 312 mg (86%), yellow solid, m.p. 208-210°C with decomp.; 1H NMR (700 MHz, DMSOd6), δ (ppm): 3.29 (s, 3 H), 3.85 (s, 3 H), 7.04 - 7.09 (m, 3 H), 7.13 (d, J=15.8 Hz, 1 H), 7.87 (d, J=8.6 Hz, 2 H), 8.14 (d, J=15.6 Hz, 1 H), 8.28 (d, J=9.0 Hz, 2 H), 8.55 (d, J=8.8 Hz, 2 H); 13C NMR (75 MHz, DMSO-d6), δ (ppm): 26.5, 55.5, 110.8, 114.6, 120.1, 123.7, 127.6, 130.7, 132.6, 141.3, 142.3, 142.5, 146.8, 161.5, 163.5, 170.1; Found, m/z: 364.1290 [M+H]+. C20H17N3O4. Calculated, m/z: 364.1292. 3-methyl-5-((Z)-4-nitrobenzylidene)-2-((E)-2-(pyridin-4-yl)vinyl)-3,5-dihydro-4H-imidazol4-one (3d). Yield 107 mg (32%), yellow solid, m.p. 258-260-164°C; 1H NMR (700 MHz, DMSO-d6), δ (ppm): 3.32 (s, 3 H), 7.21 (s, 1 H), 7.56 (d, J=15.8 Hz, 1 H), 7.84 (d, J=5.7 Hz, 2 H), 8.10 (d, J=15.8 Hz, 1 H), 8.30 (d, J=8.8 Hz, 2 H), 8.58 (d, J=8.8 Hz, 2 H), 8.70 (d, J=5.7 Hz, 2 H); 13C NMR (75 MHz, DMSO-d6), δ (ppm): 26.7, 118.5, 122.2, 122.5, 123.7, 132.9, 139.1, 140.8, 141.8, 142.0, 147.2, 150.4, 162.6, 169.9; Found, m/z: 335.1138 [M+H]+. C18H14N4O3. Calculated, m/z: 335.1139. (Z)-3-methyl-5-(quinolin-4-ylmethylene)-2-((E)-styryl)-3,5-dihydro-4H-imidazol-4-one (4b). Yield 278 mg (82%), yellow solid, m.p. 218-220°C; 1H NMR (700 MHz, DMSO-d6), δ (ppm): 3.34 (s, 3 H), 7.32 (d, J=15.8 Hz, 1 H), 7.46 - 7.52 (m, 3 H), 7.68 - 7.73 (m, 2 H), 7.82 (t, J=7.5 Hz, 1 H), 7.92 (d, J=6.9 Hz, 2 H), 8.09 (d, J=8.2 Hz, 1 H), 8.18 (d, J=15.8 Hz, 1 H), 8.40 (d, J=8.4 Hz, 1 H), 8.87 (d, J=4.6 Hz, 1 H), 9.05 (d, J=4.6 Hz, 1 H); 13C NMR (75 MHz, DMSO-d6), δ (ppm): 26.7, 113.7, 115.9, 123.2, 126.1, 127.4, 128.8, 129.0, 129.5, 129.9, 130.7, 134.9, 137.6, 142.4, 143.5, 148.3, 150.3, 163.8, 169.9; Found, m/z: 340.1441 [M+H]+. C22H17N3O. Calculated, m/z: 340.1444. (Z)-2-((E)-4-methoxystyryl)-3-methyl-5-(quinolin-4-ylmethylene)-3,5-dihydro-4H-imidazol4-one (4c). Yield 195 mg (53%), yellow solid, m.p. 205-208°C; 1H NMR (700 MHz, DMSO-d6), δ (ppm): 3.33 (s, 3 H), 3.85 (s, 3 H), 7.06 (m, J=8.6 Hz, 2 H), 7.16 (d, J=15.6 Hz, 1 H), 7.64 (s, 1 H), 7.71 (t, J=7.5 Hz, 1 H), 7.82 (t, J=7.6 Hz, 1 H), 7.89 (d, J=8.6 Hz, 2 H), 8.08 (d, J=8.2 Hz, 1 H), 8.16 (d, J=15.6 Hz, 1 H), 8.40 (d, J=8.4 Hz, 1 H), 8.89 (d, J=4.6 Hz, 1 H), 9.04 (d, J=4.6 Hz, 1 H); 13C NMR (75 MHz, DMSO-d6), δ (ppm): 26.6, 55.4, 110.8, 114.5, 114.7, 123.1, 123.2, 126.1, 127.3, 127.6, 129.4, 129.9, 130.8, 137.7, 142.6, 143.7, 148.3, 150.4, 161.5, 164.1, 169.9; Found, m/z: 370.1547 [M+H]+. C23H19N3O2. Calculated, m/z: 370.1550.

