An Acetylene-Bridged 6,8-Purine Dimer as a Fluorescent Switch-On Probe for Parallel G-Quadruplexes

Exploiting chemistry to develop compounds capable of selective recognition of biomolecules or interfering with cellular processes is the essence of chemical biology.1 One example of such biologically relevant targets are nucleic acid G-quadruplexes.2 These non-canonical structures of DNA and RNA have been widely hypothesized to play a role in the regulation of crucial genomic functions, such as telomere maintenance,3 transcription,4 and translation.5 Recently, we showed that a small G-quadruplex-interacting molecule has the ability to activate the DNA damage response machinery in human cancer cells in a replication- and transcription-dependent manner.6


S1
Experimental General All reactions were conducted under an argon atmosphere using anhydrous freshly distilled solvents unless otherwise stated. THF and acetonitrile were dried and purified by distillation from sodium/benzophenone and CaH 2 respectively. Flash chromatography was performed using 230-400 mesh silica gel from Sigma-Aldrich. Analytical thin-layer chromatography (TLC) was performed using silica gel 60 F 254 using UV light as visualizing agent. Preparative thin layer chromatography (PLC) was carried out on similar plates (1 mm) from Merck. 1 H and 13 C NMR spectra were recorded on Bruker DRX-400, DPX-400, DRX-500 instruments using residual solvent as reference. High-resolution mass spectra were obtained on a Waters Micromass® Q-Tof (ESI) spectrometer. HPLC purification was performed on a Varian ProStar instrument equipped with a Pursuit C18, 5µ column (250 × 21.2 mm) using a linear elution gradient from 90% H 2 O (containing 0.1% TFA) to 100% MeCN in 30 min at a flow rate of 12.0 ml/min. Microwave reactions were conducted using a single-mode CEM discover microwave unit. The maximum microwave power was set at 140 W and the pressure limit at 16 bar. All reactions were performed in crimp sealed thick walled glass vessels (capacity 10 mL or 30 mL). The reaction mixture was stirred using a Teflon-coated magnetic stirring bar and the temperature was monitored using an infrared sensor. Compounds 2, 3 were synthesized according to a reported procedure. [1] Synthesis of purine 4 To a suspension of 3 (100 mg, 0.28 mmol) in dry degassed THF (3 mL) in a 10 mL sealed Microwave tube was added 0.260 mL of n-amyl nitrite (1.90 mmol) via a microsyringe. The reaction mixture was stirred under microwave irradiation at 120 °C for 30 min. After reaching completion, the reaction mixture was cooled to room temperature and the solvent was removed in vacuo. The product was then purified by column chromatography on silica gel using EtOAc as the eluent, to afford 4 as a white solid (yield 65%).  155.8, 152.8, 152.7, 147.2, 134.7, 134.5, 80.0, 44.5, 39.7, 28.3 155.6, 153.5, 151.6, 148.7, 139.3, 133.6, 103.1, 94.2, 79.8, 44.0, 40.2, 28.3, 18.7, 11.2

Synthesis of purine 7
To a suspension of 2 (117 mg, 0.42 mmol) in dry degassed MeCN (3 mL) in a 10 mL sealed Microwave tube was added 1 mL of diiodomethane (12.4 mmol) and 0.260 mL of n-amyl nitrite (1.90 mmol) under argon using a microsyringe. The reaction mixture was stirred under microwave irradiation at 120 °C for 15 min. After reaching completion, the reaction mixture was cooled to room temperature and the solvent was removed in vacuo. The residue was dissolved in EtOAc and washed with saturated Na 2 S 2 O 3 and water. The organic layer was evaporated in vacuo and the crude mixture was purified by column chromatography on silica gel using EtOAc as the eluent, to afford 7 as a white solid (yield 31% Synthesis of APD 1 6 (12 mg, 0.041 mmol), 7 (14 mg, 0.036 mmol), Pd(PPh 3 ) 4 (5.5 mg, 0.005 mmol, 14 mol%), CuI (2.0 mg, 0.010 mmol, 28 mol%), and Cs 2 CO 3 (40 mg, 0.123 mmol) were suspended in dry degassed THF (3 mL) in a 5 mL RB flask under argon. The reaction mixture was stirred at rt for 5 h. The solvent was removed in vacuo and the crude mixture was purified on preparative TLC plates using EtOAc-MeOH (3:1) as the eluent, to afford the product in the N-Boc protected form. Deprotection: 20 µL of tin tetrachloride (0.116 mmol) was added to a suspension of 7 mg of N-Boc protected product (0.012 mmol) in dry EtOAc (0.5 mL). The resulting clear solution was stirred at rt for 30 min until TLC (EtOAc-MeOH 3:1) showed complete consumption of the starting material. The product was precipitated by the addition of diethylether (3 mL), collected by filtration and purified by HPLC (see general experimental for conditions) to afford 1 as yellow TFA salt (yield 40% over two steps).  : 1 H and 13 C spectra of 4 at 500 MHz at 298 K in CDCl 3 . Figure S2: 1 H and 13 C spectra of 5 at 500 MHz at 298 K in CDCl 3 . Figure S3: 1 H and 13 C spectra of 6 at 500 MHz at 298 K in CDCl 3 . Figure S4: 1 H and 13 C spectra of 7 at 500 MHz at 298 K in CDCl 3 . Figure S5: 1 H and 13 C spectra of APD (1) at 500 MHz at 298 K in D 2 O. UV-visible spectroscopy UV-Vis spectra were recorded on a Varian Cary 100-Bio at 298 K using a 10 mm path length quartz cuvette. Titrations were performed by adding a solution of pre-annealed oligonucleotide (1 mM stock solution) to a solution of APD (25 µM) in PBS buffer (50 mM, pH 7.4) containing 50 mM KCl. Samples were left to equilibrate after each addition to ensure a stable readout.

