Study On Chemoenzymatic Semi-synthesis Of Low Molecular Weight Heparin And Its Preliminary Anticoagulant Activity

Heparin(HP)is a natural anticoagulant substance that has been used clinically for more than 80 years and has played an irreplaceable and important role to date.At present,commercial heparin mainly includes unfractionated heparin and low molecular weight heparin,of which unfractionated heparin(UFH)is mainly extracted from animal organs such as porcine intestinal mucosa and bovine lung.UFH can be partially depolymerized by physical,chemical or enzymatic methods to obtain different low-molecular-weight heparins(LMWHs)with a weight-average molecular weight(Mw)of less than 8000 Da.LMWH has the advantages of long half-life in vivo,high bioavailability,low side effects and safe use,etc.In recent years,it has gradually replaced UFH as the preferred clinical anticoagulant drug.LMWH derived from animal organs has the advantages of abundant raw materials,relatively low cost,and mature production technology.However,the quality control of such animal-derived heparin with complex structure is difficult and may be contaminated,and there are greater safety risks in clinical application.Considering the importance of heparin in clinical application,the search for new non-animal-sourced heparin has received widespread attention.Microorganisms such as E.coli K5 can produce a unique capsular polysaccharide(K5CPS),which is considered to be an unmodified heparin precursor polysaccharide with different weight average molecular weights.Therefore,K5CPS is expected as a raw material to produce non-animal sources of heparin and derivatives.Therefore,we combined the partial depolymerization of heparinase III to K5CPS with the chemoenzymatic modification technology to establish a new semi-synthetic method for low molecular weight heparin that had typical structural characteristics of heparin and significant anticoagulant activity.The method can efficiently prepare non-animal-sourced low molecular weight heparin(naLMWH)with good structural uniformity and strong anticoagulant activity.The main results and conclusions of the study are as follows:1.Large-scale preparation of K5CPS and PAPSE.coli K5 was fermented in a fermenter,and the supernatant of the fermentation broth was concentrated by ultrafiltration and low-temperature alcohol precipitation to obtain crude K5CPS,which was purified by strong anion exchange column chromatography to obtain a pure product with a yield of about 503 mg/L.After characterizing K5CPS,it was found that its structure was consistent with the previous report,its purity was 95.4%,its weight average molecular weight(Mw)was 118.1 kDa and the polydispersity index(PDI)was 1.298.The preparation scale could meet the needs of further experiments.The synthesis of PAPS was improved by combining its previous synthesis process with membrane separation technology.The scale of the reaction was expanded by 10 times with only increasing to 1 time for the amount of enzyme.It was found that a small amount of PAPS in the initial solution was helpful to the reaction.The enzyme solution and effluent obtained by membrane separation could be used repeatedly,and the PAPS concentrate obtained had no enzyme,few raw materials and by-products,and could be used directly without purification.The improved method not only reduced the amount of enzyme and improved the yield of PAPS,but also reduced the purification step,and the cost was greatly reduced.2.Chemoenzymatic semi-synthesis of low molecular weight heparin and determination of anticoagulant activityThe capsular polysaccharide K5CPS was chemically N-deacetylated/N-sulfonated for the N-acetylglucosamine(GlcNAc)residues and was converted into a N-sulfated glucosamine(GlcNS)-containing NS-K5CPS with a yield of 88.4%.The disaccharide composition of NS-K5CPS was AU-GlcNAc and AU-GlcNS with a molar