Short-acting heparin-based anticoagulant compounds and methods

Abstract

The present invention relates to short-acting heparin-based anticoagulant compounds and methods. Heparin compounds and synthetic heparin analogs having short-acting anticoagulant activity are provided. Methods for synthesizing such heparin compounds using sulfotransferases, including chemoenzymatic pathways, are provided. Methods for treating a subject in need of anticoagulant activity are provided.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201880020095.X, titled "Short-Acting Heparin-Based Anticoagulant Compounds and Methods", with a filing date of March 12, 2018.

[0002] Citation of Related Applications

[0003] This application claims the benefit of U.S. Provisional Patent Application Serial No. 62 / 469,643, filed on March 10, 2017, which is incorporated herein by reference in its entirety.

[0004] Statement of Authorization

[0005] This invention was made with government support under National Institutes of Health grants GM102137, HL094463, CA207824, and GM103390. The government has certain rights in the invention. Technical Field

[0006] The subject matter disclosed herein generally relates to the synthesis of heparin compounds. More specifically, the subject matter disclosed herein relates to heparin compounds having short acting anticoagulant activity and the chemoenzymatic synthesis of heparin analogs. Background Art

[0007] Heparan sulfate (HS) is a ubiquitous component of the cell surface and extracellular matrix. It regulates a wide range of physiological and pathophysiological functions, including embryonic development and blood coagulation, and is able to promote viral infection (Esko and Selleck (2002) Annu. Rev. Biochem. 71, 435-471; Liu and Thorp (2002) Med. Res. Rev. 22, 1-25). HS exerts its biological role by interacting with the specific proteins involved in a given process (Capila and Lindhardt (2002) Angew. Chem. Int. Ed. 41, 390-412). HS is a highly charged polysaccharide that contains 1→4-linked glucosamine and glucuronic acid / iduronic acid units (containing both N- and O-sulfates). The unique sugar sequences within HS can determine the specificity of HS binding to its target proteins (Linhardt (2003) J. Med. Chem. 46, 2551-2564). Heparin, a special form of HS, is a commonly used anticoagulant drug. Thus, new methods for synthesizing heparin compounds and HS have attracted considerable interest among those developing anticoagulants and other HS-related drugs with improved pharmacological effects.

[0008] Heparin has been successfully used as an anticoagulant drug for over 50 years (Mackman, 2008). It is currently marketed in three forms: unfractionated (UF) heparin (MW 平均 ~14000 Da); low molecular weight heparin (MW 平均 ~6000 Da); and synthetic ULMW heparin pentasaccharide (MW 1508.3 Da). UF heparin is used in surgery and renal dialysis due to its relatively short half-life and safety in patients with impaired renal function (Hirsh et al., 2007).

[0009] The synthesis of HS oligosaccharides and related anticoagulant compounds remains a challenge. There is a great need for cost-effective methods and means for synthesizing new synthetic heparins. SUMMARY OF THE INVENTION

[0010] The present invention content section lists several embodiments of the disclosed subject matter, and in many cases lists variations and permutations of these embodiments. The present invention content section is merely an example of numerous different embodiments. Mentioning one or more representative features of a given embodiment is also exemplary. Such embodiments may or may not have the mentioned features; similarly, these features can be applied to other embodiments of the disclosed subject matter whether or not they are listed in the present invention content section. To avoid excessive repetition, the present invention content section does not list or suggest all possible combinations of such features.

[0011] In some embodiments, synthetic heparin analogs are provided herein that comprise 3-O-sulfated oligosaccharides 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. In some embodiments, such synthetic heparin analogs can have anticoagulant activity. In some embodiments, such synthetic heparin analogs have a binding affinity for antithrombin (from about 5 nM to about 30 nM). In some embodiments, such synthetic heparin analogs can have an -1 anticoagulant activity of from about 10 ng / mL -1 to 50 about 40 ng / mL.

[0012] In some aspects, the synthetic heparin analogs provided herein can contain at least one IdoA2S-GlcNS3S6S disaccharide unit and do not contain GlcA-GlcNS3S6S disaccharide units. In some embodiments, such synthetic heparin analogs can have a clearance rate that is about 50% to about 100% faster than other heparin compounds. In some embodiments, such synthetic heparin analogs do not cause heparin-induced thrombocytopenia (HIT). In some embodiments, such synthetic heparin analogs can be reversed by andexanet-α at a rate of 50% or higher in the presence of 20 μg / mL or less of andexanet-α.

[0013] In some embodiments, provided herein is a method for synthesizing a heparin analog, comprising providing a sugar substrate, extending the sugar substrate to a sugar of a desired or predetermined length, and performing at least one sulfation reaction using the 3-OST-3 isoform of a 3-O-sulfotransferase (3-OST) enzyme, thereby synthesizing a heparin analog. In some aspects, the sugar substrate comprises at least one IdoA2S-GlcNS3S disaccharide unit. In some aspects, the sugar substrate comprises an IdoA2S-GlcNS3S±6S disaccharide unit, wherein the method further comprises a 6-O-sulfation step using a 6-O-sulfotransferase (6-OST), wherein the 3-O-sulfation by 3-OST-3 occurs prior to the 6-O-sulfation step. In some aspects, the sugar substrate comprises a GlcA-GlcNS3S6S disaccharide unit, wherein the method further comprises a 6-O-sulfation step using a 6-O-sulfotransferase (6-OST), wherein the 3-O-sulfation by 3-OST-1 occurs prior to the 6-O-sulfation step.

[0014] In some aspects, the extension step comprises using a glucosyltransferase. In some embodiments, the glucosyltransferase 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, the extension step comprises using one or more monosaccharides selected from the group consisting of glucuronic acid (GlcUA), N-acetylated glucosamine (GlcNAc), and N-trifluoroacetyl glucosamine (GlcNTFA). In some aspects, the method for synthesizing the heparin analog has a yield of greater than about 20% to about 50%.

[0015] Also provided herein is a method for treating a subject in need of anticoagulant therapy, the method comprising providing a subject in need of anticoagulant therapy and administering to the subject a heparin analog having anticoagulant activity, wherein the heparin analog comprises at least one disaccharide unit sulfated by a 3-OST-3 enzyme and at least one IdoA2S-GlcNS3S or IdoA2S-GlcNS3S6S disaccharide unit. In some embodiments, such methods further comprise monitoring the subject for heparin-induced thrombocytopenia and, if the subject has heparin-induced thrombocytopenia, administering an antidote to the subject to reverse the anticoagulant activity of the heparin analog. In some embodiments, the antidote for reversing the anticoagulant activity of the heparin analog is andexanet-α. In some embodiments, the subject is a human subject.

[0016] In this method of treating a subject, the synthetic heparin analog can have a clearance rate that is about 50% to about 100% faster than other heparin compounds. The synthetic heparin analog can contain less than about 10% anticoagulant activity 4 hours after administration. In these methods, the subject can have an elevated bleeding risk.

[0017] The present disclosure provides pharmaceutical compositions comprising the synthetic heparin compounds disclosed herein.

[0018] The synthetic heparin analogs disclosed herein, including methods of making and / or using them, can include a structure that includes, for example:

[0019]

[0020]

[0021] wherein R is selected from the group consisting of -H, alkyl (e.g., but not limited to, -CH 3 or -CH 2 CH 3 ), substituted alkyl, aryl, and substituted aryl (e.g., but not limited to, p-nitrophenyl).

[0022] Accordingly, an object of the subject matter disclosed herein is to provide short-acting heparin-based anticoagulant compounds and methods, including novel forms of synthetic heparin.

[0023] This and other objects are achieved, in whole or in part, by the subject matter disclosed herein. In addition, after study of the following description, drawings, and embodiments, the objects, other objects, and advantages of the subject matter disclosed herein, as set forth above, will become apparent to those of ordinary skill in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The subject matter of the present disclosure can be better understood by reference to the following drawings. The components in the drawings are not necessarily drawn to scale, but rather emphasis is placed on illustrating the principles of the subject matter of the present disclosure (generally schematically). In the drawings, the same reference numerals denote corresponding parts in different views. By reference to the embodiments illustrated in the drawings, a further understanding of the subject matter of the present disclosure can be obtained. Although the illustrated embodiments are merely examples of systems for implementing the subject matter of the present disclosure, the organization and method of operation of the subject matter of the present disclosure (generally together with its further objectives and advantages) can generally be more readily understood by reference to the drawings and the following description. The drawings are not intended to limit the scope of the subject matter of the present disclosure (which is specifically set forth in the appended or subsequently amended claims), but rather are merely intended to clarify and illustrate the subject matter of the present disclosure.

[0025] For a more complete understanding of the subject matter of the present disclosure, reference is now made to the following drawings, in which:

[0026] Figures 1A to 1C The figure illustrates the synthesis of hexasaccharides and octasaccharides disclosed and tested herein, including Figure 1A , Figure 1B and Figure 1C , Figure 1A is a schematic diagram of the hexasaccharides and octasaccharides disclosed herein, Figure 1B is a schematic diagram of an exemplary method for synthesizing the hexasaccharides and octasaccharides disclosed herein, Figure 1C The figure illustrates the chemical structure of the pyranose ring of the IdoA2S residue of the hexasaccharides and octasaccharides disclosed herein.

[0027] Figure 2A is a diagram of the results of high-resolution anion-exchange HPLC, while Figure 2B shows the results of electrospray ionization mass spectrometry (ESI-MS) performed on compound 6 to demonstrate the purity and structural analysis of the oligosaccharides disclosed herein;

[0028] Figures 3A to 3D is a summary of diagrams illustrating the results of the effect of the presence of the GlcNS3S6S-IdoA2S-disaccharide unit on anticoagulant activity.

