Classifications

• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
  • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
  • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
  • C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
  • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
  • A61K31/727—Heparin; Heparan
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
  • A61P13/12—Drugs for disorders of the urinary system of the kidneys
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
  • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
  • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
  • C08L5/10—Heparin; Derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/26—Preparation of nitrogen-containing carbohydrates
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the subject matter disclosed herein relates generally to synthesis of heparin compounds. More particularly, the subject matter disclosed herein relates to chemoenzymatic synthesis of structurally homogeneous low molecular weight heparins that have a reversible anticoagulant activity.
• Heparan sulfate is a ubiquitous component of the cell surface and extracellular matrix. It regulates a wide range of physiologic and pathophysiologic functions, including embryonic development and blood coagulation, and can facilitate 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 effects 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 comprising 1 - ⁇ 4- linked glucosamine and glucuronic/iduronic acid units that contain both N- and O-sulfo groups. Unique saccharide sequences within HS can determine the specificity of the binding of HS to its target proteins (Linhardt (2003) J.
• Heparin a specialized form of HS, is a Attorney Docket No.: 421/330 PCT commonly used anticoagulant drug.
• new methods for the synthesis of heparin compounds and HS attract considerable interest for those developing anticoagulant and other HS-related drugs having improved pharmacological effects.
• UF heparin unfractionated (UF) heparin (MW avg -14000 Da); a low molecular weight heparin (MW avg -6000 Da); and the synthetic UL W heparin pentasaccharide ARIXTRA ® (MW 1508.3 Da).
• UF heparin is used in surgery and kidney dialysis due to its relatively short half-life and its safety for renal impaired patients (Hirsh et al., 2007).
• LMW heparins and the ULMW heparin ARIXTRA ® have played an increasingly important role for preventing venous thrombosis among high risk patients (Tohu et al, 2004; Weitz, 2010) because of their more predictable anticoagulant dose, long half-lives and their reduced risk of osteoporosis (Weitz and Linkins, 2007).
• Recent research on LMW heparin has resulted in the European approval of Bemiparin sodium (Martinez-Gonzalez and Rodriguez, 2010), a second-generation LMW heparin, and the United States approval of a generic LMW heparin, M-Enoxaparin.
• UF heparin is isolated from porcine intestine or bovine lung, and LMW heparins are prepared through the chemical or enzymatic degradation of this animal-sourced UF heparin.
• a worldwide outbreak of contaminated heparin has raised concerns over the reliability and safety of animal sourced heparins and LMW heparins (Guerrini et al., 2008; Liu et al., 2009).
• a cost-effective method for preparing new synthetic heparins is highly desirable (Peterson et al., 2009).
• methods of synthesizing heparin compounds are provided. Such methods can in some embodiments comprise providing a saccharide substrate, elongating the saccharide substrate to a saccharide of a desired or predetermined length, performing an epimerization reaction, and performing one or more sulfation reactions, whereby a heparin compound is synthesized.
• the elongation step can comprise employing a glycosyl transferase.
• the glycosyl transferase is selected from the group consisting of /V-acetyl glucosaminyl transferase of E.
• the elongation step can comprise employing one or more monosaccharides selected from the group consisting of: glucuronic acid (GlcUA), N-acetylated glucosamine (GlcNAc), and N-trifluoroacetyl glucosamine (GlcNTFA).
• GlcUA glucuronic acid
• GlcNAc N-acetylated glucosamine
• GlcNTFA N-trifluoroacetyl glucosamine
• methods of synthesizing heparin compounds can further comprise converting an N-sulfotransferase substrate residue on the hexasaccharide or heptasaccharide to a N-sulfo glucosamine (GlcNS) residue, comprising employing N-sulfotransferase (NST) and 3'- phosphoadenosine 5'-phosphosulfate (PAPS).
• methods of synthesizing heparin compounds can further comprise employing a base in one or more converting reactions, wherein the base is optionally a lithium hydroxide or a mixture of triethylamine, CH 3 OH, and/or H 2 0.
• the epimerization reaction comprises employing C 5 -epimerase (C 5 -epi).
• the sulfation reaction comprises employing 2-O-sulfotransferase (2-OST).
• the sulfation reaction comprises employing 6-0- sulfotransferase (6-OST), optionally 6-OST-1 and/or 6-OST-3.
• the sulfation reaction comprises employing 3-0- sulfotransferase (3-OST), optionally 3-OST-1 and/or 3-OST-5.