percentage of 4.5%:95.5%.The purity was 93.6%,Mw=29.84kDa,PDI=1.553,and its structure was correct.NS-K5CPS was partially depolymerized by heparinase III to obtain low molecular weight product(LNS-K5CPS)with a yield of 62%that had the same disaccharide composition as NS-K5CPS.Its purity was 91.7%,Mw=3.276kDa,PDI=1.178,and its structure was correct.LNS-K5CPS was catalyzed by C-5 epimerization enzyme(C5-epi)and heparin 2-O-sulfate transferase(2OST),PAPS as the sulfate donor,to convert its glucuronic acid(GlcA)residue into 2-O-sulfated iduronic acid(IdoA2S)to obtain I2S-LNS-K5CPS with a yield of 39%.The disaccharide composition of I2S-LNS-K5CPS was △U-GlcNAc,△U-GlcNS,△U2S-GlcNS with a molar percentage of 2%:33%:65%,and its purity was 90.3%.The structure was consistent with expectations.I2S-LNS-K5CPS,using PAPS as the sulfate donor,was further catalyzed by heparin 6-O-sulfate transferase(6OST),resulting in 6-O-sulfonation modification(GlcNS6S)of its glucosamine(GlcNS)residue to obtain 6S-I2S-LNS-K5CPS with a yield of 73%.Its disaccharide composition was △U-GlcNAc,△U-GlcNS,△U-GlcNS6S,△U2S-GlcNS,△U2S-GlcNS6S with a molar percentage of1%:18%:21%:8%:52%,and its purity was 92.5%.The structure was correct.Finally,6S-I2S-LNS-K5CPS,using PAPS as the sulfate donor,was catalyzed by heparin 3-O-sulfate transferase(3OST)to make its glucosamine undergo 3-O-sulfonation modification(GlcNS6S3S)to obtain non-animal-sourced LMWH(naLMWH)with a yield of 72%.The disaccharide composition of naLMWH was △U-GlcNAc,△U-GlcNS,△U-GlcNS6S,△U2S-GlcNS,△U2S-GlcNS6S with a molar percentages of 1%:18%:21%:8%:52%;and that of the enoxaparin was △U-GlcNAc,△U-GlcNS,△U-GlcNS6S,△U2S-G1cNS,△U2S-GlcNS6S with a molar percentages were 10%,5%,15%,5%,and 65%,suggesting their similar disaccharides and the main△U2S-GlcNS6S disaccharide.The Mw of naLMWH was 4.021kDa,PDI=1.076,and the proportion of Mw<8000Da was 98.9%,which was consistent with the generally specified LMWH.The Mw of enoxaparin was 5.261kDa,PDI=1.073,and the proportion of Mw<8000Da was 95.3%.HSQC spectrum found that naLMWH had the typical structural information necessary for animal-derived heparin to exhibit anti-FXa activity.In addition to the typical structure of heparin,enoxaparin also had more unnecessary structural signals,so the prepared naLMWH was more homogeneous.The anti-FXa and anti-FIIa activities of naLMWH were determined by chromogenic substrate method.The anti-FXa activity was 87.25± 1.62 IU/mg and the anti-FIIa activity was 23.02±0.26 IU/mg.The present naLMWH was prepared using raw material with controllable quality and low pollution risk under the mild and efficient reaction conditions.The resulting reaction product is easy to separate for preparing homogeneous LMWH with strong anticoagulant activity.3.Optimization of preparation process of naLMWHWe optimized C-5 epimerization/2-O-sulfonation of the preparation of naLMWH that were slightly lower yields and unsatisfactory reaction.Mainly the time of chemical N-deacetylation of K5CPS was optimized to prepare five kinds of NS-K5CPS products of N-deacetylation 5h,7h,8h,9h and 10h.The N-sulfonation ratios of the five products were 90.7%,94.4%,98.3%,99.2%and 99.9%.On this basis,we explored the effect of products containing different ratios of-GlcNAc-GlcA-and-GlcNS-GlcA-on partial depolymerization of heparinase III and C-5 epimerization/2-O-sulfonation modification.It was found that the time of chemical N-deacetylation of K5CPS was preferably 8h-10h.C-5 epimerization/2-O-sulfonation modification reaction and 6-O-sulfonation modification reaction were sequentially conducted without the purification step between the two reactions.The 6-O-sulfonation reaction was purified to obtain 6S-I2S-LNS-K5CPS directly.The combination of the two reactions simplified the LMWH semi-synthesis step,saved costs,and provided conditions for industrial mass production of naLMWH.