Cooperative Multipoint Recognition of Sialic Acid by Benzoboroxole-Based Receptors Bearing Cationic Hydrogen-Bond Donors

Sialic acid recognition remains an interesting and challenging target in molecular receptor design. Herein, we report a series of benzoboroxole-based receptors in which cationic hydrogen-bond donors have been introduced and shown to promote multipoint sialic acid recognition. One striking feature revealed by these receptors is that the carboxylate sialic acid residue is the primary binding determinant for recognition by benzoboroxole, in which the presence of charge-reinforced hydrogen bonds results in enhanced selectivity for sialic acid over other carbohydrates and a 4.5-fold increase in affinity. These findings open up wide possibilities for benzoboroxole-based receptors use in life science research, biotechnology, and diagnostics.


Isothermal titration calorimetry binding studies
ITC experiments were performed with 2 mM receptor solution (1 and 5 -11) and 80 mM solution of ligand (2 -4). Five different buffers were used: 0.1 M acetate buffer (pH 5.5), 0.1 M phosphate buffer (pH 6.5), 0.1 M phosphate buffer (pH 7.4), 0.1 M ammonium acetate buffer (pH 8.5) and 0.1 M ammonium acetate (pH 10). The buffer was degassed prior to solution preparation. The pH of the solutions was adjusted with 5 M hydrochloric acid or 5 M sodium hydroxide. The solutions were filtered and degassed prior to use. ITC experiments were performed using a VP-ITC MicroCalorimeter with the parameters reported in Table S1 by titrating the ligand into the receptor solution. The experiments were performed at 25°C. The volume of the first injection for each experiment was 2 µl and it was discarded, whilst the volume of all other injections was set at 7 µl. The spacing and the reference power selected for each experiment were based on the amount of heat realised. For experiment with a small heat release (< 2.5 µcal s −1 ), the spacing was set at 280 s and the reference power at 10 µcal s −1 . For titrations with large heat release (> 10 µcal s −1 ), the spacing was set at 480 s and the reference power at 25 µcal s −1 . Experiments with intermediate heat release had a spacing between 300 and 380 s and a reference power of 15 -20 µcal s −1 . Each experiment consists of 3 titrations and the heat of dilution was measured and subtracted for each experiment. The data was analysed with Origin software for ITC, fixing the number of binding sites to 1. The binding constants, enthalpy and entropy values are reported in Table S2 ,S3 and S4 and are the average of the three measurements, with the error calculated as twice the standard deviation.  Table S2. Isothermal titration calorimetry binding studies of receptor 1 and 2 -10 with sialic acid (2) at pH 5.5 (0.1 M acetate buffer) at 25° C. Each experiment consists of three titrations. The heat of dilution was measured and subtracted. Receptor 129.6 ± 0.8 −4.9 ± 0.1 −6.6 ± 0.2 8 141.0 ± 6.8 −4.6 ± 0.2 −5.8 ± 0.8 9 104.4 ± 5.2 −3.9 ± 0.1 −3.7 ± 0.4 10 110.2 ± 3.4 −3.3 ± 0.1 −1.8 ± 0.3 50.5 ± 2.1 −3.4 ± 0.1 −3.6 ± 0.5 10 60.9 ± 4.3 −2.9 ± 0.1 −1.7 ± 0.5 Table S4. Isothermal titration calorimetry binding studies of receptor 1 and 2 -10 with sialic acid (2) at pH 8.5 (0.1 M ammonium acetate buffer) at 25° C. Each experiment consists of three titrations. The heat of dilution was measured and subtracted. Receptor 28.1 ± 1.9 −3.2 ± 0.  Figure S1. ITC titration of 1 and 2 at pH 5.5 Figure S2. ITC titration of 5 and 2 at pH 5.5 Figure S3. ITC titration of 6 and 2 at pH 5.5 Figure S4. ITC titration of 7 and 2 at pH 5.5 S5 Figure S5. ITC titration of 8 and 2 at pH 5.5 Figure S6. ITC titration of 9 and 2 at pH 5.5 Figure S7. ITC titration of 10 and 2 at pH 5.5 Figure S8. ITC titration of 1 and 2 at pH 7.4 S6 Figure S9. ITC titration of 5 and 2 at pH 7.4 Figure S10. ITC titration of 6 and 2 at pH 7.4 Figure S11. ITC titration of 7 and 2 at pH 7.4 Figure S12. ITC titration of 8 and 2 at pH 7.4 S7 Figure S13. ITC titration of 9 and 2 at pH 7.4 Figure S14. ITC titration of 10 and 2 at pH 7.4 Figure S15. ITC titration of 1 and 2 at pH 8.5 Figure S16. ITC titration of 5 and 2 at pH 8.5 S8 Figure S17. ITC titration of 6 and 2 at pH 8.5 Figure S18. ITC titration of 7 and 2 at pH 8.5 Figure S19. ITC titration of 8 and 2 at pH 8.5 Figure S20. ITC titration of 9 and 2 at pH 8.5 S9 Figure S21. ITC titration of 10 and 2 at pH 8.5 S10 2. NMR spectra Figure

DFT calculations
All the DFT calculations were performed with the Gaussian program. All geometries of benzoboroxole derivatives and sialic acid were optimized by M06-2X/6-31+G(d,p), as shown in Figure 8 and Table S5.

Solvent models: SMD calculations and MD simulations of solutions
The binding modes of the complexes were studied in both gas phase and buffered water solvents, with the results shown in Figures S43, S44, and S45. To investigate the solvent effects on the binding modes, both implicit solvent (using SMD model in Figure S43) and explicit solvent (Figures S44 and S45) models were employed.
Using the implicit SMD model ( Figure S43), binding energies in of the optimized structures were greatly underestimated and did not agree with ITC experimental data. This indicates the limitations of implicit solvent model for this system.  Table S6. The time step was set as 1fs, and the MD trajectory was collected during 500ps, and finally get 50 frames were used for statistical analysis. For the binding between 1 and sialic acid (without counterion), we obtained some similar conformations labelled as A, B, C, and D, as shown in Figure S44, and found that the boron atom is close to the -hydroxyacid rather than the glycerol chain. The minimum distance between boron atom and carboxylate was also shown in Figure S44. For the combinations of 5 with sialic acid, due to the presence of a positive charge in 5, we used counterion chloride to neutralize the periodic cell, and found some favourable conformations (A, B, C, and D) with high occurrence in MD trajectory, as shown in Figures S45. Similarly, the B atom is closer to the -hydroxyacid, which suggests that strong binding interaction between them. We also carried out MD simulations of the possible conformations of complexes 1-2 ( Figure S44) and 5-2 ( Figure S45) in aqueous solution. It can be seen that B atom is also closer to the -hydroxyacid, similar to that in gas phase.  Figure S44. Conformations of complex 1-2 taken from MD trajectories without and with water solvent molecules. Intermolecular distance is also shown. The light blue, pink and dark blue balls represent the B, O and N atoms, respectively.
S24 Figure S45. Conformations of complex 5-2 taken from MD trajectories without and with water solvent molecules. Intermolecular distance is also shown. The light blue, pink, dark blue, and light green balls represent the B, O, N and Cl atoms, respectively

Coordinate files
Cartesian coordinates of the optimized geometries are listed as follows.
SA (the number of imaginary frequencies=0) C