Abstract
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 3d and its N-alkyl-2-methylpyridinium precursor 1c. The trans geometry of the newly synthesized (E)-4-(2-(7-bromo-10-ethyl-10H-phenothiazin-3-yl)vinyl)-1-methylpyridin-1-ium iodide 3b and (E)-1-methyl-4-(2-(10-methyl-10H-phenothiazin-3-yl)vinyl)pyridin-1-ium tetrafluoroborate 3a′ 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 3b was investigated by irradiation at 365 nm in different solvents, while the steady-state and time-resolved fluorescence properties of dye 3b and 3a′ 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.
Highlights
In the last years, the methine dyes gained special attention in bioimaging due to their high molar extinction coefficients, high fluorescence quantum yields, large range of absorption and emission wavelengths reaching near infrared (NIR) domain and a good ability to penetrate through the cell membranes
In our pursuit for finding greener synthetic alternatives to the traditional methods to carry out organic synthesis, in our previous experiments the PVP dyes were conveniently synthesized by microwaves assisted condensation of phenothiazine carbaldehyde with methylpyridinium salts in dry media, while in this work we examine the benefits of ultrasound assisted and mechanochemical synthetic procedures, as candidates for environmentally friendly protocols proficient for overcoming drawbacks such as long reaction time, nonsatisfactory yields or solvent requirements
The synthetic path presented in Scheme 1 contains two reaction steps, the preparation of the starting pyridinium salts 1a–c by the alkylation of 4-picoline with halogenated alkyl derivatives, followed by the preparation of PVP dyes 3a–d by Knoevenagel condensation of phenothiazine-carbaldehydes with the alkyl pyridinium salts
Summary
The methine dyes gained special attention in bioimaging due to their high molar extinction coefficients, high fluorescence quantum yields, large range of absorption and emission wavelengths reaching near infrared (NIR) domain and a good ability to penetrate through the cell membranes. Schiff bases-boron complexes were successfully used for the in vitro cytoplasmic staining in B16F10 murine melanoma cells [6], while fluorophores containing thiophene moieties were employed in the selective staining of cytoplasm in live mouse embryonic fibroblasts [7]. Other methine derivatives such us triphenylmethane Brilliant Blue G [8] and styryl dye LDS 820 [9], respectively, can be used as fluorescent NIR fluorescent labels for amyloid fibrils, which are associated with numerous neurodegenerative diseases. Based on the documented potential of methine dyes for practical applications as fluorescent markers the photophysical behavior of PVP dyes in biological environment (i.e., B16-F10 murine melanoma cells) was investigated via fluorescence imaging, as part of our search for novel cellular staining agents
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