• the methods of synthesizing heparin compounds can have a yield of greater than 30%.
• heparin compounds synthesized by the disclosed methods of synthesis can have a molecular weight ranging from about 1500 daltons (Da) to about 6000 Da.
• the heparin compounds can have anticoagulant activity.
• the heparin compounds can have a binding affinity to antithrombin ranging from about 5 nM to about 30 nM.
• the heparin compounds can have anti-Xa activity ranging from about 10 ngmr 1 to about 40 ngml "1 IC 50 under conditions described herein.
• the heparin compounds can have no detectable anti-lla activity.
• heparin compounds comprising providing a monosaccharide substrate, elongating the monosaccharide substrate to a hexasaccharide using enzymes /V-acetyl glucosaminyl transferase and heparosan synthase-2, and substrates glucuronic acid (GlcUA) and N-trifluoroacetyl glucosamine (GlcNTFA), converting N-trifluoroacetyl glucosamine (GlcNTFA) residue(s) on the heptasaccharide to N-sulfo glucosamine (GlcNS) residue(s) using N- sulfotransferase (NST), 3'-phosphoadenosine 5'-phosphosulfate (PAPS), and a base, epimerizing the hexasaccharide using C 5 -epimerase (GlcUA) and N-trifluoroacetyl
• the heparin compound produced by this method has anticoagulant activity.
• the heparin compound has a binding affinity to antithrombin ranging from about 5 nM to Attorney Docket No.: 421/330 PCT about 9 nM.
• the heparin compound has anti-Xa activity of about 10 ngml "1 IC 50 to about 20 ngml 1 IC 50 , optionally about 14 ngml "1 IC 50 , under. conditions described herein.
• heparin compounds comprising providing a monosaccharide substrate, elongating the monosaccharide substrate to a hexasaccharide using enzymes A/-acetyl glucosaminyl transferase and heparosan synthase-2, and substrates glucuronic acid (GlcUA) and N-trifluoroacetyl glucosamine (GlcNTFA), converting N-trifluoroacetyl glucosamine (GlcNTFA) residue(s) on the heptasaccharide to N-sulfo glucosamine (GlcNS) residues using N- sulfotransferase (NST), 3'-phosphoadenosine 5'-phosphosulfate (PAPS), and a base, optionally lithium hydroxide, epimerizing the hexasaccharide using C 5 -e
• the heparin compound produced by this method has anticoagulant activity.
• the heparin compound has a binding affinity to antithrombin ranging from about 5 nM to about 11 nM.
• the heparin compound has anti-Xa activity of about 12 ngml "1 IC 50 to about 22 ngml "1 IC 50 , optionally about 17 ngml "1 IC 50 , under conditions described herein.
• heparin compounds comprising providing a monosaccharide substrate, elongating the monosaccharide substrate to a hexasaccharide using enzymes /V-acetyl glucosaminyl transferase and heparosan synthase-2, and substrates glucuronic acid (GlcUA) and N-trifluoroacetyl glucosamine (GlcNTFA), converting N-trifluoroacetyl glucosamine (GlcNTFA) residue(s) on the heptasaccharide to N-sulfo glucosamine (GlcNS) residues using N- sulfotransferase (NST), 3'-phosphoadenosine 5'-phosphosulfate (PAPS), and a base, optionally lithium hydroxide, epimerizing the hexasaccharide using C 5 -
• the heparin compound produced by this method has anticoagulant activity.
• the heparin compound has a binding affinity to antithrombin ranging from about 4 nM to about 6 nM.
• the heparin compound has anti-Xa activity of about 10 ngml "1 IC 50 to about 20 ngml "1 IC 50 , optionally about 15 ngmr 1 IC 50 , under conditions described herein.
• the heparin compound is susceptible to Stablin-2 mediated endocytosis, wherein a heparin compound that is endocytosed at a rate of 15% or more is susceptible to Stablin-2 mediated endocytosis.
• heparin compounds comprising providing a monosaccharide substrate, elongating the monosaccharide substrate to a hexasaccharide using enzymes /V-acetyl glucosaminyl transferase and heparosan synthase-2, and substrates glucuronic acid (GlcUA) and N-trifluoroacetyl glucosamine (GlcNTFA), converting N-trifluoroacetyl glucosamine (GlcNTFA) residue(s) on the heptasaccharide to N-sulfo glucosamine (GlcNS) residues using N- sulfotransferase (NST), 3'-phosphoadenosine 5'-phosphosulfate (PAPS), and a base, optionally lithium hydroxide, epimerizing the hexasaccharide Attorney Docket No
• the heparin compound produced by this method has anticoagulant activity.
