The enzymic synthesis and N.M.R. characterisation of specifically deoxygenated and fluorinated glycogens

Abstract

The incorporation of several deoxy- and deoxyfluoro- d-glucose analogues into glycogen has been achieved through the action of rabbit muscle glycogen phosphorylase on a number of deoxy- and deoxyfluoro- analogues of α- d-glucopyranosyl phosphate. Time courses for the incorporation of these analogues into glycogen and maltopentaose have been determined, and the introduction of 4-deoxy- or 4-deoxy-4-fluoro- d-glucose units has been demonstrated to terminate after the introduction of one sugar unit per non-reducing terminus. Glycogen analogues containing sugars modified at the 3- and 4-positions have been isolated and characterised by 1H-n.m.r. and 19F-n.m.r. spectroscopy, and the extent of incorporation has been confirmed by integration of the new resonances associated with the incorporated residues. Longitudinal (T 1) relaxation times have been determined for the two 19F-n.m.r. resonances observed for 3-deoxy-3-fluoro-glycogen, and through comparison with the T 1 measured for 4-deoxy-4-fluoro-glycogen, the identity (terminal or internal) of each of these two resonances was determined. Kinetic studies indicate that neither 4-deoxy- nor 4-deoxy-4-fluoro-glycogen can serve as a substrate for glycogen phosphorylase in the direction of glycogen synthesis, proving that these glycogen analogues have been fully substituted. Both of these 4-substituted glycogens are good inhibitors of glycogen phosphorylase.