[0029] Figure 4A is a schematic diagram of the chemical structure of the AT-binding site, while Figure 4B the figure depicts the results of molecular dynamics (MD) simulations of AT in the complex;

[0030] Figures 5A to 5C The figure illustrates the substrate requirements for 3-OST-1 and 3-OST-3 using structurally homogeneous oligosaccharides of different sizes, where Figure 5A andFigure 5B are bar graphs showing the substrate requirements for 3 - OST - 1 and 3 - OST - 3, where Figure 5C the figure illustrates the chemical structures of oligosaccharides of different sizes;

[0031] Figures 6A to 6F are schematic diagrams of the chemical structures of compounds 1, 2, 3, 4, 5, and 6, respectively;

[0032] Figure 7A is a graphical summary of data demonstrating that the heparin compounds disclosed herein have a faster clearance rate than fondaparinux in a C57BL / 6J mouse model. Figure 7B is a graphical summary of data demonstrating that the anti - Fxa activity of the heparin compounds disclosed herein can be reversed by andexanet - α ( Portola Pharmaceuticals, South San Francisco, California, USA); and

[0033] Figures 8A to 8C depicts the analysis results of the 7 - mer HS compounds disclosed herein, Figure 8A is a graphical summary of data showing that the 7 - mer has anti - Xa activity, Figure 8B schematically compares the chemical structure of the 7 - mer with the 6 - mer and 8 - mer disclosed herein. Figure 8C The figure illustrates the synthetic route for synthesizing the 7 - mer. Detailed Description

[0034] The subject matter of the present disclosure will now be described more fully hereinafter, where some but not all embodiments of the present disclosure are described. In fact, the disclosed subject matter can be implemented in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements.

[0035] Sulfation at the 3-OH position of glucosamine is at least one important modification for the formation of the domain of heparan sulfate enabling it to achieve its biological functions. Seven 3-O-sulfotransferase isotypes in 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. A novel chemoenzymatic synthesis method for synthesizing six 3-O-sulfated oligosaccharides, including three hexasaccharides and three octasaccharides, is disclosed herein. The synthesis is achieved by rearranging the enzyme modification sequence to accommodate the substrate specificity of 3-O-sulfotransferase 3. The effect of 3-O-sulfation on the pyranose ring conformation of 2-O-sulfated iduronic acid was studied using NMR, and the effect of 3-O-sulfation on the correlation between the ring conformation and anticoagulant activity was also studied. Thus, a novel octasaccharide that interacts with antithrombin and exhibits anti-factor Xa activity was discovered. Interestingly, in a rat model, the octasaccharide showed a faster clearance rate than fondaparinux, an approved pentasaccharide drug, making this octasaccharide a potential short-acting anticoagulant drug candidate capable of reducing the bleeding risk. The disclosed 3-O-sulfated oligosaccharides also provide new heparan sulfate-based therapies.

[0036] Heparan sulfate (HS) is a polysaccharide consisting of disaccharide repeating units containing glucuronic acid (GlcA) or iduronic acid (IdoA) residues linked to glucosamine (GlcN) residues (each of which can be modified by sulfation). HS exhibits important physiological and pathological functions, including regulation of embryonic development, inflammatory responses, blood coagulation, and viral / bacterial infections 1-3 . Most notably, heparin, a highly sulfated form of HS, is an anticoagulant drug widely used in the clinical treatment of patients with thrombotic diseases 4 .

[0037] The functional selectivity of HS and heparin can be determined at least in part by the type of sulfation and the positions of GlcA and IdoA residues 5 . Sulfation is found at the 2-OH of IdoA (to a lesser extent GlcA) and the N-, 3-OH, and 6-OH positions of GlcN residues. In addition, the conformations of the IdoA and IdoA2S residues adopt chair ( 1 C4) and twist-boat ( 2 SO) conformations 6 . The conformations of GlcA, GlcA2S, and GlcN residues exist in the chair ( 4 C1) conformation 7,8 . The conformational flexibility of the IdoA2S residue allows binding to antithrombin and thus exhibits anticoagulant activity 9and that permits binding to fibroblast growth factor to regulate cell growth 10 .

[0038] The synthesis of HS oligosaccharides remains a challenge. Many oligosaccharides can be synthesized by purely organic synthetic methods, but it is still difficult to synthesize oligosaccharides larger than hexasaccharides with complex sulfation patterns. As an alternative, there are chemoenzymatic methods for synthesizing HS oligosaccharides using HS biosynthetic enzymes involving glucosyltransferases, C5-epimerases, and sulfotransferases 16,17 . This method enables efficient synthesis of a variety of oligosaccharides; however, due to the lack of knowledge of the substrate specificity of HS biosynthetic enzymes, the synthesis of certain oligosaccharide sequences is not yet possible.

[0039] The 3-O-sulfation in HS rarely occurs, but this type of sulfation is thought to be closely related to its biological function. 3-O-sulfation may be important for anticoagulant activity 19 , promoting herpes simplex virus entry into host cells to establish infection 20 , regulating axon guidance and growth of neurons 18 and controlling progenitor cell expansion for salivary gland development 3 . Specifically, how the 3-O-sulfated glucosamine (GlcNS3S±6S) residue plays a role in promoting the biological activity of HS is currently unclear. The GlcNS3S±6S residue is shown to be surrounded by other monosaccharide residues, forming a unique sulfated sugar sequence domain that enables HS to exert its biological role. For example, the GlcNS3S±6S residue present in the pentasaccharide domain enables HS to bind to antithrombin (AT). The octasaccharide carrying the GlcNS3S±6S residue interacts with herpes simplex virus glycoprotein D. There are 7 3-OST isotypes in the human genome that can potentially be used to prepare different 3-O-sulfated oligosaccharides 26 .

[0040] This disclosure provides protocols for the preparation of 3-O-sulfated oligosaccharide libraries using chemoenzymatic methods. It is demonstrated herein that for the synthesis of oligosaccharides containing the -IdoA2S-GlcNS3S- or -IdoA2S-GlcNS3S6S-disaccharide units, the 3-OST-3 modification must precede the 6-O-sulfation step, while the 3-OST-1 modification can only occur after 6-O-sulfation in order to generate the -GlcA-GlcNS3S6S-disaccharide unit. There is no interaction between 3-OST-3 and the 6-O-sulfonate from the tetrasaccharide substrate, which is consistent with the conclusion that the oligosaccharide substrate for 3-OST-3 does not require 6-O-sulfation. In contrast, an interaction was observed between 3-OST-1 and the 6-O-sulfonate from the heptasaccharide substrate, indicating that 6-O-sulfation is required for binding to 3-OST-1. As disclosed herein for the first time, the specific and distinct substrate requirements between 3-OST-1 and 3-OST-3 reveal that 3-O-sulfated HS modified by different isotypes of 3-OST is biosynthesized via different pathways.

[0041] It is generally accepted that the 3-OST-1 enzyme is responsible for the synthesis of anticoagulant HS, while the 3-OST-3 enzyme is not. 34,35 All of the AT-binding sequences isolated to date contain the -GlcA-GlcNS3S6S-disaccharide repeat unit, which is the product of 3-OST-1 enzyme modification. 33,39 Based on the discovery that oligosaccharides developed from the disclosed methods and which are products of 3-OST-3 enzyme modification bind to AT and exhibit anticoagulant activity, the present disclosure challenges this long-standing view. These findings suggest that 3-OST-3 is capable of synthesizing anticoagulant HS as long as the HS contains a domain similar to the domain of the compounds disclosed herein, including, for example, but not limited to compound 5.

[0042] This disclosure also provides the discovery that some of the developed HS compounds or oligosaccharides have unexpectedly rapid clearance rates. The rapid clearance of these compounds provides a potential new class of short-acting anticoagulant drug candidates with reduced bleeding risk. Short-acting anticoagulants, which can be rapidly cleared from the circulation before a major bleeding effect occurs, would be particularly beneficial for patients who have a high or elevated bleeding risk compared to normal or healthy patients / subjects. Although unfractionated heparin is an anticoagulant with a short half-life, there is concern that the drug can cause heparin-induced thrombocytopenia (HIT), a life-threatening side effect. 42 It has been found that short oligosaccharides of less than 12-linkages 43It does not bind to platelet factor 4 and thus does not exhibit the risk of HIT. As hexasaccharides and octasaccharides, the compounds disclosed herein (including, for example, compound 5) are expected to have a very low risk of HIT.

[0043] In some embodiments, as disclosed herein, HS compounds or synthetic heparin analogs can comprise 3-O-sulfated oligosaccharides containing six to eight disaccharide units, at least one disaccharide unit sulfated by the 3-OST-3 enzyme, and at least one IdoA2S-GlcNS3S or IdoA2S-GlcNS3S6S disaccharide unit. As shown in the working examples herein, such synthetic heparin analogs have anticoagulant activity, including binding affinity for antithrombin (about 5 nM to about 30 nM) and anti-Xa activity (about 10 ng / mL -1 to about 40 ng / mL -1 IC 50 ). The structure or composition of such synthetic heparin analogs is exemplified by compounds 1-11, particularly 1-6, and is included in Figures 6A to 6F .

[0044] As further discussed herein, in some embodiments, the synthetic heparin analogs, particularly compared to heparin and heparinoid compounds, can have an unexpectedly rapid clearance rate. For example, in a rat or mouse model, the clearance rate of compound 5 is at least about 50%, 75%, or 100% faster than the clearance rate of fondaparinux, 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%. This rapid clearance enables these compounds to be suitable as short-acting anticoagulant compounds, which are particularly suitable for applications and / or subjects with a high bleeding risk. Such compounds have also been demonstrated not to cause heparin-induced thrombocytopenia (HIT).

[0045] The synthetic method of the synthetic heparin analog is shown in the figure (see, for example, Figure 1A , Figure 1B and Figure 8C ) and is further discussed herein, but in some aspects can include: providing a sugar substrate, extending the sugar substrate to a desired or predetermined length of carbohydrate, and performing at least one sulfation reaction using the 3-OST-3 isoform of the 3-O-sulfotransferase (3-OST) enzyme, thereby synthesizing the synthetic heparin analog. The sugar substrate can comprise at least one IdoA2S-GlcNS3S disaccharide unit.