• the heparin compound has a binding affinity to antithrombin ranging from about 23 nM to about 37 nM.
• the heparin compound has anti-Xa activity of about 25 ngml 1 IC 50 to about 45 ngml "1 IC 50 , optionally about 35 ngml "1 IC 50 , under conditions described herein.
• the heparin compound is susceptible to Stablin-2 mediated endocytosis at a rate of 15% or more.
• the anticoagulant activity of the heparin compound is partially reversible by protamine, wherein a heparin compound having anticoagulant activity that is reversible at a rate of 50% or more in the presence of 20ug/ml or less of protamine is partially reversible.
• heparin compounds comprising providing a monosaccharide substrate, elongating the monosaccharide substrate to a hexasaccharide using enzymes /V-acetyl glucosaminyl transferase and heparosan synthase-2, and substrates glucuronic acid (GlcUA) and N-trifluoroacetyl glucosamine (GlcNTFA), converting N-trifluoroacetyl glucosamine (GlcNTFA) residue(s) on the heptasaccharide to N-sulfo glucosamine (GlcNS) residues using N- sulfotransferase (NST), 3'-phosphoadenosine 5'-phosphosulfate (PAPS), and a base, optionally lithium hydroxide, epimerizing the hexasaccharide using C 5 -
• the heparin compound produced by this method has anticoagulant activity.
• the heparin Attorney Docket No.: 421/330 PCT compound has a binding affinity to antithrombin ranging from about 12 n to about 44 nM.
• the heparin compound has anti-Xa activity of about 10 ngml "1 IC 50 to about 30 ngml "1 IC 50 , optionally about 21 ngml "1 IC 50 , under conditions described herein.
• the heparin compound is susceptible to Stablin-2 mediated endocytosis at a rate of 15% or more.
• the anticoagulant activity of the heparin compound is reversible by protamine at a rate of 50% or more in the presence of 20ug/ml or less of protamine.
• provided herein are methods of treating a subject, the methods comprising providing a subject to be treated, administering to the subject a heparin compound having anticoagulant activity, wherein the heparin compound comprises a heparin compound as disclosed herein, or wherein the heparin compound comprises a synthetic, low-molecular weight heparin compound with reversible anticoagulant activity, wherein the anticoagulant activity of the heparin compound is reversible by protamine, wherein the anticoagulant activity is reversed by about 50% or more in the presence of 1 ug/ml of protamine.
• the subject can suffer from venous thromboembolism.
• the subject can be renal-impaired.
• the subject can be a human subject.
• provided herein are methods of treating a subject in need of anticoagulant therapy, the method comprising providing a subject in need of anticoagulant therapy, administering to the subject a heparin compound having anticoagulant activity, monitoring the subject for heparin-induced thrombocytopenia, and administering to the subject an antidote to reverse the anticoagulant activity of the heparin compound if the subject suffers from heparin-induced thrombocytopenia.
• the subject suffers from venous thromboembolism.
• the subject is renal-impaired.
• the subject is a human subject.
• the antidote to reverse the anticoagulant activity of the heparin compound is protamine.
• heparin compounds with reversible anticoagulant activity wherein the Attorney Docket No.: 421/330 PCT anticoagulant activity of the heparin compound is reversible by protamine, wherein the anticoagulant activity is reversed by about 50% or more in the presence of 1 ug/ml of protamine.
• heparin compounds in some embodiments, provided herein are synthetic, low-molecular weight heparin compounds susceptible to liver-mediated clearance from circulation.
• the heparin compound can be susceptible to Stablin-2 mediated endocytosis at a rate of 15% or more.
• the heparin compound is suitable for administration to renal-impaired patients.
• the heparin compound comprises at least one 3-0 sulfate.
• the heparin compound is a dodecamer.
• Figures 1A-1 I illustrate the chemical structures of Compounds 1-9 (Figure 1A, Compound 1 ; Figure 1 B, Compound 2; Figure 1C, Compound 3; Figure 1 D, Compound 4; Figure 1 E, Compound 5; Figure 1 F, Compound 6; Figure 1G, Compound 7; Figure 1 H, Compound 8; Figure 11, Compound 9);
• FIG. 2 is schematic illustration of the synthesis of Compound 1
• FIG. 3 is a schematic illustration of the synthesis of synthetic LMWHs as disclosed herein.
