New approach to prepare fluorogenic branched dextrins for assaying glycogen debranching enzyme.

Abstract

Glycogen debranching enzyme (GDE), together with glycogen phosphorylase (GP), is responsible for the complete degradation of glycogen. GDE has distinct catalytic sites for 4-α-glucanotransferase and amylo-α-1,6-glucosidase. For the GDE sensitive assay, we previously developed the GP limit fluorogenic branched dextrin Glcα1-4Glcα1-4Glcα1-4Glcα1-4(Glcα1-4Glcα1-4Glcα1-4Glcα1-6)Glcα1-4Glcα1-4Glcα1-4GlcPA (B4/84, where Glc = D-glucose and GlcPA = 1-deoxy-1-[(2-pyridyl)amino]-D-glucitol). However, B4/84 is not widely available because of difficulties in its chemical synthesis and positional-isomer separation (0.33% yield by α-1,6-coupling of maltotetraose with Glc7-GlcPA). In this study, we attempted to develop an efficient method for the preparation of Glcα1-4Glcα1-4Glcα1-4Glcα1-4(Glcα1-4Glcα1-4Glcα1-4Glcα1-6)Glcα1-4Glcα1-4GlcPA (B3/74), which was designed to have the minimum essential dextrin structure for GDE. First, Glcα1-6Glcα1-4Glcα1-4GlcPA (B3/31) was prepared from commercially available Glcα1-6Glcα1-4Glcα1-4Glc. Using α-cyclodextrin as a donor substrate, cyclodextrin glucanotransferase elongated both the main and side branches on B3/31, while all the glycosidic bonds in B3/31 were left intact. After exhaustive digestion with GP, B3/74 was obtained from B3/31 with 16% yield, a value that is 48-fold greater than that previously reported for B4/84. GDE 4-α-glucanotransferase exhibited high activity toward both B3/74 and B4/84. In addition, we studied the efficient conversion of B3/74 into Glcα1-4Glcα1-4Glcα1-4Glcα1-4(Glcα1-6)Glcα1-4Glcα1-4GlcPA (B3/71), which has the best dextrin structure for the GDE amylo-α-1,6-glucosidase.