Affinity Enhancement by Dendritic Side Chains in Synthetic Carbohydrate Receptors

Dendritic side chains have been used to modify the binding environment in anthracene-based synthetic carbohydrate receptors. Control of length, charge, and branching enabled the positioning of side-chain carboxylate groups in such a way that they assisted in binding substrates rather than blocking the cavity. Conformational degeneracy in the dendrimers resulted in effective preorganization despite the flexibility of the system. Strong binding was observed to glucosammonium ions in water, with Ka values up to 7000 m−1. Affinities for uncharged substrates (glucose and N-acetylglucosamine) were also enhanced, despite competition from solvent and the absence of electrostatic interactions.


Synthesis of Receptors S4
Synthesis of receptor 4 S5 Synthesis of receptor 5 S12 Synthesis of receptor 6 S20 Synthesis of receptor 7 S25 Synthesis of receptor 8 S31 Synthesis of receptor 9 S38

Receptor 5
Protected receptor K 52 (20 mg, 7.08 µmol) was dissolved in DCM (5 mL) and cooled to 0 °C over ice. TFA (0.5 mL) was added dropwise over 5 min and the solution stirred under N 2 for 16 h at RT.   Figure S15. Synthesis of Receptor 6.      Figure S19. Synthesis of receptor 7.

Receptor 9
Protected receptor AA (68 mg, 18.6 µmol) was dissolved in DCM (6 mL) and cooled to 0 °C over ice.       Connections between side-chain CH2 protons M and anthracene protons A/B show that the side-chain termini can approach the entrance to the cavity. However, no connection is observed between M and E, implying that the side-chain remains outside the cavity. Note that protons M show as two signals due slow rotation about the tertiary amide CO-N bond.   (b/c) 20 mM or 154 mM NaCl. The experiment was performed as described under (a) above, except that NaCl was added to both titrant and titrand solutions to achieve the desired concentrations. Note that slightly less NaCl was required for the solution containing the glucosamine, as this solution already contained a small amount of NaCl from the neutralisation (pH = 7, ~0.13 equiv NaOD). The concentration of NaCl was thus held constant throughout these titrations. The experiments on galactosamine at 154 mM NaCl were performed similarly.
The association constants were calculated by entering the change in shift (ppm) of aromatic proton E into a specifically written non-linear least squares fitting program within Excel. This calculates the K a and the limiting change in chemical shift Δδ assuming a 1:1 binding stoichiometry. Good fits were generally observed between experimental and predicted data, supporting the assumption of 1:1 stoichiometry. The programme also calculates errors as standard deviations for K a values calculated from individual data points employing the limiting Δδ.

Fluorescence titration experiments
Fluorescence titration experiments were carried out at 298 K on a PerkinElmer LS45 spectrometer in quartz cuvette (3 mL, 10 mm path length).

S52
Titrations with glucose were performed in PBS buffer solution (pH = 7.1 , 100 mM). Titrations with glucosamine hydrochloride were performed in water at pH = 7; the pH was controlled and checked as

Control of pH and salt concentrations
As described above the titrations with aminosugars were performed at constant pH = 7 without the addition of buffer. It was expected that this should be possible because of the many carboxylic acid groups in the dendritic side chains. These should show a range pK a values from ~5 upwards and should therefore act as internal buffers, reducing the sensitivity of the solutions to adventitious acid/base. To confirm this was the case, a pH titration was performed of receptor 6 vs. NaOH. The results are shown in Fig. S41. The slope of the titration curve at pH = 7 is still quite shallow, demonstrating the buffering effect of the side-chains. For comparison, the plot from a similar titration against acetic acid is also shown (Fig. S41 inset). As expected, in this case the pH moves rapidly through 7 as NaOH is added. Figure S41. pH Titration of receptor 6 (3.4 M) vs. NaOH. The amount of added NaOH is shown in equivalents relative to 6.

S53
At pH = 7 ~14.5 equivalents have been added, implying that implying that ~3.5 carboxylic acid groups remain. The slope of the curve is still shallow at this point, reflecting the buffering effect of the unionised CO2H groups. Inset: A comparison titration against AcOH. In this case the slope is much steeper at pH = 7. Receptor 6 vs. NaOH AcOH vs. NaOH

Table of results
The full set of binding data is given in Table S1 below. In addition to the results given in the main paper, Table S1 includes binding constants to disaccharide substrates (maltose, cellobiose, lactose) and to glucose in the presence of NaCl.                                  Ka too small to be evaluated with reasonable accuracy.
Constraints were used to arrange the complexes in chosen conformations, but were then removed before final minimizations. All the structures in Figures S183-S185 are the result of such unconstrained calculations. S136 Figure S184. Models of 2 (above) and 4 (below) binding glucosammonium 13·H + , featuring salt bridge formation from sidechains on both sides of the receptor. Although the complexes remains intact, the angles of the isophthaloyl spacers (cf. Figure   3) suggest that both structures are significantly strained. S137 Figure S185. Models of 4 (above) and 8 (below) binding GlcNAc 15, both featuring hydrogen bonds (cyan) from a side-chain carboxylate to NH and OH. In 4•GlcNAc the hydrogen bonds survive minimisation but, as a consequence, an isophthaloyl spacer is pulled out of position. In 8•GlcNAc there are no indications that the complex is strained.