Relevance of the Sugars at Positions D -Dab 2 and D -Dab 7

The previous results have shown, that the conformational preorganization of cyclic peptide 20 is not responsible for the high affinities that were observed. Now the attention was turned to the role of the GlcNAc residues at positions D-Dab² and D-Dab⁷ which were not resolved in the crystal structure of 20 with WGA (Figure 13 B). A series of divalent glycopeptides was synthesized in which two GlcNAc residues were replaced by mannose which is known not to bind to WGA. By still keeping four carbohydrates in the glycopeptides the conformational properties of the ligands should be preserved comparable to the tetravalent analogs.

The divalent ligands were investigated by ITC and the results can be found in Table 1. The divalent analog 30 of cyclic peptide 20 showed a 170-fold lower affinity with a Kd of 1.43 µM. This already demonstrates the strong impact of the additional carbohydrate residues. Also the divalent linear compounds 34 and 35 showed lower affinities with a Kd of 1.25 µM and 0.85 µM compared to their tetravalent analogs 31 and 32. Compound 33 which comprises only the amino acids D-Dab⁴ and D-Dab⁵ showed a comparably low affinity of Kd = 1.44 µM.

To complete the picture also divalent ligands 36–38 were synthesized. Here, one GlcNAc residue was attached to D-Dab⁷ whereas the second one was permuted between D-Dab², D-Dab⁴, and D-Dab⁵ resulting in increased distances between the GlcNAc residues compared to divalent ligands 30 and 33–35 in which the GlcNAc residues were attached to the adjacent amino acids D-Dab⁴ and D-Dab⁵. In case of divalent ligands that are able to bridge adjacent binding sites, the binding affinity should be strongly dependent on the length of the linker unit between two carbohydrates. Indeed, the binding affinity strongly increased for ligands 36 and 37 with Kd values of 0.12 µM and 0.15 µM, respectively. The affinity

Compound

[a]  = relative inhibitory potency

A B

Mechanistic Investigation of High Affinity Glycopeptides Binding to WGA

A striking difference between the groups of tetra- and divalent ligands was also observed with respect to the stoichiometry. For tetravalent ligands 20, 31 and 32 the stoichiometry was approximately one ligand binding to one protein dimer whereas in the divalent case stoichiometries of about two ligands per WGA dimer were obtained. This already indicates that different binding modes must be in action.

Remarkably, the formation of precipitates immediately upon titration of tetravalent ligands into WGA solutions was noticed which was not the case for the divalent ligands. This furthermore emphasizes the different binding modes involving crosslinking in the tetravalent case. The binding enthalpies of 32 to 38.5 kcal mol^–1 of the tetravalent ligands are approximately twice as high as for the divalent ligands (15–

19.6 kcal mol^–1. This indicates that all four GlcNAc residues must be participating in the binding.

4.1.5 Dynamic Light Scattering

The results of the ITC experiments already gave hints to possible binding modes. As already mentioned precipitation was observed for the tetravalent ligands. To further examine what species are present in solutions of WGA and the ligands, DLS experiments were performed. DLS allows the determination of hydrodynamic radii of macromolecules or particles in solution. The ligands and WGA were incubated in a molar ratio of 1:1 (ligand to protein dimer) for the tetravalent ligands and 2:1 for divalent ligands which corresponds to the stoichiometries observed by ITC. Subsequently the samples were filtered through a 100 nm cutoff filter to remove dust and the formed precipitates which would disturb the DLS experiment by strong, diffuse scattering. With the filtrates the DLS experiments were performed. The results are shown in Figure 25 and Figure 26.

Figure 25 Typical intensity distributions of hydrodynamic radii r for tetravalent and divalent ligands, exemplarily shown for mixtures of (A) tetravalent neoglycopeptide 20 and WGA dimer in a ratio of 1:1 (red curve) and (B) divalent cyclopeptide 30 and WGA dimer in a ratio of 2:1 (green curve) in comparison to pure WGA (blue curves) determined by DLS after filtration through a 100 nm cutoff filter.

As shown in Figure 25 the hydrodynamic radius of complexes in solution rises when WGA is incubated with tetravalent ligand 20 from about 3 nm for the protein alone to about 5.2 nm for the mixture. At the same time the radii of species in solutions of WGA and divalent ligand 30 remained the same as for WGA alone.

Figure 26 (A) Mean hydrodynamic radii r (derived from intensity distributions) of species present in solutions of WGA alone (blue bars) and after addition of tetravalent (red bars) and divalent ligands (green bars)

determined by DLS after filtration through a 100 nm cutoff filter. (B) Molecular masses M of the species shown in (A) calculated from the hydrodynamic radii by OmniSIZE 3.0 using the built-in protein model.

In Figure 26 the results for all peptides are summarized. Again the groups of tetravalent and divalent ligands can be clearly distinguished. For the tetravalent ligands the radius rises to 4.5–5.4 nm dependent on the ligand (Figure 26 A). This corresponds to an increase in molecular weight (calculated from intensity distribution based on built-in protein model from the DLS analysis software) from 48 kDa for WGA to 120–177 kDa after addition of a tetravalent ligand (Figure 26 B). For divalent ligands no species with increased radius are detected, that is, no bigger aggregates are formed.

The species in solution were until now only examined at molar ratios corresponding to the stoichiometries determined in ITC. To test whether there is a concentration dependency of the size of the complexes in solution a concentration series of ligand-WGA solutions was prepared and measured using tetravalent ligand 32. The protein concentration was kept constant while the ligand concentration was varied. The results are summarized in Figure 27. At low ratios between 0.5 and 1.5 the radius is biggest with up to 5.1 nm. At higher ratios the radii decrease, yet a complete disappearance of complexes could not be observed. The stability of the complexes was also investigated over time and during a period of 4 d no change in size could be observed.

WGA

Mechanistic Investigation of High Affinity Glycopeptides Binding to WGA

Figure 27 Hydrodynamic radii of complexes of WGA and 32 at different ratios of ligand to WGA.