Research Center on Fundamental and Applied Heterochemistry, Faculty of Chemistry and Chemical Engineering, Babeş-Bolyai University, 11 Arany Janos Street, 400028 Cluj-Napoca, Romania.
Biochemistry Department, Faculty of Veterinary Medicine, University of Agricultural Science and Veterinary Medicine, 3-5 Calea Manastur Street, 400327 Cluj-Napoca, Romania.
Int J Mol Sci. 2021 Mar 15;22(6):2985. doi: 10.3390/ijms22062985.
We report here the synthesis and structural characterization of novel cationic (phenothiazinyl)vinyl-pyridinium (PVP) dyes, together with optical (absorption/emission) properties and their potential applicability as fluorescent labels. Convective heating, ultrasound irradiation and mechanochemical synthesis were considered as alternative synthetic methodologies proficient for overcoming drawbacks such as long reaction time, nonsatisfactory yields or solvent requirements in the synthesis of novel dye (E)-1-(3-chloropropyl)-4-(2-(10-methyl-10H-phenothiazin-3-yl)vinyl)pyridin-1-ium bromide and its -alkyl-2-methylpyridinium precursor . The geometry of the newly synthesized (E)-4-(2-(7-bromo-10-ethyl-10H-phenothiazin-3-yl)vinyl)-1-methylpyridin-1-ium iodide and (E)-1-methyl-4-(2-(10-methyl-10H-phenothiazin-3-yl)vinyl)pyridin-1-ium tetrafluoroborate was confirmed by single crystal X-ray diffraction. A negative solvatochromism of the dyes in polar solvents was highlighted by UV-Vis spectroscopy and explanatory insights were supported by molecular modeling which suggested a better stabilization of the lowest unoccupied molecular orbitals (LUMO). The photostability of the dye was investigated by irradiation at 365 nm in different solvents, while the steady-state and time-resolved fluorescence properties of dye and in solid state were evaluated under one-photon excitation at 485 nm. The in vitro cytotoxicity of the new PVP dyes on B16-F10 melanoma cells was evaluated by WST-1 assay, while their intracellular localization was assessed by epi-fluorescence conventional microscopy imaging as well as one- and two-photon excited confocal fluorescence lifetime imaging microscopy (FLIM). PVP dyes displayed low cytotoxicity, good internalization inside melanoma cells and intense fluorescence emission inside the B16-F10 murine melanoma cells, making them suitable staining agents for imaging applications.
我们在这里报告了新型阳离子（吩噻嗪基）乙烯基-吡啶鎓（PVP）染料的合成和结构表征，以及它们的光学（吸收/发射）性质及其作为荧光标记物的潜在适用性。对流加热、超声辐射和机械化学合成被认为是替代合成方法，可以有效地克服反应时间长、产率不理想或合成新型染料（E）-1-（3-氯丙基）-4-（2-（10-甲基-10H-吩噻嗪-3-基）乙烯基）吡啶-1-鎓溴化物及其 - 烷基-2-甲基吡啶鎓前体[1]的溶剂要求等缺点。新合成的（E）-4-（2-（7-溴-10-乙基-10H-吩噻嗪-3-基）乙烯基）-1-甲基吡啶-1-鎓碘化物和（E）-1-甲基-4-（2-（10-甲基-10H-吩噻嗪-3-基）乙烯基）吡啶-1-鎓四氟硼酸盐的几何形状通过单晶 X 射线衍射得到证实。紫外可见光谱突出显示了染料在极性溶剂中的负溶剂化变色，分子建模提供了解释性见解，表明最低未占据分子轨道（LUMO）得到了更好的稳定。在不同溶剂中用 365nm 光照射研究了染料[1]的光稳定性，而在单光子激发 485nm 下评估了固态染料[1]和[1]的稳态和时间分辨荧光性质。通过 WST-1 测定法评估了新 PVP 染料对 B16-F10 黑色素瘤细胞的体外细胞毒性，通过荧光常规显微镜成像以及单光子和双光子激发共聚焦荧光寿命成像显微镜（FLIM）评估了它们在黑色素瘤细胞内的细胞内定位。PVP 染料表现出低细胞毒性、良好的黑色素瘤细胞内化和强烈的荧光发射，使其成为适用于成像应用的染色剂。
