3.2 New Concept for Scaffolds – Linear Lectin Ligands
4.1.9 Trivalent Ligands
The preceding investigations dealt only with divalent and tetravalent ligands. Trivalent species had not been investigated. To complete the picture, two trivalent ligands, 53 and 54 have been synthesized (Figure 32).
Figure 32 Trivalent ligands 53 and 54.
A
C
B
The synthesis followed the same strategy as for the di- and tetravalent peptides. In the trivalent peptides the GlcNAc residues are either connected to directly neighboring D-Dab residues (53) or there is one D -Dab bearing mannose in between (54). Disregarding the direction of the peptide backbone these are the only two possibilities to arrange three GlcNAc residues in this sequence.
The trivalent ligands were also subjected to ITC measurements. The resulting thermograms are shown in Figure 33 B and C. As a comparison the data for divalent ligand 35 is also shown (Figure 33 A). It is obvious that the shapes of the data for the divalent and trivalent ligands differ. The divalent ligand shows a normal sigmoidal shape whereas the curves of trivalent ligands exhibit a stepwise curve with a flattened region at a molar ratio of about n = 2. The evaluation software for the ITC data provides different fitting models. The “One Set of Sites model” described in Chapter 2.4.1 is for a system that contains n identical and independent binding sites. Another available model is the “Two Sets of Sites”
model. Here a system is described that has two kinds of n and m identical and independent binding sites.
As curve fitting method for the thermograms in Figure 33 the “Two Sets of Sites” was used. As shown in Figure 34 A, the curve fit with the “One Set of Sites” model is poor compared to the fit using the
“Two Sets of Sites” model (Figure 34 B). This is due to the higher number of fitted parameters that are used in the “Two Sets of Sites” model.[183] The obtained thermodynamic data is shown in Table 2 for the “Two Sets of Sites” model and in Table 3 for the “One Set of Sites” model.
Figure 33 Thermograms recorded at 25 °C and pH = 7.0 of titrations of (A) ligand 35, (B) ligand 53, and (C) ligand 54 into a solution of WGA.
0 1 2 3 4 5 6
Mechanistic Investigation of High Affinity Glycopeptides Binding to WGA
Figure 34 ITC thermograms for trivalent ligand 53. (A) Fitting with “One Set of Sites” model, (B) Fitting with
“Two Sets of Sites” model.
Table 2 Thermodynamic data for trivalent ligands 53 and 54 derived from the “Two Sets of Sites” model.
Compound Kd (µM) n[a] H [a] Stoichiometry of binding (ligands per WGA dimer).
Table 3 Thermodynamic data for trivalent ligands 53 and 54 derived from the “One Set of Sites” model.
Compound Kd (µM) n[a] H
[a] Stoichiometry of binding (ligands per WGA dimer).
Taking a closer look at the data obtained from the “One Set of Sites” model it can be seen that for example trivalent peptide 54 exhibits a lower binding affinity (Kd = 1.1 µM) compared to divalent peptide 37 (Kd = 0.12 µM). This is remarkable because both contain the same divalent binding motif (Figure 35). Thus a higher binding affinity would be expected due to the higher valency. This further supports the assumption that the “One Set of Sites” model may not be the appropriate choice here.
-0,5 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5
Figure 35 Peptides 37 and 54 that both contain the same divalent motif (marked red).
Examining the data obtained from the “Two Sets of Sites”, fit both trivalent ligands show a similar behavior. There is one high affinity binding event with a Kd of 9 nM (ligand 53) and 32 nM (ligand 54) and a lower affinity event with a Kd of 693 nM (ligand 53) and 342 nM (ligand 54). The binding enthalpies for the higher affinity binding events are also more negative in both cases. Considering the stoichiometries, both models (“One Set of Sites” vs. “Two Sets of Sites”) match with a value of two ligands binding to one protein dimer (if the values for site 1 and site 2 are added in case of the “Two Sets of Sites” model).
The protein contains basically identical binding sites which does not explain these observations. In the ITC experiment the protein solution is placed in the sample cell and the ligand is titrated into this solution. During the first injections there is an excess of protein that means also an excess of binding sites. All free carbohydrates on the ligand should be bound by the protein with a mixed binding mode of crosslinking and chelating being in action (Scheme 7, left). In the course of the titration the point is reached when not all carbohydrates on the ligands can be saturated by the protein. The mixed crosslinking/chelating binding mode is then shifted to a chelating binding mode similar to the binding mode of the divalent peptides: Two ligands bind one protein dimer bridging two adjacent binding sites each as the observed stoichiometry already indicates (Scheme 7, right).