• the synthesis can in some aspects be initiated from a hexasaccharide (Compound 6), which can be converted to an octasaccharide (Compound 7), decasaccharide (Compound 8) and dodecasaccharide (Compound 9) to synthesize Compounds 2, 3, and 4, respectively.
• the conversion of Compound 4 to 5 can be achieved by 3- OST-5 modification;
• Figures 4A-4D are graphical depictions of the data for the determination of the clearance, anti-FXa activity and sensitivity to protamine neutralization of synthetic LMWHs.
• Figure 4A is a histogram of the percentage of 35 S-labeled synthetic LMWHs that was retained in the liver in a mouse model. In comparison to compound 1 , all tested compounds, with the exception of compound 2 and enoxaparin, showed significantly higher retention in liver (****p ⁇ 0.0001).
• Figure 4B plots FXa activity of synthetic LMWHs in the presence of different concentrations of protamine under in vitro conditions.
• Figure 4C is a histogram of ex vivo reversibility of anti-FXa activity by protamine.
• Figure 5 is a histogram of endocytosis of 35 S-labeled synthetic LMWHs in Flp-ln-293 cells.
• the cells were stably transfected with the plasmid expressing the Stabilin-2 receptor (190-HARE).
• the internalization value was calculated by subtracting the nonspecific internalization background from the total internalization of a 3 S-labeled Compound.
• Figure 6 is a line chart of anti-Xa activity of Compounds 1-5, enoxaparin and UFH.
• the phrase “A, B, C, and/or D” includes A, B, C, and D individually, but also includes any and all combinations and subcombinations of A, B, C, and D.
• heparins As used herein, the terms “heparins”, “heparin compound”, “US”, “HS- like compound”, and “HS-like molecule” are intended to refer to synthetically sulfated polysaccharides possessing one or more structural and/or functional properties of heparan sulfates.
• heparin compounds can contain glucuronic acid or iduronic acid and glucosamine with or without Attorney Docket No.: 421/330 PCT sulfo groups.
• heparin compounds include, but are not limited to, synthetic HSs, sulfated polysaccharides and heparins.
• Heparan sulfates are highly sulfated polysaccharides present on the surface of mammalian cells and in the extracellular matrix in large quantities.
• HS is a highly charged polysaccharide comprising 1 —4 4-linked glucosamine and glucuronic/iduronic acid units that contain both N- and O- sulfo groups.
• Heparin a specialized form of HS, is a commonly used anticoagulant drug.
• "heparan sulfate” includes heparin.
• Heparin is a polysaccharide that comprises a disaccharide-repeating unit of either iduronic acid (IdoA) or glucuronic acid (GIcA) and glucosamine residues, each capable of carrying sulfo groups.
• IdoA iduronic acid
• GcA glucuronic acid
• heparin is synthesized by a series of heparan sulfate (HS) biosynthetic enzymes (Figure 2).
• HS polymerase catalyzes the formation of the polysaccharide backbone, a repeating disaccharide of GlcA and N- acetylated glucosamine (GlcNAc).
• This backbone is then modified by N- deacetylase/N-sulfotransferase (NDST), C 5 -epimerase (C 5 -epi), 2-0- sulfotransferase (2-OST), 6-O-sulfotransferase (6-OST), and 3-0- sulfotransferase (3-0ST).
• heparin polysaccharides and oligosaccharides have been chemically synthesized through lengthy routes. Although several synthetic strategies have been reported, the chemical synthesis of heparin oligosaccharides is extremely challenging, especially for oligosaccharides larger than octasaccharides having a high content of sulfo groups. Chemoenzymatic methods have significantly simplified the synthesis, offering a practical approach to prepare synthetic LMWHs.
• Heparin compounds of the presently disclosed subject matter can comprise a synthetically sulfated polysaccharide possessing one or more structural and/or functional properties of heparan sulfates.
• exemplary embodiments of particular heparin compounds have been disclosed herein, the presently disclosed subject matter is not intended to be limited to the disclosed examples, but rather heparin compounds include all comparable synthetically sulfated polysaccharides as would be apparent to one of ordinary skill. Indeed, one of ordinary skill in the art, upon a review of the instant disclosure, is capable of producing numerous heparin compounds based upon the disclosed methods and compounds.
