Figure 1.
Illustration of the interactions of LecB with 1 and 2 based on the X-ray structures [9], [10], [44] of the carbohydrates with LecB.
To elucidate the individual contributions of substituents adjacent to the ring oxygen, target derivatives 3–7 were designed. A combination of moieties forming attractive interactions with LecB in hybrid structure 4 may lead to synergistic effects for binding. Hydroxy groups necessary for Ca2+-binding are shown in black. Moieties derived from l-fucose are shown in red and from d-mannose in blue. Dissociation constants (Kd) are taken from Sabin et al. [11].
Figure 2.
Synthesis of 1-deoxy glycosides 3, 5 and 7. Reagents and conditions: (a) Amberlite IR120 (H+), MeOH, 65°C, 1.5 d; (b) NaH, DMF, BnBr, 0°C - r.t., 2 - 12 h; (c) TMSOTf, Et3SiH, CH2Cl2, 0°C - r.t., 15 h; (d) Pd/C, H2, EtOH, r.t., 7 h.
Figure 3.
Synthesis of the fucose/mannose hybrid 4 and its derivative deoxyhybrid 6.
Reagents and conditions: (a) Compound 16 was synthesized as reported [20]; (b) Pt/C, H2, HCl, MeOH, r.t., 2 d; (c) NaNO2, AcOH, H2O, r.t., 1 d; (d) NaOMe/MeOH, r.t., 30 min, AcCl/EtSH, 0°C, 1.5 h, r.t., 1 h; (e) Raney Nickel, EtOH, 78°C, 30 min; (f) various conditions following the protocol of Bihovsky et al. [41].
Figure 4.
Evaluation of the complete set of LecB ligands described in this study for competitive binding to LecB using a fluorescence polarization-based assay.
By fitting a one site binding model, IC50 values were obtained (values are depicted in Figure 5). Here, one representative titration of triplicates is shown and error bars were determined by triplicates on one plate. Average IC50 values (see Figure 5) and standard deviations were determined from three independent measurements of triplicates each.
Figure 5.
Biophysical evaluation of LecB-binding to ligands of this study: comparison of isothermal microcalorimetry (ITC) with competitive binding assay.
Means and standard deviations were determined from minimum three independent titrations or from minimum three independent measurements of the competitive binding assay. Binding thermodynamics for 5 were determined by indirect titration with 3 as high affinity ligand in two independent titrations. ITC data for 1, 2, 8 and 13 and IC50 values for 2 and 8 were taken from the references indicated [11], [15], [56]. n.d. = not determined.
Figure 6.
Isothermal microcalorimetry of LecB with novel ligands 3–7 without glycosidic properties.
By fitting a one site binding model, thermodynamic data of the interactions were obtained (values are depicted in Figure 5). Here, one representative titration of independent triplicates is shown. In contrast to the direct titration of LecB with high affinity ligands 3, 4, 6, and 7, binding thermodynamics for the weak binder 5 were determined by indirect titration with 3 as high affinity ligand in two independent titrations.
Figure 7.
Correlation between calculated and experimental binding free energies.
Figure 8.
Overlays of representative structures (ns 6-15 of the molecular dynamics simulations) are shown for four LecB-ligand complexes.
The atom corresponding to C1 of fucose (or C5 of mannose, respectively) is marked as ball in the capped-sticks representation. (A) α-l-fucose (grey) and α-d-mannose (green), (B) α-l-fucose (grey) and 1-deoxy l-fucose (3, brown), (C) α-L-fucose (grey) and hybrid (4, cyan), (D) α-d-mannose (green) and hybrid (4, cyan). The calcium ions are shown as purple spheres. The complexes were aligned on the binding site residues (21–24, 45, 95–104 of the first and 114 of the second monomer).
Table 1.
Relative binding free energies calculated using thermodynamic integration.
Figure 9.
The hydrogen bond between Ser22 and Asp96 is disturbed by α-d-mannose.
Time series of the shortest distance between HG of Ser22 and OD1/OD2 of Asp96 of three independent simulations (grey, orange, blue) of the complex of LecB with α-d-mannose.
Figure 10.
Water density around the ligands in the complex of LecB with l-fucose (left), and d-mannose (closed form: middle, open form: right).
Low, medium and high probability to find a water oxygen atom at a specific position is color coded in blue, yellow and red, respectively. The calcium ions are shown as purple spheres.