3
3
Mechanistic Investigation of High Affinity Glycopeptides Binding to WGA
This behavior would also explain the step in the titration curve: when the crosslinking is broken up and replaced by a chelating binding mode the third carbohydrate of each bound ligand must be replaced by the additional ligands resulting in a decreased binding affinity and enthalpy which corresponds to the second binding event. Concerning the complex size in solution it should be expected that for substoichiometric amounts of ligand an increased complex size occurs that decreases at higher ratios of ligand to protein dimer. A precipitation assay showed that no protein precipitated, therefore DLS experiments were performed. WGA was incubated with increasing amounts of ligand 54 and after filtration the hydrodynamic radii were determined. The results are shown in Figure 36.
Figure 36 Hydrodynamic radii of WGA in complex with 54 at different ratios of ligand to WGA determined by DLS after filtration through 100 nm cutoff filter.
The hydrodynamic radius indeed increases from 3.0 for WGA alone to 3.9 nm at a ratio of 1:1 (ligand to WGA) and decreases again at higher ratios down to 3.3 nm. These findings support the above described hypothesis although it is worth mentioning that the lowest radius is reached only at a hyperstoichiometric ratio of 4:1.
ITC experiments can also be performed in an “inverse” manner, that means the macromolecule is in the titration syringe and the ligand is in the sample cell. In the specific case of the trivalent ligands a reverse ITC experiment should result in a different curve shape. As the ligand is present in excess from the beginning of the titration and the protein is immediately saturated in a chelating manner until the protein is present in excess (after a ratio of protein dimer to ligand of 0.5). Due to the proposed initial chelating binding mode one GlcNAc residue per ligand should still be free and be able to bind at the end of the titration to the excess of protein. This interaction corresponds to the interaction of a monovalent ligand to WGA. The protein concentration used in the experiment (c(WGA) = 7–9 µM) is far below the Kd
value of GlcNAc (1830 µM). Thus the binding curve should only be affected marginally and a sigmoidal curve progression is to be expected. The binding curves of inverse ITC experiments for ligand 53 and 54 are shown in Figure 37. The curves both have a sigmoidal shape that can be fitted using the “One Set
0 0.5 1
of Sites” model. The binding enthalpy of 34.2 and 37.4 kcal mol–1 (Table 4) corresponds nicely to the data obtained for tetravalent ligands confirming that at the beginning of the inverse titration four GlcNAc residues participate in the binding. Also the Kd values of 68 and 133 nM are comparable to the values obtained for the divalent peptides.
Figure 37 Thermograms of inverse ITC experiments recorded at 25 °C and pH = 7.0 of titrations of WGA into a solution of (A) ligand 53 (c = 9.2 µM) and (B) ligand 54 (c = 6.5 µM).
Table 4 Thermodynamic data of inverse ITC experiments for trivalent ligands 53 and 54 derived from the “One Set of Sites” model.
[a] Stoichiometry of binding (ligands per WGA dimer)
In this chapter a series of di- to tetravalent glycopeptides has been synthesized and the binding properties of the compounds have been investigated by different methods. The combined evidence allowed a detailed insight into the binding mechanisms and revealed relationships between structure and binding characteristics of the peptide ligands.
4.2 New Concept for Scaffolds – Linear Lectin Ligands
4.2.1 Design of Ligands
There are many diseases associated with protein aggregation and precipitation.[184] Alzheimer’s disease involves sedimentation of -amyloid protein, patients of Parkinson’s disease accumulate -synuclein aggregates in the brain[185] and hemoglobin forms precipitates in the blood vessels of sickle cell disease victims[186]. These famous examples illustrate what fatal consequences the aggregation of proteins in the human body may have. The development of high affinity lectin ligands often aims at the creation of potential inhibitors for pathogens as toxins, viruses or bacteria. Many examples of potent lectin ligands exist whose binding mechanisms involve crosslinking or precipitation of the target lectin.[11-12, 35, 79-80, 83, 116, 130-131, 153, 187-188] Reasons for the precipitation are mainly that the geometry of the scaffolds does not allow chelation of the binding sites on the protein, the valency of the lectin is not matched by the ligand or the ligand is polyvalent, as in the case of big dendrimers or polymers, and can easily bind to several copies of a lectin. Thus, it is desirable to design ligands that are capable of binding to the target lectin in a way that excludes heavy crosslinking and subsequent precipitation. This can be basically achieved by a ligand that binds in a chelating manner which should be entropically strongly favored against network formation. WGA offers at least four functional binding sites that are evenly distributed over the protein surface making it an ideal model for the testing of new ligand designs.