[0046] The disclosed method for synthesizing heparin analogs can provide surprisingly high yields of heparin compounds. By way of example and not limitation, the disclosed method for synthesizing heparin analogs can have a yield 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%.

[0047] In cases where the sugar substrate comprises an IdoA2S-GlcNS3S±6S disaccharide unit, such method can further include a 6-O-sulfation step using 6-O-sulfotransferase (6-OST), wherein 3-O-sulfation by 3-OST-3 occurs prior to the 6-O-sulfation step. In contrast, in cases where the sugar substrate comprises a GlcA-GlcNS3S6S disaccharide unit, such method can further include a 6-O-sulfation step using 6-O-sulfotransferase (6-OST), wherein 3-O-sulfation by 3-OST-1 occurs prior to the 6-O-sulfation step.

[0048] The development of the heparin compounds and synthetic heparin analogs of the present disclosure also provides treatments and methods for treating a subject therewith. For example, in some embodiments, a method for treating a subject in need of anticoagulation therapy is provided. Such methods can include: providing a subject in need of an anticoagulant treatment and administering to the subject a synthetic heparin analog having anticoagulant activity, wherein the synthetic heparin analog comprises at least one disaccharide unit sulfated by a 3-OST-3 enzyme and at least one IdoA2S-GlcNS3S or IdoA2S-GlcNS3S6S disaccharide unit. Optionally, heparin-induced thrombocytopenia in the subject can also be monitored, and if tested, if the subject has heparin-induced thrombocytopenia, an antidote can be administered to the subject to reverse the anticoagulant activity of the synthetic heparin analog. Such reversal of anticoagulant activity can be achieved, for example, in the presence of andexanet-α at 20 μg / mL or less at a rate of 50% or higher by andexanet-α ( Portola Pharmaceuticals, South San Francisco, California, USA).

[0049] Accordingly, in some embodiments of the present disclosure, a method of treating a subject in need of anticoagulant therapy is provided. In some embodiments, the method comprises: providing a subject in need of anticoagulant therapy; administering to the subject a synthetic heparin analogue having anticoagulant activity, wherein the synthetic heparin analogue comprises at least one disaccharide unit sulfated by 3-OST-3 enzyme and at least one IdoA2S-GlcNS3S or IdoA2S-GlcNS3S6S disaccharide unit.

[0050] In some embodiments, the present disclosure provides a synthetic heparin analogue having anticoagulant activity, wherein the synthetic heparin analogue comprises at least one disaccharide unit sulfated by 3-OST-3 enzyme and at least one IdoA2S-GlcNS3S or IdoA2S-GlcNS3S6S disaccharide unit, for treating and / or preventing a disease or disorder in which anticoagulant activity is beneficial. By way of example and not limitation, such diseases or disorders can include patients and individuals at high risk of developing deep vein thrombosis, including cancer patients.

[0051] In yet another embodiment, the present disclosure provides the use of a synthetic heparin analogue having anticoagulant activity, wherein the synthetic heparin analogue comprises at least one disaccharide unit sulfated by 3-OST-3 enzyme and at least one IdoA2S-GlcNS3S or IdoA2S-GlcNS3S6S disaccharide unit, for preparing a pharmaceutical composition for treating and / or preventing a disease or disorder in which anticoagulant activity is beneficial.

[0052] Exemplary synthetic heparin analogues or HS compounds are disclosed herein, including, for example, Figure 1A and Figures 6A to 6F as shown. In the structures shown, "R" can include a proton (-H), -CH 3 、-CH 2 CH 3 or other substituents similar to p-nitrophenyl. These groups can also include and / or be referred to as alkyl or lower alkyl. In some embodiments, other substituents similar to p-nitrophenyl can include aryl or substituted aryl. Alternatively or additionally, in some embodiments, "R" can include a detectable label or detectable moiety.

[0053] Definition

[0054] The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the subject matter of the present disclosure.

[0055] Although the following terms are believed to be well understood by those of ordinary skill in the art, the following definitions are set forth to facilitate the interpretation of the subject matter disclosed herein.

[0056] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques employed herein are intended to refer to techniques as commonly understood in the art, including variations of those techniques or substitutions of equivalent techniques that are obvious to those skilled in the art. Although the following terms are believed to be well understood by those of ordinary skill in the art, the following definitions are set forth to facilitate the interpretation of the subject matter disclosed herein.

[0057] In describing the subject matter disclosed herein, it should be understood that many techniques and steps are disclosed. Each of these has its respective benefits, and each can also be used in combination with one or more, or in some cases all, of the other disclosed techniques.

[0058] Accordingly, for clarity, this specification will avoid repeating each possible combination of the various steps in an unnecessary manner. However, the specification and claims should be read with the understanding that such combinations are fully within the scope of the invention and the claims.

[0059] In accordance with longstanding patent law convention, when used in this application (including the claims), the terms "a", "an", and "the" refer to "one or more". Thus, for example, reference to "a cell" includes a plurality of such cells, and so forth.

[0060] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, etc. used in the specification and claims should be understood to be modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and the appended claims are approximations that can vary depending upon the desired properties sought to be obtained by the subject matter disclosed herein.

[0061] As used herein, the term "about", when referring 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., means a variation of ±20% of the specified amount in some embodiments, a variation of ±10% of the specified amount in some embodiments, a variation of ±5% of the specified amount in some embodiments, a variation of ±1% of the specified amount in some embodiments, a variation of ±0.5% of the specified amount in some embodiments, and a variation of ±0.1% of the specified amount in some embodiments, because such variations are suitable for practicing the disclosed methods or using the disclosed compositions.

[0062] The term "comprising", which is synonymous with "including", "containing", or "characterized by", is inclusive or open-ended and does not exclude additional unrecited elements or method steps. "Comprising" is a technical term used in claim language, which means that the specified elements are necessary, while other elements can be added and still form a construct within the scope of the claim.

[0063] As used herein, the phrase "consisting of" does not include any element, step, or ingredient not specified in the claim. When the phrase "consisting of" appears in a clause of the claim body rather than immediately following the preamble, it only limits the elements specified in that clause; other elements as a whole are not excluded from the claim.

[0064] As used herein, the phrase "consisting essentially of" will limit the scope of the claim to the specified materials or steps, plus those materials or steps that do not materially affect the basic and novel characteristics of the claimed subject matter.

[0065] With respect to the terms "comprising", "consisting of", and "consisting essentially of" (where one of these three terms is used herein), the present disclosure and the claimed subject matter can include the use of either of the other two terms.

[0066] As used herein, the term "and / or", when used in the context of a list of entities, means the entities that exist either individually or in combination. Thus, for example, the phrase "A, B, C, and / or D" includes A, B, C, and D individually, but also includes any and all combinations and sub-combinations of A, B, C, and D.

[0067] As used herein, the term "alkyl" means C 1-20(including C 1 and C 20 ) a linear (i.e., "straight-chain"), branched or cyclic saturated or at least partially and in some cases completely unsaturated (i.e., alkenyl and alkynyl) hydrocarbon chain, including, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, octyl, vinyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl and allenyl. "Branched" means an alkyl in which a lower alkyl such as methyl, ethyl or propyl is attached to a straight-chain alkyl chain. "Lower alkyl" means an alkyl having 1 to about 8 carbon atoms, for example, 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms (i.e., C 1-8 alkyl). "Higher alkyl" means an alkyl having about 10 to about 20 carbon atoms, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. In certain embodiments, "alkyl" specifically refers to C 1-8 straight-chain alkyl. In other embodiments, "alkyl" specifically refers to C 1-8 branched alkyl.

[0068] The alkyl can optionally be substituted by one or more alkyl substituents ("substituted alkyl"), and the alkyl substituents can be the same or different. The term "alkyl substituent" includes, but is not limited to, alkyl, substituted alkyl, halogen, arylamino, acyl, hydroxy, aryloxy, alkoxy, alkylthio, arylthio, aralkyl-oxy, aralkylthio, carboxyl, alkoxycarbonyl, oxo and cycloalkyl. One or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms can optionally be inserted along the alkyl chain, wherein the nitrogen substituent is hydrogen, lower alkyl (also referred to herein as "alkylaminoalkyl") or aryl.

[0069] Thus, as used herein, the term "substituted alkyl" includes alkyl as defined herein, wherein one or more atoms or functional groups of the alkyl are replaced by another atom or functional group, including, for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxy, hydroxy, nitro, amino, alkylamino, dialkylamino, sulfate and mercapto.

[0070] As used herein, the term "aryl" refers to an aromatic substituent that can be a single aromatic ring or multiple aromatic rings (fused together, covalently linked, or linked to a common group such as, but not limited to, a methylene or ethylene moiety). The common linking group can also be a carbonyl, as in benzophenone, or oxygen, as in diphenyl ether, or nitrogen, as in diphenylamine. The term "aryl" specifically includes heteroaromatic compounds. The aromatic rings can include phenyl, naphthyl, biphenyl, diphenyl ether, diphenylamine, benzophenone, etc. In a specific embodiment, the term "aryl" refers to a cyclic aromatic compound containing from about 5 to about 10 carbon atoms, for example, 5, 6, 7, 8, 9, or 10 carbon atoms, and including 5- and 6-membered hydrocarbon and heteroaromatic rings.

[0071] The aryl can optionally be substituted with one or more aryl substituents that can be the same or different ("substituted aryl"), where "aryl substituent" includes alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, hydroxy, alkoxy, aryloxy, aralkyloxy, carboxyl, acyl, halogen, nitro, alkoxycarbonyl, aryloxycarbonyl, aralkyloxycarbonyl, acyloxy, acylamino, arylacylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylthio, alkylthio, alkylene, and -NR'R", where each of R' and R" can independently be hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, and aralkyl.

