Short-acting heparin-based anticoagulant compounds and methods

Abstract

To provide synthetic heparin analogues having anticoagulant activity, provide cost-effective methods of synthesizing a new synthetic heparin, and further provide methods of treating a subject in need of anticoagulant activity.SOLUTION: A synthetic heparin analogue comprises: (a) 3-O-sulfated oligosaccharide comprising six to eight saccharide units; (b) at least one disaccharide unit sulfated by a 3-OST-3 enzyme; and (c) at least one ldoA2S-GlcNS3S or ldoA2S-GlcNS3S6S disaccharide unit.SELECTED DRAWING: Figure 1A

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisional Patent Application No. 62 / 469,643, filed March 10, 2017. No. 6,399,413, which is incorporated herein by reference in its entirety. This invention was made in part in part through grant numbers GM102137, HL094463, and CA2078 awarded by the National Institutes of Health. This invention was made with U.S. Government support under grants 10 / 01 / 24 and GM103390. The U.S. Government therefore grants the present invention have certain rights. [Technical field]

[0002] This invention relates generally to the synthesis of heparin compounds. More particularly, the invention relates to the synthesis of heparin compounds in a short period of time. on the chemoenzymatic synthesis of heparin compounds and synthetic heparin analogues with type-acting anticoagulant activity do. [Background technology]

[0003] Heparan sulfate (HS) is a ubiquitous component of cell surfaces and the extracellular matrix. Heparan sulfate plays a wide range of physiological and pathophysiological functions, including embryonic development and blood coagulation. Esko and Selleck (2002) Annu. Rev. Biology ochem. 71, 435-471; Liu and Thorp (2002) Med. Res. Rev. 22, 1-25). Heparan sulfate (HS) by interacting with specific proteins involved in a given process. , exerting its biological effects (Capila and Lindhardt (2002) Angew. Chem. Int. Ed. 4 1, 390-412). Heparan sulfate (HS) is a 1→4-linked heparin that contains both N-sulfo and O-sulfo groups. It is a highly charged polysaccharide composed of glucosamine and glucuronic acid / iduronic acid units. Hep The characteristic sugar sequence within heparan sulfate (HS) can determine the binding specificity of heparan sulfate ( HS) to its target proteins (Linhardt (2003) J. Med. Chem. 46, 2551-256 4). Heparin, a special form of heparan sulfate (HS), is commonly used as an anticoagulant. Therefore, new methods for the synthesis of heparin compounds and heparan sulfate (HS) are of great interest to those developing anticoagulants with improved anticoagulant pharmacological effects and other heparan sulfate (HS)-related drugs. Heparin has been used as an anticoagulant for over 50 years (Mackman, 2008). Currently heparin is sold in three forms: unfractionated (UF) heparin (average molecular weight: ~14,000 Da); low molecular weight (LMW) heparin (average molecular weight: ~6,000 Da); and synthetic ULMW heparin pentasaccharide ARIXTRA® (molecular weight: 1508.3 Da). UF heparin is used in surgery and kidney dialysis due to its relatively short half-life and safety for patients with renal impairment (Hirsh et al., 2 007).

Summary of the Invention

Problems to be Solved by the Invention

[0004] The synthesis of heparan sulfate (HS) oligosaccharides and related anticoagulant compounds remains a challenge. A cost-effective method and approach for synthesizing new heparin are highly desired.

Means for Solving the Problems

[0005] Here, several embodiments of the present invention are shown, and in many cases, variations of these embodiments are shown, such as examples and substitution examples. These are merely illustrative of a large number of various embodiments. A reference to one or more representative features of a particular embodiment is likewise illustrative. Such embodiments can typically exist regardless of the presence or absence of the recited (plural) features, and similarly, these features can be applied to other embodiments of the present invention disclosed herein, whether or not they are shown here. To avoid excessive repetition, not all possible combinations of these features are shown here. Regardless of whether these features are shown here, they can be applied to other embodiments of the present invention disclosed herein. To avoid excessive repetition, not all possible combinations of these features are shown here. features are shown here.

[0006] In some embodiments, a synthetic heparin analog comprising a 3-O-sulfated oligosaccharide containing 6 to 8 sugar units; a disaccharide unit sulfated by at least one 3-OST-3 enzyme; and at least one IdoA2S-GlcNS3S or IdoA2S-GlcNS3S6S disaccharide unit is provided. In some embodiments, such a synthetic heparin analog can have anticoagulant activity. In some embodiments, such a synthetic heparin analog can have a binding affinity for antithrombin in the range of about 5 nM to about 30 nM. In some embodiments, such a synthetic heparin analog can have an anti-Xa activity in the range of about 10 ng / ml to about 40 ng / ml. In some embodiments, the provided synthetic heparin analog comprises at least one IdoA2S-GlcNS3S6S disaccharide unit and a GlcA-GlcNS3S6S disaccharide unit. 50 such that the IC -1 is in the range of about 10 ng / ml -1 to about 40 ng / ml.

[0007] In some aspects, the provided synthetic heparin analog comprises at least one IdoA2S-GlcNS3S6S disaccharide unit and a GlcA-GlcNS3S6S disaccharide unit. In some aspects, the provided synthetic heparin analog comprises at least one IdoA2S-GlcNS3S6S disaccharide unit and a GlcA-GlcNS3S6S disaccharide unit. ​​​It can be free of. In some embodiments, such synthetic heparin analogs The clearance rate can be about 50% to about 100 % faster than the clearance rate of other heparin compounds. In some embodiments, such synthetic heparin analogs Do not cause heparin-induced thrombocytopenia (HIT). In some embodiments , such synthetic heparin analogs are 50% or more reversibly by andexanet alpha in the presence of 20 μg / ml or less of andexanet a certain.

[0008] In some embodiments, (1) providing a glycosubstrate, (2) elongating this glycosubstrate to a sugar of a desired or predetermined length, and (3) performing at least one sulfation reaction using the 3-O-sulfotransferase (3-O ST) enzyme's 3-OST-3 isoform, whereby a synthetic heparin analog is synthesized, a method for synthesizing a heparin-like analog is provided. In some embodiments, this glycosubstrate contains at least 1 IdoA2S-GlcNS3S disaccharide unit. In some embodiments, this glycosubstrate contains the IdoA2S-GlcNS3S±6S disaccharide unit, and this method further includes a 6-O- sulfotransferase (6-OST) using 6-O-sulfation step, where before the 6-O-sulfation step, 3-O-sulfation by 3-OST-3 occurs. In some embodiments, this glycosubstrate contains the GlcA-GlcNS3S6S disaccharide unit, and this method further includes a 6-O-sulfotransferase (6-OST) using 6-O-sulfation step , where before the 6-O-sulfation step, 3-O-sulfation by 3-OST-1 occurs . This is it.

[0009] In some embodiments, this elongation step involves using a glycosyltransferase. In some embodiments, this glycosyltransferase is selected from the group consisting of N-acetylglucosaminyltransferase (KFiA) of Escherichia coli K5 and / or heparosan synthase-2 (pmHS2) from Pasteurella multocida. In some embodiments, this elongation step involves using one or more monosaccharides selected from the group consisting of glucuronic acid (GlcUA), N-acetylated glucosamine (GlcNAc), and N-trifluoroacetylglucosamine (GlcNTFA). In some embodiments, the yield of the method for synthesizing this synthetic heparin analog is from about 20% to about 50%.

[0010] Also provided herein is a method of treating a patient in need of anticoagulation therapy, the method comprising the following steps: (a) providing a patient in need of anticoagulation therapy; (b) administering to the patient a synthetic heparin analog having anticoagulant activity, the synthetic heparin analog comprising at least one disaccharide unit sulfated by at least one 3-OST-3 enzyme; and at least one IdoA2S-GlcNS3S or IdoA2S-GlcNS3S6S disaccharide unit. In some embodiments, the method further comprises monitoring the patient for heparin-induced thrombocytopenia, and administering to the patient an The antidote is andexanet alfa. In some embodiments, the patient Human. In methods of treating such patients, the synthetic heparin analogue is used in combination with other heparinized It has a clearance rate about 50% to about 100% faster than the compound. After a period of time, the anticoagulant activity of the synthetic heparin analogue may be less than about 10%. In this method, the patient may be at high bleeding risk. Also provided herein are pharmaceutical compositions comprising the synthetic heparin compounds disclosed herein.

[0011] The synthetic heparin disclosed herein, including methods of making and / or using same. Analogs can include, for example, structures including: [ka] [ka] (In the formula, R is a hydrogen atom, alkyl (-CH3 or -CH2CH3, etc., but ), substituted alkyl, aryl, and substituted aryl (p-nitrophenyl group) and the like, but are not limited to these.)

[0012] Accordingly, an object of the present invention is to provide short-acting heparin formulations, including novel forms of synthetic heparin. The present invention provides anticoagulant compounds and methods that are based on nicotinamide adenine diphosphate .... This and other objects are achieved in whole or in part by the present invention. The above objects, other objects and advantages of the present invention will become apparent from a study of the following description, drawings and examples. It will become apparent to one skilled in the art after the above. [Brief description of the drawings]

[0013] The present invention will be better understood by reference to the following drawings. The components in the drawings are not necessarily to scale nor are they emphasized, and are for the purpose of explaining the principles of the present invention (often in diagrammatic form). In these drawings, like reference numbers indicate corresponding parts for different drawings. By referring to the embodiments illustrated in the accompanying drawings , the present invention can be further understood. The illustrated embodiments are merely examples of systems for practicing the present invention, but the structure and method of operation of the present invention will both generally be more readily understood from the drawings and their description, together with their purpose and advantages. These drawings are not intended to limit the scope of the present invention as shown in the appended claims or any later amended claims, but merely to clarify and illustrate the present invention. To understand the present invention more fully, reference should be made to the following drawings: For a more complete understanding of the present invention, reference is made to the following drawings:

[0014]

Figure 1

Figure 2

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Figure 5

Figure 6

Figure 7

Figure 8

Best Mode for Carrying Out the Invention

[0015] Hereinafter, the present invention will be more fully described, where some, but not all, of the present invention Embodiments are described. In fact, the present invention can be embodied in many different forms and should not be construed as limited to the embodiments described herein. These embodiments are provided only to meet the legal requirements to which this disclosure applies.

[0016] Sulfation at the 3-OH position of glucosamine forms a structural domain of heparan sulfate and is at least one important modification for enabling its biological function. Seven 3-O-sulfotransferase isoforms of the human genome are involved in the biosynthesis of 3-O-sulfated heparan sulfate. As a rare modification present in heparan sulfate, the availability of 3-O-sulfated oligosaccharides is very limited. Herein, a novel chemoenzymatic synthetic approach for synthesizing six 3-O-sulfated oligosaccharides including three hexasaccharides and three octasaccharides is disclosed. By rearranging the enzymatic modification order to correspond to the substrate specificity of 3-O-sulfotransferase 3, these syntheses were achieved. Using NMR, the effect of 3-O-sulfation on the conformation of the pyranose ring of 2-O-sulfated iduronic acid was investigated, and the effect of 3-O-sulfation on the correlation between the ring conformation and anticoagulant activity was investigated. From this, a novel octasaccharide that interacts with antithrombin and exhibits anti-factor Xa activity was found. Interestingly, this octasaccharide shows a faster clearance rate than fondaparinux, a pentasaccharide drug approved in a rat model, indicating that this octasaccharide is a potential short-acting anticoagulant candidate that may reduce

[0017] Heparan sulfate (HS) is a polysaccharide containing disaccharide repeating units of glucuronic acid (GlcA) or iduronic acid (IdoA) residues attached to glucosamine (GlcN) residues, each of which may be modified by sulfation. Heparan sulfate (HS) exhibits important physiological and pathological functions, such as regulation of embryonic development, inflammatory response, blood coagulation, and viral / bacterial infection. Most notably, heparin, a highly sulfated form of heparan sulfate (HS), is an anticoagulant widely used in clinics for the treatment of patients with thrombotic diseases. The functional selectivity of heparan sulfate (HS) and heparin is at least partially governed by the type of sulfation and the position of GlcA and IdoA residues. Sulfation is found at the 2-OH position of IdoA (to a lesser extent but also GlcA) and at the N-, 3-OH, and 6-OH positions of GlcN residues. Furthermore, the conformations of IdoA and IdoA2S residues adopt both chair (C4) and skew boat (S0) conformations. The conformations of GlcA, GlcA2S, and GlcN residues exist in the chair (C1) conformation. 1 -3 The flexibility of the conformation of the IdoA2S residue enables it to bind to antithrombin and exhibit anticoagulant activity, and to bind to fibroblast growth factor and regulate cell growth. The synthesis of heparan sulfate (HS) oligosaccharides remains a challenge. Many oligosaccharides 4 .

[0018] The functional selectivity of heparan sulfate (HS) and heparin is at least partially governed by the type of sulfation and the position of GlcA and IdoA residues. Sulfation is found at the 2-OH position of IdoA (to a lesser extent but also GlcA) and at the N-, 3-OH, and 6-OH positions of GlcN residues. 5 Furthermore, the conformations of IdoA and IdoA2S residues adopt both chair (C4) and skew boat (S0) conformations. The conformations of GlcA, GlcA2S, and GlcN residues exist in the chair (C1) conformation. The flexibility of the conformation of the IdoA2S residue enables it to bind to antithrombin and exhibit anticoagulant activity, and to bind to fibroblast growth factor and regulate cell growth. 1 C4) and skew boat ( 2 S0) conformations. 6 The conformations of GlcA, Glc A2S and GlcN residues exist in the chair ( 4 C1) conformation. 7,8 . The flexibility of the conformation of the IdoA2S residue enables it to bind to antithrombin and exhibit anticoagulant activity, 9 and to bind to fibroblast growth factor and regulate cell growth. 10 .

[0019] The synthesis of heparan sulfate (HS) oligosaccharides remains a challenge. Many oligosaccharides They can be synthesized by a pure organic synthesis approach, but it is still very difficult to synthesize oligosaccharides larger than hexasaccharides with complex sulfation patterns. As an alternative approach, there is a chemoenzymatic method for synthesizing heparan sulfate (HS) oligosaccharides using HS biosynthetic enzymes such as glucosyltransferases, C5-epimerases, and sulfotransferases. This method can provide high-efficiency synthesis of a wide range of oligosaccharides, but the synthesis of specific oligosaccharide sequences is still impossible due to insufficient understanding of the substrate specificity of HS biosynthetic enzymes. Although 3-O-sulfation rarely occurs in heparan sulfate (HS), this type of sulfation is thought to be closely related to its biological function. This 3-O-sulfation is important for anticoagulant activity 16、17 and promotes the entry of herpes simplex virus into host cells to establish infection and regulates axon guidance and neuron growth

[0020] and controls the expansion of progenitor cells during salivary gland development. Exactly how the GlcNS3S±6S residue plays a role in contributing to the biological activity of heparan sulfate (HS) is currently unknown. This GlcNS3S±6S residue forms a unique sulfated sugar sequence domain surrounded by other monosaccharide residues, 19 indicating that heparan sulfate (HS) can exert its biological effects. For example, the GlcNS3S±6 20 S residue present in the pentasaccharide domain allows heparan sulfate (HS) to bind to antithrombin (AT). 18 An octasaccharide with the GlcNS3S±6S residue binds to herpes simplex virus glycoprotein D and 3 controls the expansion of progenitor cells during salivary gland development. Exactly how the GlcNS3S±6S residue plays a role in contributing to the biological activity of heparan sulfate (HS) is currently unknown. This GlcNS3S±6S residue forms a unique sulfated sugar sequence domain surrounded by other monosaccharide residues, indicating that heparan sulfate (HS) can exert its biological effects. For example, the GlcNS3S±6 S residue present in the pentasaccharide domain allows heparan sulfate (HS) to bind to antithrombin (AT). An octasaccharide with the GlcNS3S±6S residue binds to herpes simplex virus glycoprotein D interact. Seven isoforms of 3-OST are present in the human genome and may be used for the preparation of various different 3-O-sulfated oligosaccharides 26 .

[0021] This specification discloses a plurality of schemes for preparing a library of 3-O-sulfated oligosaccharides using a chemoenzymatic approach. Here, for generating the -GlcA-GlcNS 3S6S-disaccharide unit, 3-OST-1 modification is only possible after 6-O-sulfation, so for synthesizing oligosaccharides containing the -IdoA2S-GlcNS3S- or IdoA2S-GlcNS3S6S- disaccharide unit, it is demonstrated that 3-OST-3 modification must precede the 6-O-sulfation step. There is no interaction between the 3-OST-3 of the tetrasaccharide substrate and the 6-O-sulfo group, which is consistent with the conclusion that the oligosaccharide substrate of 3-OST-3 does not require 6-O-sulfation. In contrast, an interaction between 3-OST-1 of the heptasaccharide substrate and the 6-O -sulfo group is observed, suggesting that 6-O-sulfation is required for binding to 3-OST-1. The distinct and unique substrate requirements between 3-OST-1 and 3-O ST-3, disclosed herein for the first time, clarify that 3-O-sulfated, heparan sulfate (HS) modified by different isoforms of 3-OST is biosynthesized via different pathways. It is widely accepted that the 3-OST-1 enzyme is involved in the synthesis of the anticoagulant heparan sulfate (HS), whereas the 3- OST-3 enzyme is not. So far, all the isolated antithrombin (AT) binding sequences are products of 3-OST-1 enzyme modification.

[0022] 34,35 . ​​​​​​​​containing the -GlcA-GlcNS3S6S-disaccharide repeating unit 33,39 . This long-held belief is discussed by the present disclosure. This discussion is based on the discovery that the oligosaccharides developed by the methods disclosed herein are products of 3-OST-3 enzyme modification and bind to antithrombin (AT) and exhibit anticoagulant activity. These discoveries indicate that 3-OST-3 can synthesize anticoagulant heparan sulfate (HS) as long as heparan sulfate (HS) has a structural domain similar to that of the compounds of the present invention (e.g., including but not limited to compound 5). (AT) and exhibit anticoagulant activity. These discoveries indicate that 3-OST-3 can synthesize anticoagulant heparan sulfate (HS) as long as heparan sulfate (HS) has a structural domain similar to that of the compounds of the present invention (e.g., including but not limited to compound 5). (AT) and exhibit anticoagulant activity. These discoveries indicate that 3-OST-3 can synthesize anticoagulant heparan sulfate (HS) as long as heparan sulfate (HS) has a structural domain similar to that of the compounds of the present invention (e.g., including but not limited to compound 5). ) has a structural domain similar to that of the compounds of the present invention (e.g., including but not limited to compound 5). This shows that 3-OST-3 can synthesize anticoagulant heparan sulfate (HS).

[0023] Also disclosed herein is the discovery that some of the developed heparan sulfate (HS) compounds or oligosaccharides have unexpectedly rapid clearance. The rapid clearance of such compounds provides potential new short-acting anticoagulant candidates with reduced bleeding risk. A short-acting anticoagulant that can be rapidly removed from the bloodstream before the major bleeding effect occurs would be particularly beneficial for patients with a high bleeding risk or an increased bleeding risk compared to normal or healthy patients / subjects. Unfractionated heparin is an anticoagulant with a short half-life, but the concern is that this drug causes heparin-induced thrombocytopenia (HIT), which is a life-threatening side effect. A short-acting anticoagulant that can be rapidly removed from the bloodstream before the major bleeding effect occurs would be particularly beneficial for patients with a high bleeding risk or an increased bleeding risk compared to normal or healthy patients / subjects. Unfractionated heparin is an anticoagulant with a short half-life, but the concern is that this drug causes heparin-induced thrombocytopenia (HIT), which is a life-threatening side effect. which is a life-threatening side effect. but the concern is that this drug causes heparin-induced thrombocytopenia (HIT), which is a life-threatening side effect. 42 . Oligosaccharides shorter than 12-mers (12-mer) do not bind to platelet factors 43 and thus have been found not to pose a risk of heparin-induced thrombocytopenia (H 4 IT). As hexasaccharides and octasaccharides, the compounds of the present invention (e.g., including compound 5) have a low risk of heparin-induced thrombocytopenia (HIT). IT). As hexasaccharides and octasaccharides, the compounds of the present invention (e.g., including compound 5) have a low risk of heparin-induced thrombocytopenia (HIT). IT). As hexasaccharides and octasaccharides, the compounds of the present invention (e.g., including compound 5) have a low risk of heparin-induced thrombocytopenia (HIT). is expected to be constantly low.

[0024] The heparan sulfate (HS) compounds or synthetic heparin analogs disclosed herein may contain, in some embodiments, 3-O-sulfated oligosaccharides containing 6-8 disaccharide units, with at least one disaccharide unit (at least one IdoA2S-GlcNS3S or IdoA2S-GlcNS3S6S disaccharide unit) being sulfated by the 3-OST-3 enzyme. As shown in the examples herein, such synthetic heparin analogs have anticoagulant activity, and this anticoagulant activity includes a binding affinity for antithrombin in the range of about 5 nM to about 30 nM and an 50 IC -1 antithrombin Xa activity in the range of about 10 ng / ml -1 to about 40 ng / ml. The structure of such synthetic heparin analogs is exemplified by Compounds 1-11 (especially Compounds 1-6 (Figures 6A-6 F)).

[0025] As further discussed herein, in some embodiments, this synthetic heparin analog can have an unexpectedly fast clearance rate, especially compared to heparin and heparin-like compounds. For example, the clearance rate of Compound 5 is at least about 50%, 75% or 1 00% (or about 50% to about 100%, about 60% to about 90%, about 70% to about 80%, about 50 % to about 75%, or about 75% to about 100%) faster than the clearance rate of fondaparinux in rats or mice. Such a fast clearance can make such compounds suitable as short-acting anticoagulant compounds, which are particularly suitable for patients and / or applications where the risk of bleeding is higher. Such compounds are It has also been shown not to cause heparin-induced thrombocytopenia (HIT).

[0026] Methods for synthesizing this synthetic heparin analog are exemplified herein (see, e.g., FIGS. 1 A, 1B, and 8C) and further discussed, but in some embodiments, (1) providing a glycan substrate, (2) elongating this glycan substrate to a sugar of a desired or predetermined length, and (3) performing at least one sulfation reaction using a 3-O-sulfotransferase (3-OST) enzyme, 3-OST-3 isoform thereby synthesizing a synthetic heparin analog. This glycan substrate may contain at least one IdoA2S-GlcNS3 S disaccharide unit.

[0027] The disclosed method for synthesizing this synthetic heparin analog can provide surprisingly high yields of heparin compounds. By way of non-limiting example, the disclosed method for synthesizing this synthetic heparin analog can have a yield of greater than about 20%, greater than about 30%, greater than about 40%, greater than about 50%, from about 20% to about 50%, from about 30% to about 50%, or from about 40% to about 50%.

[0028] If this glycan substrate contains an IdoA2S-GlcNS3S±6S disaccharide unit, such a method can further include a 6-O-sulfation step using a 6-O-sulfotransferase (6-OST), where 3-O-sulfation by 3-OST-3 occurs before the 6-O-sulfation step. In contrast, if the glycan substrate contains a GlcA-GlcNS3S6S disaccharide unit, such a method can further include using a 6-O-sulfotransferase (6-OST) can include a 6-O-sulfation step, where 3-O-sulfation by 3-OST-1 occurs prior to the 6-O-sulfation step.

[0029] The development of the presently disclosed heparin compounds and synthetic heparin analogs also provides for their use in treatments and methods of treating patients. For example, in some embodiments, a method of treating a patient in need of anticoagulation therapy is provided. Such a method can comprise: (a) providing a patient in need of anticoagulation therapy; and (b) administering to the patient a synthetic heparin analog having anticoagulant activity, the synthetic heparin analog comprising disaccharide units sulfated by at least one 3-OST- 3 enzyme; and at least one IdoA2S-GlcNS3S or IdoA2S-GlcNS3S6S disaccharide unit. Optionally, the patient can also be monitored for heparin-induced thrombocytopenia, and if detected, and if the patient has heparin-induced thrombocytopenia, an antidote can be administered to the patient to reverse the anticoagulant activity of the synthetic heparin analog. Such reversal of anticoagulant activity can be achieved, for example, by andexanet alpha at 50% or greater in the presence of andexanet alpha at 20 μg / ml or less, AndexXa® (Portola Pharmaceuticals, South San Francisco, Calif ornia, USA).

[0030] Accordingly, in some embodiments of the present disclosure, a method of treating a patient in need of anticoagulation therapy is provided. In some embodiments, the method comprises: (a) providing a patient in need of anticoagulation therapy; (b) administering to the patient a synthetic heparin analog having anticoagulant activity; administering a phosphorus analog, wherein the synthetic heparin analog has at least one 3-O disaccharide unit sulfated by ST-3 enzyme; and at least one IdoA2S-Glc including a NS3S or IdoA2S-GlcNS3S6S disaccharide unit.

[0031] In some embodiments, the present invention provides a synthetic heparin analog having anticoagulant activity for use in the treatment and / or prevention of diseases or disorders where anticoagulant activity is advantageous, and this syn thetic heparin analog comprises at least one disaccharide unit sulfated by 3-OST-3 enzyme and at least one IdoA2S-GlcNS3S or IdoA2S-GlcNS 3S6S disaccharide unit. By way of non-limiting example, such diseases or disorders may include patients and individuals at high risk of deep vein thrombosis (including cancer patients).

[0032] In yet another embodiment, the present invention provides a method of using a synthetic heparin analog having anticoagulant activity for the manufacture of a pharmaceutical composition for the treatment and / or prevention of diseases or disorders where anticoagulant activity is advantageous, and in this method, the synthetic heparin analog comprises at least one disaccharide unit sulfated by 3-OST-3 en zyme and at least one IdoA2S -GlcNS3S or IdoA2S-GlcNS3S6S disaccharide unit. Examples of synthetic heparin analogs or heparan sulfate (HS) compounds are disclosed herein, including, for example, those of FIGS. 1A and 6A

[0033] to 6F. In the structures shown, "R" is , a proton (-H), -CH3, -CH2CH3, or others similar to p-nitrophenyl can include substituents. Such substituents can include alkyl or lower alkyl. This can also be the case. In some embodiments, other substituents similar to this p-nitrophenyl can include aryl or substituted aryl. Alternatively or additionally, in some embodiments, "R" can include a detectable tag or a detectable moiety.

[0034] Definition The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the invention. The following terms are believed to be well understood by those skilled in the art, but for ease of explaining the present invention, the following definitions are provided. All technical and scientific terms used herein are intended to have the same meaning as commonly understood by those skilled in the art, unless otherwise defined herein. References to techniques used herein refer to techniques commonly understood in the art, including variations of those techniques or substitutions of equivalent techniques that are obvious to those skilled in the art. The following terms are believed to be well understood by those skilled in the art, but for ease of explaining the present invention, the following definitions are provided. It will be understood that many techniques and steps are disclosed in the description of the present invention. Each of these has individual advantages and can be used alone or in combination with one or more, or in some cases all, of the other disclosed techniques. Accordingly, for clarity, this description refrains from repeating every possible combination of the individual steps in an unnecessary manner. Nevertheless, the specification and ​It should be read with the understanding that the scope of the present invention and the claims is fully within the scope of the present invention and the claims.

[0035] In accordance with the Patent Law Agreement over the years, the original text's "indefinite article - a, an" and "definite article - the" mean "one or more than two" when used in this application including the claims. Therefore, for example, "a cell" includes a plurality of such cells, and so on. If not otherwise indicated, all numbers representing amounts of components, reaction conditions, and the like used in the specification and claims should be understood to be modified in all cases by the term "about". Therefore, unless indicated to the contrary, the numerical parameters recited in this specification and the appended claims are approximate values that may vary depending on the preferred characteristics found in the subject matter disclosed herein. As used herein, the term "about" refers to a value or amount of a composition, dosage, sequence identity (e.g., when comparing two or more nucleotide or amino acid sequences), mass, weight, temperature, time, volume, concentration, percentage, etc., and in some embodiments, it includes a variation of ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1 %, in some embodiments ±0.5%, and in some embodiments ±0.1% from a particular value, and such variations are suitable for carrying out the disclosed method or adopting the disclosed composition. When referring to, etc., such variations are suitable for carrying out the disclosed method or adopting the disclosed composition. ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1 %, in some embodiments ±0.5%, and in some embodiments ±0.1% from a particular value, and such variations are suitable for carrying out the disclosed method or adopting the disclosed composition. It is meant to include variations of, and such variations are suitable for carrying out the disclosed method or adopting the disclosed composition. using.

[0036] The term "comprising" is inclusive or unrestricted, like "including", "containing", " or "characterized by", It does not exclude additional, non-enumerated elements or method steps. "Comprising" means that while the named elements are present, other elements may be added and yet the claimed scope of the composition can still be formed. It is a technical term The term "consisting of" excludes all elements, steps or contents not specified in the claims. When the phrase "consist of" appears in the body of the claim rather than immediately following the preamble, it is limited to the elements shown, but other elements are not excluded from the claim as a whole. The term "consisting essentially of" limits the claim to the specified materials or steps and further limits materials or steps that do not substantially affect the basic and novel features of the claimed invention. Regarding the terms "comprising", "consisting of" and "consisting essentially of", when one of these three terms is used in this specification, the present invention can include the use of any of the other two terms . As used herein, the term "and / or", when used to list items, indicates items that exist alone or in combination. Thus, for example, the expression "A, B, C, and / or D" includes not only A, B, C, and D individually, but also any and all combinations and sub-combinations of A, B, C, and D.

[0037] As used herein, the term "alkyl group" refers to linear (i.e., "straight-chain"), branched, or a cyclic, saturated or at least partially or in some cases completely unsaturated (i.e., alkene and alkynyl groups) hydrocarbon chain, including C 1~20 is represented, for example, by a methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, tert-butyl group, pentyl group, hexyl group, octyl group, ethenyl group, propenyl group, butenyl group, penta nyl group, hexenyl group, octenyl group, butadienyl group, propynyl group, methylpropene yl group, butynyl group, pentynyl group, hexynyl group, heptynyl group and aryl group can be mentioned "Branched" refers to an alkyl group in which a lower alkyl group such as a methyl group, an ethyl group, or a propyl group is added to a linear alkyl chain. "Lower alkyl group" refers to, for example, an alkyl group having 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms, i.e., 1 to about 8 carbon atoms. alkyl group) (i.e., C 1-8 alkyl group). "Higher alkyl group" refers to, for example, an alkyl group having about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms, such as about 10 to about 20 carbon atoms. In certain embodiments, "alkyl group" 1~8 specifically represents a linear alkyl group. In another embodiment, "alkyl group" specifically represents a branched-chain alkyl group. 1~8 The alkyl group may optionally be substituted with one or more alkyl group substituents that may be the same or different ("substituted alkyl group"). "Substituent of alkyl group" includes alkyl group, substituted

[0038] alkyl group, halo group, arylamino group, acyl group, hydroxyl group, aryloxyl group, a alkoxyl group, alkylthio group, arylthio group, aralkyloxyl group, aralkylth alkyl group, aryl group, substituted alkyl group, halo group, arylamino group, acyl group, hydroxyl group, aryloxyl group, alkoxyl group, alkylthio group, arylthio group, aralkyloxyl group, aralkylthio group, etc. Examples include, but are not limited to, an -O group, a carboxyl group, an alkoxycarbonyl group, an oxo group, and a cycloalkyl group. Optionally, one or more oxygen atoms, sulfur atoms, or substituted or unsubstituted nitrogen atoms can be inserted along the alkyl chain, where the nitrogen substituent is a hydrogen atom, a lower alkyl group (hereinafter referred to as an "alkylaminoalkyl group"), or an aryl group. As such, as used herein, the term "substituted alkyl group" includes an alkyl group in which one or more atoms or functional groups of the alkyl group are replaced by other atoms or functional groups, as defined herein, for example, an alkyl group, a substituted alkyl group, a halogen atom, an aryl group, a substituted aryl group, an alkoxyl group, a hydroxyl group, a nitro group, an amino group, an alkylamino group, a dialkylamino group, a sulfate group, and a mercapto group. . The term "aryl group" as used herein refers to an aromatic substituent that can be a single aromatic ring, or a polycyclic aromatic ring, that is fused to each other, linked by a covalent bond, or bonded to a common functional group such as a methylene group or an ethylene group portion, but not limited thereto. The common linking group can also be a carbonyl group as in the case of benzophenone, or an oxygen molecule as in diphenyl ether, or a nitrogen molecule as in diphenylamine. The term "aryl group" specifically includes heterocyclic aromatic compounds. The aromatic ring consists of, for example, a phenyl group, a naphthyl group, a biphenyl group, a diphenyl ether group, a diphenylamine group, and a benzophenone group. In a particular embodiment, the term "aryl group" consists of a cyclic aromatic ring having from about 5 to about 10 carbon atoms, for example, 5, 6, 7, 8, 9, or 10 carbon atoms.

[0039] group, a substituted aryl group, an alkoxyl group, a hydroxyl group, a nitro group, an amino group, an alkylamino group, a dialkylamino group, a sulfate group, and a mercapto group. The term "aryl group" as used herein refers to an aromatic substituent that can be a single aromatic ring, or a polycyclic aromatic ring, that is fused to each other, linked by a covalent bond, or bonded to a common functional group such as a methylene group or an ethylene group portion, but not limited thereto. The common linking group can also be a carbonyl group as in the case of benzophenone, or an oxygen molecule as in diphenyl ether, or a nitrogen molecule as in diphenylamine. The term "aryl group" specifically includes heterocyclic aromatic compounds. The aromatic ring consists of, for example, a phenyl group, a naphthyl group, a biphenyl group, a diphenyl ether group, a diphenylamine group, and a benzophenone group. In a particular embodiment, the term "aryl group" consists of a cyclic aromatic ring having from about 5 to about 10 carbon atoms, for example, 5, 6, 7, 8, 9, or 10 carbon atoms. The term "aryl group" specifically includes heterocyclic aromatic compounds. The aromatic ring consists of, for example, a phenyl group, a naphthyl group, a biphenyl group, a diphenyl ether group, a diphenylamine group, and a benzophenone group. In a particular embodiment, the term "aryl group" consists of a cyclic aromatic ring having from about 5 to about 10 carbon atoms, for example, 5, 6, 7, 8, 9, or 10 carbon atoms. -membered ring aromatic ​​means a family and also includes 5- and 6-membered hydrocarbon and heteroaromatic rings.

[0040] The aryl group may be optionally substituted with one or more aryl group substituents that are the same or different (" substituted aryl group"). As "substituents of the aryl group", there are alkyl group, substituted alkyl group, a ryl group, substituted aryl group, aralkyl group, hydroxyl group, alkoxyl group, aryloxyl group, aralkyloxyl group, carboxyl group, acyl group, halo group, nitro group, alkoxy carbonyl group, aryloxycarbonyl group, aralkoxycarbonyl group, acyloxy l group, acylamino group, aroylamino group, carbamoyl group, alkylcarbamoyl group, dialkylcarbamoyl group, arylthio group, alkylthio group, alkylene group and -NR 'R'' group, where R' and R'' are each independently a hydrogen atom, an alkyl group, a substituted alkyl group, an aryl group, a substituted aryl group and an aralkyl group.

[0041] Therefore, as used herein, the term "substituted aryl group" includes an aryl group as defined herein such that one or more atoms or functional groups of the aryl group may be replaced with other atoms or functional groups such as, for example, an alkyl group, a substituted alkyl group, a halogen atom, an aryl group, a substituted aryl group, an alkoxyl group, a hydroxyl group, a nitro group, an amino group, an alkylamino group, a dialkylamino group, a sulfate group and a mercapto group. Special examples of the aryl group include a cyclopentadienyl group, a phenyl group, a furan group, a thi ophene group, a pyrrole group, a pyran group, a pyridine group, an imidazole group, a benzimidazole group , an isothiazole group, an isoxazole group, a pyrazole group, a pyrazine group, a triazine group, a pi Examples include, but are not limited to, a lysine group, a quinoline group, an isoquinoline group, an indole group, a carbazole group, and the like. However, the invention is not limited thereto.

[0042] The structure represented by the following chemical formula as used in this specification:

Chemical formula

Chemical formula

Chemical formula

[0043] "Cyclic" and "cycloalkyl" refer to a non-aromatic mono- or multi-cyclic system having about 3 to about 10 carbon atoms, such as 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. However, the invention is not limited thereto. The cycloalkyl group can optionally be partially unsaturated. The cycloalkyl group can also optionally be substituted with an alkyl group substituent, oxo, and / or alkylene as defined herein. Along the cyclic alkyl chain, one or more oxygen atoms, sulfur atoms However, the invention is not limited thereto. However, the invention is not limited thereto. or a nitrogen substituent is a hydrogen atom, an alkyl group, a substituted alkyl group, an aryl group, or a substituted or unsubstituted nitrogen atom can be optionally inserted, thus providing a heterocyclic group. Representative monocyclic cycloalkyl rings include a cyclopentyl group, cyclohex syl group, and a cycloheptyl group. Polycyclic cycloalkyl rings include an adamantyl group , an octahydronaphthyl group, a decalin, a camphor, a camphan, and a noradamantyl are included .

[0044] The term "heterocycle" refers to a non-aromatic or aromatic, monocyclic or polycyclic ring system of about 3 to about 14 atoms wherein at least one of these atoms is a heteroatom (e.g., oxygen, nitrogen, or sulfur) . The term "N-heterocycle" refers to a heterocycle in which at least one of the heteroatoms is a nitrogen atom . Examples of N-heterocycles include azetidine, pyrrolidine, pyrrole, pyrroline, piperidine, pi ridine, piperazine, pyrazine, pyrimidine, pyridazine, morpholine, and thiazine are included, but are not limited thereto. "Aralkyl group" refers to an aryl-alkyl-group, where the aryl group and the alkyl group are as described above, and include a substituted aryl group and a substituted alkyl group. Examples of aralkyl groups include a benzyl group, a phenylethyl group, or a naphthylmethyl group. As used herein, the term "acyl group" represents an organic acid group in which the -OH group of its carboxyl group is replaced by another substituent (e.g., as represented by RC(=O)-, where R is an alkyl group, a substituted alkyl group, an aralkyl group, an aryl group or a substituted aryl group as defined herein.). Thus, the term "acyl group" specifically includes an acetylfuran group and includes arylacyl groups such as the phenacyl group. Specific examples of acyl groups are the acetyl group and the benzoyl group.

[0045] "N-acyl" refers to a group having the structure -N-C(=O)-R (wherein R is as defined for the acyl group as above).). These groups are also called amides. Modified N-acyl groups include compounds in which the oxygen of this N-acyl is replaced by S or NH, and also include compounds in which, in addition to the nitrogen atom, a second heteroatom has a carbonyl group (i.e., -C(=O)- ) bonded thereto. For example, a carbonyl group can be bonded to a second nitrogen atom to form a urea bond (i.e., -NH-C(=O)-NH-R). The term "amino" refers to -NH2, -NHR, and NR2 groups (wherein each R is independently alkyl, substituted alkyl, aryl, substituted aryl, or aralkyl.). Also refers to amino and ammonium functional groups in N-heterocycles (such as morpholine, etc.). The term "amino" as used herein refers to - NH3, - + NH(R)2, and + NH(R + )3 groups, etc., and can also refer to substituents that provide quaternary ammonium cations. (wherein each R is independently alkyl, substituted alkyl, aryl, substituted aryl, or aralkyl ).). ).

[0046] The term "ester" refers to a moiety containing an -O-C(=O)-R group (wherein R may be alkyl, substituted alkyl, aralkyl, aryl, or substituted aryl.). In some embodiments, this R can contain an amino substituent, and this ester is an amino ester. ). The term "amide" refers to a moiety containing an -N(R')-C(=O)-R group (wherein R is selected from alkyl, substituted alkyl, aralkyl, aryl, or substituted aryl, and R' is a hydrogen atom, alkyl, substituted alkyl, aralkyl, aryl, or substituted aryl). The term "urea" as used herein refers to a moiety containing an -N(R')-C(=O)-N(R')- group (wherein each R' is independently a hydrogen atom, alkyl, substituted alkyl, aralkyl, aryl, or substituted aryl). The term "hydroxyl" refers to an -OH group. When the term "independently selected" is used, the designated substituents (e.g., R groups such as R1 and R2 or X and Y groups) are the same or different. For example, both R1 and R2 are substituted alkyl groups, or R1 is a hydrogen atom and R2 is a substituted alkyl group, and so on.

[0047] Patient It should be understood that the principles of the invention disclosed herein are effective for all invertebrates and vertebrates, including mammals, for which the compositions and methods are intended to include the term "patient". However, the patient to be treated, screened, tested, or sampled is preferably a human patient. Further, mammals are understood to include any mammalian species for which screening is desirable, particularly agricultural and livestock mammalian species. The disclosed methods, compounds, and treatments are particularly useful for the examination, screening, and / or treatment of warm-blooded vertebrates. Accordingly, the present invention relates to mammals and birds.

[0048] More specifically, provided herein are mammals such as humans, and those at risk of extinction due to certain reasons (such as Siberian tigers), due to economic importance (animals raised on farms for human consumption), and / or due to social importance to humans (animals kept as pets or in zoos), important mammals, for example, carnivores other than humans (such as cats and dogs), swine (pigs, grown pigs for meat, wild boars), ruminants (cattle, steers, sheep, giraffes, deer, goats, bison, camels, etc.), horses, etc. are to be examined, screened, and / or treated. Also, treatment of birds is provided, which includes treatment of birds in danger of extinction kept in zoos, as well as domestic fowl, more specifically, domesticated fowl such as turkeys, chickens, ducks, geese, guinea fowls, etc. (since these are also economically important to humans). Therefore, provided herein is treatment of livestock including, but not limited to, domestic swine (pigs, grown pigs for meat), ruminants, horses, domestic fowl, etc. and / or due to social importance to humans (animals kept as pets or in zoos), important mammals, for example, carnivores other than humans (such as cats and dogs), swine (pigs, grown pigs for meat, wild boars), ruminants (cattle, steers, sheep, giraffes, deer, goats, bison, camels, etc.), horses, etc. are to be examined, screened, and / or treated. and / or due to social importance to humans (animals kept as pets or in zoos), important mammals, for example, carnivores other than humans (such as cats and dogs), swine (pigs, grown pigs for meat, wild boars), ruminants (cattle, steers, sheep, giraffes, deer, goats, bison, camels, etc.), horses, etc. are to be examined, screened, and / or treated. and / or due to social importance to humans (animals kept as pets or in zoos), important mammals, for example, carnivores other than humans (such as cats and dogs), swine (pigs, grown pigs for meat, wild boars), ruminants (cattle, steers, sheep, giraffes, deer, goats, bison, camels, etc.), horses, etc. are to be examined, screened, and / or treated. and / or due to social importance to humans (animals kept as pets or in zoos), important mammals, for example, carnivores other than humans (such as cats and dogs), swine (pigs, grown pigs for meat, wild boars), ruminants (cattle, steers, sheep, giraffes, deer, goats, bison, camels, etc.), horses, etc. are to be examined, screened, and / or treated. Treatment of birds is also provided, which includes treatment of birds in danger of extinction kept in zoos, as well as domestic fowl, more specifically, domesticated fowl such as turkeys, chickens, ducks, geese, guinea fowls, etc. (since these are also economically important to humans). Treatment of birds is also provided, which includes treatment of birds in danger of extinction kept in zoos, as well as domestic fowl, more specifically, domesticated fowl such as turkeys, chickens, ducks, geese, guinea fowls, etc. (since these are also economically important to humans). Treatment of birds is also provided, which includes treatment of birds in danger of extinction kept in zoos, as well as domestic fowl, more specifically, domesticated fowl such as turkeys, chickens, ducks, geese, guinea fowls, etc. (since these are also economically important to humans). Therefore, provided herein is treatment of livestock including, but not limited to, domestic swine (pigs, grown pigs for meat), ruminants, horses, domestic fowl, etc. Therefore, provided herein is treatment of livestock including, but not limited to, domestic swine (pigs, grown pigs for meat), ruminants, horses, domestic fowl, etc.

[0049] In some embodiments, the patient used according to the present invention is a patient in need of treatment and / or diagnosis. In some embodiments, the patient may be in need of anticoagulant therapy or may be a condition or phenotype related thereto. In some embodiments, the patient in need of anticoagulant therapy may be a patient at high risk of bleeding. In some embodiments, the patient used according to the present invention is a patient in need of treatment and / or diagnosis. In some embodiments, the patient may be in need of anticoagulant therapy or may be a condition or phenotype related thereto. In some embodiments, the patient in need of anticoagulant therapy may be a patient at high risk of bleeding. In some embodiments, the patient used according to the present invention is a patient in need of treatment and / or diagnosis. In some embodiments, the patient may be in need of anticoagulant therapy or may be a condition or phenotype related thereto. In some embodiments, the patient in need of anticoagulant therapy may be a patient at high risk of bleeding. In some embodiments, the patient in need of anticoagulant therapy may be a patient at high risk of bleeding.

[0050] Preparation In some embodiments, the composition of the present invention comprises a composition comprising a pharmaceutically acceptable carrier. Any suitable pharmaceutical formulation can be used to prepare an adenovirus vector for administration to a patient. In some embodiments, the composition of the present invention comprises a composition comprising a pharmaceutically acceptable carrier. Any suitable pharmaceutical formulation can be used to prepare an adenovirus vector for administration to a patient. Any suitable pharmaceutical formulation can be used to prepare an adenovirus vector for administration to a patient. Suitable formulations may include, for example, aqueous and non-aqueous sterile injection solutions, which may be An agent, buffer, bacteriostatic agent, bactericidal antibiotic, and solutes that render the formulation isotonic with the body fluid of the patient recipient, and may include aqueous and non-aqueous sterile suspensions that may contain suspending and thickening agents. This formulation can be provided in unit dose or multi-dose containers (e.g., sealed ampoules and vials), and can be stored in a refrigerated or lyophilized state that requires only the addition of a sterile liquid carrier (e.g., water for injection) immediately before use. Some exemplary components are SDS, mannitol or other sugars, and phosphate buffered saline (PBS). It should be understood that the formulations of the present invention can include other agents commonly used in the art, in addition to the components specifically mentioned above, taking into account the type of formulation in question. For example, sterile aqueous and non-aqueous solutions free of pyrogens can be used. Administration of the compositions of the present invention can be by any method known to those skilled in the art, including, but not limited to, intravenous administration, intra-articular administration, transdermal administration, intramuscular administration, subcutaneous administration, topical administration, rectal administration, vaginal administration, intratumoral administration, oral administration, buccal administration, nasal administration, parenteral administration, inhalation, and gas infusion. In some embodiments, methods suitable for administration of the compositions of the present invention include, but are not limited to, intravenous administration. Alternatively, the composition may be deposited at the site in need of treatment by any other method. The specific mode of administration of the compositions of the present invention depends on various factors.

[0051] Administration An effective dosage of the compositions of the present invention is administered to a patient in need thereof. A "therapeutically effective amount"

[0052] Dosage is the amount of the composition sufficient to cause a measurable reaction (e.g., anticoagulation). In some embodiments, a therapeutically effective amount is an amount sufficient to prevent coagulation (i.e., anticoagulate). In some embodiments, a therapeutically effective amount is an amount sufficient to improve the health, well-being, prognosis, and / or survival rate of a patient in need of anticoagulation therapy. The actual dosage level of the active ingredient in the composition of the present invention can be varied so as to administer an amount of the active compound effective to achieve the desired therapeutic response for a particular patient. The selected dosage level may depend on the activity of the therapeutic composition, the route of administration, the combination with other drugs or treatments, the severity of the treatment condition, and the condition and prior medical history of the patient being treated. However, it is within the discretion of those skilled in the art to start the dosage of the composition at a level lower than that required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. After carefully examining the disclosure of the present invention presented herein, those skilled in the art can adjust the dosage for an individual patient taking into account the particular formulation, the method of administration used, and the severity of the symptoms. In further calculations of dosage, the height and weight of the patient, the severity and stage of the symptoms, and the presence of additional adverse physical conditions can be considered. Such adjustments or variations, as well as the

[0053] evaluation of the timing and manner of making such adjustments or variations, are well known to those skilled in the medical arts.

Examples

[0053] The following examples are included to further illustrate various embodiments of the present invention. However, those skilled in the art can make many changes in the specific embodiments It should be understood that the same or similar results can be obtained without departing from the spirit and scope of the present invention.

[0054] Materials and Methods Expression and Purification of Enzymes In SF9 cells (Invitrogen), using Sf-900 TM III SFM (Life Technologies), 6- O-sulfotransferase 1 (6-OST-1) and 6-O-sulfotransferase 3 (6-OST-3) were expressed. Insect cells at a concentration of 2.0×10 6 cells / ml were infected with a recombinant virus expressing mouse 6-OST-1 and human 6-OST-3 and incubated in a shaker at 27°C for 96 hours. This culture solution was centrifuged at 4,000 rpm for 10 minutes to pellet the cells. Next, a fresh 100 mM stock solution of phenylmethanesulfonyl fluoride (PMSF, in 95% ethanol) was prepared.), Triton X-100 and 0.2% glycerol The supernatant containing was centrifuged at 8,000 rpm for 30 minutes and filtered through a 1.5 μM membrane. Next, the resulting medium was mixed with an equal volume of 40 mM 3-(N- morpholino)propanesulfonic acid (MOPS) buffer (pH 7.0) containing 0.05% Triton-100 and 2% glycerol. Heparin Toyopearl gel (Tosoh Bioscience) column was used with two buffers (buffer A contains 20 mM MOPS, pH 7.0, 100 mM NaCl, 2% glycerol, and 0.1% reduced triton X-100 (Sigma) and buffer B contains 20 mM MOPS, pH 7.0, 1 M NaCl, 2% glycerol, and 0.1% reduced tr ​​ It contains Triton X-100. 6-OST-1 and 3 were purified by (this method). After loading this medium, 4 mL At a flow rate of / min, the column was washed with buffer solution A until the absorbance at 280 nm reached the baseline A gradient elution of 0-100% B was applied over 60 minutes, and the column was eluted with 100% B at a flow rate of 1.5 mL / min for 60 minutes. The purification of the enzyme was carried out at 4 °C .

[0055] The expression of 3-OST-1 and 3-OST-3 in Escherichia coli was carried out using BL21 cells . This transformed cell was grown in LB medium containing 50 μg / L kanamycin for 3-OST-1 and 3-OST-3, and incubated at 37 °C until the OD600 reached 0.6-0.8 . Isopropylthiogalactopyranoside (IPTG) with a final concentration of 0.2 mM was added to induce the expression of 3-OST-1 and 3. This bacterial culture was maintained with shaking overnight at 22 °C . The bacterial cells were harvested by centrifugation at 3,400 rpm for 15 minutes. These cells were resuspended in 25 mL of a buffer containing 25 mM Tris, pH 7.5, 30 mM imidazole, and 500 mM NaCl . This suspension was sonicated and centrifuged at 14,000 rpm for 30 minutes. Before purification , this supernatant was filtered through a 0.45 μm membrane. A nickel agarose (GE Healthcare) column was used to purify the protein using two buffer solutions (Buffer C contains 25 mM Tris, pH 7.5, 30 mM imidazole, and 500 mM NaCl, and Buffer D contains 25 mM Tris, pH 7.5, 300 mM imidazole, and 500 mM NaCl. After loading this medium, at a flow rate of 2 mL / min ). Immediately, the column was washed with buffer C until the absorbance at 280 nm reached the baseline. Next, buffer D was applied to elute the protein. The synthesis of compounds 1 - 3 started from the hexasaccharide (GlcNS - GlcA - GlcNS - IdoA2S - GlcNS - GlcA - pNP) (referred to as the "NS2S 6 - mer substrate").

[0056] Chemoenzymatic Synthesis of Oligosaccharides (Compounds 1 - 6) The synthesis of compounds 1 - 3 started from the hexasaccharide (GlcNS - GlcA - GlcNS - IdoA2S - GlcNS - GlcA - pNP) (referred to as the "NS2S 6 - mer substrate"). To synthesize compound 1, this substrate (25 mg) was incubated with 1.8 mM 3'-phosphoadenosine 5'-phosphosulfate (PAPS) in a buffer containing 50 mM MOPS (pH 7.0), 10 mM MnCl2, 7 mM MgCl2, and 2 mL 3 - OST - 3 (0.11 mg / mL) with a total volume of 100 mL. The reaction mixture was incubated at 37°C overnight. A small amount of the reaction mixture was injected into an anion - exchange HPLC (TSKgel DNA - NPR - column (4.6 mM x 7.5 cm, 2.5μm, obtained from Tosoh Bioscience)) to monitor the completion of the reaction. If the reaction was less than 60%, more 3 - OST - 3 enzyme and PAPS were added, and the reaction mixture was maintained at 37°C for an additional 18 - 24 hours. When the reaction was complete, the reaction mixture was subjected to Q - Sepharose chromatography (GE Healthcare). To synthesize compound 1, this substrate (25 mg) was incubated with 1.8 mM 3'-phosphoadenosine 5'-phosphosulfate (PAPS) in a buffer containing 50 mM MOPS (pH 7.0), 10 mM MnCl2, 7 mM MgCl2, and 2 mL 3 - OST - 3 (0.11 mg / mL) with a total volume of 100 mL. The reaction mixture was incubated at 37°C overnight. A small amount of the reaction mixture was injected into an anion - exchange HPLC (TSKgel DNA - NPR - column (4.6 mM x 7.5 cm, 2.5μm, obtained from Tosoh Bioscience)) to monitor the completion of the reaction. If the reaction was less than 60%, more 3 - OST - 3 enzyme and PAPS were added, and the reaction mixture was maintained at 37°C for an additional 18 - 24 hours. When the reaction was complete, the reaction mixture was subjected to Q - Sepharose chromatography (GE Healthcare). To synthesize compound 1, this substrate (25 mg) was incubated with 1.8 mM 3'-phosphoadenosine 5'-phosphosulfate (PAPS) in a buffer containing 50 mM MOPS (pH 7.0), 10 mM MnCl2, 7 mM MgCl2, and 2 mL 3 - OST - 3 (0.11 mg / mL) with a total volume of 100 mL. The reaction mixture was incubated at 37°C overnight. A small amount of the reaction mixture was injected into an anion - exchange HPLC (TSKgel DNA - NPR - column (4.6 mM x 7.5 cm, 2.5μm, obtained from Tosoh Bioscience)) to monitor the completion of the reaction. If the reaction was less than 60%, more 3 - OST - 3 enzyme and PAPS were added, and the reaction mixture was maintained at 37°C for an additional 18 - 24 hours. When the reaction was complete, the reaction mixture was subjected to Q - Sepharose chromatography (GE Healthcare). To synthesize compound 1, this substrate (25 mg) was incubated with 1.8 mM 3'-phosphoadenosine 5'-phosphosulfate (PAPS) in a buffer containing 50 mM MOPS (pH 7.0), 10 mM MnCl2, 7 mM MgCl2, and 2 mL 3 - OST - 3 (0.11 mg / mL) with a total volume of 100 mL. The reaction mixture was incubated at 37°C overnight. A small amount of the reaction mixture was injected into an anion - exchange HPLC (TSKgel DNA - NPR - column (4.6 mM x 7.5 cm, 2.5μm, obtained from Tosoh Bioscience)) to monitor the completion of the reaction. If the reaction was less than 60%, more 3 - OST - 3 enzyme and PAPS were added, and the reaction mixture was maintained at 37°C for an additional 18 - 24 hours. When the reaction was complete, the reaction mixture was subjected to Q - Sepharose chromatography (GE Healthcare). To synthesize compound 1, this substrate (25 mg) was incubated with 1.8 mM 3'-phosphoadenosine 5'-phosphosulfate (PAPS) in a buffer containing 50 mM MOPS (pH 7.0), 10 mM MnCl2, 7 mM MgCl2, and 2 mL 3 - OST - 3 (0.11 mg / mL) with a total volume of 100 mL. The reaction mixture was incubated at 37°C overnight. A small amount of the reaction mixture was injected into an anion - exchange HPLC (TSKgel DNA - NPR - column (4.6 mM x 7.5 cm, 2.5μm, obtained from Tosoh Bioscience)) to monitor the completion of the reaction. If the reaction was less than 60%, more 3 - OST - 3 enzyme and PAPS were added, and the reaction mixture was maintained at 37°C for an additional 18 - 24 hours. When the reaction was complete, the reaction mixture was subjected to Q - Sepharose chromatography (GE Healthcare). To synthesize compound 1, this substrate (25 mg) was incubated with 1.8 mM 3'-phosphoadenosine 5'-phosphosulfate (PAPS) in a buffer containing 50 mM MOPS (pH 7.0), 10 mM MnCl2, 7 mM MgCl2, and 2 mL 3 - OST - 3 (0.11 mg / mL) with a total volume of 100 mL. The reaction mixture was incubated at 37°C overnight. A small amount of the reaction mixture was injected into an anion - exchange HPLC (TSKgel DNA - NPR - column (4.6 mM x 7.5 cm, 2.5μm, obtained from Tosoh Bioscience)) to monitor the completion of the reaction. If the reaction was less than 60%, more 3 - OST - 3 enzyme and PAPS were added, and the reaction mixture was maintained at 37°C for an additional 18 - 24 hours. When the reaction was complete, the reaction mixture was subjected to Q - Sepharose chromatography (GE Healthcare). To synthesize compound 1, this substrate (25 mg) was incubated with 1.8 mM 3'-phosphoadenosine 5'-phosphosulfate (PAPS) in a buffer containing 50 mM MOPS (pH 7.0), 10 mM MnCl2, 7 mM MgCl2, and 2 mL 3 - OST - 3 (0.11 mg / mL) with a total volume of 100 mL. The reaction mixture was incubated at 37°C overnight. A small amount of the reaction mixture was injected into an anion - exchange HPLC (TSKgel DNA - NPR - column (4.6 mM x 7.5 cm, 2.5μm, obtained from Tosoh Bioscience)) to monitor the completion of the reaction. If the reaction was less than 60%, more 3 - OST - 3 enzyme and PAPS were added, and the reaction mixture was maintained at 37°C for an additional 18 - 24 hours. When the reaction was complete, the reaction mixture was subjected to Q - Sepharose chromatography (GE Healthcare). To synthesize compound 1, this substrate (25 mg) was incubated with 1.8 mM 3'-phosphoadenosine 5'-phosphosulfate (PAPS) in a buffer containing 50 mM MOPS (pH 7.0), 10 mM MnCl2, 7 mM MgCl2, and 2 mL 3 - OST - 3 (0.11 mg / mL) with a total volume of 100 mL. The reaction mixture was incubated at 37°C overnight. A small amount of the reaction mixture was injected into an anion - exchange HPLC (TSKgel DNA - NPR - column (4.6 mM x 7.5 cm, 2.5μm, obtained from Tosoh Bioscience)) to monitor the completion of the reaction. If the reaction was less than 60%, more 3 - OST - 3 enzyme and PAPS were added, and the reaction mixture was maintained at 37°C for an additional 18 - 24 hours. When the reaction was complete, the reaction mixture was subjected to Q - Sepharose chromatography (GE Healthcare). To synthesize compound 1, this substrate (25 mg) was incubated with 1.8 mM 3'-phosphoadenosine 5'-phosphosulfate (PAPS) in a buffer containing 50 mM MOPS (pH 7.0), 10 mM MnCl2, 7 mM MgCl2, and 2 mL 3 - OST - 3 (0.11 mg / mL) with a total volume of 100 mL. The reaction mixture was incubated at 37°C overnight. A small amount of the reaction mixture was injected into an anion - exchange HPLC (TSKgel DNA - NPR - column (4.6 mM x 7.5 cm, 2.5μm, obtained from Tosoh Bioscience)) to monitor the completion of the reaction. If the reaction was less than 60%, more 3 - OST - 3 enzyme and PAPS were added, and the reaction mixture was maintained at 37°C for an additional 18 - 24 hours. When the reaction was complete, the reaction mixture was subjected to Q - Sepharose chromatography (GE Healthcare).

[0057] To synthesize compound 2, compound 1 (5 mg) was incubated with 6 - OST - 1, 6 - OST - 3 enzymes and 1.3 mM PAPS in a buffer containing 100 mM MOPS (pH 7.0) and 1 mL of the enzyme cocktail of 6 - OST - 1 and 3 at 37°C overnight. A small amount of the reaction mixture was injected into an anion - exchange HPL To synthesize compound 2, compound 1 (5 mg) was incubated with 6 - OST - 1, 6 - OST - 3 enzymes and 1.3 mM PAPS in a buffer containing 100 mM MOPS (pH 7.0) and 1 mL of the enzyme cocktail of 6 - OST - 1 and 3 at 37°C overnight. A small amount of the reaction mixture was injected into an anion - exchange HPL To synthesize compound 2, compound 1 (5 mg) was incubated with 6 - OST - 1, 6 - OST - 3 enzymes and 1.3 mM PAPS in a buffer containing 100 mM MOPS (pH 7.0) and 1 mL of the enzyme cocktail of 6 - OST - 1 and 3 at 37°C overnight. A small amount of the reaction mixture was injected into an anion - exchange HPL By injecting into C, the completion of the reaction was monitored and the product was purified by Q-Sepharose . To synthesize compound 3, compound 2 (4 mg) was added to a buffer solution with a total volume of 100 mL containing 18 mM MOPS (pH 7.0 ), 5 mM MnCl2, 5 mM MgCl2, and 6 mL of 3-OST-1 (4 μg / mL), and incubated with 1.3 mM P APS. The reaction mixture was incubated overnight at 37 °C. A small amount of the reaction mixture was injected into anion-exchange HPLC to monitor the completion of the reaction, and the product was purified by Q- Sepharose.

[0058] For the synthesis of compounds 4 - 6, the octasaccharide (GlcNS-GlcA-GlcNS-IdoA2S-GlcNS-IdoA2S-GlcNS-GlcA-pNP )(referred to as the NS2S 8-mer substrate) was used as the starting material. To synthesize compound 4, 30 mg of this substrate was added to a buffer solution with a total volume of 90 mL containing 33 mg of MOPS (pH 7.0), 1 0 mM MnCl2, 5 mM MgCl2, and 4 mL of 3-OST-3 (0.11 mg / mL), and incubated with 2 mM PAP S. The reaction mixture was incubated overnight at 37 °C. A small amount of the reaction mixture was injected into anion-exchange HPLC to monitor the completion of the reaction. If the reaction was less than 60 %, more 3-OST-3 enzyme and PAPS were added, and the reaction mixture was maintained at 37 °C for an additional 18 - 24 hours . After the reaction was completed, the reaction mixture was subjected to Q-Sepharose chromatography (GE Hea lthcare).