(Z)-3-methyl-2-((E)-2-(pyridin-4-yl)vinyl)-5-(quinolin-4-ylmethylene)-3,5-dihydro-4Himidazol-4-one (4d). Yield 54 mg (16%), yellow solid, m.p. 225-227°C; 1H NMR (700 MHz, DMSO-d6), δ (ppm): 3.36 (s, 3 H), 7.58 (d, J=15.8 Hz, 1 H), 7.73 (t, J=7.6 Hz, 1 H), 7.78 (s, 1 H), 7.84 (t, J=7.5 Hz, 1 H), 7.86 (d, J=5.5 Hz, 2 H), 8.08 - 8.13 (m, 2 H), 8.42 (d, J=8.6 Hz, 1 H), 8.70 (d, J=5.5 Hz, 2 H), 8.83 (d, J=4.6 Hz, 1 H), 9.06 (d, J=4.4 Hz, 1 H); 13C NMR (201 MHz, DMSO-d6), δ (ppm): 26.7, 117.5, 118.5, 120.8, 122.2, 123.3, 123.3, 127.4, 129.5, 129.9, 137.4, 139.3, 141.8, 143.3, 148.3, 150.3, 150.4, 163.2, 169.7; Found, m/z: 341.1394 [M+H]+. C21H16N4O. Calculated, m/z: 341.1397. 5-((Z)-2,5-dimethoxybenzylidene)-3-methyl-2-((E)-styryl)-3,5-dihydro-4H-imidazol-4-one (5b). Yield 132 mg (38%), yellow solid, m.p. 185-188°C; 1H NMR (700 MHz, DMSO-d6), δ (ppm): 3.29 (s, 3 H), 3.83 (s, 3 H), 3.86 (s, 3 H), 7.00 - 7.05 (m, 2 H), 7.27 (d, J=15.8 Hz, 1 H), 7.35 (s, 1 H), 7.44 7.50 (m, 3 H), 7.84 (d, J=7.2 Hz, 2 H), 7.97 (d, J=15.8 Hz, 1 H), 8.58 (d, J=2.3 Hz, 1 H); 13C NMR (75 MHz, DMSO-d6), δ (ppm): 26.5, 55.3, 56.2, 112.4, 114.1, 116.2, 117.7, 117.9, 123.3, 128.5, 129.0, 130.3, 135.0, 139.1, 140.3, 153.0, 153.2, 160.5, 170.1; Found, m/z: 349.1543 [M+H]+. C21H20N2O3. Calculated, m/z: 349.1547. 5-((Z)-2,5-dimethoxybenzylidene)-2-((E)-4-methoxystyryl)-3-methyl-3,5-dihydro-4Himidazol-4-one (5c). Yield 227 mg (60%), yellow solid, m.p. 178-180°C; 1H NMR (700 MHz, DMSOd6), δ (ppm): 3.27 (s, 3 H), 3.82 (s, 3 H), 3.83 (s, 3 H), 3.85 (s, 3 H), 7.00 - 7.05 (m, 4 H), 7.10 (d, J=15.8 Hz, 1 H), 7.31 (s, 1 H), 7.80 (d, J=8.8 Hz, 2 H), 7.94 (d, J=15.8 Hz, 1 H), 8.58 (d, J=2.7 Hz, 1 H); 13C NMR (75 MHz, DMSO-d6), δ (ppm): 26.4, 55.3, 55.4, 56.1, 111.3, 112.3, 114.5, 116.2, 116.8, 117.6, 123.4, 127.7, 130.3, 139.2, 140.3, 153.0, 153.1, 160.8, 161.1, 170.1; Found, m/z: 379.1651 [M+H]+. C22H22N2O4. Calculated, m/z: 379.1652. 