Fluorescence spectroscopy and quantum yield calculations
Fluorescence measurements were performed using a Varian Cary Eclipse Spectro-Fluorometer. Emission spectra were recorded using a 10 mm path length quartz cuvette by exciting the samples at 430 nm and recording the emissions over the spectral range of 480-600 nm. (slit emission and excitation were set at 10 nm). Fluorescence titrations were performed by adding solutions of pre-annealed oligonucleotides (100 µM and 1 mM) to a solution of APD (0.5 µM) in PBS buffer (50 mM, pH 7.4) containing 50 mM KCl. Samples were left to equilibrate after each addition to ensure a stable readout. All titrations were performed in triplicates.
Quantum yields were calculated using fluorescein as standard (φ = 0.95). Emission spectra of APD and fluorescein were recorded (25 µM) in a 50 mM potassium phosphate buffer (pH 7.4) containing 50 mM KCl using a λ ext = 430nm. Oligonucleotides pre-anelled in this buffer were added to the APD solution to the final concentration of 100 µM. ODs of these samples were recorded at 430 nm and the quantum yield values were calculated according to the following equation:

φsample = φref (A sample /A Ref )*(OD Ref/ OD Sample )
Where: φ ref is the quantum yield of the reference; A sample and A ref are the area underneath the emission spectra of the sample and the reference respectively and OD ref and OD sample are the optical density of the reference and the sample respectively measured at the excitation wavelength.
All the experiments have been performed in triplicates.
Circular dichroism spectroscopy CD spectra were recorded on an Applied Photo-physics Chirascan circular dichroism spectropolarimeter using a 1 mm path length quartz cuvette. CD measurements were performed at 298 K over a range of 200-340 nm using a response time of 1 s, 1 nm pitch and 0.5 nm bandwidth. The recorded spectra represent a smoothed average of three scans, zero-corrected at 320 nm and normalized (Molar ellipticity θ is quoted in S10 10 5 deg cm 2 dmol −1 ). The absorbance of the buffer was subtracted from the recorded spectra. Oligonucleotides were dissolved in lithium cacodilate buffer (100 mM, pH 7.2) containing 100 mM of KCl and 1 mM EDTA to the concentration of 10 μM. The oligonucleotides were annealed following the abovementioned conditions prior to measurements.
FRET-melting assays 100 μM stock solutions of oligonucleotides were prepared in molecular biology grade DNase-free water. ) which is a dual-labeled 20-mer oligonucleotide comprising a self-complementary sequence with a central polyethylene glycol linker able to fold into a hairpin. The donor fluorophore was 6-carboxyfluorescein (FAM) and the acceptor fluorophore was 6carboxytetramethylrhodamine (TAMRA). The dual-labeled oligonucleotides were annealed at a concentration of 400 nM by heating at 94 ºC for 10 min followed by slow cooling to rt at a controlled rate of 0.1 ºC/min. 96well plates were prepared by addition of 50 μl of the annealed DNA solution to each well, followed by 50 μl solution of APD at the appropriate concentration. Measurements were made in duplicate with an excitation wavelength of 483 nm and a detection wavelength of 533 nm. Final analysis of the data was carried out using Prism 5 data analysis and graphing software (Prism ® ).

Nuclear magnetic resonance spectroscopy
Titration with 1 was performed on a 500 MHz TCI-ATM Cryo instrument at 298 K and the spectra were recorded immediately after each addition. Water suppression was achieved using excitation sculpting.