[0059] For the synthesis of compound 5, compound 4 (22 mg) was added to a buffer solution with a total volume of 100 mL containing 100 mM MOPS (pH 7.0 In a buffer containing 3 mL of a mixture of 6-OST-1 and 6-OST-3 and 3, 6-OST-1, 6-OST-3 enzymes and 0.8 mM PA were incubated overnight at 37 °C with PS. A small amount of the reaction mixture was injected into anion-exchange HPLC to monitor the completion of the reaction, and the product was purified by Q-Sepharose. To synthesize compound 6, compound 5 (6.5 mg) was added to a buffer with a total volume of 100 mL containing 18 mM MOPS (pH 7 .0), 5 mM MnCl2, 5 mM MgCl2, and 4.5 mL of 3-OST-1 (4 μg / mL) and incubated with 1 .3 mM PAPS. The reaction mixture was incubated overnight at 37 °C. The completion of the reaction was monitored by injecting a small amount of the reaction mixture into anion-exchange HPLC, and the generated product was purified by Q-Sepharose.

[0060] Substrate Specificity of 3 - OST - 1 and 3 - OST - 3 To determine the substrate requirements of 3-OST-1 and 3-OST-3, a plurality of structurally uniform oligosaccharides of different sizes were used. These oligosaccharides include N-sulfated and 2-O-sulfated 6-mer to 12-mer (6-mer2S to 12-mer2S) and N-sulfated, 2-O-sulfated, and 6-O-sulfated 6-mer to 12-mer (6-mer2 S6S to 12-mer2S6S). These oligosaccharides were prepared by chemoenzymatic method 1, and their structures are shown in Figure 5C. Oligosaccharides (0.033 mM) were mixed with 50 mM MOP (pH 7.0), 10 mM MnCl2, 5 mM MgCl2, 2.5 μL of 3- OST-1 or 3-OST-3, and [35S] PAPS (1 - 3 x 10 5 cpm) and 30 μM PAPS in 100 It was incubated at 37 °C for 1 hour in μL reaction buffer. To this reaction mixture, 900 μL of 3 M urea containing 1.4 mM EDTA, 5 0 mM sodium acetate, and 150 mM NaCl was added to stop the reaction. Subsequently, this reaction mixture was purified using a DEAE column.

[0061] Kinetic Analysis of 3 - OST - 3 against Multiple Different Oligosaccharide Substrates The enzyme reaction rate was characterized by incubating a mixture of 30 μL of purified 3-OST-3 for N-sulfated and 2-O-sulfated oligosaccharides or 60 μL of purified 3-OST-3 for N-sulfated, 2-O-sulfated, and 6-O-sulfated oligosaccharides. PAPS (120 μM) was mixed with 1 × 1 0 cpm of [35S] PAPS as a sulfate donor and various concentrations of oligosaccharides from 0 to 200 μM at 37 °C for 1 hour. 3 0 5 To purify the 35S-labeled oligosaccharide product, the reaction solution was applied to a DEAE column. The amount of 35S-labeled oligo saccharide product was plotted against the substrate concentration, and curve fitting of the Michaelis-Menten graph was performed using Sigma Plot software to obtain the Km and Vmax values.

[0062] Purification of Compounds 1 - 6 by Q - Sepharose Purification of sulfated oligosaccharides was performed using a Q-Sepharose column. Mobile phase A was 25 mM Tris at pH 7.5, and mobile phase B contained 25 mM Tris and 1 M NaCl at pH 7.5. The elution gradient was synthesized at a flow rate of 1 mL / min based on the number of sulfate groups of the synthesized oligosaccharides. Absorbance at 310 nm and 260 nm was scanned and recorded. After purification, the sample was dialyzed twice against a buffer containing 5 mM sodium phosphate (pH 7.5) using a 1000 MWCO membrane.

[0063] HPLC Analysis of Synthetic Oligosaccharides Using a TSKgel DNA-NPR column, the completion degree of the reaction and the purity of the synthesized oligosaccharide after purification were detected. Mobile phase A was 25 mM Tris, pH 7.5, and mobile phase B was 25 mM Tris and 1 M NaCl, pH 7.5. The gradient step was 0 - 100% B in 100 minutes at a flow rate of 0.4 mL / min. The eluent was monitored using the absorption at 310 nm and 260 nm.

[0064] ESI - MS Analysis of Oligosaccharides The molecular weight structure of the synthesized oligosaccharide was determined by ESI-MS (Thermo LCQ-Deca). ESI -MS analysis was performed in the negative ion mode using the following parameters: Spray voltage 3.0 kV, curved desolvation line temperature 120 °C, mass range 300 - 1000.

[0065] Preparation of 3 - O - [34S] Sulfated Compound 3 To synthesize 3-O-[34S]sulfated compound 4, the substrate NS2S 8-mer (2 mg) was incubated in a total volume of 20 mL buffer containing 38 mM MOPS ( pH 7.0), 10 mM MnCl2, 5 mM MgCl2, and 6 mL 3-OST-3 with 3-OST-3 enzyme (0.11 mg / mL) and 0.1 mM [34S]PAPS. This reaction mixture was incubated overnight at 37 °C. The purification of the product was carried out using a Q-Sepharose column.

[0066] Tandem MS Analysis of 3 - OST - 3 Modified Octasaccharides Tandem mass spectrometry analysis was acquired on a Thermo LTQ-FT instrument in the negative ion mode using the following para meters: Spray voltage 3.5 kV, capillary voltage -40 kV, tube lens - 50 V, capillary temperature 275 °C. In the case of tandem mass, the selected precursor ion was run with the following parameters: IsoWidth (m / z): 3.0, normalized collision energy (%): 50.0 , Act.Q: 0.250, Act. time: 30, maximum injection time (ms): 500.000. MS and MS / MS data were recorded and processed using Xcalibur 2.2 software.

[0067] Structural Analysis of Compounds 1 - 6 by NMR NMR experiments were performed at 298 K using a Bruker Avance 700 MHz and 850 MHz spectrometer equipped with Topsin 3.2 software. Multiple samples (0.5 - 3.0 mg) were each dissolved in 0.5 ml D2O (99.9 96%, Sigma - Aldrich), and freeze - dried three times to remove exchangeable protons. These samples were redissolved in 0.5 ml D2O and transferred to NMR microtubes (outer diameter 5 mM, Norrell). Chemical shifts were referenced to external sodium 2,2 - dimethyl - 2 - silapentane - 5 - sulfonate salt (DSS, Sigma. Co.). Deuterated EDTA (Sigma. Co.) was added to remove the effects of paramagnetic ions. 1 D 1 The 1H - NMR experiment "zg" pulse sequence was run with 64 scans and an acquisition time of 3.8 seconds. 1 D 13 The 13C - NMR experiment "zgdc30" pulse sequence was run with 10,000 scans and an acquisition time of 1.0 second. 2 D 1 H - 13 The 1H - 13C HSQC experiment "hsqcgpph" pulse sequence was run with 48 scans, 512 increments, a relaxation delay of 1.5 seconds, and an acquisition time of 120 milliseconds. 2D The spectrum was recorded using GARP carbon decoupling. 48 dummy - scans were used before acquisition started. A total of 2048 points were collected at f2. 13 C transmitter off The set was set at 90.0 ppm.

[0068] Pretreatment of Molecular Dynamics The existing crystal structure of antithrombin (AT)-pentasaccharide complex (PDB ID: 3EVJ) was adopted as the starting structure of the system . Amino acid residues 25 - 33 and 396 were not resolved in this crystal structure and were therefore generated using the chimera interface to Modeller 47 . N-glyc ans were removed as they were distal (>15 Å) from the fondaparinux ligand. This original 48-51 ligand was modified for various simulations and manually adjusted using chimera . The parameters for amino acid and carbohydrate residues were obtained from the ff14SB7 and GLYCAM06(J-1) force fields . The system was neutralized with Na ions and solvated with a TIP3P water model in a truncated octahedral box of 12 Å around the complex . 53, 54 . The system was neutralized with Na + ions and solvated with a TIP3P water model in a truncated octahedral box of 12 Å around the complex .

[0069] Molecular Dynamics Protocol Energy minimization and MD simulations were performed using the pmemd.cuda module of AMBER14 . The Cα atoms of the protein backbone were restricted by Cartesian constraints 52 (10 kcal / molÅ ) at all stages of the process. In two simulations, 2 C4 or 1 S0 stereochemistry 2 was used Internal constraints of the IdoA2S residue d (Figure 4A) are required to maintain the seat, in accordance with the settings described above was executed 55 Before switching to the conjugate gradient in the remaining 24,000 cycles, the system was minimized using the steepest descent method in the first 1000 cycles To assist in generating a stable arrangement of the antithrombin (AT)-oligosaccharide complex Two minimizations were performed with various atomic constraints First, all solute atoms were restrained. Subsequently, the ligand restraint and the protein side chains were removed. Electrostatic interactions were treated with the particle-mesh Ewald algorithm and an 8A cutoff for non-bonded interactions was adopted 56 The SHAKE algorithm was applied to hydrogen-containing bonds, enabling an integration time step of 2 fs. The system was heated to 300 K under NVT conditions for over 60 ps using a Berendsen thermostat with a coupling time constant of 1 ps and equilibrated for a total of 50 ns under NPT conditions Under the same NPT conditions, an additional dataset after equilibration was collected for an additional 150 ns Under the same NPT conditions, an additional dataset after equilibration was collected for an additional 150 ns The interaction energy was calculated using the MPBSA.py.MPI module with the single-trajectory molecular mechanics-generalized

[0070] Simulation Data Analysis Born Solvent Accessible Surface Area (MM-GBSA) method Before this analysis 57 All water molecules and ions were removed from each complex, and the contribution from the desolvation energy was approximated by the GB implicit solvent model (igb = 2) This simulation was divided into 5 ns bins and an ensemble of 100 snapshots evenly distributed within each bin was used 58 This simulation was divided into 5 ns bins ) and from an ensemble of 100 snapshots evenly distributed within each bin The contribution of the average interaction energy was calculated. GraphPad Prism for Windows version 5.04 was used for statistical analysis. By t-test analysis [Number] Significance was determined based on. Images were created using the Visual Molecular Dynamics program completed 59 .

[0071] In Vitro Anti - FXa Activity Measurement Factor Xa (Enzyme Research Laboratories, South Bend, IN) was diluted to 60 nM in phosphate-buffered saline (PBS) containing 1 mg / mL bovine serum albumin (BSA). Human antithrombin (AT) (obtained from Cutter Biological) at a concentration of 0.65 μM in PBS containing 1 mg / mL BSA was prepared. The chromogenic substrate S-2765 (Diapharma) was dissolved in water to create a 1 mg / mL stock solution . Multiple oligosaccharides at various concentrations (10 - 200 nM) were prepared in PBS. This solution was a mixture of 60 μL of antithrombin (AT) and 15 μL of the sample solution, which was stirred and incubated at room temperature for 2 minutes. Then 90 μL of Factor Xa was added to this and incubated at room temperature for 4 minutes , and then 30 μL of S-2765 was added. The absorbance of this reaction mixture at 405 nm was measured continuously for 2 minutes. The calculation of the IC value was plotted as a function of the sample concentration and the initial reaction rate . All studies were conducted in accordance with the Public Health Service guidelines on the care and use of laboratory animals 50 .

[0072] In Vivo Anti - FXa Effect Measurement ​​Under an Institutional Animal Care and Use Committee (IACUC) approved protocol at the University of North Carolina Sixteen 400-g Lewis rats were evenly divided into four groups. Each group was administered fondaparinux without endotoxin (0.46 μM / kg -1 ), compound 11 (0.43 μM / kg -1 ), compound 5 (0.42 μM / kg -1 ), or saline by intravenous injection. Next, one sample was collected prior to the administration of the compound, and blood was drawn at designated time points (0.5, 1, 2, 4, 8 hours) after the administration of the compound. One sample was collected immediately before the injection of the compound, and 0.8 mL of blood was drawn from the contralateral femur / saphenous vein 0.5 , 1, 2, 4, and 8 hours after the injection of the compound and placed in 150 mM citrate. These samples were centrifuged to obtain approximately 400 μL of plasma. These blood samples were subjected to FXa activity analysis. Saline was administered subcutaneously periodically to maintain fluid volume . The concentration of the compound in the reaction mixture was read using the calibration curve prepared for each compound , and the corresponding percentage of FXa activity (%) was obtained. The mean plasma

[0073] Determination of Oligosaccharide Clearance from Animals Treated with Drugs concentration and standard deviation among the four rats in each group at each time point were calculated. Using these values, a graph of plasma concentration versus time was plotted . In this study, the synthesis of hexasaccharides (compounds 1 - 3, Figure 1A) and octasaccharides (compounds 4 - 6, Figure 1A) was achieved .

[0074] Example 1 Chemoenzymatic Synthesis of Oligosaccharides with GlcNS3S and GlcNS3S6S Residues Two 3-O-sulfotransferase (3-OST) isoforms, 3-OST-1 and 3-O ST-2, were involved Using ST-3, GlcNS3S±6S residues were introduced into different sugar sequences. The 3-OST-1 enzyme introduces sulfation to form GlcNS3S6S residues that bind to the GlcA residue at the non-reducing end, forming a disaccharide unit of -GlcA-GlcNS3S6S-. On the other hand, the 3-OST-3 enzyme introduces sulfation to form GlcNS3S residues that bind to the IdoA2S residue at the non-reducing end, forming a disaccharide unit of -IdoA2S-GlcNS3S-. 3-OST-1 has been used in the synthesis of oligosaccharides in many studies, but the synthesis of oligosaccharides containing the -IdoA2S-GlcNS3S- disaccharide unit using 3-OST-3 has not been reported. introduces sulfation to form GlcNS3S6S residues that bind to the GlcA residue at the non-reducing end, forming a disaccharide unit of -GlcA-GlcNS3S6S-. While, the 3-OST-3 enzyme introduces sulfation to form GlcNS3S residues that bind to the IdoA2S residue at the non-reducing end, forming a disaccharide unit of -IdoA2S-GlcNS3S-. forming a disaccharide unit of -IdoA2S-GlcNS3S-. 3-OST-1 has been used in the synthesis of oligosaccharides in many studies However, the synthesis of oligosaccharides containing the -IdoA2S-GlcNS3S- disaccharide unit using 3-OST-3 has not been reported. 16,17,27,28 , using 3-OST-3 to synthesize oligosaccharides containing the -IdoA2S-GlcNS3S- disaccharide unit has not been reported. disaccharide unit has not been reported.

[0075] Disclosed herein is the discovery that 3-OST-3 and 3-OST-1 have different substrate requirements. The 3-OST-1 enzyme sulfates oligosaccharide substrates that are already 6-O-sulfated, but shows very low reactivity towards oligosaccharide substrates that are not 6-O-sulfated (Figures 5A and 5B). This is consistent with the above conclusion. sulfates oligosaccharide substrates that are already 6-O-sulfated, but shows very low reactivity towards oligosaccharide substrates that are not 6-O-sulfated (Figures 5A and 5B). This is consistent with the above conclusion. consistent with the above conclusion. 16 . In contrast, 3-OST-3 preferentially sulfates oligosaccharides that are not 6-O-sulfated (Figures 5A and 5B), but has low reactivity towards the 3-OST-3 modification of these 6-O-sulfated oligosaccharide substrates (Figures 5A, 5B and 5C). The results of kinetic analysis show that 3-OST-3 has a higher catalytic efficiency (determined by the value of kcat / Km) towards oligosaccharide substrates that are not 6-O-sulfated (Table 1). sulfates oligosaccharides that are not 6-O-sulfated (Figures 5A and 5B), but has low reactivity towards the 3-OST-3 modification of these 6-O-sulfated oligosaccharide substrates (Figures 5A, 5B and 5C). The results of kinetic analysis show that 3-OST-3 has a higher catalytic efficiency (determined by the value of kcat / Km) towards oligosaccharide substrates that are not 6-O-sulfated (Table 1). , has a higher catalytic efficiency (determined by the value of kcat / Km) towards oligosaccharide substrates that are not 6-O-sulfated (Table 1).

[0076]

Table 1

[0077] The discovery that the substrate requirements for 3-OST-1 and 3-OST-3 are different is described in this specification as disclosed herein, led to the development of two different schemes for synthesizing multiple oligosaccharides with different 3-O-sulfated sugar sequences. For the synthesis of oligosaccharides containing the IdoA2S-GlcNS3S±6S disaccharide unit (compounds 1, 2, 4, and 5 shown in Figures 6A, 6B, 6D, and 6E respectively), 3-O-sulfation by 3-OST-1 is introduced before the 6-O-sulfation step (Figure 1B). On the other hand, for the synthesis of oligosaccharides containing the GlcA-GlcNS3S6S disaccharide unit, 3-O-sulfation by 3-OST-3 is carried out after the 6-O-sulfation step (Figure 1B)(compounds 3 and 6 shown in Figures 6C and 6F). The antithrombin (AT) binding domain contains the pentasaccharide unit of -GlcNS(or Ac)6S-GlcA-GlcNS3S±6S-IdoA2S-GlcA-GlcNS6S-, and the 3-O-sulfation there might be necessary for high binding affinity . Among the oligosaccharides tested in this study, only compounds 8 and 11 contain this pentasaccharide unit. 24,29

[0078] Example 2 Structural and Conformational Analysis of Oligosaccharides Purity measurement and structural analysis of compounds 1 - 6 were performed. Representative data from the analysis of compound 6 (Figure 6F) are shown in Figures 2A and 2B. Compound 6 eluted as a single peak by high-resolution anion-exchange HPLC , indicating that this compound is pure (Figure 2A). The molecular weight of compound 6 was determined to be 2449.43±0.74 by electrospray ionization mass spectrometry (ESI-MS). This is very close to the calculated molecular weight of 2448.92 (Figure 2B). The 1H-NMR spectrum of compound 6 shows eight anomeric 1 ​​​- Protons are clearly shown, and it is confirmed that the product is an octasaccharide. For compound 6 13 C-NMR and the complete NMR assignments are shown in Supplementary Figure 25 and Supplementary Table 1, respectively. To identify the 3-O-sulfate group of compound 4 tandem MS analysis was performed. In this analysis, a stable isotope-labeled [34S] sulfate group was introduced by the 3-OST-3 enzyme, and it was clearly identified that there is a 3-O-sulfate group in residue d of compound 4 Since compound 4 is an intermediate between compounds 5 and 6, the tandem MS analysis of compound 4 was also useful for the identification of the IdoA2S-GlcNS3S6S disaccharide unit of compounds 5 and 6.

[0079] The pyranose ring of the IdoA2S residue is in the chair form ( 1 C4 and 4 C1) and the skew boat form ( 2 S0) and interconverts between different conformations (Figure 1C). The effect of 3-O sulfation on the conformation of adjacent IdoA2S residues was investigated. Conformational analysis was completed by NMR by measuring the three-bond proton-proton coupling constant (3JH -H). Comparing with the IdoA2S residue (compound 7) of the hexasaccharide without the GlcNS3S6S residue the number of 2SO conformational isomers of the IdoA2S residue in the hexasaccharide increases due to 3-O sulfation (compounds 1-3, Table 2 and Figure 1A). In compound 3, the IdoA2S residue shows almost exclusively 30 the S0 conformation. In the case of the octasaccharide (compounds 4-6, Table 2 and Figure 1A), there are two IdoA2S residues called residues c and e. The effect of 3-O sulfation on the number (proportion) of S0 is various For example, 3-O sulfation, compared with the IdoA2S residue (compound 10) of the octasaccharide without the GlcNS3S6S residue exclusively 2 shows the S0 conformation. In the case of the octasaccharide (compounds 4-6, Table 2 and Figure 1A), there are two IdoA2S residues called residues c and e. exist. 2 The effect of 3-O sulfation on the number of S0 is various For example, 3-O sulfation, compared with the IdoA2S residue (compound 10) of the octasaccharide without the GlcNS3S6S residue That is, as shown in Compound 5, the number (proportion) of the population of S0 of the IdoA2S residues on both sides is increased. 2 In Compound 11, due to the 3-O sulfation of residue f, the number (proportion) of the population of S0 of the adjacent IdoA2S residue (residue e) increases, but it has no effect on the distant IdoA2S residue (residue c) compared with the corresponding residue of Compound 10. In Compound 6, due to the 3-O sulfation of residues d and f, the number (proportion) of the population of S0 of residue e increases, but the number (proportion) of the population of S0 of residue c decreases from 71% to 58% compared with the corresponding residue of Compound 5 (Table 2). These observations suggest that 3-O-sulfation regulates the structure of heparan sulfate (HS) through its influence on the conformation of IdoA2S residues. 2 In Compound 11, due to the 3-O sulfation of residue f, the number (proportion) of the population of S0 of the adjacent IdoA2S residue (residue e) increases, but it has no effect on the distant IdoA2S residue (residue c) compared with the corresponding residue of Compound 10. In Compound 6, due to the 3-O sulfation of residues d and f, the number (proportion) of the population of S0 of residue e increases, but the number (proportion) of the population of S0 of residue c decreases from 71% to 58% compared with the corresponding residue of Compound 5 (Table 2). These observations suggest that 3-O-sulfation regulates the structure of heparan sulfate (HS) through its influence on the conformation of IdoA2S residues. In Compound 6, due to the 3-O sulfation of residues d and f, the number (proportion) of the population of S0 of residue e increases, but the number (proportion) of the population of S0 of residue c decreases from 71% to 58% compared with the corresponding residue of Compound 5 (Table 2). These observations suggest that 3-O-sulfation regulates the structure of heparan sulfate (HS) through its influence on the conformation of IdoA2S residues. 2 In Compound 6, due to the 3-O sulfation of residues d and f, the number (proportion) of the population of S0 of residue e increases, but the number (proportion) of the population of S0 of residue c decreases from 71% to 58% compared with the corresponding residue of Compound 5 (Table 2). These observations suggest that 3-O-sulfation regulates the structure of heparan sulfate (HS) through its influence on the conformation of IdoA2S residues. 2 In Compound 6, due to the 3-O sulfation of residues d and f, the number (proportion) of the population of S0 of residue e increases, but the number (proportion) of the population of S0 of residue c decreases from 71% to 58% compared with the corresponding residue of Compound 5 (Table 2). These observations suggest that 3-O-sulfation regulates the structure of heparan sulfate (HS) through its influence on the conformation of IdoA2S residues. In Compound 6, due to the 3-O sulfation of residues d and f, the number (proportion) of the population of S0 of residue e increases, but the number (proportion) of the population of S0 of residue c decreases from 71% to 58% compared with the corresponding residue of Compound 5 (Table 2). These observations suggest that 3-O-sulfation regulates the structure of heparan sulfate (HS) through its influence on the conformation of IdoA2S residues. In Compound 6, due to the 3-O sulfation of residues d and f, the number (proportion) of the population of S0 of residue e increases, but the number (proportion) of the population of S0 of residue c decreases from 71% to 58% compared with the corresponding residue of Compound 5 (Table 2). These observations suggest that 3-O-sulfation regulates the structure of heparan sulfate (HS) through its influence on the conformation of IdoA2S residues.

[0080]

Table 2

[0081] Example 3 The presence of the - GlcNS3S6S - IdoA2S - disaccharide unit affects anticoagulant activity. Oligosaccharides of heparan sulfate (HS) exert anticoagulant activity by interacting with antithrombin (AT). Therefore, oligosaccharides showing anti-FXa activity should bind to AT. The effects of sugar residues surrounding the GlcNS3S6S residue on AT binding and anti-FXa activity were investigated. These data can clarify the role played by the presence of the -GlcNS3S6S-IdoA2S- disaccharide unit (i.e., IdoA2S is located at the reducing end of the GlcNS3S6S residue) in the determination of anticoagulant activity (Table 3). Oligosaccharides of heparan sulfate (HS) exert anticoagulant activity by interacting with antithrombin (AT). Therefore, oligosaccharides showing anti-FXa activity should bind to AT. The effects of sugar residues surrounding the GlcNS3S6S residue on AT binding and anti-FXa activity were investigated. These data can clarify the role played by the presence of the -GlcNS3S6S-IdoA2S- disaccharide unit (i.e., IdoA2S is located at the reducing end of the GlcNS3S6S residue) in the determination of anticoagulant activity (Table 3). Oligosaccharides of heparan sulfate (HS) exert anticoagulant activity by interacting with antithrombin (AT). Therefore, oligosaccharides showing anti-FXa activity should bind to AT. The effects of sugar residues surrounding the GlcNS3S6S residue on AT binding and anti-FXa activity were investigated. These data can clarify the role played by the presence of the -GlcNS3S6S-IdoA2S- disaccharide unit (i.e., IdoA2S is located at the reducing end of the GlcNS3S6S residue) in the determination of anticoagulant activity (Table 3). Oligosaccharides of heparan sulfate (HS) exert anticoagulant activity by interacting with antithrombin (AT). Therefore, oligosaccharides showing anti-FXa activity should bind to AT. The effects of sugar residues surrounding the GlcNS3S6S residue on AT binding and anti-FXa activity were investigated. These data can clarify the role played by the presence of the -GlcNS3S6S-IdoA2S- disaccharide unit (i.e., IdoA2S is located at the reducing end of the GlcNS3S6S residue) in the determination of anticoagulant activity (Table 3). Oligosaccharides of heparan sulfate (HS) exert anticoagulant activity by interacting with antithrombin (AT). Therefore, oligosaccharides showing anti-FXa activity should bind to AT. The effects of sugar residues surrounding the GlcNS3S6S residue on AT binding and anti-FXa activity were investigated. These data can clarify the role played by the presence of the -GlcNS3S6S-IdoA2S- disaccharide unit (i.e., IdoA2S is located at the reducing end of the GlcNS3S6S residue) in the determination of anticoagulant activity (Table 3). Compounds 2 and 8 have six residues, eight sulfonate groups, one IdoA2S, and two GlcA residues. However, only compound 8 showed anti-FXa activity (Figure 3A and Table 3). As expected, antithrombin (AT) strongly binds to compound 8 (Kd value 7 ± 2 nM), but AT does not bind to compound 2 (Table 3). These compounds are structurally different in the position of the IdoA2S residue. In compound 8, the IdoA2S residue is adjacent to the reducing end of the 3-O-sulfated glucosamine (GlcNS3S6S) residue, and compound 8 has a -GlcNS3S6S-IdoA2S- disaccharide unit. On the other hand, the GlcNS3S6S of compound 2 has a GlcA residue adjacent to the reducing end of the residue and has a -GlcNS3S6S-GlcA- disaccharide unit. Thrombin (AT) strongly binds to compound 8 (Kd value 7 ± 2 nM), but AT does not bind to compound 2 (Table 3). Structurally, these compounds differ in the position of the IdoA2S residue. In compound 8, the IdoA2S residue is adjacent to the reducing end of the 3-O-sulfated glucosamine (GlcNS3S6S) residue, and compound 8 has a -GlcNS3S6S-IdoA2S- disaccharide unit. On the other hand, the GlcNS3S6S of compound 2 has a GlcA residue adjacent to the reducing end of the residue and has a -GlcNS3S6S-GlcA- disaccharide unit.

Table 3

[0082] consistent with the previously published conclusion that the SO structure affects anticoagulant activity.

[0083] The IdoA2S residue in compound 8 shows the SO conformation. 2 On the other hand, in compound 2, this position is occupied by a GlcA residue that takes the C1 conformation. The X-ray crystal structure and NMR solution structure of the complex of antithrombin (AT) and fondaparinux show that the IdoA2S residue is present in the SO conformation of the complex. 9 The IdoA-containing hexamer (6-mer) in which the IdoA2S residue was replaced with an IdoA residue showing the SO conformation showed anti-FXa activity (Table 3). 2 However, the GlcA2S-containing hexamer in which the IdoA2S residue was replaced with a GlcA2S residue showing the C1 conformation did not show anti-FXa activity. This position is 4 occupied by a GlcA residue that takes the C1 conformation. Antithrombin (AT) The X-ray crystal structure and NMR solution structure of the complex of antithrombin (AT) and fondaparinux show that the IdoA2S residue is present in the SO conformation of the complex. 2 indicating that the IdoA2S residue is present in the SO conformation. 31,32 The IdoA2S residue 2 is replaced with an IdoA residue showing the SO conformation. The IdoA-containing hexamer (6-mer) showed anti-FXa activity (Table 3). 30 However, the IdoA2S residue is 4 replaced with a GlcA2S residue showing the C1 conformation. The GlcA2S-containing hexamer did not show anti-FXa activity. (Table 3).8 (Table 2). Compound 3, this hexamer (6-mer) is composed of two GlcNS3S6S residues, therefore, shows strong anti-FXa activity (Figure 3A and Table 3), consistent with the results previously published for an octasaccharide containing a similar pentasaccharide domain. 32 Residues d and c of compound 3 constitute the -GlcNS3S6S-IdoA2S-disaccharide unit.

[0084] Example 4 The GlcA residue in the antithrombin (AT) binding domain can be replaced by the IdoA2S residue These studies revealed a new AT-binding sugar sequence. The currently known AT-binding sequence contains the disaccharide -GlcA-GlcNS3S±6S- unit. 33 . Replacing the -GlcA-GlcNS3S6S- disaccharide unit in heparan sulfate (HS) with the -IdoA2S-GlcNS3S6S- disaccharide unit was recognized to abolish the AT-binding affinity. 34,35 . Data from the anti-FXa activity and AT-binding analysis of the 8-mer clearly challenged this claim. Compound 5 containing the IdoA2S-GlcNS3S6S- (not -GlcA-GlcNS3S6S-) disaccharide unit shows strong anti-FXa activity (Table 3). Isothermal titration calorimetry (ITC) was used for the AT-binding affinity analysis, which also confirmed that compound 5 binds firmly to antithrombin (AT) (Figure 3B and Table 3). Compound 6, this octamer contains the -GlcA-GlcNS3S6S- disaccharide unit, therefore, shows anti-FXa activity and high AT-binding affinity (Table 3).

[0085] The anticoagulant activity of compound 5 was further confirmed in an in vivo experiment using a rat model. For this purpose, compound 5 was administered, and its anti-FXa effect was compared with fondaparinux and previously anticoagulant Compared with compound 11, which was reported to be an octasaccharide 17 . The results showed that compound 5 had an efficacy comparable to that of fondaparinux and compound 11 against FXa within 30 minutes after drug administration , as demonstrated in (Figure 3C). However, the anti-FXa effect of compound 5 decreased after 4 hours. On the other hand the anti-FXa effects of fondaparinux and compound 11 persisted even after 8 hours (Figure 3C). Blood samples were taken to determine the in vivo clearance rate of each compound (Figure 3D ). Compared with fondaparinux and compound 11, compound 5 was rapidly removed from the animals within the first 2 hours .

[0086] The structural disorder of residue d in the antithrombin (AT) binding site (Figure 4A) is supported by molecular dynamics (MD ) simulations of antithrombin (AT) in complex with heparin sulfate (HS) pentasaccharide. This computational technique was first verified by replacing parts known to be essential for AT binding within the co-crystal structure of AT and fondaparinux . Calculations of the free energy of binding showed that removing the 3-O sulfate on residue c or replacing residue b with GlcA destabilized the complex, as indicated by a significant weakening of the interaction energy by 29% and 17% respectively , which was qualitatively consistent with the experimental data. In contrast, replacing GlcA (residue d) with IdoA2S in the C4 24,36 conformation had no effect on the free energy (Figure 4B). Interestingly , replacement of the IdoA2S residue in the SO conformation enhanced the binding energy by only 12% , as confirmed 1 and was in qualitative agreement with the experimental data. In contrast, replacing GlcA (residue d) with IdoA2S in the C4 conformation had no effect on the free energy (Figure 4B). Interestingly, replacement of the IdoA2S residue in the SO conformation enhanced the binding energy by only 12% . 2 Substitution of the IdoA2S residue in the SO conformation enhanced the binding energy by only 12% ​Figure 4B), which indicates that if residue d is in the 2SO conformation, IdoA2S, a more stable antitron would be obtained. Both conformations of IdoA2S are involved in the formation of fondaparinuclide. As found for the GlcA residue in the simulation of the protein, In summary, these The molecular dynamics (MD) data showed that the substitution of residue d with the IdoA2S residue did not result in antithrombin (AT). This supports the conclusion that compound 5 does not decrease the binding affinity of antithrombin (AT). Explains how it acts as an active anticoagulant despite lacking the canonical pentasaccharide sequence do.

[0087] Example 5 Analysis of 7 - mer Heparan Sulfate (HS) Compounds Using the synthetic methods disclosed herein, 7-mer heparan sulfate (HS) compounds or heparin Analogs were developed. The 7-mer, the structure of which is shown in FIG. 8B, was tested for anti-Xa activity. The anti-Xa activity was demonstrated (Figure 8A). Figure 8B shows the chemical structure of the 7-mer in the 6-mer as shown therein. As an example and not by way of limitation, the following are some examples for synthesizing 7-mers: One synthetic route is shown in FIG. 8C. In some embodiments, the costs associated with the synthesis It may be preferable to have relatively short oligosaccharides, such as 7-mers, as this allows for a reduction in There is.

[0088] Example 6 Discussion of Results Herein, we present a method for preparing a 3-O-sulfated oligosaccharide library using a chemoenzymatic approach. Multiple schemes for production are disclosed. In this specification, -IdoA2S-GlcNS3S- or IdoA2S-G To synthesize oligosaccharides containing the lcNS3S6S- disaccharide unit, 3-OST-3 modification must precede the 6-O-sulfation step. On the other hand, it has been shown that for the production of the -GlcA-GlcNS3S6S- disaccharide unit, 3-OST-1 modification must occur only after 6-O-sulfation. This discovery is supported by the ternary co-crystal structures of 3-O ST-1 / heptasaccharide / PAP 37 and 3-OST-3 / tetrasaccharide / PAP 38 There is no interaction between 3-OST-3 and the 6-O-sulfo group of the tetrasaccharide substrate, which is consistent with the conclusion that the oligosaccharide substrate of 3-OST-3 does not require 6-O-sulfation. In contrast, an interaction is observed between 3- OST-1 and the 6-O-sulfo group of the heptasaccharide substrate, indicating that 6- O-sulfation is required for binding to 3-OST-1. This clear and unique substrate requirement between 3-OST-1 and 3-OST-3 raises the possibility that 3-O-sulfated heparan sulfate (HS) modified by different isoforms of 3-OST is biosynthesized via different pathways. It has been widely accepted that the 3-OST-1 enzyme is involved in the synthesis of the anticoagulant heparan sulfate (HS), while the 3-OST-3 enzyme is

[0089] not. All of the AT-binding sequences isolated so far were composed of the -GlcA-GlcNS3S6S- disaccharide repeat unit, which is the product of 3-OST-1 enzyme modification. 34,35 Compound 5, which is the product of 3-OST-3 enzyme modification, does not contain the -GlcA-GlcNS3S6S- disaccharide unit, but 3 3,39 ​​, bind to AT and exhibit anticoagulant activity. These findings suggest that 3-OST-3 can synthesize anticoagulant heparan sulfate (HS) as long as it contains a structural domain similar to that of compound 5 's structural domain. (HS

[0090] The rapid clearance of compound 5 provides a potential new short-acting anticoagulant candidate with a reduced bleeding risk. A short-acting anticoagulant that can be rapidly removed from the circulation before the major bleeding effect occurs is particularly beneficial for patients at high risk of bleeding . 41 Un fractionated heparin is an anticoagulant with a short half-life, but there is concern that this drug may cause heparin-induced thrombocytopenia (HIT), which is a life-threatening side effect 42 . Short oligosaccharides smaller than 12-mers do not bind to platelet factor 4 and thus have been shown not to pose a risk of heparin-induced thrombocytopenia (HIT). Therefore, compound 5, as an octasaccharide 43 , is expected to have a very low risk of HIT. The availability of 3-O-sulfated oligosaccharides also opens up an opportunity to investigate the casual relationship between sugar sulfation / conformation and biological function . This is a major advance in the detailed investigation of the relationship between the structure and function of heparan sulfate (HS). Heparan sulfate (HS) 3-OST exists in seven different isoforms. In this study , it was demonstrated that different chemoenzymatic schemes are required for 3-OST-1 and 3-OST-3 to prepare different 3-O-sulfated oligosaccharide sequences. The conclusion of this study leads others to develop chemoenzymatic methods using different 3-OST isoforms, which is 3-O-sulfated glu . In this study, it was demonstrated that different chemoenzymatic schemes are required for 3-OST-1 and 3-OST-3 to prepare different 3-O-sulfated oligosaccharide sequences. The conclusion of this study leads others to develop chemoenzymatic methods using different 3-OST isoforms, which is 3-O-sulfated glu cose ​​Enable the preparation of more complex heparan sulfate (HS) saccharides with a broader sulfation pattern containing sialic acid residues. These studies enrich the oligosaccharide library of heparan sulfate (HS) and support HS-related research.

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Claims

**Claim 1** a 3-O-sulfated oligosaccharide containing 6 to 8 sugar units, at least one disaccharide unit sulfated by 3-OST-3 enzyme, and at least one IdoA2S-GlcNS3S or IdoA2S-GlcNS3S6S disaccharide unit comprising [wherein R is selected from the group consisting of -H, alkyl, substituted alkyl, aryl, and substituted aryl] and having a structure of a synthetic oligosaccharide. **Claim 2** The following structure: [wherein R is selected from the group consisting of -H, alkyl, substituted alkyl, aryl, and substituted aryl] The synthetic oligosaccharide according to claim 1, consisting of.

3. The alkyl is -CH ３ or -CH ２ CH ３ or the substituted aryl is a p-nitrophenyl group, the synthetic oligosaccharide according to claim 1 or 2. **Claim 4** A pharmaceutical composition for treating a subject in need of anticoagulant therapy, comprising a synthetic oligosaccharide of [wherein R is selected from the group consisting of -H, alkyl, substituted alkyl, aryl, and substituted aryl] ​

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