5-((Z)-2,5-dimethoxybenzylidene)-3-methyl-2-((E)-2-(pyridin-4-yl)vinyl)-3,5-dihydro-4Himidazol-4-one (5d). Yield 261 mg (75%), yellow solid, m.p. 218-220°C; 1H NMR (700 MHz, DMSOd6), δ (ppm): 3.82 (s, 3 H), 3.86 (s, 3 H), 7.05 (s, 2 H), 7.41 (s, 1 H), 7.51 (d, J=15.8 Hz, 1 H), 7.79 (d, J=5.7 Hz, 2 H), 7.90 (d, J=15.8 Hz, 1 H), 8.53 (s, 1 H), 8.67 (d, J=5.7 Hz, 2 H); 13C NMR (75 MHz, DMSO-d6), δ (ppm): 26.5, 55.3, 56.1, 112.4, 116.3, 118.2, 118.8, 119.0, 122.1, 123.0, 137.3, 138.9, 142.0, 150.3, 153.0, 153.4, 159.8, 169.9; Found, m/z: 350.1498 [M+H]+. C20H19N3O3. Calculated, m/z: 350.1499. (Z)-5-((3,7-dimethoxynaphthalen-2-yl)methylene)-3-methyl-2-((E)-styryl)-3,5-dihydro-4Himidazol-4-one (6b). Yield 246 mg (62%), yellow solid, m.p. 262-265°C; 1H NMR (700 MHz, DMSOd6), δ (ppm): 3.91 (s, 3 H), 3.96 (s, 3 H), 7.14 - 7.20 (m, 1 H), 7.27 (d, J=15.8 Hz, 1 H), 7.34 (s, 1 H), 7.42 - 7.53 (m, 5 H), 7.73 (d, J=8.8 Hz, 1 H), 7.93 (d, J=7.2 Hz, 2 H), 8.22 (d, J=15.6 Hz, 1 H), 9.37 (s, 1 H); 13 C NMR (75 MHz, DMSO-d6), δ (ppm): 26.5, 55.3, 55.7, 105.8, 107.2, 113.8, 117.6, 120.4, 124.3, 128.0, 128.6, 128.9, 129.1, 130.3, 131.9, 135.1, 139.8, 141.1, 154.1, 156.1, 161.1, 170.2; Found, m/z: 399.1701 [M+H]+. C25H22N2O3. Calculated, m/z: 399.1703. (Z)-5-((3,7-dimethoxynaphthalen-2-yl)methylene)-2-((E)-4-methoxystyryl)-3-methyl-3,5dihydro-4H-imidazol-4-one (6c). Yield 255 mg (60%), yellow solid, m.p. 223-225°C; 1H NMR (700 MHz, DMSO-d6), δ (ppm): 3.31 (br. s., 3 H), 3.85 (s, 3 H), 3.92 (s, 3 H), 3.97 (s, 3 H), 7.07 (d, J=8.8 Hz, 2 H), 7.14 (d, J=15.6 Hz, 1 H), 7.18 (dd, J=8.9, 2.6 Hz, 1 H), 7.35 (s, 1 H), 7.44 (s, 1 H), 7.49 (d, J=2.3 Hz, 1 H), 7.74 (d, J=9.0 Hz, 1 H), 7.92 (d, J=8.8 Hz, 2 H), 8.21 (d, J=15.8 Hz, 1 H), 9.37 (s, 1 H); 13C NMR (75 MHz, DMSO-d6), δ (ppm): 26.5, 55.3, 55.4, 55.7, 105.8, 107.2, 111.0, 114.4, 116.6, 120.3, 124.4, 127.9, 128.0, 129.1, 130.2, 130.5, 131.8, 140.0, 141.1, 154.1, 156.1, 161.2, 161.4, 170.2; Found, m/z: 429.1806 [M+H]+. C26H24N2O4. Calculated, m/z: 429.1809. (Z)-5-((3,7-dimethoxynaphthalen-2-yl)methylene)-3-methyl-2-((E)-2-(pyridin-4-yl)vinyl)3,5-dihydro-4H-imidazol-4-one (6d). Yield 133 mg (33%), yellow solid, m.p. 195-198°C; 1H NMR (700

