Fine-tuning alkyne cycloadditions: Insights into photochemistry responsible for the double-strand DNA cleavage via structural perturbations in diaryl alkyne conjugates

Hybrid molecules combining photoactivated aryl acetylenes and a dicationic lysine moiety cause the most efficient double-strand (ds) DNA cleavage known to date for a small molecule. In order to test the connection between the alkylating ability and the DNA-damaging properties of these compounds, we investigated the photoreactivity of three isomeric aryl–tetrafluoropyridinyl (TFP) alkynes with amide substituents in different positions (o-, m-, and p-) toward a model π-system. Reactions with 1,4-cyclohexadiene (1,4-CHD) were used to probe the alkylating properties of the triplet excited states in these three isomers whilst Stern–Volmer quenching experiments were used to investigate the kinetics of photoinduced electron transfer (PET). The three analogous isomeric lysine conjugates cleaved DNA with different efficiencies (34, 15, and 0% of ds DNA cleavage for p-, m-, and o-substituted lysine conjugates, respectively) consistent with the alkylating ability of the respective acetamides. The significant protecting effect of the hydroxyl radical and singlet oxygen scavengers to DNA cleavage was shown only with m-lysine conjugate. All three isomeric lysine conjugates inhibited human melanoma cell growth under photoactivation: The p-conjugate had the lowest CC50 (50% cell cytotoxicity) value of 1.49 × 10−7 M.

General information. 1 H, 13 C NMR spectra were recorded on a Varian Gemini 300 MHz, Bruker 400 MHz and 600 MHz NMR spectrometer. Mass spectrometry data was collected on a Jeol JMS-600H. UV spectra were recorded on a Shimadzu UV-2100. Fluorescence spectra were obtained with SPEX FluoMax spectrofluorimeter using right-angle geometry. pH was monitored with AB 15 plus pH meter (Accument) after standardization at 25 ºC. All buffers were prepared and pH was adjusted with HCl (aq.) and NaOH (aq.) at room temperature (25 °C).
Singlet excitation lifetime. The singlet excitation lifetimes were measured using the timecorrelated single photon counting (TCSPC) technique. The samples were excited at 295 nm wavelength with LED operating at a repetition rate of 1MHz. The emission decay was observed at emission λ max of each sample and data were recorded with 10,000 counts in the peak channel.
The timescale of the experiment was 200 ns (29.19 ps/channel). The decay data were analyzed with DAS6 software.
Plasmid DNA photocleavage. pBR322 plasmid DNA (4,361 b/p; from BioLabs Inc., 1μg/μL solution in 10 mM Tris-HCl (pH 8.0), and 1mM EDTA buffer) was diluted to a concentration of S3 0.01 μg/μL. The solution containing the cleavage agent, DNA (30 μM/bp) in 20 mM sodium phosphate buffer was incubated for 1 hour at 30 °C. Samples were placed in ice at a distance of 20 cm from 200 W Hg-Xe lamp (Spectra-Physics, Laser & Photonics Oriel Instruments with long pass filter with 300 nm cut-on wavelength). Electrophoretic analysis. The gel electrophoresis was carried out in 1x TBE buffer at 80 V using Miligel FisherBiotech Horizontal Electrophoresis System. All gels were run on 1% agarose slab gels. Before loading, the DNA samples were mixed with 0.33 volume of tracking dye containing bromophenol blue (0.25%) and glycerol (30%) in water. After staining in ethidium bromide solution (2 μg/ml) for 1 hour, the gel was washed with water and pictures were taken.
The relative quantities of the supercoiled, nicked, and linear DNA were calculated by integrating the "area" of each spot by the image analyzer software Total/Lab (Nonlinear Dynamics Ltd., UK). The amount of supercoiled DNA was multiplied by factor of 1.4 to account for reduced ethidium bromide intercalation into supercoiled DNA.
Cell culture. The human cancer cell line used in this report was obtained from the American Type Culture Collection (ATCC ® ). The human mesothelioma cell line MSTO-211H (CRL-2081™) was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (Life Technologies™). Cells were propagated according to ATCC guidelines and maintained in a 37 °C incubator with 5% CO 2 atmosphere.
MTT assay. A375 cells were seeded at 2000 cells per well in a 96-well plate. Seven 2-fold serial dilutions of the compounds were added to the cells in triplicate. Concentrations were from 1 to .016 M of compounds. After incubation for 4h with the compounds, cells were UV-irradiated for ten minutes at 365 nm (UV transluminator, Spectronomics Corp. model TR-365R). After 72hs of incubation, MTT (Sigma® cat# M2128) was added to cells to give a final concentration of 1.25 mg/ml and incubated for 60 minutes. Cells were centrifuged at 900 g for 5 minutes at room temperature. Media was replaced with DMSO and absorbance measured in a plate reader at 570 nm.

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Synthesis of compounds. All reagents used were obtained from commercial sources and were of the highest grade available.