Chloride Binding Modulated by Anion Receptors Bearing Tetrazine and Urea

Abstract Modulation and fine‐tuning of the strength of weak interactions to bind anions are described in a series of synthetic receptors. The general design of the receptors includes both a urea motif and a tetrazine motif. The synthetic sequence towards three receptors is detailed. Impacts of H‐bond strength and linker length between urea and tetrazine on chloride complexation are studied. Binding properties of the chloride anion are examined in both the ground and excited states using a panel of analytical methods (NMR spectroscopy, mass spectrometry, UV/Visible spectroscopies, and fluorescence). A ranking of the receptors by complexation strength has been determined, allowing a better understanding of the structure‐properties relationship on these compounds.


General experimental procedures and Materials
Unless otherwise noted, all starting materials were obtained from commercial suppliers and used without purification. N,N-Dimethylformamide (100mL, Anhydrous, 99.8%) was purchased at Sigma-Aldrich. Dichloromethane was distilled over Sodium and under argon. For NMR titrations, deuterated acetonitrile (99.80% D) was purchased in 0.75mL precoated bulbs from Eurisotop®. For photophysical analysis, acetonitrile RS -SPECTROSOL -For optical spectroscopy was purchased from Carlo Erba®. Dichlorotetrazine reagent was synthesized according to a published procedure. [1] Reaction progress was carried out using pre-coated TLC sheets ALUGRAM® Xtra SIL G/UV254 (0.20mm) from Macherey-Nagel® and visualized under 254 and 365 nm UV lamp from Fisher Bioblock Scientific®. Flash chromatography were proceeded using Silica 60M (0.04-0.063mm) for column chromatography silica gel from Macherey-Nagel®.
Crystals suitable for X-ray analysis were obtained by slow evaporation in an NMR tube of a saturated solution of the desired compound in deuterated acetone.

Instrumentation
1 H NMR spectra were recorded with Bruker AV-I 300MHz spectrometer at 298K, referenced to TMS signal and were calibrated using residual proton in Acetone d6 (δ=2.05ppm) or Acetonitrile d3 (δ=1.94ppm), according to the literature. [2] 19 F NMR spectra were recorded with Bruker AV-I 300MHz spectrometer at 282MHz and 298K and were not calibrated. 13 C NMR spectra were recorded with a Bruker AV-I 300MHz spectrometer at 75MHz and 298 K and were calibrated using Acetone d6 (δ = 30.60 ppm). [2] 1 H NMR spectroscopic data are reported as follow: chemical shift δ [parts per million] (multiplicity, coupling constants in Hertz, integration). Multiplicities are reported as follow: s = singlet, d = doublet, t = triplet, q = quadruplet, quint = quintuplet, sext = sextuplet, hept = heptuplet, dd = doublet of doublet, td = triplet of doublet, tt = triplet of triplet, ddd = doublet of doublet of doublet, m = multiplet. 13 C NMR spectroscopic data are reported in terms of chemical shifts δ [ppm] and when it is necessary multiplicity and coupling constant in Hertz.
To check the structure of the product obtained during the synthesis, high resolution mass spectra (HRMS) were obtained with a Waters Xevo QTOF instrument fitted with an electrospray ionization source (ESI+), using Leucine Enkephaline solution as internal calibrant.
Interactions of receptors with the various anions occurring in the gas phase were studied with a 3D ion trap instrument (Bruker Amazon Speed ETD). Complexes were generated in the gas phase by electrospray. To this end, equimolar mixtures of receptors/NBu4Cl were prepared. Starting from 5 10 -2 M stock solutions of receptors and NBu4Cl solubilized in acetonitrile (ACN) and purified water, respectively, 10 -4 M mixtures of receptors/ NBu4Cl (90/10 ACN/H2O) were introduced in the electrospray source by a syringe pump (3 μL/min). Typical experimental conditions were as followed: Capillary voltage: -4500 V; End plate offset : -500 V; Dry gas: 4 L/min / Dry gas temperature: 180 °C, Nebuliser gas : 7.3 PSI ; Cap exit: -140 V; Trap Drive 49.5.
All spectra were recorded in the "Maximum Resolution mode" MS analysis : ICC mode : "off" and acquisition time : manual. MS n analysis : ICC mode off / accumulation time 1 to 5 ms / Isolation window 1 to 7 Da / Fragmentation delay 40 ms/ amplitude of fragmentation : 0.20-1.0 depending on the ions. UV-Visible spectra were recorded at 25°C on a Cary 400 (Agilent) double-beam spectrometer using a 10 mm path quartz cell.
Emission spectra were measured on a Fluoromax-3 (Horiba) or a Fluorolog-3 (Horiba) spectrofluorometer. An angle configuration of 90° was used. Optical density of the samples was checked to be less than 0.1 to avoid reabsorption artifacts.
Fluorescence decay curves were obtained using an Edinburgh instrument LP920 laser flash photolysis spectrometer combined with an Nd:YAG laser (Continuum) doubled at 530 nm via non linear crystals. This second harmonic is optimized to pump an OPO. The fluorescence photons were detected at 90° through a long pass filter (GG385 SCHOTT) and a monochromator by means of a Hamamatsu R928 photomultiplier. The Levenberg-Marquardt algorithm was used for nonlinear least square fit (tail fit) as implemented in the L900 software (Edinburgh instrument). In order to estimate the quality of the fit, the weighted residuals were calculated.
Theoretical UV-Visible spectra were calculated on optimized geometries structures by an energy calculation using time dependant DFT calculation at TD PBE0/6-311+g(d,p)//APFD/6-31+g(d,p) level and solving on 24 first singlet states. A standard solvation model (IEFPCM) for acetonitrile was used. PBE0 was chosen for evaluation of the absorption properties because it gives good estimate for the vertical transition values for a broad range of organic dyes. [3] We have verified previously that it is performing accurately on the tetrazine and urea receptors. [4] Electrostatic Potentials Surfaces (ESP) were thus calculated using Gaussview® software from optimized structures using a fine grid for Total Density and a medium grid of ESP. NCIplots were generated using the NCI method [5] implemented into the MultiWfn Software using a fine grid. [6] Visualization of NCIplots was performed using the Visual Molecular Dynamics VMD Software. [7]