MHz, DMSO-d6), δ (ppm): 3.34 (s, 3 H), 3.92 (s, 3 H), 3.98 (s, 3 H), 7.20 (dd, J=8.9, 2.4 Hz, 1 H), 7.37 (s, 1 H), 7.49 (d, J=2.3 Hz, 1 H), 7.53 - 7.59 (m, 2 H), 7.75 (d, J=9.0 Hz, 1 H), 7.89 (d, J=5.7 Hz, 2 H), 8.18 (d, J=16.0 Hz, 1 H), 8.70 (d, J=5.7 Hz, 2 H), 9.37 (s, 1 H); 13C NMR (75 MHz, DMSO-d6), δ (ppm): 26.6, 55.3, 55.8, 106.0, 107.3, 119.1, 119.2, 120.6, 122.5, 124.1, 128.0, 129.1, 130.5, 132.2, 137.8, 139.6, 143.0, 149.6, 154.2, 156.1, 160.5, 170.0; Found, m/z: 400.1654 [M+H]+. C24H21N3O3. Calculated, m/z: 400.1656. General procedure for the synthesis of the compounds 1e-6e

Scheme 3. Synthesis of the compounds 1e-6e. A solution of imidazo[1,2-a]pyridin-3(2H)-one hydrochloride (0.5 g, 2.7 mmol) was refluxed in phosphorus trichloride (5 mL) for 4 h in an inert atmosphere. The solution was evaporated. The residue was dissolved in dry freshly distilled pyridine (4 mL) in an inert atmosphere. Aldehyde (2 mmol) and triethylamine (0.5 mL) was added, and the mixture was stirred at room temperature for 1.5 h. The solution was evaporated. The residue was dissolved in CHCl3 (100 mL), washed with K2CO3 solution (10%, 2 × 50 mL), water (2 × 50 mL) and brine (2 × 50 mL) and dried over Na2SO4. The solvent was evaporated, and the product was purified by column chromatography (eluent - CHCl3). The residue was washed with 5 ml of ether and dried in vacuo. Imidazo[1,2-a]pyridin-3(2H)-one hydrochloride was synthesized as described previously.[21] Methyl (Z)-4-((3-oxoimidazo[1,2-a]pyridin-2(3H)-ylidene)methyl)benzoate (1e). Yield 89 mg (16%), red solid, m.p. 183-186°C with decomp.; 1H NMR (DMSO-d6), δ (ppm): 3.87 (s, 3 H), 6.43 (t, J=7.0 Hz, 1 H), 7.03 (d, J=9.4 Hz, 1 H), 7.21 (s, 1 H), 7.41 (ddd, J=9.3, 6.5, 1.0 Hz, 1 H), 7.78 (d, J=6.9 Hz, 1 H), 8.01 (d, J=8.5 Hz, 2 H), 8.39 (d, J=8.3 Hz, 2 H); 13C NMR (DMSO-d6), δ (ppm): 52.2, 109.9, 118.4, 123.8, 126.7, 129.2, 129.8, 132.1, 139.0, 140.0, 140.0, 157.7, 165.8, 167.1; Found, m/z: 281.0918 [M+H]+. C16H12N2O3. Calculated, m/z: 281.0921. (Z)-4-((3-oxoimidazo[1,2-a]pyridin-2(3H)-ylidene)methyl)benzonitrile (2e). Yield 84 mg (17%), red solid, m.p. 210-213°C; 1H NMR (DMSO-d6), δ (ppm): 6.44 (t, J=6.6 Hz, 1 H), 7.03 (d, J=9.4 Hz, 1 H), 7.20 (s, 1 H), 7.43 (ddd, J=9.2, 6.5, 1.1 Hz, 1 H), 7.79 (d, J=6.9 Hz, 1 H), 7.90 (d, J=8.3 Hz, 2 H), 8.45 (d, J=8.5 Hz, 2 H); 13C NMR (DMSO-d6), δ (ppm): 110.0, 111.1, 118.3, 118.8, 122.7, 126.8, 132.3, 132.3 (2 C), 139.1, 140.4, 140.6, 158.1, 167.1; Found, m/z: 248.0817 [M+H]+. C15H9N3O. Calculated, m/z: 248.0818. (Z)-2-(4-nitrobenzylidene)imidazo[1,2-a]pyridin-3(2H)-one (3e). Yield 110 mg (15%), dark solid, m.p. 255°C with decomp.; 1H NMR (700 MHz, DMSO-d6), d (ppm): 6.46 (t, J=6.7 Hz, 1 H), 7.05 (d, J=9.3 Hz, 1 H), 7.25 (s, 1 H), 7.46 (ddd, J=9.4, 6.3, 1.2 Hz, 1 H), 7.81 (d, J=6.9 Hz, 1 H), 8.29 (m, J=9.0 Hz, 2 H), 8.52 (m, J=9.0 Hz, 2 H); 13C NMR (201 MHz, DMSO-d6), d (ppm): 110.1, 118.2, 121.8, 123.5, 126.7, 132.5, 140.6, 140.9, 141.0, 146.9, 158.4, 167.0; Found, m/z: 368.0719 [M+H]+. C14H9N3O3. Calculated, m/z: 368.0717. (Z)-2-(quinolin-4-ylmethylene)imidazo[1,2-a]pyridin-3(2H)-one (4e). Yield 27 mg (5%), red solid, m.p. 148-151°C; 1H NMR (700 MHz, DMSO-d6), δ (ppm): 6.68 (d, J=8.2 Hz, 1 H), 6.72 (ddd, J=6.6, 5.6, 0.8 Hz, 1 H), 7.07 (s, 1 H), 7.50 (ddd, J=8.4, 7.0, 1.9 Hz, 1 H), 7.54 - 7.59 (m, 2 H), 7.76 (ddd, J=8.2, 7.0, 1.2 Hz, 1 H), 8.01 - 8.05 (m, 2 H), 8.07 (d, J=8.0 Hz, 1 H), 8.85 (d, J=4.6 Hz, 1 H), 8.86 (s, 1 H); 13C NMR (75 MHz, DMSO-d6), δ (ppm): 109.9, 114.9, 115.2, 120.5, 124.6, 125.9, 126.6, 129.5,

