Competitive ITC with Tetravalent Ligands

All tetravalent ligands that have been studied within this work had a binding affinity between 1 and 10 nM. Although the structural differences between for example ligand 32 and 117 and are tremendous and also the binding modes are evidently different there is only a small absolute difference in Kd values (6.2 nM for ligand 32 and 1.6 nM for ligand 117). The c-values with 1300 for 32 and 3200 for 117 are high and the corresponding binding isotherms are very steep. This is suspicious and calls for a closer examination.

As described above competitive ITC allows the investigation of high affinity ligands. In order to apply this on a high affinity multivalent system, ligand 134 (Figure 57, synthesized by Philipp Lohner in his bachelor thesis) was used for a first test experiment as it was easier accessible than the tetravalent glycopeptides. Ligand 134 served as high affinity ligand and as competitor divalent ligand 25 was chosen as it has a lower Kd and a less negative binding enthalpy than ligand 134. The ITC measurement of ligand 135 without added competitor yielded in the data given in Table 10. The binding affinity is similarly high as for the other tetravalent ligands. This makes it a suitable candidate for competitive experiments. Competitive ligand 25 in different concentrations was incubated with WGA. The ratio of 25 to WGA dimer ranged from 0.2:1 (Figure 58 B) up to 1.7:1 (Figure 58 E), i.e. it was in all cases lower than the stoichiometry of 1.8:1 that had been determined for 25 and WGA (Table 5). The resulting solutions were used for the ITC experiments with ligand 134. The results are shown in Figure 58.

Figure 57 Tetravalent ligand 134 based on a tetrahydroxybenzene scaffold.

Table 10 Thermodynamic binding parameters for tetravalent ligand 134 binding to WGA at pH 7.0, 25 °C [a] Corresponding to dimeric WGA, [b] relative binding affinities compared to GlcNAc.

-30

Competitive ITC Experiments

Interestingly, the competing ligand has no effect on the slope of the binding isotherm which remains steep for all measurements. However, the curve is shifted with respect to the stoichiometry. At low concentration of 25 there is almost no difference to a normal ITC experiment (Figure 58 A). At the highest concentration of competing ligand 25, saturation is reached already after the third injection of 134 (Figure 58 D). One explanation would be that the competing ligand is binding so strongly in a chelating manner that ligand 134 is not able to replace it. The divalent substructures of 134 do not have the optimal distance for a chelation of adjacent binding sites and should have a lower affinity than 25 in a divalent system. Only the presence of the additional sugars is responsible for the high affinity of 134 when measured alone. Another reason can be found in the competing ligand. A requirement for the competing ligand is not only a lower affinity but also the binding enthalpy must differ clearly from the ligand to be measured. If this is not the case, the competing ligand may be replaced by the high affinity ligand but all binding enthalpy is then used to release the competing ligand. This results in a net heat of zero that cannot be detected. Here, in case of the divalent ligand 25, four GlcNAc residues in total bind to the protein (due to the stoichiometry of 2:1, ligand to protein dimer). Also the tetravalent ligand bears four GlcNAc moieties. These all bind with basically the same binding enthalpy to the protein which results in a situation similar to the one described above although the absolute binding enthalpies of the individual ligands are different. Since the competing ligand is present in substoichiometric amounts there are still unoccupied binding sites. The binding of 134 to these free sites is responsible for the signals in the thermograms. However, with increasing concentration of competitor the number of free binding sites decreases and saturation is reached earlier during the titration (Figure 58 B–D).

Although the same problem should occur using GlcNAc, competitive experiments with GlcNAc as competitor were performed. The binding isotherm for ligand 134 binding to WGA in presence of GlcNAc (12 mM) is shown in Figure 59.

Figure 59 Thermogram of 134 binding to WGA in presence of GlcNAc. [134] = 175 µM, [WGA] = 21 µM,

Now the binding isotherm is not shifted along the x-axis. The slope is flat and the binding enthalpy is considerably reduced as it would be expected for a normal competitive experiment. Until now no suitable model exists to analyze and fit such a multivalent competitive system. Yet the reduced apparent Kd values obtained from a “One Set of Sites” fit of different ligands could be compared. All tetravalent ligands were examined using GlcNAc as a competitor and evaluated using the “One Set of Sites” model.

The resulting Kd values are summarized in Table 11.

Table 11 Apparent Kd values of tetravalent glycopeptides (20, 31, 32) and LLLs (111–117) Compound Kd, app (µM) n^[a]L:P tetravalent glycopeptides. The stoichiometries stay constant, indicating that the binding mode does not change compared to the normal ITC experiments. The experiments for glycopeptides and LLLs (Table 11) had been performed independently and the conditions (concentrations of protein and competitor) differ. Therefore, the experiment has been repeated exemplarily for compound 31, 111, and 117 at the same conditions. The results are shown in Table 12.

Table 12 Apparent Kd values of selected tetravalent ligands (31, 111, 117) binding to WGA (c = 36.2 µM) in presence of 10 mM GlcNAc.

Compound Kd, app (µM) ^[a]Kd,app

31 602 1

111 124 9

117 13 46

Competitive ITC Experiments

In the competitive experiment linear lectin ligand 117 shows a 46-fold higher affinity than tetravalent linear glycopeptide 31. This confirms the assumption that the resolution of the ITC in the low nanomolar range is problematic. It also demonstrates the power of the new ligand design that increases the binding affinity compared to the tetravalent glycopeptide system significantly.