Chemoenzymatic Modification Of Heparosan And Preliminary Study On In Vitro Anti-adhesion Effect

Heparin(HP)is a member of glycosaminoglycans(GAG)that is composed of disaccharide units either glucuronic acid(GlcA)or iduronic acid(IdoA)linked with glucosamine(GlcN),in which different sites of disaccharide units carried with sulfations,resulting in complex structure of heparin.In clinical practice,heparin is important anticoagulant drugs,and at the same time it was found that heparin could significantly prolong the survival time of cancer patients.Pharmacological studies showed that heparin and its derivatives had significantly anti-metastasis effect in vivo,and the main mechanism was to inhibit tumor cell adhesion,angiogenesis etc.However,the animal-source heparin has a big hidden risk,and the chemical desulfation and acetylation modification may lead to unreliable preparation and products with more complex structure.Therefore,it is urgent to develop a safer and more reliable method for the preparation of heparin and its derivatives.The capsular polysaccharide pro,duced by Escherichia coli K5 is a non-sulfated GAG,whose disaccharide repeating units is composed of GlcA and acetyl glucosamine(GlcNAc),and was regarded as heparin precursor(named as heparosan or N-acetylheparosan,NAH).Therefore,this study is to prepare heparosan from fermentation liquid of E.coli K5,and then selectively modify NAH by chemical treatment and enzyme catalytic reaction to synthesize heparin derivatives with different modification patterns.Finally,the effect of five heparin derivatives on the adhesion of tumor cells in vitro was determined to reveal their structure-activity relationship which will provide a reference for the study of derived heparin of non animal sources as the anti-cancer innovative drugs.The main results and conclusions of the study are as follows:1.Preparation and characterization of heparosan polysaccharidesE.coli K5 was fermented for 18h in a glucose defined medium by shaking flask.The fermentation supernatant was treated by ultrafiltration and alcohol precipitation to obtain the crude heparosan.The crude product was furher purified by strong anion exchange chromatography to obtain purified heparosan.Disaccharide analysis and nuclear magnetic resonance(NMR)confirmed the structure identity of the resulting heparosan.Gel permeation chromatography(GPC)analysis indicated that the purity of heparosan was 98.2%.Its molecular weight was 69.22 kDa based on multi-angle laser light scattering(MALLS)dection.And the preparation scale reached gram level.2.Preparation and characterization of heparin derivatives from non-animal sources.Heparosan was thoroughly N-deacetylated under alkaline conditions,and then N-sulfated with sulfur trioxide-trimethylamine complex to obtain a heparin derivative containing N-sulfated glucosamine(GlcNS)(named as NS-NAH).And according to the diaccharide analysis of the complete hydrolysate by heparinase,the ratio of the unsaturated disaccharide ?U-GlcNS was 97.9%,indicating that most of the GlcNAc residues were modified to GlcNS.Under the catalysis of C5-epimerase(C5-epi)and 2-O-sulfotransferase(2-OST),the GlcA residues of NS-NAH were converted to 2-O-sulfated IdoA(IdoA2S)residues in one step,and the IdoA2S-containing heparin derivatives(named I2S-NS-NAH)was purified by the Q Sepharose strong anion exchange chromatography.The disaccharide analysis showed that the content of?U2S-GlcNAc and AU2S-GlcNS were 0.7%and 90.4%,indicating that the conversion rate of GlcA was over 90%.Under the catalysis of 6-O-sulfotransferase 1 and 3(6-OST1/3),the GlcNS residues of I2S-NS-NAH were 6-O-sulfated(6S),and the reaction was purified by ion exchange column chromatography to give heparin derivatives(named as 6S-I2S-NS-NAH)containing GlcNS6S and GlcNAc6S.The contents of ?U-GlcNAc6S??U-GlcNS6S ?AU2S-GlcNS6S were 0.5%,13.5%and 66.4%respectively,indicating that the 6S modification rate of G1cN was>80%.Another heparin derivative(named as 6S-NS-NAH)was prepared from NS-NAH by 6-OST1/3 catalytic modification and purified by ion exchange chromatography.The content of AU-GlcNAc6S and ?U-GlcNS6S was 0.4%and 87.4%,indicating that the 6S modification rate of GlcN was 87.8%.In addition,6S-I2S-NS-NAH in 0.5M NaOH was frozen at-20 ? for 3d,and then lyophilizated to prepare the IdoA-containing derivatives(named as 6S-I-NS-NAH).The disaccharide analysis showed that 90.5%of IdoA2S was 2-O-desulfated.The structural characteristics of five heparin derivatives were identified by NMR.GPC analysis showed that except 6S-I-NS-NAH,the purity of the other four heparin derivatives was>90%.Each derivative was prepared at milligram scale.3.Anticoagulant activity of heparin derivatives from non animal sources and its effect on tumor cell adhesionThe anticoagulant activity of heparosan and five heparin derivatives was determined by anti-FXa kit.The results showed that the potency of all samples were less than 1%of the reference heparin sodium,suggesting the synthetic products were non-anticoagulant heparin derivatives.Human umbilical vein endothelial cells(HUVECs)were extracted from neonatal umbilical cord and identified by flow cytometry.The cytotoxicity test showed that heparosan and five heparin derivatives with concentration of<200 ?g/ml had no significant cytotoxicity to HUVECs and tumor cells(P>0.05).The five heparin derivatives with a concentration of 200 ?g/ml showed some cytotoxicity(P<0,05)to the B16-F10 murine melanoma cells,while heparosan could not.For human breast cancer cells MDA-MB-231,only 6S-I2S-NS-NAH,6S-I-NS-NAH showed cytotoxicity(P<0.05).And I2S-NS-NAH showed cytotoxicity to HUVECs(P<0.05).The tumor cell adhesion test showed that heparosan and NS-NAH at low,middle and high concentrations(50 ?g/ml,100 ?g/ml,200 ?g/ml),did not exhibit obvious anti-adhesion effects.I2S-NS-NAH exhibited the inhibition of B16-F10 adhesion to HUVECs only at high concentration,while 6S-I2S-NS-NAH,6S-I-NS-NAH,6S-NS-NAH were demonstrated anti-adhesion effect at middle and high concentrations.And heparin sodium showed significant anti-adhesion effect at low,medium and high concentrations.The 6S-I2S-NS-NAH at medium concentration had obvious anti-tumor adhesion(P<0.05),while I2S-NS-NAH did not(P>0.05);6S-NS-NAH had obvious anti-tumor adhesion(P<0.05)at medium and high concentrations(P<0.05),while NS-NAH had no action(P>0.05).Therefore,6-O-sulfation was crucial to the anti-adhesion effect of heparin derivatives.There was no significant difference in the anti-adhesion effects between 6S-I-NS-NAH and 6S-I2S-NS-NAH at difference concentrations(P>0.05),suggesting that the 2-O-sulfation of IdoA had no significant effect on anti-adhesion.In addition,there was also no significant difference in the anti-adhesion effect between 6S-I-NS-NAH and 6S-NS-NAH(P>0.05).Their main difference was uronic acid,which showed that GlcA and IdoA had little effect on the anti-adhesion effect of heparin derivatives.Compared with other derivatives,the synthesis of heparin derivative 6S-NS-NAH from heparosan as raw material was simpler and more productive.Therefore,it is more worthy of further research and development.