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Heparin molecule containing AT binding sequence and continuous 2-O-glucuronic acid residues, preparation method and application thereof

A technology of residues and heparin, which can be applied in drug combinations, extracellular fluid diseases, blood diseases, etc., can solve the problems of poor pharmacokinetics of compounds, catalytic modification efficiency lower than that of IdoA residues, unrealistic problems, etc., to achieve good results Industrial application prospects, strong and specific anti-FXa activity, and the effect of high-quality anticoagulant and antithrombotic drugs

Active Publication Date: 2021-11-02
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the unique elastic conformation of IdoA, the IdoA2S-GlcNS6S-rich polyanionic sugar chains are prone to specific or nonspecific binding to different proteins, which may lead to poor pharmacokinetics of the compound subcutaneous injection and potential side effects related to heparin therapy (such as thrombocytopenia, nonspecific bleeding), etc.
[0006] According to literature reports (US9951149; J Biol Chem, 2014,289:13407-18), heparin 2-O-sulfotransferase (2OST) can recognize sugar chain-specific IdoA or GlcA residues and carry out 2-O-sulfation Modification, based on the substrate specificity of the enzyme, it has been possible to efficiently prepare heparin molecules containing multiple 2-O-sulfated IdoA (IdoA2S) residues; however, the efficiency of catalytic modification of GlcA residues by 2OST enzymes is much lower than that of IdoA residues Therefore, it is unrealistic to efficiently prepare heparin molecules with different numbers of consecutive GlcA2S residues by using the existing chemoenzymatic strategies
Whether "rare" heparin sequences that do not contain consecutive IdoA2S residues can exhibit anticoagulant activity that can be reversed by protamine has not yet been reported.

Method used

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  • Heparin molecule containing AT binding sequence and continuous 2-O-glucuronic acid residues, preparation method and application thereof
  • Heparin molecule containing AT binding sequence and continuous 2-O-glucuronic acid residues, preparation method and application thereof
  • Heparin molecule containing AT binding sequence and continuous 2-O-glucuronic acid residues, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1: Preparation of rare heparin pentasaccharide 5 containing 2 consecutive GlcA2S

[0080] Weigh 500mg of nitrophenyl-β-D-glucuronide (GlcA-PNP, 1) and dissolve it in ~200mL 50mmol / LTris-HCl buffer (containing 6mmol / L MnCl 2 , pH=7.5), while adding 1.2 times the equivalent of UDP-GlcNTFA and 5mL KfiA enzyme, stirring at room temperature overnight, the reaction was detected by PAMN-HPLC, and the chromatographic conditions were 0→100% KH within 45min 2 PO 4 Gradient elution, the flow rate is 0.5mL / min, and the detection wavelength is 310nm. When the yield is ≥95%, adjust the pH to 2-3 with trifluoroacetic acid (TFA) to stop the reaction, and the reaction solution is purified with a C18 chromatography column (3.0×50 cm), eluting with methanol-water containing 0.1% TFA, and the yield is to the target component. The obtained disaccharide skeleton was placed in 200 mL of the same buffer as above, while 1.2 equivalents of UDP-GlcA and 5 mL of PmHS2 enzyme were added,...

Embodiment 2

[0085] Example 2: Preparation of rare heptasaccharide 7 containing 3 consecutive GlcA2S

[0086] Take ~200mg of pentasaccharide skeleton 3, and refer to Example 1 to carry out enzymatic sugar chain extension by KfiA and PmHS2 in sequence to obtain heptasaccharide skeleton 6, whose molecular weight as measured by ESI-MS is consistent with the theoretical value; then LiOH is used to remove trifluoroacetyl, NST catalyzed N-sulfation modification to obtain heptasaccharide 7, the purity of which was >99% as measured by PAMN-HPLC, and the molecular weight as measured by ESI-MS was 1566.32Da.

[0087] The obtained heptasaccharide 7 was modified by 2OST enzymatically with reference to Example 1, the amount of PAPS added was changed to 4 times, and the reaction solution was purified to obtain product 8, whose purity was >99% as measured by PAMN-HPLC. ESI-MS measured that its component was 1805.92Da, and compared with substrate 7, three sulfuric acid groups were added. by 7 1 H-NMR (6...

Embodiment 3

[0090] Example 3: Preparation of rare heparin nonasaccharide 13 containing 4 consecutive GlcA2S

[0091] Take 100 mg of rare heparin pentasaccharide 5 containing 2 GlcA2S residues, carry out enzymatic sugar chain extension by KfiA and PmHS2 to form heparin nonasaccharide 11 with reference to Example 1, purify on a Q Sepharose chromatography column (30×1.6 cm), and perform ESI- The molecular weight measured by MS is consistent with the theoretical value; then LiOH is treated to remove trifluoroacetyl, NST is catalyzed for N-sulfation modification to obtain heparin nonasaccharide 12, and its purity is >99% as measured by PAMN-HPLC, and its The molecular weight is consistent with the theoretical value.

[0092] The obtained heparin nonasaccharide 12 was modified by 2OST enzymatic method with reference to Example 1, and the reaction solution was purified by Q Sepharose chromatography column (30×1.6cm) to obtain product 13, whose component was 2303.2Da as measured by ESI-MS, indica...

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Abstract

The invention relates to a heparin molecule containing an AT binding sequence and a continuous 2-O-glucuronic acid residue, a preparation method and application thereof. The heparin molecule comprises an AT binding sequence and a continuous 2-O-sulfated glucuronic acid (GlcA2S) residue, wherein the structural formula is shown in a formula II. According to the invention, the novel heparin molecule with the determined structure has high-effect specific anti-FXa activity, and does not have obvious anti-IIa activity, the activity of the novel heparin molecule can be reversed by protamine, and the novel heparin molecule is suitable for preparing safer and high-quality anticoagulant and antithrombotic drugs, and has a very good industrial application prospect.

Description

technical field [0001] The invention relates to a heparin molecule containing an AT binding sequence and a continuous 2-O-glucuronic acid residue, a preparation method and application thereof, and belongs to the technical field of biomedicine. Background technique [0002] Heparin is an ancient natural polysaccharide anticoagulant, which belongs to the family of highly sulfated glycosaminoglycans, composed of α-L-iduronic acid (IdoA, ≥80%) or β-D-glucuronide Acid and α-D-glucosamine (GlcN) are polymerized alternately with disaccharide units linked by 1,4-glycosidic bonds, in which IdoA residues are usually 2-O-sulfated (IdoA2S, ≥85%), and GlcA rarely undergoes 2-O-sulfated GlcA (GlcA2S, <5%); more than 80% of GlcN residues are N-sulfated (GlcNS), and a small amount is N-acetylated (GlcNAc) or N-non-substituted ( GlcNH 3 + ), and 6-O-sulfation (GlcNS6S, GlcNAc6S) and / or a small amount of 3-O-sulfation (GlcNS6S3S) commonly occur, making its structure highly heterogeneous...

Claims

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Application Information

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IPC IPC(8): C08B37/10C12P19/26C12P19/18A61P7/02
CPCC08B37/0075C12P19/26C12P19/18A61P7/02Y02P20/55
Inventor 刘纯慧马亚卿张桂姣
Owner SHANDONG UNIV
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