Computational Data
Energies reported, unless noted, are expressed in Hartree.   Figure S1 : General procedure for the synthesis of 1, 2, 3, S1, S2 and S3 compounds 3.1 General procedure A for the synthesis of S1, S2 and S3

Synthetic procedures and characterization data
Inspired by our previously published procedure. [4] Solids (if any) and a magnetic stirrer were added to a round bottom flask under argon atmosphere, followed by dry N,N-dimethylformamide (DMF). Then, liquid reagents were added dropwise to the solution cooled down to 0°C using an ice-bath. Mixture was stirred for a night. Then, the mixture was diluted with water and HCl 1M solution, followed by extraction using ethyl acetate. Organic layers were gathered, dried over MgSO4 and volatiles were evaporated. The crude product was directly purified by flash chromatography.

General procedure B for the synthesis of 1, 2 and 3
Inspired by our previously published procedure. [4] Chloride binding modulated by anion receptors bearing tetrazine and urea -Supplementary information R. Plais

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Compound S (1.0eq), dichlorotetrazine (1.0eq) and a magnetic stirrer were added to a round bottom flask under argon atmosphere. Distilled dichloromethane was added to the flask, followed by 2,4,6-collidine (1.05eq). The mixture was stirred at rt. Volatiles was evaporated and the crude product was directly purified by flash chromatography.

3D ion trap experiments
All spectra were recorded in the "Maximum Resolution mode"

Practical analysis procedure
Previously to each analysis, anion salts were solubilized into acetone and precipitated by addition of diethylether to remove water. Salts were then dried to remove residual solvents and stored in the dessicator until use.
2mL of a solution containing the anion receptor was prepared (3.5mmol/L). 500μL were placed into a new NMR tube. 1mL of stock solution was taken and desired amount of anionic guest as the tetrabutylammonium (NBu4 + ) salt was added.

General practical analysis procedure
Previously to each analysis, anion salts were solubilized into acetone and precipitated by addition of diethylether to remove water. Salts were then dried to remove residual solvents and stored in the dessicator until use.
2.5mL of solution were added to a 1cm quartz glass cuvette. Aliquots of the solution containing the anion and the receptor are subsequently added to the sample cuvette for each measurement.
After blank subtraction, absorbance spectra were measured from 200 to 700nm. From the absorbance spectra were determined the absorbance maximum (510nm), that corresponds to the excitation wavelength for the emission spectra.
Emission spectra were measured from 520 nm (λabs,max+10) to 700nm using the wavelength determined before as excitation wavelength. All experiments were proceeded in temperature-controlled room at 300K.
Fluorescence decay data were analyzed using the Globals software package developed at the Laboratory for Fluorescence Dynamics at the University of Illinois at Urbana-Champaign, which includes reconvolution analysis and global non-linear least-squares minimization method.
Experimental measurements were plotted using Excel software. Determination of binding constants was done using a method developed by Valeur et al. [9] using non-linear least-squares minimization method.

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Emission spectra of reference and compounds were recorded using the maximum absorption wavelength of the reference, Rhodamine-6G, as excitation wavelength. Fluorescence quantum yields ФF were determined using Rhodamine-6G as reference (ФF= 0.91 in ethanol). [12] Figure S49: Quantum yield measurement for 1 (here "compound 3" refers to 1)  Figure S51: Quantum yield measurement for 3 (here "compound 2" refers to 3)                   The molar fraction of the complex xC can be calculated from the expression of the association constant K: