Multistep enzymatic synthesis of nucleotide-activated sugars

UDP-glukoza sintetizirana je četverostupanjskom reakcijom i dalje, kao supstrat, korištena u proizvodnji UDP-glukuronske kiseline. Označavanje UDP-glukoze izotopima nije imalo utjecaja na ovu reakciju i slični prinosi proizvoda dobiven su s neoznačenim i označenim supstratom. Iako se UDP-glukoza proizvela u trećoj reakciji, provedena je četverostupanjska reakcija. Četvrta reakcija u nizu je provedena jer pirofosfat utječe na prinos UDP-glukoze. Uklanjanje pirofosfata pomaknulo je ravnotežu reakcije prema sintezi UDP-glukoze. Sinteza UDP-glukoze bila je vrlo učinkovita uz prinos proizvoda između 86-99%. Konverzija UDP-glukoze u UDP-glukuronsku kiselinu pomoću humane UDP-glukoza dehidrogenaze provedena je uz prinos od oko 100%. Proizvedena UDP-glukuronska kiselina je pročišćena kao amonijeva sol te kao natrijeva sol. Obje soli korištene su u reakciji koju katalizira UDP-apioza/ksiloza sintaza (AXS). U reakciji s amonijevom solju manje UDP-apioze je nastalo nego u reakciji s natrijevom solju. AXS je jednako koristila sve testirane supstrate, neoznačene i označene UDP-glukuronske kiseline.Four-step synthesis of UDP-Glc was performed and synthesized UDP-Glc was used as substrate in production of UDP-GlcUA. In the synthesis of UDP-Glc, isotope-labeling did not have any influence on the reaction and similar yields were obtained with unlabeled and labeled UDP-Glc. Although UDP-Glc was produced in the 3rd reaction, four-step reaction was performed. Since pyrophosphate affects yield of produced UDP-Glc, 4threaction was performed. Removal of a pyrophosphate dragged equilibrium toward UDP-Glc synthesis. Synthesis of UDP-Glc was very successful with theoretical yield between 86-99%. Conversion of UDP-Glc into UDP-GlcUA by human UDP-glucose dehydrogenase was performed with yield of approximately 100%. Produced UDP-GlcUA was purified as ammonium salt and exchanged into sodium salt. Both salts were used for reaction catalyzed by UDP-apiose/xylose synthase (AXS). In AXS catalyzed reaction with ammonium salt less UDP-Api was formed than in the reaction with sodium salt. AXS accepted all substrates, unlabeled and labeled UDP-GlcUA