Precision Profiling of α-1,4-vs α-1,6-glycosidase activity using synthetic linear and branched glycogen tetramers using HPLC-MS
Year: 2026
Type: preprint
Abstract: Accurately defining glycosidase specificity is often limited by access to well-defined oligosaccharide probes and analytical tools capable of resolving regioisomers at low concentrations. We synthesized two linear and one branched glycogen-mimetic tetrasaccharides, bearing exclusively 1,2-cis, namely α-1,4 or α-1,6 linkages using a reagent-controlled stereoselective glycosylation strategy. Their enzymatic degradation by Homo sapiens glycogen debranching enzyme (HsGDE) and two commercial α-1,4-glucosidases was assessed by HILIC-MS and high-resolution cyclic ion mobility mass spectrometry (cIMS-MS), enabling separation and quantification of substrates and products in the micromolar range. HsGDE selectively cleaved the α-1,6-branch while showing low activity on terminal α-1,6 linkages. The Saccharomyces cerevisiae α-1,4-glucosidase showed high α-1,4 selectivity, whereas the rice enzyme displayed broad promiscuity toward all regioisomers. The cIMS-MS provided rapid baseline separation of the synthetic tetrasaccharides and confirmed the absence of inhibition by non-substrate isomers. This integrated synthetic-analytical workflow offers an efficient platform for rapid annotation of glycosidase specificity and mechanistic studies in glycogen metabolism.
Source: ChemRxiv
Language: English
Subfield: Organic Chemistry
Field: Chemistry
Domain: Physical Sciences
Open Access status: green
OpenAlex ID: W7128793471