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

Glycogen debranching enzyme (GDE), together with glycogen phosphorylase (GP), is responsible for the complete degradation of glycogen. GDE has distinct catalytic sites for 4-alpha-glucanotransferase and amylo-alpha-1,6-glucosidase. For the GDE sensitive assay, we previously developed the GP limit fluorogenic branched dextrin Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4(Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4Glc alpha 1-6)Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4GlcPA (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 alpha-1,6-coupling of maltotetraose with Glc(7)-GlcPA). In this study, we attempted to develop an efficient method for the preparation of Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4(Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4Glc alpha 1-6)Glc alpha 1-4Glc alpha 1-4GlcPA (B3/74), which was designed to have the minimum essential dextrin structure for GDE. First, Glc alpha 1-6Glc alpha 1-4Glc alpha 1-4GlcPA (B3/31) was prepared from commercially available Glc alpha 1-6Glc alpha 1-4Glc alpha 1-4Glc. Using alpha-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-alpha-glucanotransferase exhibited high activity toward both B3/74 and B4/84. In addition, we studied the efficient conversion of B3/74 into Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4Glc alpha 1-4(Glc alpha 1-6)Glc alpha 1-4Glc alpha 1-4GlcPA (B3/71), which has the best dextrin structure for the GDE amylo-alpha-1,6-glucosidase.