129.6, 134.8, 137.3, 140.2, 147.2, 148.1, 150.2, 155.3, 165.9; Found, m/z: 274.0974 [M+H]+. C17H11N3O. Calculated, m/z: 274.0975. (Z)-2-(2,5-dimethoxybenzylidene)imidazo[1,2-a]pyridin-3(2H)-one (5e). Yield 124 mg (22%), red solid, m.p. 180-183°C; 1H NMR (DMSO-d6), δ (ppm): 3.76 (s, 3 H), 3.86 (s, 3 H), 6.38 - 6.43 (m, 1 H), 6.99 - 7.07 (m, 3 H), 7.35 (ddd, J=9.3, 6.5, 1.0 Hz, 1 H), 7.50 (s, 1 H), 7.76 (d, J=6.9 Hz, 1 H), 8.54 (d, J=2.4 Hz, 1 H); 13C NMR (DMSO-d6), δ (ppm): 55.4, 56.1, 109.7, 112.1, 117.4, 117.6, 118.6, 119.2, 123.6, 126.3, 137.9, 138.8, 153.0, 153.3, 156.1, 166.8; Found, m/z: 283.1074 [M+H]+. C16H14N2O3. Calculated, m/z: 283.1077. (Z)-2-((3,7-dimethoxynaphthalen-2-yl)methylene)imidazo[1,2-a]pyridin-3(2H)-one (6e). 1 Yield 33 mg (5%), red solid, m.p. 273°C with decomp.; H NMR (700 MHz, DMSO-d6), d (ppm): 3.89 (s, 3 H), 3.98 (s, 3 H), 6.44 (t, J=6.4 Hz, 1 H), 7.08 (d, J=9.3 Hz, 1 H), 7.18 (dd, J=8.9, 2.0 Hz, 1 H), 7.27 (d, J=2.1 Hz, 1 H), 7.36 (s, 1 H), 7.40 (t, J=7.7 Hz, 1 H), 7.63 (s, 1 H), 7.74 (d, J=8.8 Hz, 1 H), 7.80 (d, J=6.7 Hz, 1 H), 9.32 (s, 1 H); 13C NMR (201 MHz, DMSO-d6), d (ppm): 55.1, 55.7, 105.8, 106.7, 109.7, 118.4, 119.1, 120.5, 124.5, 126.4, 127.9, 129.0, 130.3, 132.2, 138.5, 139.0, 154.1, 156.1, 156.6, 166.8; Found, m/z: 333.1236 [M+H]+. C20H16N2O3. Calculated, m/z: 333.1234. 2.3 Fluorescent imaging in living cells 2.3.1 General HeLa Kyoto, HEK293 cell lines were obtained from established frozen stocks of our laboratory. Rat cardiomyoblasts H9c2 cell line was obtained from EMBL. Mouse myoblasts C2C12 were a kind gift of N. Podkiychenko. Cells were seeded into 35-mm glass bottom dishes (SPL) and cultured in Dulbecco’s Modified Essential Medium (DMEM) with 2 mM glutamine and 4.5 g/L glucose (PanEco) supplemented with 10% fetal bovine serum (HyClone, ThermoScientific) and 1% Penicillin+Streptomycin (5000 U/mL + 5000 µg/mL, PanEco) in a humidified atmosphere under 37℃ and 5% CO2. After 24 h, cells were transfected by a mixture of 1.5-2 ng DNA and 5 µL FuGENE HD transfection reagent (Promega) in 100 µL OptiMEM (Gibco) solution per dish. After the 24-36 h cells were imaged in 1 mL Hank’s Balanced Salt Solution with calcium and magnesium (PanEco), supplemented with 20 mM HEPES (pH 7.1, Sigma-Aldrich) at room temperature. For imaging of dyes, cells were incubated with 1.0-10 µM of a dye (added from 5 mM stock solution in DMSO) for 1 min. For co-localization analysis, cells were incubated with 1 µM of ER-tracker Red (added from 1 mM stock solution in DMSO) for 5 min. Wide-field images were acquired with Leica DMI6000B inverted microscope (Leica Microsystems) equipped with HCX PL Fluotar 63x1.25 oil immersion lens and HC PL Apo 40x0.85 lens (Leica Microsystems), GFP filter cube (excitation filter 470/40, emission filter 525/50) (Leica Microsystems) and mCherry/TFT filter cube (excitation filter 578/21, emission filter 641/75, Semrock). CoolLED pE-300white (CoolLED Ltd) with illumination intensity of about 2.5-5 mW for 40x lens and 0.5-3 mW for 63x lens was used as a light source. Images were acquired with Andor Zyla 5.5 CL 10 Tap sCMOS camera (Andor Technology) controlled by the Micromanager software (ver 1.4.23). For imaging of b and d dyes, GFP cube was used, while for imaging of e and ER Tracker Red (Invitrogen) mCherry/TFT cube was used. Confocal images were acquired with TSC SP2 confocal system (Leica Microsystems) installed on inverted fluorescent microscope Leica DM IRE equipped with HCX PL APO Lbd.BL 63x1.40 oil immersion lens and argon (458/488 nm) laser. Image acquisition was performed using the 458 nm line of the argon laser (≈6.5 µW) with the emission 470-530 nm for mTurquoise2 imaging; 0.7 µW of 458 nm line with the emission 560-600 for 2b and 5d imaging; 2.5 µW of 458 nm line with the emission 560-600

for 1b imaging; 488 nm line of the argon laser (≈9 µW) with the emission 499-540 nm for EGFP or emission 570-600 for 1e. Images were processed and analyzed for co-localization using Fiji ImageJ distribution (ver. 1.52n)[22] and NanoJ plug-in (ver. 1.14stable1).[23] 2.3.2 Photobleaching We performed a photobleaching analysis of 1b, 2b and 5d in HeLa Kyoto and HEK293 cells with mTurquoise2 as a reference. Similarly, we assessed the photobleaching of 1e in HeLa Kyoto and HEK293 cells with EGFP as a reference. Cells were transfected as described in the previous paragraph. Subset of HeLa Kyoto and HEK293 dishes passed through the same procedure without transfection step and incubated with 5 µM of 1b/2b/5d/1e (added from 1 mM DMSO with 20% F-127 pluronic, Sigma Aldrich) for 1 min. Images were acquired with TSC SP2 based on inverted fluorescent microscope Leica DM IRE equipped with HCX PL APO Lbd.BL 63x1.40 oil lens and argon (458/488 nm) laser. For bleaching of mTurquoise2, 1b, 2b and 5d 14*103 µm2, region was scanned at 0.3 fps with 9 µW of 458 nm line of argon laser. For bleaching of EGFP and 1e, a similar region at the same fps was scanned with 25 µW of 488 nm line of an argon laser. Images were processed and analyzed in Fiji. Graphs in Fig. 7 were processed using custom scripts in Python 3.5.

