Design, synthesis and application of carbazole macrocycles in anion sensors

Carboxylate sensing solid-contact ion-selective electrodes (ISEs) were created to provide a proof-of-concept ISE development process covering all aspects from in silico ionophore design to functional sensor characterization. The biscarbazolylurea moiety was used to synthesize methylene-bridged macrocycles of different ring size aiming to fine tune selectivity towards different carboxylates. Cyclization was achieved with two separate strategies, using either amide synthesis to access up to –[CH2]10– macrocycles or acyl halides to access up to –[CH2]14– macrocycles. Seventy-five receptor–anion complexes were modelled and studied with COSMO-RS, in addition to all free host molecules. In order to predict initial selectivity towards carboxylates, 1H NMR relative titrations were used to quantify binding in DMSO-d6/H2O solvent systems of two proportions – 99.5%:0.5% m/m and 90.0%:10.0% m/m, suggesting initial selectivity towards acetate. Three ionophores were selected for successful sensor prototype development and characterization. The constructed ion-selective electrodes showed higher selectivity towards benzoate than acetate, i.e., the selectivity patterns of the final sensors deviated from that predicted by the classic titration experiments. While the binding constants obtained by NMR titration in DMSO-d6/H2O solvent systems provided important guidance for sensor development, the results obtained in this work emphasize the importance of evaluating the binding behavior of receptors in real sensor membranes.


Chemicals and raw materials
All starting materials and solvents used in syntheses were acquired from commercial sources (Sigma-Aldrich, Acros, Alfa Aesar, Fluka) with at least 97% purity and used as received.
DMSO-d6 for NMR was obtained from Sigma-Aldrich with at least 99.9% purity. Dry solvents were prepared at least 72 h before use in round-bottomed flasks supplied with 3 Å molecular sieves under an argon atmosphere.
All inert gases used for syntheses were of at least 5.0 purity. Water used in this work was obtained from a MilliQ Advantage A10 system or from an ELGA PURELAB® Ultra system.
For sensor development, the following chemicals were of Selectophore TM grade: bis ( [3]

Relative binding affinity measurements
Binding of anions in solution was studied using our previously published relative 1 H NMR titration method. [4] For sample preparation, a few milligrams of receptors were weighed into a NMR tube alongside an anchor molecule, to which binding had previously been quantified using absolute binding measurements. [4][5][6] The compounds were dissolved in 700 µL DMSO- During the titration, a blank spectrum of pure receptors was initially obtained. Then, the analyte was added using an automatic titration syringe. The chemical shifts of the formed host-guest complexes were recorded for each titration step. The dilute solution was used during the first part of the titration, where complexation was partial. The concentrated sample was used towards the end of the titration to ensure full complexation of all host molecules. The recorded chemical shifts of all titration steps were used to calculate the differences of affinity to the anchor molecules, as described in reference [4]. Spectra of all titration experiments are available as supporting information.

Preparation of the membrane cocktails
The solvent polymeric membranes were prepared using PVC, DOS as the plasticiser, and TDMACl as the anion exchanger. The ion-selective membranes (ISMs) were prepared with the following composition as the target: 2 wt % ionophore, 50 mol % TDMACl relative to the ionophore, 65 wt % DOS, and 32 wt % PVC. A control membrane was prepared with no ionophore, 0.7 wt % TDMACl, 66 wt % DOS, and 33 wt % PVC. The membrane components were dissolved in THF to produce membrane cocktails containing 83 wt % THF. See Table S1 for the final compositions of the membrane cocktails.

Preparation of the sensor prototypes
Glassy carbon rods (3 mm diameter) encased in PVC (8 mm

Potentiometric measurements
The sensors were conditioned in 0.01 M sodium acetate prior to all of the potentiometric measurements. Liquid junction potentials were estimated using the Henderson equation.
Activity coefficients were estimated using the extended Debye-Hückel equation.

Synthesis and characterisation of compounds
Compound 2 was prepared as in reference [7]. Compounds 6-8 were prepared based on reference [8], but with slight modifications (please refer to Scheme 1 in main text).
[10] was modified as follows: A 100-mL pressure tube (withstanding a maximum pressure of 10 bar at 120 °C) was charged with a stirring bar, 1,8-dibromo-3,6-di-tert-butyl-9H-carbazole (compound 4, 2.00 g, 4.57 mmol) and CuI (261 mg, 1.37 mmol). DMSO (24 mL) was added and argon gas was bubbled through for 5 minutes while the mixture was magnetically stirred. Under argon counterflow, N,N'-dimethylethylenediamine (221 µL, 2.06 mmol) was added, and argon was bubbled through the mixture for an additional minute. After that, the argon flow was stopped and the flask was closed with a screwcap. The contents were magnetically stirred for 20 minutes and then cooled in a water bath (10-15 °C). The stirring was stopped and the screwcap removed, then under an argon counterflow, 12 mL of 33% aqueous NH3 solution were carefully S9 added to the mixture by tube sidewalls without disturbing the contents. Argon flow was removed and the pressure tube was tightly closed with a screwcap. The mixture was vigorously stirred in an oil bath at 130 °C for 18 h. After that, the tube was cooled to approximately 15 °C and its contents were diluted with 100 mL of ethyl acetate. The mixture was filtrated and brine (250 mL) was added to the filtrate. Phases were separated and the aqueous phase was extracted with ethyl acetate (3 × 50 mL). The combined organic phases were washed with brine, dried over anhydrous Na2SO4, filtered and solvents removed by evaporation. The resulting residue was triturated with 10 mL DCM, then 100 mL of n-hexane was added and the mixture cooled in an ice bath. The precipitate was filtered and washed three times with cold n-hexane/ DCM 10:1 mixture to yield tan crystals (1.20 g, 3.88 mmol, 85%), the structure of which was confirmed to be identical with literature data [8].
General procedure for preparation of diacyl chlorides (ClCO(CH2)nCOCl, n = 11-14): An oven-dried 10-mL round-bottomed flask was charged with a dicarboxylic acid (1 mmol) and a magnetic stirring bar. Then, 2 mL of dry DCM and 1 drop of dry DMF were added. The flask was flushed with argon and closed with a septum through which an argon-filled balloon was connected. The mixture was stirred and thionyl chloride (0.29 ml, 4 mmol) was added dropwise through the septum via syringe. After 4 h of stirring at room temperature the reaction was complete by 1 H NMR analysis. Solvents and residual SOCl2 were evaporated, the residue dried in vacuo and used in further experiments without additional purification.

Preparation of macrocyclic receptors MC011-MC014
An oven-dried 100-mL round-bottomed flask was charged with a magnetic stirring bar and 1,3bis (8-amino-3,6-di-tert-butyl-9H-carbazol-1-yl)urea (8)  was washed with 20 mL of 10% aqueous K2CO3 and the phases were separated. The aqueous phase was extracted with DCM (3 × 10 mL) and the combined organic phases were dried on anhydrous Na2SO4, filtered, and the solvent evaporated. The residue was purified by column chromatography eluting with DCM-MeOH to yield the desired macrocycles as white to offwhite crystals in 25-44% yields. Please refer to Table S2 for exact reaction conditions for MC011-MC014.