• Heparins play roles in a variety of important biological processes, including assisting viral infection, regulating blood coagulation and embryonic development, suppressing tumor growth, and controlling the eating behavior of test subjects by interacting with specific regulatory proteins (Liu, J., and Thorp, S. C. (2002) Med. Res. Rev. 22: 1-25; Rosenberg, R. D., et al., (1997) J. Clin. Invest. 99:2062-2070; Bernfield, M., et al., (1999) Annu. Rev. Biochem. 68:729-777; Alexander, C. M., et al., Attorney Docket No.: 421/330 PCT
• the unique sulfated saccharide sequences can determine to which specific proteins heparins bind, thereby regulating biological processes.
• heparin occurs in the Golgi apparatus. It can initially be synthesized as a copolymer of glucuronic acid and /V-acetylated glucosamine by D-glucuronyl and /V-acetyl-D-glucosaminyltransferase, followed by various modifications (Lindahl, U., et al., (1998) J. Biol. Chem. 273:24979-24982).
• modifications can include /V-deacetylation and N- sulfation of glucosamine, C 5 epimerization of glucuronic acid to form iduronic acid residues, 2-O-sulfation of iduronic and glucuronic acid, as well as 6-0- sulfation and 3-O-sulfation of glucosamine.
• Several enzymes that are responsible for the biosynthesis of HS have been cloned and characterized (Esko, J. D., and Lindahl, U. (2001) J. Clin. Invest. 108: 169-173).
• HS /V-deacetylase//V-sulfotransferase, 3-O-sulfotransferase, and 6- O-sulfotransferase are present in multiple isoforms.
• Each isoform is believed to recognize a saccharide sequence around the modification site in order to generate a specific sulfated saccharide sequence (Liu, J., et al., (1999) J. Biol. Chem. 274:5185-5192; Aikawa, J.-l., et al. , (2001) J. Biol. Chem.
• HS D-glucosaminyl 3-O-sulfotransferase (3-OST) isoforms generate 3-O-sulfated glucosamine residues that are linked to different sulfated iduronic acid residues.
• 3-OST isoform 1 (3-OST-1 ) transfers sulfate to the 3-OH position of an /V-sulfated glucosamine residue that is linked to a glucuronic acid residue at the nonreducing end (GlcUA-GlcNS ⁇ 6S).
• 3-OST isoform 3 (3-OST-3) transfers sulfate to the 3-OH position of an /V-unsubstituted glucosamine residue that is linked to a 2-O-sulfated iduronic acid at the nonreducing end (ldoUA2S-GlcNH 2 ⁇ 6S) (Liu, J., et al., (1999) J. Biol. Chem. 274:38 55-38162).
• the difference in the substrate specificity of 3-OSTs results in distinct biological functions.
• the HS modified by 3-OST-1 binds to antithrombin (AT) and possesses anticoagulant activity (Liu, J., et al., (1996) J. Biol. Chem. 271 :27072-27082).
• the HS modified by 3-OST-3 (3-OST-3A and 3-OST-3B) binds to glycoprotein D (gD) of herpes simplex virus, type 1 , (HSV-1) thus mediating viral entry (Shukla, D., et al. , (1999) Cell 99:13-22).
• gD glycoprotein D
• HSV-1 herpes simplex virus
• HS-regulated anticoagulation mechanisms have been studied extensively. It is now known that HS, including heparin, interact with AT, a serine protease inhibitor, to inhibit the activities of thrombin and factor Xa in the blood coagulation cascade (Rosenberg, R. D., et al., (1997) J. Clin. Invest. 99:2062-2070). Anticoagulant-active HS (HS act ) and heparin contain one or multiple AT-binding sites per polysaccharide chain.
• This binding site Attorney Docket No.: 421/330 PCT contains a specific pentasaccharide sequence with a structure of -GlcNS(or Ac)6S-GlcllA-GlcNS3S( ⁇ 6S)-ldoUA2S-GlcNS6S-.
• the 3-O-sulfation of glucosamine for generating GlcNS3S( ⁇ 6S) residue which is carried out by 3- OST-1 , plays a role in the synthesis of HS act (Liu, J., et al., (1996) J. Biol. Chem. 271 :27072-27082; Shworak, N. W. , et al., (1997) J. Biol. Chem. 272:28008-28019).
• heparin compounds of the presently disclosed subject matter can have a strong binding affinity for AT.
• the binding constant (K d ) of heparin compounds of the presently disclosed subject matter can range from about 3 to about 100 nM.
