Two novel pyrene-quinoline conjugates differing in the linker flexibility between aryls were prepared. In comparison with referent pyrene derivative, both conjugates showed intramolecular pyrene-quinoline stacking in aqueous medium, much more efficient for rigid conjugate. Consequently, only rigid conjugate showed excimer fluorescence with exceptionally strong bathochromic shift (+ 55 nm) of emission maximum in respect to referent pyrene analogue and flexible conjugate. All studied compounds showed similar, 10 µmol dm −3 affinity toward ds-DNA, characterised in general by fluorescence quenching. The flexible conjugate showed at large excess of DNA over dye formation of pyrene-quinoline excimer, while rigid conjugate retained excimer emission throughout all DNA concentrations. Lack of significant thermal stabilisation of ds-DNA by studied compounds and minor changes in CD spectrum of DNA supported non-specific agglomeration of both conjugates and referent pyrene analogue within hydrophobic DNA grooves as the dominant binding mode.
Unless otherwise stated, all starting materials and solvents required for the synthesis of the DABCO cyanine dyes were purchased from Sigma-Aldrich, Organica Feinchemie GmbH Wolfen, Fluka, Alfa-Aesar, TCI Europe, Deutero GmbH, and used without any further purification. The solvents used for the spectroscopic analyses were purchased form Macron Fine Chemicals TM. All other starting materials and solvents were commercial products of analytical grade and were used without further purification. 2.2 Synthesis and spectroscopic analysis of the dyes 2.3. Analysis methods and equipment All products were characterized using various spectroscopic techniques. The progress of the reactions was monitored employing TLC (Merck F 254 silica gel; chloroform: methanol: acetic acid-80:15:5). Recrystallization from methanol yielded analytical samples of the title cyanines. 1 H-NMR and APT-NMR spectra of the compounds were recorded on a Brucker Avance III 500 MHz instrument in DMSO-d6 at room temperature. Chemical shifts were reported in ppm in δ-values with respect to tetramethylsilane (TMS) as an internal reference, or the corresponding peak of the deuterated solvent. Coupling constants JH-H were expressed in Hz. The structures of all intermediates were also evaluated on the target cyanine dyes. Melting point temperatures were evaluated on a Kofler bench and are uncorrected. Absorption and steady state fluorescence spectra of the unbound dyes were recorded in methanol solutions at room temperature, using 10-mm path-length quartz cuvettes on a Cecil Aurius CE 3021 UV-Vis spectrophotometer and Perkin Elmer LS45 fluorescence spectrometer (fixed slits 10-10 nm) at room temperature. 2.4. Synthetic approach to the DABCO monomethine cyanines and intermediate products 2.4.1. Preparation of intermediates 2a-2c
Newly designed and synthesized diarylethene (DAE) derivatives with aliphatic amine sidearms and one with two pyrenes, revealed excellent photo-switching property of central DAE core in MeOH and water. The only exception was bis-pyrene analogue, its DAE core very readily photochemically closed, but reversible opening completely hampered by aromatic stacking interaction of pyrene(s) with cyclic DAE. In this process, pyrene fluorescence showed to be a reliable monitoring method, an open form characterized by strong emission at 480 nm (typical for pyrene-aggregate), while closed form emitted weakly at 400 nm (typical for pyrene-DAE quenching). Only open DAE-bis-pyrene form interacted measurably with ds-DNA/RNA by flexible insertion in polynucleotide grooves, while self-stacked closed form did not bind to DNA/RNA. For the same steric reasons, flexible open DAE-bis-pyrene form was bound to at least three different binding sites at bovine serum albumin (BSA), while rigid, self-stacked closed form interacted dominantly with only one BSA site. Preliminary screening of antiproliferative activity against human lung carcinoma cell line A549 revealed that all DAE-derivatives are non-toxic. However, bis-pyrene analogue efficiently entered cells and located in the cytoplasm, whereby irradiation by light (315–400 nm) resulted in a strong, photo-induced cytotoxic effect, typical for pyrene-related singlet oxygen species production.
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