Chemoenzymatic Synthesis Of Heparan Sulfate Oligosaccharides And Their Preliminary Analysis By HPLC-MS

Heparan Sulfate (HS) belongs to glycosaminoglycan (GAGs) family, and anticoagulant Heparin (HP) is a special form of HS. HS is widely found on the mammalian cell surface and in the extra-cellular matrix.One or more HS chains are covalently linked to serine/threonine residues of the core protein to form heparan sulfate proteoglycan (HSPG). HS participates in or regulates various biological processes, including blood coagulation, inflammation, virus infection and cell proliferation via selective interaction of various fine oligosaccharides with different heparin binding proteins (HBPs). So, HS oligosaccharideswith well-defined structure would be leading compounds for innovative drug discovery. Because of the complexity in structure, chemical synthesis of HS/HP oligosaccharides has many disadvantages such as more reaction steps, low yield. Recently, a new and promising chemoenzymatic method for diverse HS/HP oligosaccharides has developed by Professor Jian Liu from the University of North Carolina at Chapel Hill based on elucidation of HS/HP biosynthetic pathway.Therefore, we prepared sugar donors including Uridine 5’-diphosphate-N-(trifluoro) acetylglucosamine (UDP-GlcNAc/TFA), uridine 5’-diphosphate glucuronic acid (UDP-GlcA(?)), as well as sulfo donor 3’-phosphoadenosine-5’-phosphosulfate (PAPS) on a large scale using enzymatic methods, chemoenzymatically synthesized a variety of well-defined HS backbone oligosaccharides, as well as N-sulfated and (or) 6-O-sulfated HS oligosaccharides.Finally, a new HPLC-CAD-MS analytical method was established for HS oligosaccharide analysis. The main results of the study are listed as follows:1. Enzymatic synthesis and large-scale preparation of sugar donorsUDP-GlcNAc (or UDP-GlcNTFA) was enzymatically synthesized by N-acetylhexosamine 1-kinase (NahK), N-acetylglucosamine-1-phosphate uridyltransferase (GlmU), and inorganic pyrophosphatase (PPA) expressed by E. coli using N-acetylglucosamine (GlcNAc) (or trifluoroacetylglucosamine, GlcNTFA) as starting material in one step. UDP-GlcA was enzymatically synthesized by UDP-Glc dehydrogenase (UDP-Glc DH) from bovine liver and L-lactate dehydrogenase (LDH). The reaction mixtures were purified by anion exchange chromatography to give the compounds of interest at a purity of>95%. All donors were prepared in gram scale.2. Enzymatic synthesis and large-scale preparationof sulfate donorPAPS were enzymatically synthesized using ATP and sulphate as starting material by adenosine phosphokinase and ATP sulfurylase (KAST-APSK) co-expressed by E coli and PPA in one step.The reaction mixture was purified by anion exchange chromatography to give the compounds of interest at a purity of>90%. PAPS were prepared in gram scale.3. Enzymatic synthesis and characterization of HS backbone oligosaccharidesHS backbone disaccharide was synthesized from p-nitrophenol (PNP) glucuronide (GlcA-PNP) catalyzed by N-acetylglucosamine transferase (KfiA) using UDP-GlcNAc(or UDP-GlcNTFA) as sugar donors.The reaction was monitored by high performance liquid chromatography (HPLC) on a YMC polyamine (PAMN) column, followed by purification by C18 reverse phase chromatography to obtain dimer when the reaction was complete (products> 95%). Then, the resultant disaccharide was elongated by Pasteurella multocida heparosan synthetase (PmHS2) using UDP-GlcA as sugar donor.The reaction mixture was further purified by C18 reverse phase chromatography upon the complete consumption of dimer. Further elongation to the desired size of oligosaccharides was achieved by an additional reaction with the incubation of UDP donors, KfiA, or PmHS2 following the same procedure as described above. The structures were elucidated by ESI-MS and 1H NMR.4. Chemoenzymatic synthesis of N-sulfated HS oligosaccharidesThe HS backbone trisaccharide with GlcNTFA was hydrolized by LiOH to remove the TFA group, then N-sulfated by N-sulfotransferase (NST) using PAPS as sulfate donor. The reaction was monitored by PAMN-HPLC to make sure the yield was>95% and was purified by anion exchange chromatography to obtain N-sulfated trimer (NS-3mer). Its purity was above 99% determined by HPLC.The structure was elucidated by ESI-MS and 1H NMR.5. Enzymatic synthesis of 6-O-sulfated HS oligosaccharidesThe NS-3mer was further 6-O-sulfated by 6-O-sulfotransferase 1/3 (6OST1/3) using PAPS as sulfate donor, the reaction was monitored by PAMN-HPLC to make sure the yield was>95% and purified by anion exchange chromatography to obtain 6-O-sulfated NS-3mer (NS6S-3mer) whose purity was above 99% determined by HPLC. Its structure was elucidated by ESI-MS and1H NMR.Similarly, the N-/6-O-sulfated pentamer and hexamer were also synthesized by following the same procedures as above. Each product had a purity of more than 95% detemined by HPLC.6. Establishment of a HPLC-CAD/MS method for HS backbone oligosacchride analysisThe chromatographic behavior of HS oligosaccharides (dimer to octamer) was analyzed on a PGC column and CAD detector. A new isocratic HPLC-CAD method was established for simultaneous analysis of 7 oligosaccharides.The optimal mobile phase was composed of acetonitrile/H2O/methanol/TFA(48/48/4/0.2, v/v) with 0.4ml/min flow rate at 25 ℃. This method was compatible with mass spectrometry for simultaneous quantification and identification of 7 HS backbone oligosaccharides.