• the binding constant (K d ) of a heparin compound of the presently disclosed subject matter can range from about 5 to about 40 nM. Any suitable approach to determine binding affinity can be employed as would be appreciated by one of ordinary skill in the art upon review of the instant disclosure.
• heparin compounds disclosed herein can have a molecular weight ranging from about 1 ,500 daltons (Da) to about 6,000 Da.
• the term low molecular weight (LMW) heparin is intended to refer to a heparin compound ranging in size from 3,500 to 6,000 Da and having from about 12 to 20 saccharide units, and in some embodiments having anticoagulant activity.
• a LMW heparin of the presently disclosed subject matter can be about 3,500 Da, about 4,000 Da, about 4,500 Da, about 5,000 Da, about 5,500 Da, or about 6,000 Da.
• a LMW heparin of the presently disclosed subject matter can have 12, 14, 16, 18 or 20 saccharide units. In some embodiments, a LMW heparin of the presently disclosed subject matter can have anticoagulant activity that is reversible in the presence of an antidote, such as for example protamine.
• the anticoagulant activity can be measured by determining anti-Xa and anti-Ma activities. In some embodiments, the anti- Xa and anti-Ma activities can be determined in the presence of antithrombin.
• Heparin compounds with a strong binding affinity for AT and/or a high anticoagulant activity can have high anti-Xa and anti-lla activities.
• heparin compounds with high anticoagulant activity can have IC 50 values for anti-Xa activity ranging from about 1 to about 20 nM.
• heparin compounds with high anticoagulant activity can have IC 50 values for anti-Xa activity ranging from about 1 to about 10 nM.
• heparin compounds with high anticoagulant activity can have IC 50 values for anti-Xa activity ranging from about 1 to about 5 nM.
• heparin compounds with high anticoagulant activity can have IC 50 values for anti-Xa activity ranging from about 10 ngmr 1 to about 40 ngm 1 , under the conditions disclosed herein. In some embodiments, heparin compounds with high anticoagulant activity can have IC 50 values for anti-Xa activity ranging from about 15 ngml "1 to about 35 ngm 1 , under the conditions disclosed herein. As would be appreciated by one of ordinary skill in the art, IC 50 values are relative to the conditions under which the measurements are taken. As such, the IC 50 values expressed herein are relative to the conditions under which the measurements were taken and can therefore be adjusted accordingly as would be understood by one of ordinary skill in the art. Any suitable approach to determine anticoagulant activity can be employed as would be appreciated by one of ordinary skill in the art upon review of the instant disclosure.
• the disclosed heparin compounds can have an anticoagulant activity that is reversible in the presence of a reversing agent, such as for example protamine.
• a heparin compound can have an anticoagulant activity that is completely reversible, substantially completely reversible, or at least partially reversible by protamine.
• a heparin compound having anticoagulant activity that is reversible at a rate of 50% or more in the presence of 20ug/m! or less of protamine is partially reversible.
• a heparin compound of the present disclosure is considered to have a reversible anticoagulant activity if the anticoagulant activity of the heparin compound is reversible at a rate similar to that of unfractionated heparin, as would be appreciated by one of ordinary skill in the art upon a review of the instant disclosure.
• Unfractionated heparin (UFH) and larger chained heparin compounds can bind to stabilin-2, a scavenger receptor present on liver sinusoidal endothelial cells that mediates their clearance.
• the disclosed heparin compounds displayed significant endocytosis mediated by Stabilin-2 as measured in a cell-based assay.
• heparin compounds with a 3-0 sulfation can be more efficiently taken up and cleared by liver. See also Yu et al, 2014.
• the sulfation pattern of the presently disclosed heparin compounds can impact the clearance of such compounds through the liver.
• a synthetic, low-molecular weight heparin compound susceptible to liver-mediated clearance from circulation comprises a 3-0 sulfation.
• heparin compounds disclosed herein that are dodecamers or longer with a 3-0 sulfation can have such enhanced liver clearance.
• a sulfation pattern such as a 3-0 sulfation pattern, in a synthetic, low-molecular weight heparin compound, such as a dodecamer or larger synthetic oligosaccharide, which matches the requirements for rapid liver clearance.
• heparin compounds of the presently disclosed subject matter can be susceptible to Stablin-2 mediated endocytosis, wherein a heparin compound that is endocytosed at a rate of 15% or more is susceptible to Stablin-2 mediated endocytosis.
