High-Affinity Anion Binding by Steroidal Squaramide Receptors

Exceptionally powerful anion receptors have been constructed by placing squaramide groups in axial positions on a steroidal framework. The steroid preorganizes the squaramide NH groups such that they can act cooperatively on a bound anion, while maintaining solubility in nonpolar media. The acidic NH groups confer higher affinities than previously-used ureas or thioureas. Binding constants exceeding 1014 m−1 have been measured for tetraethylammonium salts in chloroform by employing a variation of Cram’s extraction procedure. The receptors have also been studied as transmembrane anion carriers in unilamellar vesicles. Unusually their activities do not correlate with anion affinities, thus suggesting an upper limit for binding strength in the design of anion carriers.


Synthesis General methods
All reagents were purchased from commercial suppliers and used without further purification, unless otherwise stated. Anhydrous DCM was dried by passing through a modified Grubbs system, with an alumina column manufactured by Anhydrous Engineering. Anhydrous MeOH was purchased from a commercial supplier and used as received.
Flash column chromatography was performed using silica gel (Fisher brand silica 60 Å particle size 35-70 micron) as the absorbent. Routine monitoring of reactions was performed using precoated silica gel TLC plates (Merck silica gel 60 F 254 ). Spots were visualised under UV light or by staining with phosphomolybdic acid, potassium permanganate, or ninhydrin; R f values are given under these conditions. 1 H, 13 C and 19 F NMR spectra were recorded using a ECP 300, ECP 400, Varian 400, Varian System 500A (carbon sensitive probe) or Varian System 500B (proton sensitive probe) spectrometer. All spectra were recorded at 298 K unless otherwise stated. Chemical shifts (δ) are quoted in parts per million (ppm), coupling constants (J) are quoted in Hz and spectra are referenced to the appropriate residual solvent peak. NMR spectra for characterisation of the bis-squaramide receptors were obtained in CDCl 3 in the presence of two equivalents of Et 4 N + Cl -, conditions which gave wellresolved and reproducible spectra. Mass spectra were recorded on a Bruker microTOF II (ESI), VG Analytical Quattro (ESI) or VG Analytical Autospec (EI). IR spectra were recorded on a Perkin-Elmer Spectrum 100 FT-IR spectrometer. Elemental analysis was carried out by the microanalysis department at the School of Chemistry, University of Bristol. The carbon numbering system for steroids is as below:

Synthesis of new squaramide receptors
The six new squaramides described in this communication were synthesised as outlined in Scheme S1 below, starting from previously reported intermediate A. [1] Intermediates B, C, and 13 were prepared from intermediate A as previously described. [1,2] Known intermediate 14 [3] was prepared according to a modified literature procedure and novel compound D was prepared from intermediate

Binding studies Preparation of Et 4 N + EtSO 3 by neutralisation
While monitoring the pH with an electronic pH meter, an aqueous solution of EtSO 3 H was added dropwise to an aqueous solution of Et 4 N + OHuntil pH 7.00 was observed. The resulting aqueous solution was concentrated in vacuo, maintaining the temperature below 50 °C (this precaution was taken in order to prevent elimination of a Et 4 N + ethyl group). The solution was further dried by lyophilisation yielding Et 4 N + EtSO 3 as a white solid.
General procedure for extraction studies in chloroform by Cram's extraction method [7,8] Et 4 N + EtSO 3 was obtained as above, other Et 4 N + salts were obtained from commercial suppliers. Due to their hygroscopic nature, the salts were dried overnight under high vacuum before use. Receptors were also dried under high vacuum overnight before use. All host solutions were prepared using chloroform that had been deacidified by passage through a flash chromatography column containing activated basic alumina. Guest solutions were prepared using deionised water that had been passed through a Millipore filtration system.
The following is a typical example of an extraction experiment involving receptor 11 and Et 4 N + Cl -.
A solution of squaramide 11 (8.1 × 10 -6 M) in deacidified chloroform was prepared and a known volume (100 mL) was added to a 1 L round bottomed flask. To this organic solution was added an aqueous solution of Et 4 N + Cl -(2.6 × 10 -5 M, 500 mL). A magnetic stirring bar was added to the flask and the flask was immersed in a water bath that was heated to 303 K. After 30 s the flask was stoppered and the contents were stirred vigorously. After 30 minutes stirring was stopped and the two phases were allowed to separate. The majority of the aqueous phase was removed by decantation and the remaining mixture was poured into a separating funnel. The organic phase was separated and filtered through Whatman 1PS hydrophobic filter paper to remove any trace of aqueous phase. The filtrate was concentrated in vacuo and the resulting solid was dried on a high vacuum line. The solid was then dissolved in acetone-d 6 and an excess of tetraphenylphosphonium bromide (TPhP-Br, ~7.5 mM) was added to sharpen peaks and facilitate integration. A 1 H NMR spectrum was collected at 298 K and referenced to the residual solvent peak (δ = 2.05 ppm). The S20 CH 2 signal of Et 4 N + Clwas integrated with respect to the receptor signals to give the guest:host ratio (R), allowing the value of K a to be determined (see equations below).
For the strongest receptors (8-11) the concentration of host solution used was between 7 × 10 - The value for R obtained from the 1 H NMR spectra was used to calculate the extraction equilibrium constant K e : We have to take into account the equilibrium between the unbound Et 4 N + Xguest in the organic phase and the dissociated Et 4 N + and Xions in the aqueous phase, described by: . [8] The binding constant K a is then calculated from K e and K d :

S21
K a values for chloride anion binding by extraction method Table S1. Extraction data and derived association constants of squaramides receptors 6-11 to Et 4 N + Clin CHCl 3. 1 H NMR titrations were also attempted in DMSO-d 6 + 0.5% H 2 O with the aim of obtaining association constants in this medium. However the initial spectra were too broad to obtain accurate peak positions, and hence the data could not be fitted to a binding isotherm.

Transport Studies
General procedure for transport measurements Chloride ion transport was measured using large unilamellar vesicles (LUVs, 200 nm average diameter) composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and cholesterol at a ratio of 7:3. POPC was obtained from Avanti® Polar Lipids, Inc. Extrusion apparatus and 200 nm polycarbonate membranes, were obtained from GC Technology Ltd. All lipid and receptor solutions were prepared using chloroform that had been purified by passage through basic alumina, and all aqueous solutions were prepared using deionised water that had been passed through a Millipore filtration system.
The following is a typical example of an anion transport experiment involving bis-squaramide

S27
Half-lives of fluorescence decay were obtained by fitting the reciprocal transport curves (F 0 /F) from 0-500 seconds to a single exponential decay function (Eq. 1) using Origin 9.0. The half-life was calculated using fit parameter 'b' (Eq. 2). (1) Initial rates of fluorescence decay were obtained by fitting the reciprocal transport curves from 0-500 seconds to a double exponential decay function (Eq. 3) using Origin 9.0, and differentiating at t = 0 (Eq. 4). (3) S28 Chloride transport into vesicles by eicosyl ester squaramides 6-11 Figure S12. Chloride transport into 200 nm vesicles by squaramide receptors 6-11 at a receptor:lipid ratio of 1:2500, and by squaramides 10 and 11 at 1:25000 (broken lines). Transport rates at 1:25000 are roughly 10 times lower than those at 1:2500, suggesting that the relatively poor transport by 10 and 11 is not caused by self-association.