Chemoenzymatic Synthesis Of Heparin Oligosaccharides With Different Modification Patterns And Investigation Into The Substrate Specificities Of Heparin Lyases

Heparin and Heparan sulfate(HS)are sulfated glycosaminoglycans(GAGs)that have disaccharide repeating units of 1?4-linked a-D-glucosamine(GlcN)and either?-D-glucuronic acid(GlcA)or a-L-iduronic acid(IdoA),each of which is capable of carrying sulfo groups,so their structure is very complex.The diverse repertoire of biological activities and functions result from the abundant oligosaccharide fragments in heparin and HS.Considering the animal sources and structural heterogeneities of heparin,it has been paid more and more attention to synthesize structurally defined heparin and HS oligosaccharides.Although chemical synthesis of oligosaccharides has made great progress,still faces many challenges,such as many steps,low-yield.The newly chemoenzymatic method is expected to be an ideal technology for synthesis of heparin and HS oligosaccharides due to its stereoselectivity and high-yield.Therefore,the present study synthesized heparin oligosaccharides with different modification patterns by using chemoenzymatic method.On the other hand,bacterial heparinases or heparin lyases that can depolymerize heparin and HS by a ?-elimination mechanism are powerful tools in sequencing HS and heparin and in generating oligosaccharide fragments.Heparin and its derivatives were used as substrates for investigation into the catalytic mechanism and cleavage activity of heparinases.The heterogeneity of polymeric substrates could interfere enzymatic degradation activity and increase the difficulty in product analysis,which make it impossible to uncover the detailed differences of heparin lyases in cleaving different cleavage sites.Thus,based on the possible cleavage sites,we synthesized a tailored library of HS oligosaccharides as substrates for more complete understanding of specificity of the enzymes,which will lay the basis for extending the use of heparin lyases.The main results and conclusions of the study are as follows:1.Large-scale preparation of sugar donors and sulfate donorUDP-GlcNTFA or UDP-GlcNAc were synthesized under the catalysis of N-acetylhexosamine 1-kinase(NaHK),N-acetylglucosamine-1-phosphate uridyltransferase(GlmU)and inorganic pyrophosphatase(PPA)by using GlcNAc or GlcNTFA,ATP,UTP as starting material.UDP-GlcA was enzymatically synthesized by UDP-Glc dehydrogenase(UDP-Glc DH)and L-lactate dehydrogenase(LDH)using UDP-Glc as starting material.The sugar donors were prepared at gram scale with a purity of more than 98%.PAPS were synthesized under the catalysis of adenosine phosphokinase and ATP sulfurylase by using Na2SO4 and ATP as starting material.The purified product was at a purity of up to 99%,and prepared in gram scale.2.Synthesis,purification and structure characterization of heparin hexasaccharidesThe synthesis of HS hexasaccharide backbone was initiated from p-nitrophenol glucuronide(GlcA-PNP),and GlcNTFA(GlcNAc)or GlcA was alternately added to the oligosaccharides at the non-reducing end by using N-acetylglucosamine transferase(KfiA)and heparosan synthetase(PmHS2).The yield of every step was up to 98%,the purity was up to 90%after purification by C18 reverse phase chromatography,and preparation reached gram scale.After removal of TFA by LiOH,N-sulfated hexasaccharide was synthesized by using N-sulfotransferase(NST),and PAPS as sulfate donor.And then GlcA residue flanked with two GlcNS residues in oligosaccharide was converted to IdoA2S in one step by using C5-epimerase(C5-epi)and 2-Osulfotransferase(2-OST).At last,6-O-sulfation of GlcNS or GlcNAc of oligosaccharide was completed by the catalysis of 6-O-sulfotransferase-1/3(6-OST-1/3).The yield of heparin hexasaccharides with different sulfated modification was 99%,72%,99%respectively.The purity was up to 99%after purification by ion exchange chromatography and preparation was up to milligram level.The structure was confirmed by ESI-MS and NMR.3.The study of heparin lyases substrate specificityA serial of structurally defined heparin and HS oligosaccharides was synthesized using chemoenzymatic method according to the possible cleavage sites of heparin lyases.Oligosaccharides were determined by HPLC,and confirmed the structure by ESI-MS.And then we analyzed the cleavage of heparin lyases towards HS oligosaccharides under different conditions to investigate the specificity.Meanwhile,the interactions of Hep ? with oligosaccharides substrates were studied preliminarily by SPR.The results were as follows:(1)Hep ? can not cleave the glycosidic bond sites between GlcN and nonsulfated uronic acids(GlcN-GlcA or GlcN-IdoA),and can only cleave the glycosidic bond between-GlcNS-IdoA2S-,-GlcNS6S3S-IdoA2S-and-GlcNS-GlcA2S-.The results indicated that UA2S was recognized as an essential for the scission and 6-0-or 3-0-sulfation had little influence on cleavage of Hep ?.(2)Hep ? was capable of hydrolyzing two HS trisaccharides(Tri-NAc and Tri-NS)that contained one single primary cleavage linkage(GlcNAc-GlcA or GlcNS-GlcA),but the catalytic efficiency on Tri-NAc was much higher than that on Tri-NS.Hep ? can tolerate the secondary cleavage glycosidic linkage between different modification pattern and GlcA,with much lower catalytic efficiency than the counterpart primary trisaccharide.The data suggested that N-unsubstituted or 6-O-sulfated modification decreased reactivity of HS oligosaccharides to enzymatic cleavage of Hep ?,Hep ?had a less preference towards the secondary cleavage GlcNS-IdoA over primary cleavage site GlcNS-GlcA at the beginning of catalytic reaction,but there were a slight difference as a whole.The IdoA2S residues of oligosaccharides significantly decreased the reactivity of Hep ? towards its adjacent GlcNS-GlcA at reducing end,but showed no any influence on its nearby cleavage site at non-reducing end.The susceptibility of the substrate to the digestion of Hep ? was size-dependent,and the catalytic efficiency increased with increasing size of HS oligosaccharides.Hep ?could randomly acted on multiple cleavage sites of HS oligomers,which is consistent with endolytic activity of heparinase,nevertheless,Hep ? showed a preference for cleavage of the internal glycosidic linkages over those near to non-reducing/reducing ends.SPR analysis showed that it was not entirely possible to determine the cleavage efficiency by simply comparing the affinity between the enzyme and the substrates because the complex molecular surface charge and conformation of oligosaccharide,which allowed for incorrect binding of substrates to the enzyme and finally reduced catalytic activity of enzyme on HS oligosaccharides.(3)Hep ? can cleave the oligosaccharides with cleavage sites of both Hep? and Hep?.Hep? acted on oligosaccharides containing the cleavage sites of Hep ?,except the oligosaccharide with-GlcNS6S-GlcA-site,as lower efficiency than Hep ?.Hep? cleaved oligosaccharide bearing cleavage sites of Hep ? as almost the same efficiency,and as higher efficiency than towards GlcNS-GlcA.