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

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 (alpha-D-mannopyranosyl)-(1–>3)-{[}(alpha-D-mannopyranosyl)-(1–>6)]-be ta-D-mannopyranoside (6) and methyl (2-deoxy-2-fluoro-alpha-D-mannopyranosyl)-(1–>3)-{[}(2-deoxy-2-fluoro-a lpha-D-mannopyranosyl)(1–>6)]-{[}beta-D-mannopyranoside (8) were rapidly synthesized from unprotected methyl beta-D-mannopyranoside (12). Methyl (2-deoxy-2-fluoro-alpha-D-mannopyranosyl)-(1–>3)-{[}(alpha-D-mannopyran osyl)-(1–>6)]-beta-D-mannopyranoside (7) and methyl (alpha-D-mannopyranosyl)-(1–>3)-{[}(2-deoxy-2-fluoro-alpha-D-mannopyran osyl)-(1–>6)]-beta-D-mannopyranoside (9) were synthesized from the common orthogonally protected precursor methyl 2-O-acetyl-4,6-O-benzylidene-beta-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. (C) 2004 Elsevier Ltd. All rights reserved.