[0072] Thus, as used herein, the term "substituted aryl" includes aryl as defined herein, where one or more atoms or functional groups of the aryl are replaced by another atom or functional group (including, for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxy, hydroxy, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto).

[0073] Specific examples of aryl include, but are not limited to, cyclopentadienyl, phenyl, furan, thiophene, pyrrole, pyran, pyridine, imidazole, benzimidazole, isothiazole, isoxazole, pyrazole, pyrazine, triazine, pyrimidine, quinoline, isoquinoline, indole, carbazole, and the like.

[0074] As used herein, a structure generally represented by the following formula, such as:

[0075]

[0076] is a ring structure, for example, but not limited to, aliphatic and / or aromatic cyclic compounds containing 3-carbon, 4-carbon, 5-carbon, 6-carbon, etc. groups with substituent R groups, where the R groups can be present or absent, and when present, one or more R groups can each be substituted on one or more available carbon atoms of the ring structure. The presence or absence of the R groups and the number of R groups are determined by the value of the integer n. Each R group (if more than one) is substituted on the available carbon of the ring structure rather than on another R group. For example, the structure:

[0077]

[0078] where n is an integer from 0 to 2, including compound groups, including but not limited to:

[0079] etc.

[0080] "Cyclic" and "cycloalkyl" refer to non-aromatic monocyclic or polycyclic ring systems of about 3 to about 10 carbon atoms, for example, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. The cycloalkyl can optionally be partially unsaturated. The cycloalkyl can also optionally be substituted by alkyl substituents, oxy groups, and / or alkylene groups as defined herein. One or more oxygen, sulfur, or substituted or unsubstituted nitrogen atoms can optionally be inserted along the cycloalkyl chain, where the substituents of the nitrogen are hydrogen, alkyl, substituted alkyl, aryl, or substituted aryl, thereby providing a heterocyclic group. Representative monocyclic cycloalkyl rings include cyclopentyl, cyclohexyl, and cycloheptyl. Polycyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decalin, camphor, camphene, and noradamantyl.

[0081] The term "heterocyclic" refers to non-aromatic or aromatic monocyclic or polycyclic ring systems of about 3 to about 14 atoms, where at least one of the atoms is a heteroatom (e.g., oxygen, nitrogen, or sulfur). The term "N-heterocyclic" refers to a heterocyclic where at least one heteroatom is a nitrogen atom. Examples of N-heterocyclics include, but are not limited to, azetidine, pyrrolidine, pyrrole, pyrroline, piperidine, pyridine, piperazine, pyrazine, pyrimidine, pyridazine, morpholine, and thiazine.

[0082] "Aralkyl" refers to an aryl-alkyl-group, where aryl and alkyl are as described above, and includes substituted aryl and substituted alkyl. Exemplary aralkyls include benzyl, phenethyl, and naphthylmethyl.

[0083] As used herein, the term "acyl" refers to an organic carboxylic acid group in which the -OH of the carboxyl group has been replaced by another substituent (i.e., as represented by RC(=O)-, where R is an alkyl, substituted alkyl, aralkyl, aryl, or substituted aryl as defined herein). Thus, the term "acyl" specifically includes arylacyls such as acetylfuran and benzoyl. Specific examples of acyl include acetyl and benzoyl.

[0084] "N-acyl" refers to a group having the structure -N-C(=O)-R, where R is as defined for acyl. These groups can also be referred to as amides. Modified N-acyls include compounds in which the oxygen of the N-acyl is replaced by S or NH, and compounds in which the carbonyl group (i.e., -C(=O)-) is linked to a second heteroatom other than nitrogen. For example, the carbonyl can be linked to a second nitrogen atom to form a urea linking group (i.e., -NH-C(=O)-NH-R).

[0085] The term "amino" refers to -NH 2 , -NHR and -NR 2 groups, where each R is independently an alkyl, substituted alkyl, aryl, substituted aryl, or aralkyl, and the amino and ammonium functional groups in N-heterocycles (e.g., morpholine, etc.). As used herein, the term "amino" can also refer to a substituent that provides a quaternary ammonium cation, such as - + NH 3 , - + NH(R) 2 and - + N(R) 3 groups, where each R is independently an alkyl, substituted alkyl, aryl, substituted aryl, or aralkyl.

[0086] The term "ester" refers to a moiety containing an -O-C(=O)-R group, where R can be an alkyl, substituted alkyl, aralkyl, aryl, or substituted aryl. In some embodiments, the R group can include an amino substituent, and the ester is an amino ester.

[0087] The term "amide" refers to a moiety containing an -N(R')-C(=O)-R group, where R is selected from alkyl, substituted alkyl, aralkyl, aryl, or substituted aryl, and R' is H, alkyl, substituted alkyl, aralkyl, aryl, or substituted aryl.

[0088] The term "urea" as used herein can refer to a moiety containing an -N(R')-C(=O)-N(R')- group, where each R' is independently H, alkyl, substituted alkyl, aralkyl, aryl, or substituted aryl.

[0089] The term "hydroxy" refers to the -OH group.

[0090] When the term "independently selected" is used, the substituents so mentioned (e.g., R groups such as group R 1 and R 2 , or groups X and Y) can be the same or different. For example, both R 1 and R 2 can be substituted alkyl, or R 1 can be hydrogen while R 2 can be substituted alkyl, etc.

[0091] Subject

[0092] The subject in which treatment, screening, testing or sample collection is carried out is preferably a human subject, although it should be understood that the principles of the disclosed subject matter indicate that the compositions and methods are effective for invertebrates and for all vertebrate species, including mammals, which are all intended to be included within the term "subject". In addition, mammals should be understood to include any mammalian species in which screening is desired, particularly agricultural and domestic mammalian species.

[0093] The disclosed methods, compounds and treatments are particularly applicable to the testing, screening and / or treatment of warm-blooded vertebrates. Accordingly, the subject matter of the present disclosure relates to mammals and birds.

[0094] More specifically, provided herein are the testing, screening and / or treatment of mammals such as humans and those mammals that are of importance due to being endangered (e.g., Siberian tiger), of economic importance to humans (animals raised on farms for human consumption) and / or of social importance (animals kept as pets or in zoos), e.g., carnivores other than humans (such as cats and dogs), swine (domestic pigs, hogs and wild boars), ruminants (such as cows, beef cattle, sheep, giraffes, deer, goats, bison and camels) and horses. Also provided herein is the treatment of birds, including the treatment of those species of birds that are endangered and kept in zoos, as well as fowl, particularly poultry, i.e., meat poultry, such as turkeys, chickens, ducks, geese, guinea fowl, etc., as they are also of economic importance to humans. Accordingly, provided herein is the treatment of domestic animals, including but not limited to, domestic pigs (domestic pigs and hogs), ruminants, horses, poultry, etc.

[0095] In some embodiments, the subject used in accordance with the subject matter of the present disclosure is a subject in need of treatment and / or diagnosis thereof. In some embodiments, the subject may be in need of anticoagulant therapy or related conditions or phenotypes. In some embodiments, the subject in need of anticoagulant treatment can be a subject with a high bleeding risk.

[0096] Preparation

[0097] In some embodiments, the compositions of the subject matter of the present disclosure include compositions containing a pharmaceutical carrier. Any suitable pharmaceutical formulation can be used to prepare the adenoviral vector to be administered to a subject.

[0098] For example, suitable formulations can include aqueous and non-aqueous sterile injection solutions, which can contain antioxidants, buffers, bacteriostatic agents, antibiotics, and solutes that render the formulation isotonic with the body fluids of the intended recipient; aqueous and non-aqueous sterile suspensions, which can include suspending agents and thickening agents. The formulations can be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and can be stored under frozen or freeze-dried (lyophilized) conditions and require only the immediate addition of a sterile liquid carrier, such as water, prior to use for injection. Some exemplary components are SDS, mannitol or another sugar, and phosphate-buffered saline (PBS).

[0099] It should be understood that, in addition to the components specifically mentioned above, the formulations of the subject matter of the present disclosure can include conventional other reagents in the art for the type of formulation discussed. For example, sterile pyrogen-free aqueous and non-aqueous solutions can be used.

[0100] Administration

[0101] Administration of the compositions of the subject matter of the present disclosure can be carried out by any method known to those of ordinary skill in the art, including but not limited to, intravenous administration, intra-synovial administration, transdermal administration, intramuscular administration, subcutaneous administration, topical administration, rectal administration, intravaginal administration, intratumoral administration, oral administration, buccal administration, nasal administration, parenteral administration, inhalation, and insufflation. In some embodiments, suitable methods for administering the compositions of the subject matter of the present disclosure include, but are not limited to, intravenous administration. Alternatively, the composition can be placed on the site in need of treatment in any other manner. The specific manner of administering the composition of the subject matter of the present disclosure depends on various factors.

[0102] Dose

[0103] An effective dose of the compositions of the subject matter of the present disclosure is administered to a subject in need thereof. A "therapeutically effective amount" is an amount of the composition sufficient to produce a measurable response (e.g., an anticoagulant effect). In some embodiments, a therapeutically effective amount is an amount sufficient to prevent blood clotting, i.e., an anticoagulant effect. In some embodiments, a therapeutically effective amount is an amount sufficient to improve the health, maintain the well-being, prognosis, and / or viability of a subject in need of treatment with an anticoagulant.

[0104] The actual dosage level of the active ingredient in the compositions of the present disclosure may vary so as to administer an amount of the active compound effective to achieve the desired therapeutic response for a particular subject. The selected dosage level will depend upon the activity of the therapeutic composition, the route of administration, the combination with other drugs or therapies, the severity of the condition being treated, and the condition and prior medical history of the subject being treated. However, it is within the skill of the art to initiate 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.

