The synthesis of a series of modified mannotrisaccharides as probes of the enzymes involved in the early stages of mammalian complex N-glycan formation

Abstract

A series of mannotrisaccharides were synthesized by two distinct chemical pathways as probes of the enzymes involved in the early stages of mammalian complex N-glycan formation. Methyl (α- d-mannopyranosyl)-(1→3)-[(α- d-mannopyranosyl)-(1→6)]-β- d-mannopyranoside ( 6) and methyl (2-deoxy-2-fluoro-α- d-mannopyranosyl)-(1→3)-[(2-deoxy-2-fluoro-α- d-mannopyranosyl)-(1→6)]-β- d-mannopyranoside ( 8) were rapidly synthesized from unprotected methyl β- d-mannopyranoside ( 12). Methyl (2-deoxy-2-fluoro-α- d-mannopyranosyl)-(1→3)-[(α- d-mannopyranosyl)-(1→6)]-β- d-mannopyranoside ( 7) and methyl (α- d-mannopyranosyl)-(1→3)-[(2-deoxy-2-fluoro-α- d-mannopyranosyl)-(1→6)]-β- d-mannopyranoside ( 9) were synthesized from the common orthogonally protected precursor methyl 2- O-acetyl-4,6- O-benzylidene-β- d-mannopyranoside ( 15). The 2-deoxy-2-fluoro substitution common to trisaccharides 7– 9 renders these analogues resistant to enzyme action in two distinct ways. Firstly the fluorine serves as a non-nucleophilic isostere for the acceptor hydroxyl in studies with glycosyl transferases GnT-I and GnT-II ( 7 and 9, respectively). Secondly it should render trisaccharide 8 stable to hydrolysis by the mannosidases Man-II and Man-III by inductive destabilization of their oxocarbenium ion-like transition states. These analogues should be useful for structural studies on these enzymes.