“CinNapht” dyes: a new cinnoline/naphthalimide fused hybrid fluorophore. Synthesis, photo-physical study and use for bio-imaging

Six-membered-diaza ring of cinnoline has been fused on naphthalimide dye to give a donor–acceptor system called CinNapht. This red shifted fluorophore, that can be synthesised in gram scale, exhibits a large Stoke shift and a fluorescence quantum yield up to 0.33. It is also characterized by a strong solvatochromic effect from green to red emission as well and can be used for bio-imaging.


III. Experimental section General
Unless otherwise noted, all commercially available reagents and solvents were used without further purification. TLC were carried out on silica gel aluminum plates with F-254 indicator; The spots were directly visualized or through illumination with UV lamp ( = 254/365 nm). Flash-column chromatography purifications were performed on silica gel (40-63 μm) from Macherey-Nagel. Organic solvents for spectroscopy were purchased from Acros Organics or Sigma Aldrich. Absolute EtOH was provided by Carlo Erba. The HPLC grade MeCN used for RP-HPLC analyses was obtained from Carlo Erba. Formic acid (FA, puriss p.a., ACS reagent, reag. Ph. Eur., ≥98%) was provided by Merck-Millipore (brand Sigma-Aldrich). Aq. mobile-phases for HPLC were prepared using water purified with a Milli-Q Integral 3 system from Merck-Millipore (purified to 18.2 MW.cm).

Instrument and methods
1 Proton NMR ( 1 H) spectra were recorded on a Bruker Avance 500 or 300 MHz and proton-decoupled carbon 13 NMR spectra were recorded at 125 MHz. NMR experiments were carried out in CDCl3 or DMSO-d6 and chemical shifts are expressed in parts per million (ppm) from the residual nondeuterated solvent signal. Calibration was made by using residual signals of partially deuterated solvent summarized in 2010 by Fulmer et al. 3 The following abbreviations are used for the multiplicities: s: singlet; d: doublet; t: triplet; q: quadruplet; qt: quintuplet; m: multiplet or overlap of nonequivalent resonances; br s: broad singlet; Coupling constants (J) are reported in hertz (Hz). High resolution mass spectra were determined on an AEI MS-9 using electrospray ionization (ESI) and a time-offlight (TOF) analyzer. IR spectra were recorded with a PerkinElmer Spectrum BX FT-IR spectrometer directly from the substance via attenuated total reflectance (ATR-IR) and bond vibration frequencies are expressed in reciprocal centimeters (cm -1 ). HPLC-MS analyses were performed on an Alliance W2690 system (Waters, USA) Melting points were measured with using a Büchi B-540 melting point apparatus.

Spectroscopy Studies
UV-vis absorption measurements (scan mode) were conducted on a Shimadzu UV-visible spectrophotometer UV-2600 using rectangular 10 mm path length quartz cuvettes from Thuet, at 25 °C. Fluorescence spectroscopic studies (scan and kinetics modes) were performed with a Fluoromax-4 spectrofluorometer (Jobin-Yvon; Horiba) at 25 °C, with rectangular 10 mm path length quartz cuvettes from Thuet. The absorption spectra of isoA were recorded with concentrations in the range 47-2.4 µM (total volume = 10 mL volumetric flask, six distinct dilutions for the accurate determination of molar extinction coefficients). For all the solvents, dilution were made from a stock solution in chloroform at 0.477 mM. The emission and excitation spectra were recorded with a diluted solution with 1% in volume of chloroform and lower than 0.1 absorbance values. For the excitation and emission spectra the slits width were adjusted between 2-5 nm, integration time = 0.1 s, 1 nm step, HV (S1) = 950 V). For chloroform, dichloromethane, ethanol, methanol and DMSO the excitation were performed at 488nm, at 478 nm for toluene, at 437 nm for n-hexane and at 470 nm for DMSO. For the excitation spectra, the emission was observed at 590 nm for DCM and CHCl3, at 427 nm for ethanol, at 577 nm for toluene, at 600 nm for methanol and DMSO, at 518 nm for hexane and for dioxane. All fluorescence spectra were corrected from the lamp fluctuations and the apparatus response fluctuations. Fluorescence quantum yields were measured at 25 °C, using three diluted solution of isoA in the corresponding solvent (A<0.1) and three diluted solution of Ru(bpy)3 2+ in air-saturated miliQ water (A<0.1). For each QY measurements, slidth width, excitation wavelength, scan rate, integration time and emission range were keep the same for the reference and the sample. The fluorescence quantum yield was determined thanks to the following formula: Where S is the slope of the linear plot of the integrated fluorescence intensity in function of the absorbance value, n is the refractive index of the solvents (at 25 °C) used in measurements, and the subscripts s and x represent standard and unknown, respectively.
The fluorescence quantum yield for the reference, Ru(bpy)3 2+ in air-saturated miliQ water, was measured to be 0.040 in aerated water with the Horiba K-sphere accessory, in good correlation with the literature values. 4 For solid-state fluorescence, a solution of isoA in DCM was drop-casted on a microscope slide. The emission spectrum was then measured on a XENIUS, SAFAS fluorimeter. The excitation wavelength was fixed at 493nm, the band-width is 10 nm in excitation and emission, integration time = 0.1 s, 1 nm step.

