Selective parallel G-quadruplex recognition by a NIR-to-NIR two-photon squaraine

A selective and efficient nonlinear squaraine fluorescent probe for parallel G-quadruplexes suitable for NIR-to-NIR two-photon imaging procedures is reported.


S3
The UV/Vis measurements were performed in conventional quartz cell cuvettes with path lengths of 0.01 -50.0 mm. ECD spectra were measured with a Jasco J-815 spectropolarimeter (JascoInc, USA) equipped with the JascoPeltier-type temperature controller (CDF-426S/15) and are presented as a sum of 3 accumulations.
ECD measurements were performed at 25 °C in the wavelength range of 210-310 nm at different CAS-C1 / G4 ratios. Before use, the optical chamber of the CD spectrometer was deoxygenated with dry nitrogen and was held under nitrogen atmosphere during the measurements. Appropriate references were subtracted from the obtained CD spectra.
TCSPC and singlet lifetime. The buffered solution of squaraine and G4 was prepared as for the titration studies. The suitable amount of G4 was added in order to obtain a complete formation of the complex. The formation of the complex was monitored by UV/Vis spectroscopy, then emission and excitation spectra were recorded. Lifetimes were obtained by monitoring the decay in the emission maximum upon irradiation of the pulsed laser at 670 nm (laser pulse 100 ns, emission slit 6.0 nm).
Data were fitted with a monoexponential function (Eq. S1) or a biexponential function (Eq. S2): (Eq. S1)  Table S1.  12 where TPEF of the sample is compared with the signal of a standard under the same experimental conditions. We used styryl 9M as a standard. 13 Data processing. All the data were corrected for the dilution upon titration with the G4 stock solution (500 µM). Binding constants were obtained with Bindfit (available on the website http://supramolecular.org/) by using multiple global fitting methods (Nelder-Mead method) of both the UV/Vis data in the range of 600 -740 nm and fluorescence data in the range 690 -764 nm. Dilution corrections option was included in the fitting option, as described in the references. 14,15 In order to ensure to find the minima in the fitting analyses, all the fittings were confirmed with three different start values.

Synthesis and characterization
Squaraine CAS-C1 was synthesized according to the route depicted in Scheme S1. The staring compounds 1 20 and 5 21 were synthesized by literature-known procedures and 2-methylbenzothiazole 3 was obtained from commercial supplier.

Synthesis of 2
To a solution of benzyl alcohol derivative 1 (1.03 g, 1.73 mmol) in anhydrous dichloromethane (5 mL) PBr 3 (0.086 mL, 0.905 mmol) was added at 0 under nitrogen atmosphere. The reaction mixture was stirred for 2 h at 0 . Afterwards, water (50 mL) was added and the organic layer was separated. The aqueous phase was extracted with dichloromethane (100 mL × 3). The combined organic layer was washed with brine and dried over sodium sulfate. The solvent was removed under reduced pressure to obtain benzyl bromide 2 as a light brown liquid in nearly quantitative yield (1.14 g, 99%

Synthesis of CAS-C1
A round-bottom flux, equipped with a water-removing Dean-Stark apparatus, was charged with 4 mL nbutanol and 4 mL toluene. Subsequently, dicyanomethylene squaric ester 5 (13.0 mg, 44.6 µmol) and     The apparent extinction coefficient was plotted against the temperature and fitted well with a temperature-dependent isodesmic aggregation model (Eq. S3), 22 with an estimated T m = 64 °C and assuming an enthalpy of each step of polymerization of ΔH pol = -55.9 kJ mol -1 .

G-quadruplex studies Kinetics
The binding of CAS-C1 with various G4s revealed no significant kinetics.                Figure S18 Global fitting of CAS-C1 titration with A) ckit-87up, C) ckit-2 monitored in the absorption spectra at 632 nm (aggregate maximum wavelength, grey), 699 nm (complex maximum, black) and in the fluorescence spectra at the maximum emission wavelength (red) and at 740 nm (blue). The fitting is shown for 1:1 (dotted) and 1:2 (filled) binding model. The covariance is shown on the right (B, D for the respective G4s) for the 1:1 fitting of the absorption (black) and fluorescence (red) data, and the 1:2 fitting (grey for the absorption and orange for the fluorescence).