Modifying the terminal phenyl group of monomethine cyanine dyes as a pathway to brighter nucleic acid probes

Three novel monomethine cyanine dyes were synthesized carrying electron donating groups to obtain even brighter nucleic acids probes.


Experimental Procedures
All reagents were used as purchased apart from 1,2-dichloroethane which was distilled and stored in fridge under 3 Å molecular sieves.K2CO3 was dried at 120 °C overnight to make sure it was anhydrous.NMR spectra were measure with Bruker Avance III 500 MHz NMR spectrometer.Accurate MS spectra were measured with Agilent 6560 LC-IMMS-TOF mass spectrometer.
Spectroscopy measurements were done in room temperature with Hellma 110-QS-absoption cuvettes or 111-QS-fluoresence cuvettes with 10 mm light path.pH was kept at constant 7.6 in TE buffer and nucleic acid solutions.Calf thymus (ct) DNA was purchased from Sigma-Aldrich and stored in fridge.Ethanol was chosen as an organic solvent to investigate the absorption coefficient to ensure stable dye concentration by choosing a solvent with higher boiling point than previously used DCM. 1 Ethanol is also used to extract the SYBR Green I from DNA after imaging is completed. 2Absorption spectra were measured with Perkin Elmer 650 UV-vis spectrometer.Varian Cary Eclipse fluorescence spectrometer was used to collect emission and excitation data.Dry DMSO was used to prepare stock solutions of the dyes in 19.6 mM concentration.Viral RNA was extracted as previously described. 12. Synthesis Procedures 2.1.General synthesis procedure for the quinolines Synthesis was performed based on procedure reported by Ying. 3 4-methylcarbostyril (6, 1.0 g, 6.3 mmol), copper powder (7, 2.3 g, 36.5 mmol), anhydrous K2CO3 (8, 0.88 g, 6.3 mmol) and substituted 4-iodobenzene (9-11)* were mixed in oven dried flask equipped with reflux condenser and sealed with CaCl2-tube.Dry DMF (15 ml) was added, and mixture was stirred vigorously to dissolve all compounds.Resulting mixture was heated to 160 °C on an oil bath and stirred overnight.In the morning, mixture was allowed to cool to room temperature and filtered through a pad of Hyflo Super gel.Solvent was evaporated resulting to a black oil.Water and ethyl acetate were introduced to the oil.Mixture was filtered again through a pad of Hyflo Super gel to remove formed solid.After filtering, organic layer was washed with water and brine and dried with Na2SO4.Solvent was evaporated and resulting solid was dissolved again in a mixture of 1:4 hexane and ethyl acetate and purified with flash column chromatography using the Hex/EtOAc mixture as eluent.*Used compounds are listed below for each individual synthesis.

1-(4-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)phenyl)-4-methylquinolin-2(1H)-one (17)
Tosylation of triethylene glycol monomethyl ether (PEG) was performed as described by Nguyen et al. 4 PEG (13, 1.724 ml, 10.0 mmol) was dissolved in pyridine (1.724 ml) at 0 °C.ptoluenesulfonyl chloride (14, 2.2878 g, 12.0 mmol) was dissolved in 3.6 ml of pyridine and added to the reaction mixture.Resulting pale yellow solution was stirred at 0 °C for four hours.After mixing, ice and 25 ml of 6 N HCl was added to the thick orange solution.DCM was introduced to the mixture and the water phase was extracted three times with more DCM.Organic phases were combined and finally washed with 2 N HCl.Resulting organic phase was dried with Na2SO4 overnight.Solution was filtered and solvent was evaporated.Product 15 was collected as a pale yellow oil.Yield 3.101 g (9.7 mmol, 97 %) Tosylated PEG (15) was used as such without further purification steps.Pegylation of 4-iodophenol was done according to the procedure published by Nguyen et al. 4 4-iodophenol (10, 2.2 g, 10.0 mmol) was dissolved in 70 ml of acetonitrile.Formed light pink solution was heated to 80 °C on an oil bath.To the hot mixture, K2CO3 (8, 1.6 g, 12.0 mmol) was added.Resulting cloudy mixture was stirred vigorously before adding the tosylated PEG (15, 3.1 g, 9.7 mmol).Resulting mixture was refluxed overnight under N2-atmosphere.In the morning white solution was allowed to cool to room temperature after which solvent was evaporated under reduced pressure.Water and DCM were introduced to the residue.Aquatic phase was extracted three times with DCM.Organic phases were combined and washed three times with NaOH-solution (pH 12).Organic layer was dried with Na2SO4 after which the solvent was evaporated.Desired product was collected as pale yellow oil.Yield 3.18 g (8.7 mmol, 86.8 %).Pegylated 4-iodophenol was used as such in the next step without further purification.