3.0 Results discussion In order to assess the solvatochromic behavior of the obtained compounds, their optical properties were studied in five different solvents - water, methanol, acetonitrile, ethyl acetate and dioxane (see Table 1). Table 1. Solvatochromic properties of compounds 1a-6e. Absorbancea H2O

MeOH

CH3CN

Emissionb EtOAc

dioxane

H2Oc

MeOH

CH3CN

EtOAc

dioxane

413 (3.0)

450 (6.4)

442 (19.2)

438 (7.9)

1a

355 (16)

358 (16)

364 (16)

365 (16)

367 (15)

464 (<0.1)

1b

399 (18)

418 (17)

422 (18)

420 (17)

423 (14)

591 (0.5)

553 (2.5)

555 (3.1)

551 (5.9)

547 (8.6)

1c

438 (20)

433 (19)

434 (20)

431 (21)

432 (15)

584 (0.5)

568 (0.9)

575 (2.0)

568 (1.6)

568 (2.5)

550 (7.3)

546 (8.5)

541 (11.2)

543 (14.4)

595 (1.7)

580 (8.9)

568 (8.8)

566 (6.3)

447 (2.5)

454 (6.0)

446 (20.8)

411 (21.7)

1d

409 (12)

416 (12)

417 (12)

419 (12)

421 (9)

547 (2.7)

1e

495 (12)

498 (12)

508 (13)

508 (13)

511 (10)

2a

355 (14)

361 (12)

365 (13)

365 (14)

366 (12)

2b

461 (19)

423 (19)

423 (19)

422 (20)

424 (7)

597 (1.7)

558 (1.7)

561 (2.3)

554 (4.2)

551 (6.4)

2c

443 (21)

436 (21)

436 (21)

433 (21)

435 (12)

596 (0.2)

576 (0.4)

585 (0.5)

576 (0.7)

574 (1.1)

2d

409 (16)

415 (15)

418 (16)

419 (16)

420 (12)

547 (3.7)

543 (6.2)

547 (7.3)

544 (8.9)

514 (12.3)

2e

497 (11)

505 (11)

512 (10)

512 (11)

514 (9)

604 (0.4)

587 (6.2)

574 (6.7)

570 (5.3)

3a

370 (16)

375 (16)

382 (15)

383 (19)

384 (16)

588 (1.1)

528 (2.7)

485 (0.5)

480 (0.2)

3b

423 (12)

427 (12)

431 (13)

428 (13)

430 (8)

619 (0.4)

614 (1.5)

587 (1.9)

568 (4.3)

544 (7.6)

3c

450 (23)

448 (22)

452 (24)

449 (24)

452 (11)

636 (0.2)

632 (0.5)

618 (1.3)

594 (1.5)

585 (1.8)

3d

414 (18)

424 (17)

429 (17)

429 (18)

430 (10)

637 (0.3)

583 (2.4)

574 (2.8)

558 (7.3)

534 (11.3)

3e

507 (-e)

513 (6)

520 (3)

520 (6)

522 (4)

-d (0)

634 (0.8)

630 (2.5)

604 (5.4)

588 (4.7)

481 (<0.1) 600 (<0.1)

466 (<0.1)

462 (<0.1)

453 (0.3)

449 (0.5)

570 (0.8)

577 (1.0)

572 (3.8)

570 (5.6)

608 (<0.1) 456 (<0.1)

616 (<0.1) 635 (<0.1)

4a

360 (30)

369 (29)

377 (28)

377 (32)

380 (24)

4b

428 (16)

428 (15)

429 (14)

429 (15)

428 (11)

4c

454 (20)

447 (18)

444 (22)

441 (20)

442 (10)

592 (0.2)

595 (0.3)

597 (0.4)

590 (0.6)

588 (0.9)

4d

414 (11)

425 (12)

425 (11)

426 (11)

428 (10)

575 (0.9)

575 (1.9)

568 (3.6)

563 (6.6)

563 (8.1)

4e

503 (0.7)

514 (0.8)

520 (0.6)

520 (0.8)

522 (0.7)

622 (0.2)

612 (0.9)

600 (2.8)

583 (3.8)

580 (2.8)

5a

392 (14)

396 (12)

392 (11)

391 (14)

393 (4)

571 (0.7)

541 (1.2)

499 (6.6)

475 (6.5)

472 (5.8)

560 (18.4)

564 (18.5)

5b

455 (15)

445 (14)

443 (14)

441 (16)

445 (10)

581 (0.9)

566 (3.1)

558 (15.9)

5c

462 (25)

449 (24)

447 (25)

445 (26)

448 (15)

587 (0.4)

564 (1.6)

565 (1.9)

528 (3.8)

532 (6.5)

601 (0.8)

582 (8.7)

567 (34.7)

564 (41.1)

550 (0.2)

555 (5.4)

550 (5.3)

554 (4.9)

600 (0.3)

545 (8.4)

508 (16.5)

504 (22.3) 561 (11.3)

e

5d

450 (- )

454 (15)

450 (15)

448 (17)

452 (11)

5e

496 (14)

499 (14)

505 (14)

505 (14)

509 (13)

533 (<0.1) 606 (<0.1) 506 (<0.1)

6a

371 (17)

370 (17)

371 (17)

369 (16)

371 (8)

6b

439 (18)

434 (16)

438 (18)

438 (19)

439 (12)

596 (0.8)

626 (0.2)

601 (1.5)

560 (9.8)

6c

448 (22)

444 (21)

446 (22)

445 (22)

446 (14)

588 (0.5)

615 (0.2)

577 (1.2)

565 (1.8)

566 (3.6)

563 (0.2)

585 (1.9)

575 (5.9)

562 (2.0)

556 (3.8)

559 (3.2)

6d 6e

e

396 (- ) 467 (9) a

432 (16) 498 (10)

437 (18) 509 (6)

438 (18) 509 (9)

584 (<0.1) 600 (<0.1)

d

440 (8)

- (0)

512 (3)

610 (<0.1) -1

-1

-3

b

Maxima position in nm (Extinction coefficient in M × cm × 10 ); Emission position in nm (FQY in %); cEmission in water is very weak for many chromophores and maxima position is determined crudely; dEmission is extremely low, no peak found; eExtremely low solubility, extinction coefficient couldn’t be measured properly.

