Synthesis Of Long-Chain Heparin Oligosaccharides And Their Interactions With Basic Fibroblast Growth Factor

The anticoagulant drugs heparin(HP)and its structural analogs,heparan sulfate(HS),are sulfated glycosaminoglycans(GAGs),widely distributed on the surface of mammalian cells and extracellular matrix.HP/HS interacts with heparin-binding proteins(HBPs)through a large number of fine oligosaccharide fragments of different structures in the sugar chain.It plays an important regulatory role in coagulation,embryonic development,cell differentiation and tumor development.More than 300 HBPs have been discovered so far,among them are those related to angiogenesis including FGFs,VEGF,HGF,ES,etc.Exploring the structural basis of the interaction between heparin and angiogenesis factors helps to elucidate the antitumor metastasis mechanism of heparin.However,the highly heterogeneous microstructure has always been a great challenge to further elucidate the role of natural heparin in angiogenesis.Recently,the synthesis of heparin oligosaccharides has made considerable progress,many new synthetic methods and strategies have emerged,laying a solid foundation for the preparation of structurally diverse heparin oligosaccharides.In the present study,a series of structurally complex long-chain heparin oligosaccharides were designed and synthesized using the chemoenzymatic strategy established previously.The affinity interaction between heparin oligosaccharides and basic FGF(bFGF)was then investigated using affinity chromatography to reveal the structural basis of the interaction between heparin and bFGF,laying the foundation for the discovery of anti-tumor heparin oligosaccharide novel drugs targeting angiogenesis.The main results and conclusions of the study are as follows:1.Large-scale preparation and structure characterization of sugar donors and sulfate donorUridine 5'-diphosphate glucuronic acid(UDP-G1cA)was synthesized from UDP-Glc(UDP-G1c)with catalysis of UDP-Glc dehydrogenase(UDP-Glc DH)and L-Lactate dehydrogenase(LDH).Uridine 5'-diphosphate-N-trifluoroacetylglucosamine(UDP-G1cNTFA)and uridine 5'-diphosphate-N-acetylglucosamine,(UDP-GlcNAc)was synthesized from N-trifluoroacetyl glucosamine(G1cNTFA)or N-acetylglucosamine(G1cNAc)catalyzed by N-acetylhexosamine 1-kinase(NahK),N-acetylglucosamine-1-phosphate uridyltransferase(G1mU),and inorganic pyrophosphatase(PPA)in one pot.Using adenosine triphosphate(ATP)and sodium sulfate as starting materials,3'-phosphoadenosine-5'-phosphosulfate(PAPS)was synthesized catalyzed by ATP sulfatase-APS kinase(KAST-APSK)and PPA.All the above four donors were purified by strong anion exchange column chromatography.All donors were prepared in gram scale,and the purity of each product by HPLC was>95%.2.Synthesis,purification and structure characterization of heparin hexasaccharidesThe synthesis of heparin hexasaccharide backbone was initiated from p-nitrophenol glucuronide(G1cA-PNP),and G1cNTFA(G1cNAc)or GlcA was alternately added to the oligosaccharides at the non-reducing end by using N-acetylglucosamine transferase(KfiA)and heparosan synthetase(PmHS2).The purity of backbone hexasaccharide was up to 95%after purification by C18 reverse phase chromatograhhy.N-sulfate hexasaccharides(6mer-1)was synthesized by using N-sulfotransferase(NST),and PAPS as sulfate donor.Next,the G1cA residue flanked by N-sulfated glucosamine(G1cNS)was directly converted into 2-O-iduronic acid(IdoA2S)under the catalysis of C5-epi and 2-O-sulfatotransferase(20ST)to give an 2-O-sulfated hexasaccharide(6mer-2)with a yield of 66%.Finally,using PAPS as donor,6-O-sulfate transferase(60ST)catalyzes the sulfation of C6-OH of GlcNS or GlcNAc to obtain 6-O-sulfated heparin hexasaccharide(6mer-3)with a yield of 65%.The 6mer-1,6mer-2,and 6mer-3 were purified from reaction by a strong anion exchange column chromatography respectively.The purity for each product by HPLC was>99%.The scale of preparation was all in the order of one hundred milligrams.Their structures were confirmed by ESI-MS and NMR analysis.3.Synthesis,purification and structure characterization of heparin octasaccharidesThe synthesis of heparin octasaccharide backbone also was initiated from GlcA-PNP,and catalyzed by KfiA and PmHS2.The purity was up to 95%after purification by C18 reverse phase chromatograhhy.N-sulfate octasaccharides(8mer-1)was synthesized by using N-sulfotransferase(NST)with a yield of 66%.Next,we tried to simultaneously modify two reactive GlcA in heparin octasaccharide and successfully obtained 2-O-sulfated HS octasaccharide(8mer-2)containg two IdoA2S residues with a yield of 40%.Finally,under the catalysis of 60ST,6-O-sulfated heparin octasaccharide(8mer-3)was obtained with a yield of 37%.Besides,we directly modified 8mer-1 by 60ST to obtain 6-O-sulfated heparin octasaccharide containing G1cA(8mer-4)with a yield of 64%.Each product was purified by a strong anion exchange column.The purity by HPLC was over 95%.Their structures were confirmed by ESI-MS and NMR analysis.4.Synthesis,purification and structure characterization of heparin decasaccharidesThe heparin nonasaccharide backbone also was initiated from GlcA-PNP,and catalyzed by KfiA and PmHS2.The resulting nonamer was N-sulfated by NST and then elongated by KfiA to obtain heparin decasaccharides(10mer-1)in a 60%yield.Finally,under the catalysis of 60ST,6-O-sulfated heparin decasaccharide(10mer-2)was obtained from 10mer-1 in 52%yield.Each product was purified by a strong anion exchange column,and the purity was over 95%by HPLC.Their structures were confirmed by ESI-MS and NMR analysis.5.The interaction between heparin oligosaccharide and FGF2The differences in the binding force between the long-chain oligosaccharides and the heparin-binding domain of FGF2 was determined by affinity chromatography.Preliminary revealed the possible structural features crucial for binding of heparin oligosaccharides with FGF2,that is,The size,2-O or(and)6-O-sulfate groups of oligosaccharides might affect in various degrees the affinity of oligosaccharides with FGF2.