Figure 38 (A) Divalent ligand 25. (B) WGA in complex with 25 (PDB ID: 2X52). The ligand is represented by a stick model (black) and the protein is shown as surface representation (gray).
The crystal structure of WGA with divalent ligand 25 that was obtained previously[11] served as a starting point for the investigations (Figure 38). Here, divalent ligand 25 neatly bridges two adjacent primary binding sites making it an ideal fragment for a tetravalent ligand. As mentioned above, the usual way to create ligands of higher valency is to connect the carbohydrates to a central scaffold bearing suitable linkers with functional groups. This scaffold can be manifold reaching from small branched organic molecules, dendrimers, calixarenes, or nanoparticles to polymer scaffolds. In Figure 39 two possible
A B
New Concept for Scaffolds – Linear Lectin Ligands
linkers (structure 55, Figure 39 A). In Figure 39 B a potential ligand based on a central benzene scaffold is shown (structure 56).
Figure 39 Possible tetravalent ligand designs (A) based on ligand 25 and (B) a central benzene scaffold.
Yet there is also another ligand design conceivable. A close examination of the crystal structure of WGA with 25 revealed that the 6-OH groups of the GlcNAc residues are pointing away from the binding pocket. This makes them readily accessible and a modification here should not affect the binding affinity. The feasibility of modifications at this position has also already been demonstrated by using the 6-OH position as an attachment point for spin labels.[133] The easiest and most efficient way to create a tetravalent ligand would be to connect these two 6-OH groups by a linker unit as schematically shown in Figure 40 A. By doing so, two GlcNAc residues would become part of the backbone and all carbohydrates would be lined up in a linear fashion (Figure 40 B). The new concept is therefore termed
“Linear Lectin Ligands” (LLL). This design has so far not been employed for the creation of multivalent lectin ligands. It is especially efficient in molecular size since the integrated carbohydrates fulfil the role of scaffold as well as binding epitope. Simultaneously, the saccharides are anchored to the scaffold at two positions hindering dissociation once bound to the binding pockets.
The implementation of the new ligand design should be carried out in two steps. First, the length of linker 1 (Figure 40 B) of the divalent fragment should be optimized and then the connection of these fragments (linker 2) should be addressed.
Figure 40 (A) Schematic representation of linker between 6-OH positions (dashed line). (B) Design of new tetravalent ligand.
4.2.2 Optimization of Linker 1
In previous studies[11] ligand 25 exhibited a lower affinity in terms of IC50 values than ligand 26 which was one atom shorter (Figure 12). In order to identify the ligand which is most suitable for chelating two binding sites, a series of divalent ligands with different linker lengths was synthesized according to Scheme 8. Active carbonate 39 was coupled to diamines 57–62. The resulting products 63–68 were deacetylated using NaOMe in MeOH resulting in the final products in yields of 17–91 % over two steps.
The ligands were then investigated by isothermal titration calorimetry (ITC). The results are summarized in Table 5.
Table 5 Thermodynamic binding parameters for divalent ligands (25, 26, 69–72) binding to WGA at pH 7.0, 25 °C determined by ITC.
Compound Kd (µM) n[a]L:P H
(kcal mol–1) –TS
(kcal mol–1) G
(kcal mol–1) Kd[c]
GlcNAc 1830 ± 81 4[b] –7.1 ± 0.5 4.4 ± 0.8 –2.6 ± 0.2 1
69 0.697 ± 0.007 2.18 ± 0.10 –11.2 ± 0.3 2.8 ± 0.3 –8.40 ± 0.01 2 630
70 1.92 ± 0.06 1.90 ± 0.04 –13.8±0.1 6.0 ± 0.1 –7.8 ± 0.03 950
26 0.128 ± 0.006 1.51 ± 0.01 –18.2 ± 0.1 8.8 ± 0.1 –9.40 ± 0.02 14 300 25 0.102 ± 0.011 1.79 ± 0.08 –19.3 ± 0.1 9.7 ± 0.03 –9.55 ± 0.05 17 940 71 0.208 ± 0.018 1.55 ± 0.06 –17.2 ± 0.5 8.1 ± 0.5 –9.12 ± 0.04 8 800 72 0.730 ± 0.011 1.70 ± 0.01 –17.6 ± 0.6 9.2 ± 0.6 –8.39 ± 0.01 2 510
New Concept for Scaffolds – Linear Lectin Ligands
Scheme 8 Synthesis of divalent ligands 25, 26 and 69–72 by coupling of carbonate 39 to diamines 57–62.