Thus, as it is clearly seen from Table 1, proposed modifications in all cases lead to a significant red-shift of the absorption and emission maxima (Table 1, Figure 3).

Figure 3. Fluorescence and absorption spectra of compounds 1a-e and 5a-e in acetonitrile. Maximum shifts were achieved for compounds with an additional cyclic fragment containing two double bonds. In the series of compounds b-d, which are analogues of the Kaede chromophore, the bathochromic shift increased with an increase in the donor nature of the substituent - the highest shifts were achieved in the case of derivatives containing a methoxy group. This distinguishes them from previously synthesized derivatives of the Kaede chromophore containing donor groups in the benzylidene fragment, for which the bathochromic shifts increased with an increase in the acceptor nature of the groups in styrene moiety.[13] We note, that an increase in the conjugated pi-system leads to red-shift only in case of the addition of double bonds in the imidazolone part of the molecules. Thus, the introduction of naphthalene moiety (compounds 4 and 6) into the benzylidene part does not result in the red-shift in comparison with corresponding benzene derivatives. Moreover, such compounds often characterized by an extremely low extinction coefficient. Similarly to chromophore derivatives containing the pyridinium ring,[11] all synthesized compounds were characterized by a rather weak solvatochromic variation of the absorption maxima positions. In contrast, the solvatochromic variations of the emission maxima positions in some cases reached 100 nm (Table 1, Figure 4). The FQY values were also remarkably varied - typically the difference in emission intensity between water and dioxane exceed an order of magnitude (Table 1). The largest red-shift, as well as the smallest FQY, was observed in polar and protic media for all compounds. However, as was previously shown for pyridine derivatives, compounds with two additional double bonds (group e), in some cases, demonstrated a atypical direction of solvent-dependent FQY variation. Thus, compounds 1e, 2e, and 5e were more emissive in aprotic, but polar media (e.g., acetonitrile).

Figure 4. Fluorescence and absorption spectra of the compounds 1e and 5d in various solvents. The bathochromic shifts of emission and fluorescence quenching in polar media suggest that these chromophores have large dipole moments in their first excited state, which are remarkably larger than in the ground state. It is also confirmed by the fact that the absorption maxima are weakly dependent on the solvent nature, while the emission maxima vary significantly. These properties lead to a huge Stokes shift (in the range of 100-150 nm for several compounds). It was already observed earlier for similar compounds and explained by the charge redistribution in the main excited state that also accompanied by the significant change in the geometry of the molecule and, as a consequence, a change in the energy of the reverse transition to the ground state. [11] Remarkable solvatochromism of the emission maxima and FQY suggests that all these dyes can be used for local polarity sensing or as a fluorogenic dye for organelles labeling. Thus, on the next step, we check the behaviour of created dyes in the cells. We excluded the compounds of group c from this test because they showed very low FQY variation and the “parent” compounds of the group a since they are characterized by very a short-wave maxima position. In all cases, the addition of the dyes into the cell media in the concentration of 1-10 µM resulted in an instant appearance of fluorescence associated predominantly with ER structures (see SI Part 4), excluding 4e compound, that showed slight fluorescence increase in a cell without any compartment localization. However, only four dyes (1b, 2b, 5d, and 1e – Figure 5) showed high selectivity in ERstaining without the formation of an excessive amount of fluorescent droplets and with the high signal-tonoise ratio.

COOMe

CN

1b

2b

N

N

N

O

O

N

COOMe

OMe 5d

MeO

1e

N O

N

N N O

N

Figure 5. Compounds 1b, 2b, 5d and 1e. We showed that all these dyes could be used for ER staining in various cell lines (Figure 6). In HeLa Kyoto and HEK293 ER, contacting with the nucleus was detected as well as peripheral tubes. Also, longitudinal tubules of sarcoplasmic reticulum were detected in myoblasts (C2C12) and cardiomyoblasts (H9c2).

Figure 6. Widefield fluorescence microscopy. ER-staining of HeLa Kyoto, HEK293, C2C12 and H9c2 cells by chosen dyes: 1b, 2b, 5d and 1e. Cells stained with 5 µM of corresponding dye (added from 5 mM stock in DMSO). Images acquired with Leica DMI6000B inverted microscope (Leica Microsystems) equipped with HCX PL Fluotar 63x1.25 oil lens. 1b, 2b and 5d: GFP filter cube; 1e: mCherry/TFT filter cube. Scale bars are 10 µm.