[0105] After reading the disclosure of the present disclosure provided herein, one of ordinary skill in the art can customize the dosage administered to an individual patient considering the specific formulation, the method of administration to be used with the composition, and the severity of the condition. Further calculations of the dosage can take into account the patient's height and weight, the severity and stage of the symptoms, and the presence of other adverse medical conditions. Such adjustments or variations, and the evaluation of when and how to make such adjustments or variations, are well known to one of ordinary skill in the medical arts.

[0106] Examples

[0107] The following examples are included to further illustrate various embodiments of the present disclosure. However, one of ordinary skill in the art should understand, in light of the present disclosure, that many changes can be made in the specific embodiments disclosed without departing from the spirit and scope of the present disclosure and still obtain the same or similar results.

[0108] Materials and Methods

[0109] Expression and Purification of Enzymes

[0110] Expression of 6-O-sulfotransferase 1 (6-OST-1) and 6-O-sulfotransferase 3 (6-OST-3) was carried out in Sf9 cells (Invitrogen) using Sf-900 TM III SFM (Life Technologies) at a concentration of 2.0×10 6Insect cells at [number of cells] / mL were infected with recombinant viruses expressing mouse 6-OST-1 and human 6-OST-3 and incubated at 27 °C for 96 hours in an oscillator. The culture solution was centrifuged at 4,000 RPM for 10 min to pellet the cells. Then the supernatant containing 1 mM phenylmethylsulfonyl fluoride (PMSF, freshly prepared from a 100 mM stock solution in 95% ethanol), 0.05% Triton X-100, and 0.2% glycerol was centrifuged at 8,000 RPM for 30 min and filtered through a 1.5 μm membrane. Then the resulting culture 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. 6-OST-1 and 6-OST-3 were purified using a heparin Toyopearl gel (Tosoh Bioscience) column with the following two buffers: buffer A, containing 20 mM MOPS, pH 7.0, 100 mM NaCl, 2% glycerol, and 0.1% reduced triton X-100 (Sigma); and buffer B, containing 20 mM MOPS, pH 7.0, 1 M NaCl, 2% glycerol, and 0.1% reduced triton X-100. After loading the culture medium, the column was washed with buffer A at a flow rate of 4 mL / min until the UV absorbance at 280 nM reached the baseline. A gradient elution of 0–100% B over 60 min was applied, and the column was additionally eluted with 100% B at a flow rate of 1.5 mL / min for 60 min. Enzyme purification was carried out at 4 °C.

[0111] Expression of 3-OST-1 and 3-OST-3 was carried out in Escherichia coli using BL21 cells. For 3-OST-1 and 3-OST-3, the transformed cells were grown in LB medium containing 50 μg / L kanamycin and incubated at 37 °C until the OD600 reached 0.6 - 0.8. Isopropyl thiogalactopyranoside (IPTG) with a final concentration of 0.2 mM was added to induce the expression of 3-OST-1 and 3-OST-3. The bacterial culture was kept shaking overnight at 22 °C. The bacterial cells were harvested by spinning at 3400 RPM for 15 min. The cells were resuspended in 25 mL of a buffer solution containing 25 mM Tris (pH 7.5), 30 mM imidazole, and 500 mM NaCl. The suspension was sonicated and centrifuged at 14,000 RPM for 30 min. Before purification, the supernatant was filtered through a 0.45 μm membrane. The protein was purified using a nickel-agarose (GE Healthcare) column with the following two buffer solutions: Buffer C, containing 25 mM Tris (pH 7.5), 30 mM imidazole, and 500 mM NaCl; Buffer D, containing 25 mM Tris (pH 7.5), 300 mM imidazole, and 500 mM NaCl. After loading the medium, the column was washed with Buffer C at a flow rate of 2 mL / min until the UV absorbance at 280 nM reached the baseline. Then the protein was eluted with Buffer D.

[0112] Chemoenzymatic synthesis of oligosaccharides (Compounds 1 - 6)

[0113] The synthesis of Compounds 1 - 3 was started from the hexasaccharide (GlcNS-GlcA-GlcNS-IdoA2S-GlcNS-GlcA-pNP) called "NS2S 6-linker substrate". To synthesize Compound 1, the substrate (25 mg) was combined with 1.8 mM 3'-phosphoadenosine 5'-phosphosulfate (PAPS) in a solution containing 50 mM MOPS (pH 7.0), 10 mM MnCl 2 , 7 mM MgCl 2Incubate in a total volume of 100 mL of buffer containing 2 mL of 3-OST-3 (0.11 mg / mL). The reaction mixture is incubated overnight at 37 °C. The completion of the reaction is monitored by injecting a small amount of the reaction mixture into an anion-exchange HPLC (TSKgel DNA-NPR-column (4.6 mm × 7.5 cm, 2.5 μm, from Tosoh Bioscience)). If the reaction is less than 60% complete, additional 3-OST-3 enzyme and PAPS are added, and the reaction mixture is kept at 37 °C for another 18 - 24 hours. If the reaction is complete, the reaction mixture is subjected to Q-Sepharose chromatography (GE Healthcare).

[0114] For the synthesis of compound 2, compound 1 (5 mg) is incubated overnight at 37 °C with 6-OST-1, 6-OST-3 enzymes and 1.3 mM PAPS in a buffer with a total volume of 100 mL containing 100 mM MOPS (pH 7.0) and 1 mL of an enzyme mixture of 6-OST-1 and 6-OST-3. The completion of the reaction is monitored by injecting a small amount of the reaction mixture into an anion-exchange HPLC, and the product is purified by Q-Sepharose. For the synthesis of compound 3, compound 2 (4 mg) is incubated with 1.3 mM PAPS in a buffer with a total volume of 100 mL containing 18 mM MOPS (pH 7.0), 5 mM MnCl 2 , 5 mM MgCl 2 and 6 mL of 3-OST-1 (4 μg / mL). The reaction mixture is incubated overnight at 37 °C. The completion of the reaction is monitored by injecting a small amount of the reaction mixture into an anion-exchange HPLC, and the product is purified by Q-Sepharose.

[0115] For the synthesis of compounds 4 - 6, an octasaccharide (GlcNS-GlcA-GlcNS-IdoA2S-GlcNS-IdoA2S-GlcNS-GlcA-pNP) called the NS2S8-linker substrate is used as the starting material. For the synthesis of compound 4, 30 mg of the substrate is incubated with 2 mM PAPS in a buffer with a total volume of 100 mL containing 33 mM MOPS (pH 7.0), 10 mM MnCl 2 , 5 mM MgCl 2Incubate in a buffer with a total volume of 90 mL containing 4 mL of 3-OST-3 (0.11 mg / mL). The reaction mixture is incubated overnight at 37 °C. The completion of the reaction is monitored by injecting a small amount of the reaction mixture into an anion-exchange HPLC. If the reaction is less than 60% complete, additional 3-OST-3 enzyme and PAPS are added, and the reaction mixture is maintained at 37 °C for an additional 18 - 24 hours. If the reaction is complete, the reaction mixture is subjected to Q-Sepharose chromatography (GE Healthcare).

[0116] For the synthesis of compound 5, compound 4 (22 mg) is incubated with 6-OST-1, 6-OST-3 enzymes, and 0.8 mM PAPS in a buffer with a total volume of 100 mL containing 100 mM MOPS (pH 7.0) and 3 mL of a 6-OST-1 and 6-OST-3 mixture at 37 °C overnight. The completion of the reaction is monitored by injecting a small amount of the reaction mixture into an anion-exchange HPLC, and the product is purified by Q-Sepharose. For the synthesis of compound 6, compound 5 (6.5 mg) is incubated with 1.3 mM PAPS in a buffer with a total volume of 100 mL (4 μg / mL) containing 18 mM MOPS (pH 7.0), 5 mM MnCl 2 , 5 mM MgCl 2 and 4.5 mL of 3-OST-1. The reaction mixture is incubated overnight at 37 °C. The completion of the reaction is monitored by injecting a small amount of the reaction mixture into an anion-exchange HPLC, and the product is purified by Q-Sepharose.

[0117] Substrate specificities of 3-OST-1 and 3-OST-3

[0118] To determine the substrate requirements of 3-OST-1 and 3-OST-3, structurally homogeneous oligosaccharides of different sizes were used. These oligosaccharides include N-sulfated and 2-O-sulfated 6-mer to 12-mer (6-mer 2S to 12-mer 2S) and N-sulfated, 2-O-sulfated, and 6-O-sulfated 6-mer to 12-mer (6-mer 2S6S to 12-mer 2S6S). These oligosaccharides were prepared by the chemoenzymatic method 1, and their structures are as Figure 5C shown. These oligosaccharides (0.033 mM) were incubated in a solution containing 50 mM MOP (pH 7.0), 10 mM MnCl 2 , 5 mM MgCl 2 , 2.5 μL of 3-OST-1 or 3-OST-3, and 30 μM of [35S]PAPS ((1 - 3)×10 5Incubate for 1 h at 37 °C in 100 μL of reaction buffer containing PAPS. Quench the reaction by adding 900 μL of 3 M urea containing 1.4 mM EDTA, 50 mM sodium acetate, and 150 mM NaCl to the reaction mixture. Then purify the reaction mixture using the DEAE-column.

[0119] Kinetic analysis of 3-OST-3 against different oligosaccharide substrates

[0120] The enzyme kinetics was characterized by incubating a mixture of 30 μL of purified 3-OST-3 for oligosaccharides with N-sulfation and 2-O-sulfation or 60 μL of purified 3-OST-3 for oligosaccharides with N-sulfation, 2-O-sulfation, and 6-O-sulfation. Mix [35S]PAPS (120 μM) as a sulfate donor with 5 5 cpm and different concentrations of oligosaccharides from 0 to 200 μM at 37 °C for 1 h. Load the reactants onto a DEAE column to purify the 35S-labeled oligosaccharide product. Plot the amount of 35S-labeled oligosaccharide product against the substrate concentration, and then perform curve fitting on the Michalis-Menten curve using Sigma Plot software to obtain the Km and Vmax values.