Luminescence lifetime
Lifetime experiments were done using 10 mm path length quartz cuvette and a Fluoromax-4 spectrofluorometer (Jobin-Yvon; Horiba) equipped with a Horiba Scientific Nanoled source N485L. For the excitation we used a LED at 483 nm (950 V) with a pulse duration <200 ps. For MeOH, DCM and CHCl3, the excitation was done with a 370 nm (950 V) Nanoled-370, pulse duration 1,3 ns. Each measurement was made with a pre-set peak of 12 000 hits and the emission was recorded at the fluorescence maximum. The prompt of the LED was recorded using a Ludox® HS-40 colloidal silica from Sigma Aldrich, a solution composed of silica nanoparticles (40% weight) in water, to diffuse the incident light. The experimental decay were fitted with a single or a biexponantial function (for MeOH, Dixoane, and DMSO), and the non-linear least-squares fit was obtained thanks to the Levenberg-Marquardt algorithm. The fit quality was estimated thanks the calculated χ² (The variance of weigh residuals). The fit were estimate relevant for χ² values lower than 1.2.
In case of bi-exponential fit the average lifetime was determined using: with B the pre-exponential factor and T the lifetime of the fit.

Quantum chemical calculations
Ground state geometries were optimized at the B3LYP/6-31+g(d) level of calculation followed by a frequency calculation to confirm the convergence to a local minimum. A TDDFT calculation was done at the PBE0/6-311+g(d,p) level of theory. Then first singlet excited state geometries was optimized at the PBE0/6-311+g(d,p) level followed by a frequency calculation at the same level. Finally, a TDDFT calculation was done on this S1 optimized geometries at the PBE0/6-311+g(d,p) level of theory. Alternatively the N,N-dimethylamino substituent was twisted to 90°C and the TICT state optimized in its first excited state at the PBE0/6-311+g(d,p) level of theory followed by a frequency calculation. All calculations were done with Gaussian 16 (Revision B.01) software. Data were analyzed with GaussView 6.0 software.

Cell culture
Human A549 cancer cell line was obtained from the American type Culture Collection (Rockville, USA) and was grown in RPMI 1640 supplemented with 10% fetal calf serum (FCS) and 1% glutamine according to the supplier's instructions. 2.10 4 A459 cells were plated in Lab-Teck ® chambers and after 48 hours of incubation at 37 °C in a humidified atmosphere containing 5% CO2, the medium was removed and replaced by fresh RPMI 1640 containing the fluorophore at 5 µM final concentration. The cells were incubated for 2 h at 37 °C in a humidified atmosphere containing 5% CO2. The fluorophore solution was then removed and the cells were washed thrice with PBS at RT. A 4% solution of PFA in PBS was then added and the cells were incubated at RT for 15 min until fixation. After removing the PFA solution, cells were washed thrice with PBS (1X) then prepared for microscopy.

Fluorescence microscopy
Once cell fixation was achieved, the culture chambers were removed and the microscope slide was prepared using a Fluoroshield mounting medium. Fluorescence images were acquired using a Leica SP8-X inverted confocal microscope with a 40× oil immersion objective (HC PL APO CS2 Leica). Excitation was performed using a White laser pulsed at 80MHz and excitation was applied at 475 nm. Detection was carried out by using PMT detector (Hamamatsu 6357) collecting photons from 500 nm to 700 nm. Recorded image size was 1024x1024 pixels and a pixel size of 65nm was reach using the zoom factor according sampling laws.

Cytotoxicity Study
Cell viability was determined by a luminescent assay according to the manufacturer's instructions (Promega, Madison, WI, USA). Briefly, A549 cells were seeded in 96-well plates (2.5 × 103 cells/well) containing 90 μL of growth medium. After 24 h of culture, the cells were treated with the tested compounds at 5, 10, 20 and 50 µM final concentrations. Control cells were treated with the vehicle. After 72 h of incubation, 100 μL of CellTiter Glo Reagent was added for 15 min before recording luminescence with a spectrophotometric plate reader PolarStar Omega (BMG LabTech). The percent viability index was calculated from three experiments.