As one may note, there were no correlations between the structure of compounds and their ER staining properties. Thus, as we previously claim, the ability to stain the ER selectively is based on the combination of fluorogenic properties and a suitable degree of polarity that provides an accumulation of dye only in ER membranes.[11a] The dyes with an excessive lipophilicity stain other membranes and forms fluorescent droplets, while too polar dyes do not accumulate in cells and give lousy signal-to-noise ratio. The possible exchange of fluorogen molecules between membranes and solution typically results in extremely high photostability.[11a] However, compounds 1b and 2b bleached relatively fast under widefield fluorescence microscopy conditions. Such behaviour can be explained by the precipitation of the dyes from cell media that leads to the decrease of dye concentration and prevent the possible recovery of the signal. Contrary, compounds 1e and 5d showed a very bright and contrast signal, which turned out to be comparable in photostability with fluorescent proteins. Using confocal microscopy, we confirmed high selectivity of ER staining by co-localization experiment with commercially available ER-tracker Red (Invitrogen). (Figure 7)

Figure 7. Live-cell confocal microscopy with fluorogenic dyes. (A) ER-staining of HeLa Kyoto cells by 5 µM of 5d (added from 1 mM stock in DMSO with 20% F-127 pluronic). Image processed with Fiji Lookup Tables “NanoJ-orange.” (B) ER-staining of H9c2 cells by 5 µM of 1e (added from 1 mM stock in DMSO with 20% F-127 pluronic). Image processed with Fiji Lookup Tables “NanoJ-orange.” Co-localization analysis of 5 µM 5d (C) and 1e (D) dyes in H9c2 cells compared to 1 µM ER-tracker Red (Invitrogen, added from 1mM stock in DMSO, incubation 5 min). Pearson co-localization analysis

followed with Costses randomized test for 1e resulted 0.87±0.03 (n=4) in H9c2 cells and 0.88±0.06 (n=3) in C2C12 cells. Similarly for 5d 0.69±0.05 (n=4) in H9c2 cells and 0.81±0.02 (n=3) in C2C12 cells. Photostability analysis in HeLa Kyoto cells stained with 5 µM 5d and 1e (added from 1 mM stock in DMSO with 20% F-127 pluronic) in comparison with cytoplasm-localized fluorescent proteins: (E) mTurquoise2, 9 µW of 458 nm laser line and (F) EGFP, 25 µW of 488 nm laser line in a time-lapse regime. Data are shown as mean ± SD. Scale bars are 10 µm. In order to clarify the possible mechanism of such selective staining, we perform additional experiments on the study of solvatochromic behavior of dyes 5d and 1e (see SI Part 3). We found that the viscosity of the medium does not affect the quantum yield of fluorescence of these compounds, but otherwise, their behavior is very well described by the Kamlet-Taft model. This model associates the spectral characteristics (X) of the dye with solvent parameters such as acidity (α), basicity (β), and polarity (π*) (see SI Part 3).[24] X= X0 + pπ* + aα + bβ

(1)

This model has already been successfully used for other fluorogenic derivatives of the GFP chromophore [11] and also allows analyzing the effect of solvent parameters on fluorescence quantum yield. The parameters obtained from this model (p, a, and b) reflects the sensitivity of a chromophore to the corresponding solvent’s property and are presented in Table 2. Table 2. Solvatochromic spectral (in 103/cm) and FQY (in %) parameters of compounds 5d and 1e. Compound

1e

5d

Characteristic

a

b

p

νo/FQYo

R

Absorbance Emission FQY Absorbance Emission FQY

0.6 -0.5 -7.0 1.55 -2.3 -20

0 -0.5 -1.5 0.8 -1.1 -20

-0.3 -1.1 -3.0 -1.1 -1.4 -35

19.8 18.4 8.0 22.3 19.0 58.5

0.97 0.94 0.82 0.89 0.95 0.90

An increase of parameter p upon excitation reflects a significant increase of dipole moment, [11] and correlates well with the observed value of the Stokes shift. The revealed change in the sign of parameter a is typical of many BDIs and indicates changes in proton-accepting properties upon excitation.[6,11] We also found that an increase in all three parameters of the solvent leads to a decrease in the emission intensity of both dyes. This behavior is also typical of many BDIs, including those that have already demonstrated selective staining of the endoplasmic reticulum[11a] and appears to be the key characteristic responsible for such selectivity.

4.0 Conclusion Thus, we present the technique of the creation of red-shifted fluorogenic dyes based on the GFP chromophore core. This technique allows creating a large family of environment-sensitive compounds that can be used as the polarity sensors. Most of these dyes are characterised by a large Stokes shift and a high degree of environment-dependent FQY variation. A unique combination of lipophilic properties and fluorogenic behaviour enables using two of synthesized compounds as ER-staining dyes in eukaryotic cells.

5.0 Acknowledgments This work was supported by the Russian Science Foundation grant 16-14-10364. Appendix A. Supplementary data Supplementary data to this https://doi.org/10.1016/j.dyepig.0000.000000.

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Highlights - Novel red-shifted fluorogenic dyes based on the GFP chromophore are presented; - Dyes demonstrated high solvent-dependent variations of the fluorescence quantum yield; - Created compounds can be used for fluorescent staining of Endoplasmic Reticulum

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: