The design, synthesis and evaluation of high affinity macrocyclic carbohydrate inhibitors

Abstract

Carbohydrate-protein interactions have been investigated for a model system of a monoclonal antibody, SYA/J6, which binds a trisaccharide epitope of the O-polysaccharide of the Shigella flexneri variant Y lipopolysaccharide. The thermodynamics of binding for the methyl glycoside of the native trisaccharide epitope, Rha-Rha-GlcNAc () to SYA/J6 over a range of temperatures exhibits strong, linear enthalpy-entropy compensation and a negative heat capacity change (DeltaC(p)=-152 cal mol(-1) degree(-1)). At 293 K the free energy of association is the sum of favourable enthalpy and entropy contributions (DeltaH=-3.9 kcal mol(-1) and -TDeltaS=-2.9 kcal mol(-1)). Crystal structures for SYA/J6 Fab detailed the position of the native trisaccharide epitope, Rha-Rha-GlcNAc, and facilitated a strategy to design a tighter binding, low molecular weight ligand. This involved pre-organization of the native trisaccharide in its bound conformation by addition of intramolecular constraints (a beta-alanyl or glycinyl tether). ELISA measurements indicated that the glycinyl tethered trisaccharide was not an optimal candidate for further analysis, while microcalorimetry provided data showing that the beta-alanyl tethered trisaccharide displayed a 15-fold increase in affinity for SYA/J6. Tethering resulted in a favourable entropic contribution to binding, relative to the native trisaccharide (-TDeltaDeltaS=-1.2 kcal mol(-1)). Potential energy and dynamics calculations using the AMBER Plus force fields indicated that trisaccharide adopted a rigid conformation similar to that of the bound conformation of the native trisaccharide epitope. While this strategy resulted in modest free energy gains by minimizing losses due to conformational entropy, thermodynamic data are consistent with significant contributions from solvent reorganization.

Conformational constraint in protein ligand design and the inconsistency of binding entropy

“…[33][34][35][36][37] Since then, a handful of studies have appeared in which constrained systems have been examined in a controlled fashion, and which have taken advantage of the availability of calorimetric methods to measure the underlying thermodynamic binding parameters; these have included carbohydrates 38,39 as well as peptides [40][41][42][43] and peptidomimetic scaffolds. [44][45][46] FIGURE 1 Theoretical behavior of conformational entropy in the case of a flexible ligand and corresponding constrained analog.…”

The binding sites of monoclonal antibodies to the non‐reducing end of Francisella tularensis O‐antigen accommodate mainly the terminal saccharide

“…Constraining more glycosidic bonds would be expected to carry a larger entropic penalty. However, studies estimating the penalty, performed with tethered antigens that are restrained to the bound state conformation in solution, have resulted only in modest gains in affinity . Therefore, other factors, such as solvent reorganization, may have a larger effect on protein–carbohydrate binding than conformational restraint.…”

“…However, studies estimating the penalty, performed with tethered antigens that are restrained to the bound state conformation in solution, have resulted only in modest gains in affinity. 52,53 Therefore, other factors, such as solvent reorganization, may have a larger effect on proteincarbohydrate binding than conformational restraint. Hence, the increased affinity of the terminally binding mAbs could be attributed to the complete sequestration of the epitope from solvent, whereas the internal binders would have much of the epitope and antibody still exposed to solvent.…”

Disaccharide‐Containing Macrocycles by Click Chemistry and Intramolecular Glycosylation

“…[1,2] This increasing demand for carbohydrate-containing molecules for biological, pharmaceutical and medicinal studies has led to tremendous efforts to develop novel and facile methods for the synthesis of diverse glycosides. [8] They have also found application in host-guest chemistry, in the study of carbohydrate-carbohydrate interactions [9] or in cyclodextrins or cyclodextrin mimetics, for instance, in glycophanes or their hybrids. [8] They have also found application in host-guest chemistry, in the study of carbohydrate-carbohydrate interactions [9] or in cyclodextrins or cyclodextrin mimetics, for instance, in glycophanes or their hybrids.…”