2255-14-3Relevant articles and documents
Toward Automated Enzymatic Glycan Synthesis in a Compartmented Flow Microreactor System
Heinzler, Raphael,Fisch?der, Thomas,Elling, Lothar,Franzreb, Matthias
supporting information, p. 4506 - 4516 (2019/08/20)
Immobilized microfluidic enzyme reactors (IMER) are of particular interest for automation of enzyme cascade reactions. Within an IMER, substrates are converted by paralleled immobilized enzyme modules and intermediate products are transported for further conversion by subsequent enzyme modules. By optimizing substrate conversion in the spatially separated enzyme modules purification of intermediate products is not necessary, thus shortening process time and increasing space-time yields. The IMER enables the development of efficient enzyme cascades by combining compatible enzymatic reactions in different arrangements under optimal conditions and the possibility of a cost-benefit analysis prior to scale-up. These features are of special interest for automation of enzymatic glycan synthesis. We here demonstrate a compartmented flow microreactor system using six magnetic enzyme beads (MEBs) for the synthesis of the non-sulfated human natural killer cell-1 (HNK-1) glycan epitope. MEBs are assembled to build compartmented enzyme modules, consisting of enzyme cascades for the synthesis of uridine 5′- diphospho-α- d-galactose (UDP-Gal) and uridine 5′-diphospho-α-d-glucuronic acid (UDP-GlcA), the donor substrates for the Leloir glycosyltransferases β4-galactosyltransferase and β3-glucuronosyltransferase, respectively. Glycan synthesis was realized in an automated microreactor system by a cascade of individual enzyme module compartments each performing under optimal conditions. The products were analyzed inline by an MS-system connected to the microreactor. The high synthesis yield of 96% for the non-sulfated HNK-1 glycan epitope indicates the excellent performance of the automated enzyme module cascade. Furthermore, combinations of other MEBs for nucleotide sugars synthesis with MEBs of glycosyltransferases have the potential for a fully automated and programmed glycan synthesis in a compartmented flow microreactor system. (Figure presented.).
β-Glucose-1,6-Bisphosphate stabilizes pathological phophomannomutase2 mutants in vitro and represents a lead compound to develop pharmacological chaperones for the most common disorder of glycosylation, PMM2-CDG
Monticelli, Maria,Liguori, Ludovica,Allocca, Mariateresa,Andreotti, Giuseppina,Cubellis, Maria Vittoria
, (2019/10/22)
A large number of mutations causing PMM2-CDG, which is the most frequent disorder of glycosylation, destabilize phosphomannomutase2. We looked for a pharmacological chaperone to cure PMM2-CDG, starting from the structure of a natural ligand of phosphomannomutase2, α-glucose-1,6-bisphosphate. The compound, β-glucose-1,6-bisphosphate, was synthesized and characterized via 31P-NMR. β-glucose-1,6-bisphosphate binds its target enzyme in silico. The binding induces a large conformational change that was predicted by the program PELE and validated in vitro by limited proteolysis. The ability of the compound to stabilize wild type phosphomannomutase2, as well as frequently encountered pathogenic mutants, was measured using thermal shift assay. β-glucose-1,6-bisphosphate is relatively resistant to the enzyme that specifically hydrolyses natural esose-bisphosphates.
Facile enzymatic synthesis of sugar 1-phosphates as substrates for phosphorylases using anomeric kinases
Liu, Yuan,Nishimoto, Mamoru,Kitaoka, Motomitsu
, p. 1 - 4 (2015/02/19)
Three sugar 1-phosphates that are donor substrates for phosphorylases were produced at the gram scale from phosphoenolpyruvic acid and the corresponding sugars by the combined action of pyruvate kinase and the corresponding anomeric kinases in good yields. These sugar 1-phosphates were purified through two electrodialysis steps. α-d-Galactose 1-phosphate was finally isolated as crystals of dipotassium salts. α-d-Mannose 1-phosphate and 2-acetamido-2-deoxy-α-d-glucose 1-phosphate were isolated as crystals of bis(cyclohexylammonium) salts.