Fig. S3. Pegylation of 4-iodophenol 10.
Pegylated quinolinone was synthesized accordingly to the general method.Pegylated 4iodophenol from previous step was used as substituted 4-iodobenzene (16, 3.18 g, 8.7 mmol).Column was not needed to purify the compound.After washing with water and drying organic phase, solvent was evaporated, and product was isolated by filtering the residue through cotton.Product 17 was collected as brown oil.Resulted product was used in the next step without further purifications.Yield with small impurities 1.

General method for the dye synthesis
Previously synthesized quinoline* (12, 17 or 18, 0.438 mmol) was dissolved in distilled 1,2-DCE in N2 atmosphere.To the stirred solution, POCl3 was added (19, 123 µl, 1.31 mmol).Resulting mixture was heated to 70 °C on an oil bath and mixed overnight.On the morning, solvent was evaporated and resulting oil (20, 21 or 22) was dissolved again in dry DCM.Oxazolium (23, 0.1399 g, 0.438 mmol) was prepared as described previously 1 and added to the stirred solution under N2 atmosphere.Resulting mixture was stirred at room temperature for four hours.After that, solvent was evaporated and resulting oil was purified with flash column chromatography using 10 % MeOH/DCM as eluent.Eluent was evaporated from the fractions containing product.Resulting oil was dissolved in acetonitrile and excess HNMe2 (5 ml) was added to stirred solution.Resulting mixture was stirred overnight.Water and DCM was added to the solution and aquatic phase was extracted three times with DCM.Organic phases were combined, dried with Na2SO4 and finally solvent was evaporated.Product was collected from flash column chromatography using 10 % MeOH/DCM as eluent.*Synthesis reported above for compounds 12, 17 and 18.

X-ray Diffraction
Suitable single crystals for X-ray diffraction analysis were obtained via slow evaporation of DCM / CH3CN mixture (1:1 for 1) or p-xylene / CH3CN (1:1 for 3) at rt. Single-crystal X-ray data were collected at 120 K by a Rigaku XtaLAB Synergy-R diffractometer equipped with a HyPix-Arc100 detector and an Oxford Cryostream 800 cooling system using mirrormonochromated Cu-Kα radiation (λ = 1.54184Å).Data collection and reduction for all complexes were performed with the program CrysAlisPro 5 and analytical face-index absorption correction method was applied. 5The structure was solved with intrinsic phasing method (SHELXT) 6 and refined by full-matrix least-squares based on F 2 using SHELXL-2019. 7nisotropic displacement parameters (ADPs) were introduced for all non-hydrogen atoms.The hydrogen atoms were placed in idealized positions and included as riding, except H atoms of water molecules were found from the electron density maps.Isotropic displacement parameters for all H atoms were constrained to multiples of the equivalent displacement parameters of their parent atoms with Uiso(H) = 1.2 Ueq (C) or Uiso(H) = 1.5 Ueq (O).In structure 1•3H2O antibumping restraints 6 (DFIX −1.4,s = 0.02) were applied for the H-O-H angles in water molecules to prevent H atoms to end up in too close distances from each other.The O-H distances were also restrained to be more equal (DFIX 0.84, s = 0.01).In 3•0.325CH3CN•0.5H2Othe bond distances of CH3CN molecule were made structurally reasonable by hard restraints (DFIX 1.46 and 1.16, s = 0.001).The X-ray single crystal data and experimental details as well as CCDC numbers are given below.

General
The geometry calculations for OxN-NMe2 (1), OxN-OMe (3) and PyrON 8 were done at the M06-2X/def2-TZVP level of theory 9 and the SPARTAN20 program 10 with acetonitrile (dielectric = 37.50) as a solvent using conductor like polarizable continuum model (C-PCM). 11,12The initial models were built using SPARTAN20 and optimized at MM-level before the DFT calculations.The optimized structures with relevant geometrical parameter are given in Fig. S17.

Fig. S16 .
Fig. S16.The residual electron density map around dye 1.The anomalous residual electrondensity was treated as positionally disordered chloride anion (90:10) and three water molecules (one of the with 0.9 occupancy).The 10% chloride anion and 90% water molecule occupy nearly the same position of the unit cell.The charge of the mono-cationic 1 is thusbalanced with two chloride anion positions with 90% and 10% occupancy.

Fig. S18 .
Fig. S18.The residual electron density map around two molecules of dye 3 in the asymmetric unit.The anomalous residual electron density was treated as positionally disordered chloride and iodide anions (1.6:0.4),one water and 0.65 acetonitrile molecules.The residual densities close to the disordered acetonitrile molecule are too high to be chloride anions, and they are thus modelled as disordered iodide anions.The iodide anion results in from one of the

Fig. S26 .
Fig. S26.Processing of data from Fig. S21 and Fig. S23 gave Scatchard plots presenting the