[0121] Purification of Compounds 1-6 by Q-Sepharose

[0122] Purify the sulfated oligosaccharides using a Q-Sepharose column. Mobile phase A is 25 mM Tris, pH 7.5. Mobile phase B contains 25 mM Tris and 1 M NaCl, pH 7.5. The elution gradient is based on the number of sulfate ester groups of the synthetic oligosaccharides, and the flow rate is 1 mL / min. Scan and record the absorbance at 310 nM and 260 nM. After purification, dialyze the sample twice against a buffer (pH 7.5) containing 5 mM disodium hydrogen phosphate using a 1000 MWCO membrane.

[0123] HPLC analysis of synthetic oligosaccharides

[0124] Use a TSKgel DNA-NPR-column to detect the completion of the reaction and the purity of the synthetic oligosaccharides after purification. Mobile phase A is 25 mM Tris, pH 7.5. Mobile phase B is 25 mM Tris and 1 M NaCl, pH 7.5. The gradient step is 0-100% B in 100 min, and the flow rate is 0.4 mL / min. Monitor the eluate using the absorbance at 310 nM and 260 nM.

[0125] ESI-MS analysis of oligosaccharides

[0126] The molecular weight conformation of the synthesized oligosaccharides was determined by ESI-MS (Thermo LCQ-Deca). The ESI-MS analysis was carried out in the negative ion mode with the following parameters: a spray voltage of 3.0 kV and a curved desolvation line temperature of 120 °C. The mass range was set at 300 - 1000.

[0127] Preparation of 3-O-[34S]-sulfated compound 3

[0128] To synthesize 3-O-[34S]-sulfated compound 4, the substrate NS2S 8-mer (2 mg) was incubated with 3-OST-3 enzyme (0.11 mg / mL) and 0.1 mM [34S]PAPS in a buffer with a total volume of 20 mL containing 38 mM MOPS (pH 7.0), 10 mM MnCl 2 , 5 mM MgCl 2 and 6 mL of 3-OST-3. The reaction mixture was incubated overnight at 37 °C. The product was purified using a Q-Sepharose column.

[0129] Tandem MS analysis of 3-OST-3 modified octasaccharide

[0130] The tandem mass spectra analysis was obtained on a Thermo LTQ-FT instrument in the negative ion mode with the following instrument parameters: spray voltage (kV), -3.5 kV; capillary voltage (kV), -40 kV; tube lens (V), -50 V; and capillary temperature, 275 °C. For the tandem mass spectra, the selected precursor ions were carried out with the following parameters: Iso width (m / z): 3.0, normalized collision energy (%): 50.0, Act.Q: 0.250, Act. time: 30, maximum injection time (ms): 500.000. The MS and MS / MS data were recorded and processed using Xcalibur 2.2 software.

[0131] Structural analysis of compounds 1 - 6 by NMR

[0132] The NMR experiments were carried out at 298 K on Bruker Avance 700 MHz and 850 MHz spectrometers with Topsin 3.2 software. Each sample (0.5 - 3.0 mg) was dissolved in 0.5 mL of D 2It was dissolved in O (99.996%, Sigma - Aldrich) and freeze - dried three times to remove exchangeable protons. The sample was redissolved in 0.5 mL D 2 O and transferred to an NMR microtube (O.D. 5 mm, Norrell). Chemical shifts were referenced to external sodium 2,2 - dimethyl - 2 - silapentane - 5 - sulfonate (DSS, Sigma, Co.). Deuterated EDTA (Sigma, Co.) was added to eliminate the influence of paramagnetic ions. The 1D 1H - NMR experiment with the "zg" pulse sequence was carried out with 64 scans and a collection time of 3.8 seconds. The 1D 13C - NMR experiment with the "zgdc30" pulse sequence was carried out with 10,000 scans and a collection time of 1.0 second. The 2D 1H - 13C HSQC experiment with the "hsqcgpph" pulse sequence was carried out with 48 scans, 512 increments, a relaxation delay of 1.5 seconds, and a collection time of 120 milliseconds. The 2D spectra were recorded using GARP carbon decoupling. Collection started after 48 dummy scans. 2048 full points were collected in f2. 13 The 13C transmitter offset was set at 90.0 ppm.

[0133] Molecular dynamics pre - treatment

[0134] The existing crystal structure of the AT - pentasaccharide complex (PDB ID: 3EVJ) was used as the starting structure of the system 47 . Amino acid residues 25 - 33 and 396 were not resolved in the crystal structure, so they were generated using the Chimera interface of Modeller 48-51 . The N - glycans were removed because they were far from the fondaparinux ligand The original ligand was modified using tleap for various simulations and manually adjusted using Chimera. The parameters of the amino acid and carbohydrate residues were obtained from the ff14SB7 and GLYCAM06(J - 1) force fields 53,54 . The system was neutralized with Na+ ions and solvated in a truncated octahedral box around the complex using the TIP3P water model .

[0135] Molecular dynamics protocol

[0136] Energy minimization and MD simulations were carried out using the pmemd.cuda module from AMBER14 52 . At each step of the whole process, the Cα atoms of the protein backbone were restricted by Cartesian constraints (10 kcal / mol ). The two simulations required for the IdoA2S residue d(Figure 4A )Internal constraints are imposed to maintain 1 C 4 or 2 S O conformations and implemented according to the settings described previously 55 . After minimizing the system using the steepest descent method in the first 1000 cycles, the conjugate gradient is switched to in the remaining 24,000 cycles. To help generate a stable conformation of the AT-oligosaccharide complex, minimization is performed twice with different atomic constraints. Initially, each solute atom is constrained. Subsequently, the constraints on the ligands and side chains of the protein are removed. Electrostatic interactions are treated using the Particle-Mesh Ewald algorithm 56 , and a cutoff value for non-bonded interactions is used. The SHAKE algorithm is applied to hydrogen-containing bonds to enable the use of an integration time step of 2 fs. The system is heated to 300 K in 60 ps under NVT conditions using a Berendsen thermostat with a coupling time constant of 1 ps, and allowed to equilibrate for a total of 50 ns under NPT conditions. Also under NPT conditions, an equilibrated data set is collected within an additional 150 ns

[0137] Simulation data analysis

[0138] Using the MPBSA.py.MPI module 52 , the single-trajectory Molecular Mechanics–Generalized Born Solvent Accessible Surface Area method (MM-GBSA) is used 57 to calculate the interaction energy. Before analysis, all water molecules and ions are removed from each complex, and the GB implicit solvation model (igb = 2) 58Contribution of approximate desolvation energy. The simulation was divided into 5-ns bins and the average interaction energy contribution was calculated from an ensemble of 100 snapshots evenly distributed within each bin. Statistical analysis was performed using GraphPad Prism for Windows version 5.04. Significance was determined based on a t-test analysis with a p-value ≤ 0.0001. Images were created using the Visual Molecular Dynamics program 59 .

[0139] Determination of in vitro anti-FXa activity

[0140] Factor Xa (Enzyme Research Laboratories, South Bend, IN) was diluted to 60 nM with PBS containing 1 mg / mL bovine serum albumin (BSA). Human antithrombin (AT) (from Cutter Biological) was prepared in PBS at a concentration of 0.65 μM containing 1 mg / mL BSA. The chromogenic substrate S-2765 (Diapharma) was dissolved in water to prepare a 1-mg / mL stock solution. The oligosaccharides were made at different concentrations (10 - 200 nM) in PBS. The solution was a mixture of 60 μL of AT and 15 μL of the sample solution, which was vortexed and incubated at room temperature for 2 min. Then factor Xa (90 μL) was added and the mixture was incubated at room temperature for 4 min, and then 30 μL of S-2765 was added. The reaction mixture was continuously measured for up to 2 min based on its absorbance at 405 nm. The calculated results of the IC 50 values were plotted as a function of sample concentration relative to the initial reaction rate

[0141] Determination of in vivo anti-FXa effect

[0142] All studies were conducted in accordance with the Public Health Service guidelines for the care and use of laboratory animals under a protocol approved by the Institutional Animal Care and Use Committee (IACUC) of the University of North Carolina at Chapel Hill. Sixteen 400-g Lewis rats were divided into 4 groups on average. Each group was administered intravenously with endotoxin-free fondaparinux (0.46 μM kg -1 ), compound 11 (0.43 μM kg -1 ), compound 5 (0.42 μM kg -1) or saline. Blood was drawn at specified time points (0.5, 1, 2, 4, 8 hours) after administration of the compound, and a sample was collected immediately before dosing. At 0.5, 1, 2, 4, and 8 hours after injection of the compound, 0.8 mL of blood was drawn from the contralateral femoral / saphenous vein into 150 mM citrate, and a sample was drawn immediately before injection. The samples were centrifuged to obtain approximately 400 μL of plasma. The blood samples were analyzed for FXa activity. Saline was administered subcutaneously regularly to maintain fluid volume.

[0143] Determination of Oligosaccharide Clearance Rate in Drug-Treated Animals

[0144] Using the calibration curves generated for each compound, the concentration of the compound in the reaction mixture was read out for each corresponding percentage of FXa activity. The average plasma concentration and the standard deviation between each group of the 4 rats were calculated for each time point; these values were used to plot a graph of plasma concentration versus time.