The Kd values showed a strong dependency on the linker length. The highest Kd of 102 nM was found for ligand 25 that had also been co-crystallized with the protein. Ligand 26, that had performed slightly better than 25 in an ELLA assay[11], now had a somewhat lower affinity of Kd = 128 nM compared to compound 25. Ligands 71 and 72, that again have longer linkers than 25, showed a decreasing affinity of Kd = 208 nM and 730 nM. Interestingly, ligand 69 with the shortest linker 1 had an affinity of 697 nM which is higher than for ligand 70 (1.92 µM). Also the ITC thermogram of ligand 69 (Figure 41 A) shows a different behavior as there is not the usual plateau at the beginning of the titration (Figure 41 B).
Figure 41 ITC thermograms for (A) ligand 69 and (B) ligand 70.
The stoichiometries for all divalent ligands are approximately two ligands binding to one protein dimer or somewhat lower. This suggests a chelating binding mode with only the primary binding sites being occupied as it was also observed for divalent glycopeptide ligands[131]. Precipitation of protein could not be observed. Also the binding enthalpies are about twice the value of GlcNAc further supporting this model. However, to exclude that the ligands do not form crosslinks leading to aggregates, DLS experiments were performed.
The ligands were incubated with WGA at a stoichiometry of two ligands to one protein dimer and a protein concentration of 20 µM. All samples were filtered through a 100 nm cutoff filter prior to measurement. The results are shown in Figure 42. Compounds 25, 26, and 70–72 show a radius of 3.1–
3.3 nm which is similar to the radius of the protein alone (3.1 nm). Only ligand 69 shows a different behavior and forms complexes with a radius of 4.9 nm.
0 1 2 3 4 5
New Concept for Scaffolds – Linear Lectin Ligands
Figure 42 Hydrodynamic radii of WGA (blue), WGA incubated with compound 69 (red) and WGA incubated with compounds 24, 25, 70–72 (green).
Molecular modeling revealed that ligand 69 is not capable of chelating the two binding sites since its linker is too short to reach the second binding site as it is illustrated in Figure 43. The elevated radius observed in DLS measurements confirms this and gives rise to the question how a crosslinked binding mode could be achieved.
Figure 43 Superimposition of ligand 69 (red) onto the crystal structure of 25 (green) with WGA.
To test the feasibility of crosslinked binding modes for ligand 69 molecular modeling was performed (Figure 44). Two X-ray structures of WGA (PDB ID: 2X52) were arranged in close proximity to enable a crosslinking by ligand 69. The carbohydrate residues of two copies of 69 were superimposed on the GlcNAc residues in the binding pockets in a way that each ligand was binding to both proteins (Figure 44 A and B).
WGA WGA + 69
WGA + 70 WGA + 26
WGA + 25 WGA + 71
WGA + 72 0
1 2 3 4 5 6
r / nm
Figure 44 Model of ligand 69 (red, spheres) binding to WGA (gray, surface representation) in a crosslinked binding mode (based on PDB ID: 2X52), remaining binding sites marked black. (A) front view, (B) back view.
It can be seen that the remaining binding pockets are now arranged in a way that does not allow any further crosslinking of the two proteins by ligand 69 (Figure 44 B). That means that two WGA dimers cannot be crosslinked by four molecules of 69 in order to fulfil the observed stoichiometry of two ligands to one protein dimer and at least three WGA dimers must be interconnected by 69 in a crosslinking binding mode. The molecular weight of the complex of WGA with 69 that can be calculated from the hydrodynamic radius is 140 kDa whereas for WGA alone a value of 50 kDa is achieved. This further supports a binding mode involving three WGA dimers as it is depicted in Figure 45 A.
Figure 45 (A) Binding mode of ligand 69 and WGA with a stoichiometry of 3:3 (ligand to protein dimer). (B) Chelating binding mode of divalent ligands. WGA dimers represented by gray ellipses, ligands shown in black.
The linker lengths do not represent the actual dimensions.
In summary all divalent ligands with exception of ligand 69 bind in a chelating binding mode (Figure 45 B) with ligand 25 having the highest affinity. This makes compound 25 an optimal candidate to use as a building bock in the further synthesis.
A B
A B
New Concept for Scaffolds – Linear Lectin Ligands