Probing the Origin of Affinity in the GM1-Cholera Toxin Complex through Site-Selective Editing with Fluorine.

Carbohydrates regulate an inimitable spectrum of biological functions, yet successfully leveraging this therapeutic avenue continues to be frustrated by low affinities with glycan-specific proteins. A conspicuous exception is the interaction of monosialotetrahexosylganglioside (GM1) with the carbohydrate-recognition domain of cholera toxin from Vibrio cholerae : this is one of the strongest protein-carbohydrate interactions known. To establish the importance of a long-discussed key hydrogen bond between C2 of the terminal galactose of GM1 and the B subunit pentamer of cholera toxin (CTB 5 ), the total synthesis of a selectively fluorinated GM1 epitope was conducted in 19 steps. This process of molecular editing (O δ- H → F δ- ) strategically deletes the hydrogen bond donor while retaining the localized partial charge of the substituent. Comparison of the binding affinity of F-GM1/CTB 5 with native GM1, the GM1 carbohydrate epitope, and meta -mononitrophenyl-α-galactoside (MNPG) revealed a trend that fully supports the importance of this key interaction. These NMR data suggest that F-GM1 binds in a closely similar conformation as native GM1. Crystallographic analyses of the complex also confirm that the OH → F bioisosteric exchange at C2 of the terminal galactose induces a ring conformation that eliminates key hydrogen bonds: these interactions are compensated for by inter- and intramolecular fluorine-specific interactions.