[0145] Example 1

[0146] Chemoenzymatic Synthesis of Oligosaccharides Carrying GlcNS3S and GlcNS3S6S Residues

[0147] In this study, the synthesis of hexasaccharides (compounds 1 - 3, Figure 1A ) and octasaccharides (compounds 4 - 6, Figure 1A ) was completed. Two 3-O-sulfotransferase (3-OST) isotypes (3-OST-1 and 3-OST-3) were used to install GlcNS3S±6S residues into different sugar sequences. The 3-OST-1 enzyme introduced sulfation to form GlcNS3S6S residues, which were linked to GlcA residues at the non-reducing end to form a disaccharide unit of -GlcA-GlcNS3S6S-; while the 3-OST-3 enzyme introduced sulfation to form GlcNS3S residues, which were linked to IdoA2S residues at the non-reducing end to form a disaccharide unit of -IdoA2S-GlcNS3S-. Although 3-OST-1 has been successfully used in the synthesis of oligosaccharides in many studies 16,17,27,28 , there has been no report on the synthesis of oligosaccharides containing the -IdoA2S-GlcNS3S- disaccharide unit using 3-OST-3.

[0148] The discovery that 3-OST-3 and 3-OST-1 have different substrate requirements is disclosed herein. The 3-OST-1 enzyme sulfates oligosaccharide substrates carrying 6-O-sulfation, while showing very low reactivity towards oligosaccharide substrates without 6-O-sulfation ( Figure 5A andFigure 5B ), which is consistent with the previously described conclusion 16 . In contrast, 3-OST-3 preferentially sulfates oligosaccharides that do not carry 6-O-sulfation ( Figure 5A and Figure 5B ), but these 6-O-sulfated oligosaccharide substrates have lower reactivity towards 3-OST-3 modification ( Figure 5A , Figure 5B and Figure 5C ). The results of kinetic analysis showed that 3-OST-3 has a higher catalytic efficiency for oligosaccharide substrates without 6-O-sulfation, which was determined by the k cat / K m value (Table 1).

[0149] Table 1. Kinetic parameters of 3-OST-3 for oligosaccharide substrates

[0150]

[0151] As disclosed herein, the discovery of the different substrate requirements of 3-OST-1 and 3-OST-3 led to the development of two separate protocols for synthesizing oligosaccharides containing different 3-O-sulfated sugar sequences. To synthesize oligosaccharides containing the IdoA2S-GlcNS3S±6S disaccharide unit (Compounds 1, 2, 4, and 5, as shown in Figure 6A , Figure 6B , Figure 6D and Figure 6E respectively), the 3-O-sulfation by 3-OST-3 was introduced before the 6-O-sulfation step ( Figure 1B ), and to synthesize oligosaccharides containing the GlcA-GlcNS3S6S disaccharide unit, the 3-O-sulfation by 3-OST-1 was carried out after the 6-O-sulfation step ( Figure 1B )(Compounds 3 and 6, as shown in Figure 6C and Figure 6F respectively). The AT-binding domain contains a pentasaccharide unit of -GlcNS(or Ac)6S-GlcA-GlcNS3S±6S-IdoA2S-GlcA-GlcNS6S-, where the 3-O-sulfation may be required for high binding affinity 24,29 . Among all the oligosaccharides tested in this study, only Compounds 8 and 11 contain the pentasaccharide unit.

[0152] Example 2

[0153] Structural and conformational analysis of oligosaccharides

[0154] The purity and structure of Compounds 1-6 were analyzed. Representative data of Compound 6 shown in the analysis Figure 6E are as shown in Figure 2A andFigure 2B as shown. By high-resolution anion-exchange HPLC, compound 6 eluted as a single peak, indicating that the compound was pure ( Figure 2A ). The molecular weight of compound 6 was determined by electrospray ionization mass spectrometry (ESI-MS) to be 2449.43 ± 0.74, which was very close to the calculated molecular weight of 2448.92 ( Figure 2B ). The 1H-NMR spectrum of compound 6 clearly showed eight anomeric protons, confirming that the product was an octasaccharide. The 13C-NMR and full NMR assignments of compound 6 are shown in Supplementary Table 1, respectively. To localize the 3-O-sulfo group in compound 4, tandem MS analysis was performed. In this analysis, a stable isotope-labeled [34S] sulfo group was introduced by the 3-OST-3 enzyme, enabling us to unambiguously identify the presence of the 3-O-sulfo group at residue d in compound 4. Since compound 4 was an intermediate of compounds 5 and 6, the tandem MS analysis of compound 4 also helped to localize the IdoA2S-GlcNS3S6S disaccharide unit in compounds 5 and 6.

[0155] The pyranose ring of the IdoA2S residue interconverted between different conformations, including chair (1C4 and 4C1) and twist-boat (2SO) ( Figure 1C ). The effect of 3-O-sulfation on the conformation of the neighboring IdoA2S residue was investigated. The conformational analysis was done using NMR by measuring the three-bond proton-proton coupling constant (3JH-H) 30 . The 3-O-sulfation increased the population of the 2SO conformer of the IdoA2S residue in the hexasaccharide compared to those of the IdoA2S residue in the hexasaccharide (compound 7) without the GlcNS3S6S residue (compounds 1 - 3, Table 2 and Figure 1A ). In compound 3, the IdoA2S residue showed almost exclusively the 2SO conformation. For the octasaccharide (compounds 4 - 6, Table 2 and Figure 1A) There are two IdoA2S residues, designated as residue c and e. The effect of 3-O-sulfation on the 2SO population is different. For example, compared to those of the IdoA2S residues in the octasaccharide (Compound 10) without GlcNS3S6S residue, 3-O-sulfation increases the 2SO population of the flanking IdoA2S residues shown in Compound 5. In Compound 11, the 3-O-sulfation on residue f increases the 2SO population of the adjacent IdoA2S residue (residue e), but has no effect on the distal IdoA2S residue (residue c) compared to the corresponding residue in Compound 10. In Compound 6, the 3-O-sulfation on residues d and f increases the 2SO population of residue e, but the 2SO population of residue c decreases from 71% to 58% compared to the corresponding residue from Compound 5 (Table 2). These observations suggest that 3-O-sulfation is able to modulate the structure of HS through its effect on the conformation of IdoA2S residues.

[0156]

[0157] Example 3

[0158] The presence of the -GlcNS3S6S-IdoA2S-disaccharide unit affects the anticoagulant activity.

[0159] HS oligosaccharides achieve their anticoagulant activity through interaction with AT, so oligosaccharides showing anti-FXa activity should bind to AT. The effect of the sugar residues surrounding the GlcNS3S6S residue on the AT-binding and anti-FXa activity was examined. These data indicate that the presence of the -GlcNS3S6S-IdoA2S-disaccharide unit (i.e., IdoA2S located at the reducing end of the GlcNS3S6S residue) can play a role in determining the anticoagulant activity (Table 3). Compounds 2 and 8 have 6 residues, 8 sulfates, 1 IdoA2S and 2 GlcA residues, but only Compound 8 exhibits anti-FXa activity ( Figure 3A and Table 3). As expected, the Kd value for the tight binding of AT to Compound 8 is 7 ± 2 nM, but it does not bind to Compound 2 (Table 3). Structurally, the compounds are different in the position of the IdoA2S residue. In Compound 8, the 3-O-sulfated glucosamine (GlcNS3S6S) residue is flanked by the IdoA2S residue on the upper side of the reducing end, thus making it have the -GlcNS3S6S-IdoA2S-disaccharide unit, while the GlcNS3S6S in Compound 2 is flanked by the GlcA residue on the upper side of the reducing end, thus making it have the -GlcNS3S6S-GlcA-disaccharide unit.

[0160]

[0161] The findings of this article are consistent with previously published conclusions that the 2 SO conformation affects anticoagulant activity 9 . The IdoA2S residue in compound 8 exhibits the 2 SO conformation; while in compound 2, this position is occupied by a GlcA residue adopting the 4 C1 conformation. The X-ray crystal structure and NMR solution structure of the complex of AT and fondaparinux indicate that the IdoA2S residue exists in the 2 SO conformation in the complex 31,32 . Among them, the IdoA-containing hexasaccharide in which the IdoA2S residue is replaced by an IdoA residue exhibiting the 2 SO conformation exhibits anti-FXa activity 30 (Table 3). However, the GlcA2S-containing hexasaccharide in which the IdoA2S residue is replaced by a GlcA2S residue exhibiting the 4 C1 conformation does not exhibit anti-FXa activity 8 (Table 2). Compound 3 exhibits strong anti-FXa activity because the hexasaccharide is composed of two GlcNS3S6S residues ( Figure 3A and Table 3), which is consistent with the results of an octasaccharide composed of a similar pentasaccharide domain reported previously 32 . Residues d and c in compound 3 form the -GlcNS3S6S-IdoA2S- disaccharide unit.

[0162] Example 4

[0163] The GlcA residue in the AT-binding domain can be replaced by an IdoA2S residue

[0164] These studies have revealed a new AT-binding sugar sequence. The currently known AT-binding sequences contain the disaccharide -GlcA-GlcNS3S±6S- units 33 . Replacing the -GlcA-GlcNS3S6S- disaccharide unit with the -IdoA2S-GlcNS3S6S- disaccharide unit in HS is considered to abolish the AT-binding affinity 34,35 . The data from the anti-FXa activity and the AT-binding analysis of the octasaccharide clearly challenge this assertion. Compound 5 containing the -IdoA2S-GlcNS3S6S- (but not -GlcA-GlcNS3S6S-) disaccharide unit shows strong anti-FXa activity (Table 3). The AT-binding affinity analysis using isothermal titration calorimetry (ITC) also confirmed that compound 5 binds tightly to AT ( Figure 3Band Table 3). Compound 6 exhibited anti-FXa activity and high AT-binding affinity (Table 3) because the 8-mer contains the -GlcA-GlcNS3S6S- disaccharide unit.

