*2.2. Anion Binding Studies*

Binding affinities with chloride were determined for Compounds **1**–**8** using 1H-NMR spectroscopy titration techniques, with the results displayed in Table 1. Studies for the quinone receptors **5**–**8** were conducted in pure acetonitrile-*d*3 and for the hydroquinones **1**–**4** in acetonitrile-*d*3/1% DMSO-*d*6 to assist with solubility. The amide hydrogen of the quinone species **5**–**8** was followed and fitted to a 1:1 binding model using Bindfit [40]. For titrations with the hydroquinone species, the change in chemical shift of both the hydroxyl and amide hydrogens were followed where possible. The resultant binding for these compounds proved complex and the data could not be fitted adequately to a 1:1 model. However, fitting to a 2:1 binding model provided a greater than 10 times increase in the quality of fit (covfit, see supporting information), which is evidence in support of the formation of a 2:1 complex in the presence of small amounts of chloride. It is likely that a 1:1 complex is favored as the concentration of chloride in solution is increased following the equilibrium:

$$\text{2H} \overset{+\text{G}}{\rightleftharpoons} \text{H}\_2\text{G} \overset{+\text{G}}{\rightleftharpoons} 2[\text{HG}] \tag{1}$$

The interaction parameter, α, was calculated for the series **1**–**4** and was found to have a value of α > 14 in all cases. Values of α > 1 describe positive cooperative binding [41], and this can be taken as further evidence for the initial favorable formation of a 2:1 complex at low chloride equivalents.

Compound **5** was also titrated with TBACl in acetonitrile-*d*3/1% DMSO-*d*6, the solvent mixture used in the hydroquinone receptor experiments. The more complex 2:1 binding exhibited by receptor **1** means direct comparison is difficult, however both K21 and K11 for **1** are larger than the association constant for equivalent quinone Compound **5** with chloride. Interestingly, the hydroquinone series do not follow the expected trend where increasing the electron-withdrawing power of the motif appended to the amide increases the binding affinities, because a more polarized N-H bond should result in stronger hydrogen bond formation.

Receptor **4** possesses the most strongly electron-withdrawing substituents and the X-ray crystal structure of the free receptor revealed the presence of a surprising alternate conformation, which can be seen in Figure 3b. In this case, a stronger amide hydrogen bond results in a premature switch in the molecule due to the formation of an intramolecular hydrogen bond between the amide proton and the hydroxyl oxygen. This competing interaction blocks the availability of the binding site, adversely affecting chloride affinity with increasing magnitude for receptors with the highest degree of electron-withdrawing substitution. The chloride binding event reverts the conformation back to the anticipated binding mode, which is evidenced by the downfield shift of both hydroxyl protons during the titration experiments. One hydroxyl group is involved in convergen<sup>t</sup> chloride binding with the amide, while the other shifts due to the formation of another intramolecular hydrogen bond with the

amide carbonyl upon altering conformation. The second hydroxyl peak shift would not be expected to be as significant if due only to inductive effects of chloride binding. This secondary hydrogen bond can also be considered to play a part in the reduction in chloride affinity as stronger electron-withdrawing groups are appended. Removing electron density from the amide carbonyl will deplete the strength of this stabilizing secondary hydrogen bond and hence diminish the favorability of the binding mode.

**Table 1.** Overview of the 2:1 association constants for the complexation of hydroquinone receptors **1**–**4** and Cl− (as TBA salt) in CD3CN/1% DMSO-*d*6, their interaction parameters (α), and the 1:1 association constants for the complexation of quinone Compounds **5**–**9** with Cl− in pure CD3CN. The 1:1 association constant for 5 and Cl− in CD3CN/1% DMSO-*d*6 is also reported.


a All errors < 12%. b The interaction parameter (α) is calculated by multiplying *K*21 by 4 and dividing by *K*11. c Titrations performed in CD3CN/1% DMSO-*d*6 at 298K. d Titrations performed in pure CD3CN at 298K.

In comparison, the quinones **5**–**8** have a stronger chloride affinity with increasingly electron-withdrawing appendages. Repulsion between the quinone carbonyls and the approaching chloride anion inhibits binding, and repulsion is reduced when electron density is pulled away from the quinone system. The anticipated intramolecular hydrogen bond between the amide and hydroquinone carbonyl is still expected to interfere with binding, however in this case the chloride binding event is not enhanced by the formation of a new intramolecular hydrogen bond with the amide carbonyl. Instead, it is hindered by additional repulsion between the amide and quinone carbonyls, which is diminished in the presence of more potent electron-withdrawing groups.

The converse trends in binding strengths between the reduced and oxidised forms of the receptors highlight the importance of the relationship between the hydroquinone/quinone couple and the amide carbonyl in dictating chloride affinity. 1H-NMR titrations were also performed for the dimethoxybenzamide Compounds **9**–**12** with TBACl and fitted to a 1:1 binding model. The association constants were all found to be greater than 7 <sup>M</sup>−1, highlighting the importance of the hydroquinone hydroxyl proton in creating a convergen<sup>t</sup> anion binding site. Full titration data and fitting for these species can be found in the Supplementary Materials. The likely presence of an amide intramolecular hydrogen bond in the lowest energy conformers of both the hydroquinone and quinone forms of the receptors sugges<sup>t</sup> that this singular contribution cannot result in the differences in binding affinity reported. A combination of the relative strengths of this intramolecular bond, the convergen<sup>t</sup> nature of the hydroquinone binding cleft and the influence of the amide carbonyl over the stability of the binding mode all must be factored in when considering the stronger binding exhibited by the hydroquinone species.