[0165] The anticoagulant activity of compound 5 was further confirmed in in vivo experiments using a rat model. To this end, compound 5 was administered and the anti-FXa effect was compared with fondaparinux and compound 11, an anticoagulant octasaccharide previously reported. 17 . The results confirmed that compound 5 had comparable anti-FXa potency to fondaparinux and compound 11 ( Figure 3C ) within 30 min after administration of the drug. However, the anti-FXa effect from compound 5 decreased after 4 h, while the anti-FXa effects from fondaparinux and compound 11 persisted after 8 h ( Figure 3C ). Drug concentrations in blood samples were also obtained to determine the clearance rate of each compound in vivo ( Figure 3D ). Compared with fondaparinux and compound 11, compound 5 was cleared faster from the animals within the first 2 h.

[0166] The structural promiscuity of residue d in the AT-binding site ( Figure 4A ) was supported by molecular dynamics (MD) simulations of AT in complexes with various modified HS pentasaccharides. The computational technique was first validated by substituting parts known to be important for AT binding in the existing co-crystal structure of AT and fondaparinux 24,36 . Calculations of the binding free energy confirmed that removal of the 3-O-sulfate from residue c or substitution of residue b with GlcA destabilized the complex, as indicated by a significant 29% and 17% decrease in the interaction energy, respectively, which was qualitatively consistent with the experimental data. In contrast, substitution of GlcA (residue d) with IdoA2S in the 1 C4 conformation had no effect on the free energy ( Figure 4B ). Interestingly, substitution of the IdoA2S residue in the 2 SO conformation moderately enhanced the binding energy by 12% ( Figure 4B ), indicating the possibility of a more stable AT / pentasaccharide complex if residue d is IdoA2S in the 2 SO conformational isomer. IdoA2S in both conformations was able to maintain the carboxyl moiety in a position relative to the protein similar to that found for the GlcA residue in the fondaparinux mimic. Overall, the MD data support the conclusion that substitution of residue d with an IdoA2S residue does not reduce the binding affinity for AT and explains how compound 5 acts as an active anticoagulant despite the lack of a classical pentasaccharide sequence for AT.

[0167] Example 5

[0168] Analysis of 7-link HS compounds

[0169] Using the disclosed synthetic methods, 7-link HS compounds or heparin analogs were developed. The 7-link (the structure of which is as Figure 8B shown), and its anti-Xa activity ( Figure 8A ) was tested and confirmed. Figure 8B Schematically, the chemical structure of the 7-link was compared with the 6-link and 8-link disclosed herein. By way of example and not limitation, at least one synthetic route for synthesizing the 7-link is as Figure 8C shown. In some embodiments, it is possible to preferably have relatively short oligosaccharides, such as 7-links, as this can reduce the cost associated with synthesis.

[0170] Example 6

[0171] Discussion of results

[0172] A protocol for preparing a library of 3-O-sulfated oligosaccharides using a chemoenzymatic method is disclosed herein. It is confirmed herein that, in order to synthesize oligosaccharides containing the -IdoA2S-GlcNS3S- or -IdoA2S-GlcNS3S6S- disaccharide unit, the 3-OST-3 modification must be carried out before the 6-O-sulfation step, while the 3-OST-1 modification can only occur after 6-O-sulfation, thereby generating the -GlcA-GlcNS3S6S- disaccharide unit. This finding is supported by the ternary cocrystal structure of 3-OST-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 from 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 was observed between 3-OST-1 and the 6-O-sulfo group from the heptasaccharide substrate, indicating that 6-O-sulfation is required for binding to 3-OST-1. The different and unique substrate requirements between 3-OST-1 and 3-OST-3 increase the possibility of biosynthesizing 3-O-sulfated HS modified by different isotypes of 3-OST through different pathways.

[0173] It is generally believed that the 3-OST-1 enzyme is responsible for synthesizing the anticoagulant HS, while the 3-OST-3 enzyme is not. 34,35。All of the AT-binding sequences isolated so far are composed of the -GlcA-GlcNS3S6S- disaccharide repeating unit, which is a product of 3-OST-1 enzyme modification. 33,39 。Although compound 5, which is a product of 3-OST-3 enzyme modification, does not contain the -GlcA-GlcNS3S6S- disaccharide unit, it binds to AT and exhibits anticoagulant activity. These findings suggest that 3-OST-3 is capable of synthesizing anticoagulant HS as long as the HS contains a domain with a structure similar to that of compound 5.

[0174] The rapid clearance of compound 5 provides a potential new short-acting anticoagulant drug candidate with a reduced bleeding risk. Short-acting anticoagulants, which can be rapidly cleared from the circulation before a major bleeding effect occurs, would be particularly beneficial for patients at high risk of bleeding. 41 。Although unfractionated heparin is an anticoagulant with a short half-life, there is concern that the drug can cause heparin-induced thrombocytopenia (HIT), a life-threatening side effect. 42 。It has been found that short oligosaccharides less than 12-linkers 43 do not bind to platelet factor 4 and thus do not exhibit the HIT risk. Therefore, as an octasaccharide, compound 5 is expected to have a very low HIT risk. The availability of 3-O-sulfated oligosaccharides also provides an opportunity to study the casual relationship between carbohydrate sulfation / conformation and biological function, which is an important step in dissecting the structure-function relationship of HS. HS 3-OST exists in seven different isotypes. In this study, we demonstrated that 3-OST-1 and 3-OST-3 require different chemoenzymatic protocols to prepare different 3-O-sulfated oligosaccharide sequences. The conclusions of this study will guide others to develop chemoenzymatic methods using different 3-OST isotypes, allowing the preparation of more complex HS sugars with a wider range of sulfation patterns involving 3-O-sulfated glucosamine residues. These studies will enrich the HS oligosaccharide library and thus assist HS-related research.

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Claims

1. A synthetic heparin analogue, comprising: 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; and further, wherein the synthetic heparin analogue is:

2. The synthetic heparin analogue according to claim 1, wherein the synthetic heparin analogue has anticoagulant activity.

3. The synthetic heparin analogue according to claim 1, wherein the synthetic heparin analogue has a binding affinity for antithrombin of 5 nM to 30 nM.

4. The synthetic heparin analogue according to claim 1, wherein the synthetic heparin analogue has an anti-Xa activity of 10 ng / mL -1 to 40 ng / mL -1 IC 50 activity.

5. The synthetic heparin analogue according to any one of claims 1 to 4, wherein the synthetic heparin analogue contains at least one IdoA2S-GlcNS3S6S disaccharide unit and does not contain GlcA-GlcNS3S6S disaccharide unit.

6. The synthetic heparin analogue according to any one of claims 1 to 4, wherein the synthetic heparin analogue has a clearance rate 50% to 100% faster than other heparin compounds.

7. The synthetic heparin analogue according to any one of claims 1 to 4, wherein the synthetic heparin analogue does not cause heparin-induced thrombocytopenia (HIT).

8. The synthetic heparin analogue according to any one of claims 1 to 4, wherein in the presence of andexanet α at 20 μg / mL or less, the anticoagulant activity of the synthetic heparin analogue can be reversed by andexanet α at a rate of 50% or higher.

9. A method for synthesizing a synthetic heparin analogue, comprising: providing a sugar substrate; extending the sugar substrate to a sugar of a desired or predetermined length; and performing at least one sulfation reaction using the 3-OST-3 isoform of 3-O-sulfotransferase (3-OST), thereby synthesizing a synthetic heparin analogue; and wherein the synthetic heparin analogue is:

10. The method according to claim 9, wherein the synthetic heparin analogue contains at least one IdoA2S-GlcNS3S disaccharide unit.

11. The method according to claim 9, wherein the synthetic heparin analogue contains IdoA2S-GlcNS3S±6S disaccharide unit, and the method further comprises a 6-O-sulfation step using 6-O-sulfotransferase (6-OST), wherein the 3-O-sulfation by 3-OST-3 occurs before the 6-O-sulfation step.

12. The method according to claim 9, wherein the extension step comprises using a glycosyltransferase.

13. The method according to claim 12, wherein the glycosyltransferase is selected from the group consisting of N-acetylglucosaminyltransferase (KfiA) of Escherichia coli (E. coli) K5 and / or heparin synthase-2 (pmHS2) from Pasteurella multocida.

14. The method according to claim 9, wherein the extension step comprises using one or more monosaccharides selected from the group consisting of glucuronic acid (GlcUA), N-acetylglucosamine (GlcNAc), and N-trifluoroacetylglucosamine (GlcNTFA).

15. The method according to claim 9, wherein the synthetic method of the synthetic heparin analog has a yield greater than 20%.

16. The method according to claim 9, wherein the synthetic method of the synthetic heparin analog has a yield of 20% to 50%.

17. A synthetic heparin analog produced by the method according to any one of claims 9 to 16, wherein the synthetic heparin analog has anticoagulant activity.

18. A synthetic heparin analog produced by the method according to any one of claims 9 to 16, wherein the synthetic heparin analog has a binding affinity for antithrombin of 5 nM to 30 nM.

19. A synthetic heparin analogue produced by the method according to any one of claims 9 to 16, wherein the synthetic heparin analogue has an anti-Xa activity of 10 ng / mL -1 to 40 ng / mL -1 IC 50 activity.

20. A synthetic heparin analog produced by the method according to any one of claims 9 to 16, wherein the synthetic heparin analog is:

21. A synthetic heparin analog produced by the method according to claim 9, wherein the synthetic heparin analog contains at least one IdoA2S-GlcNS3S6S disaccharide unit and does not contain a GlcA-GlcNS3S6S disaccharide unit.

22. A synthetic heparin analog produced by the method according to claim 9, wherein the synthetic heparin analog has a clearance rate that is 50% to 100% faster than other heparin compounds.

23. A synthetic heparin analog produced by the method according to claim 9, wherein the synthetic heparin analog does not cause heparin-induced thrombocytopenia (HIT).

24. A synthetic heparin analog produced by the method according to claim 9, wherein in the presence of andexanet α at 20 μg / mL or less, the anticoagulant activity of the synthetic heparin analog can be reversed by andexanet α at a rate of 50% or higher.

25. A pharmaceutical composition comprising the synthetic heparin analog according to any one of claims 1 to 4.

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