• a heparin compound that is endocytosed at a rate of 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 90% or more is susceptible to Stablin-2 mediated endocytosis.
• the heparin compounds can be susceptible to Stablin-2 mediated endocytosis at a rate of 15% or more. Due to these characteristics, in some aspects the disclosed heparin compounds can be suitable for administration to renal-impaired patients. In some aspects, such heparin compounds that are susceptible to liver-mediated clearance from circulation comprises a sulfation pattern, such as a 3-0 sulfation pattern, such at least one 3-0 sulfate, and in some aspects can be a dodecamer.
• LMWHs Five homogenous LMWHs (Compounds 1-5, Figures 1A-1 E, respectively), ranging from hexa- to dodeca-saccharide, can be synthesized.
• the structures of Compounds 1-4 differ at their reducing ends, containing a different number of -ldoA2S-GlcNS6S- repeating units.
• Dodecasaccharide 5 differs from 4, as it has two 3-O-sulfo groups ( Figure 1 D and 1 E).
• the synthesis of Compound 1 can in some embodiments be initiated from a commercially available monosaccharide, l-O-(para-nitrophenyl) glucuronide (GlcA-pnp) ( Figure 2).
• Elongation of GlcA-pnp to a hexasaccharide can be accomplished using two bacterial glycosyltransferases: KfiA (N- acetylglucosaminyl transferase from E. coli K5 strain) and heparosan synthase 2 (pmHS2) from Pasteurella multocida.
• KfiA N- acetylglucosaminyl transferase from E. coli K5 strain
• pmHS2 heparosan synthase 2
• the approach can also afford for the synthesis of hexasaccharide 6, an intermediate for subsequent syntheses.
• Figure 3 is a schematic illustration of the synthesis of synthetic LMWHs as disclosed herein, and particularly the synthesis of Compounds 2 to 5.
• the synthesis can be initiated from a hexasaccharide 6.
• the hexasaccharide can be converted to an octasaccharide (7), decasaccharide (8) and dodecasaccharide (9) to synthesize Compounds 2, 3, and 4, respectively.
• the conversion of Compounds 4 to 5 can be achieved by 3- Attorney Docket No.: 421/330 PCT
• GIcA to ldoA2S involves two steps: the C 5 -epi catalyzed epimerization of a GIcA to an IdoA; and 2-OST transferred a sulfo group to the IdoA residue.
• C 5 -epi catalyzes both the forward and reverse reactions, leading to the incomplete conversion of GIcA to ldoA2S 26 and a complex mixture of products 27 .
• the conversion of Compound 6 to 7, involving the compound of the pentasaccharide domain recognized by C 5 -epi, can be completed in four steps.
• the GlcNTFA residue (Residue F) of hexasaccharide 6 can first be converted to a GlcNS residue (Step a, Figure 3B).
• the hexasaccharide can then elongated to an octasaccharide in two enzymatic steps (step b and c, Figure 3B), to obtain the desired pentasaccharide domain, as depicted in the dashed box ( Figure 3B).
• step d The conversion of GIcA (Residue E) to ldoA2S to yield 7 can then achieved by C 5 -epi and 2-OST (step d, Figure 3B).
• the formation of ldoA2S residue can remove its reactivity towards further Cs-epi modification. Repeating these steps (step a to d, Figure 3B) once or twice can afford 8 and 9, respectively.
• the products of the disclosed and illustrated reactions can contain a pnp tag with a strong absorbance at 310 nm, facilitating purity analysis by high performance liquid chromatography (HPLC). Structures were determined by electrospray ionization mass spectrometry (ESI-MS), one-dimensional (1 D) and two-dimensional (2D) nuclear magnetic resonance (NMR) analysis. Diethylaminoethyl (DEAE)- Attorney Docket No.: 421/330 PCT
• HPLC analysis demonstrates that 5 is of high purity, MS affords an observed mass of 3562.8755, very close to the calculated exact mass of 3562.9087 and observed isotopic distribution for C80H-121 N7O 15S17 matches the simulated distribution.
• 1 H-NMR spectrum of 5 depicts the presence of 12 anomeric protons. Additional 1-D NMR and 2-D NMR analysis confirmed the correct glycosidic linkages and led to the spectral assignment of 5.
• MS- assisted sequence analysis pinpointed the residues carrying 3-O-sulfo groups.
• Figures 4A-4D illustrate that Compounds 3, 4 and 5 are internalized in mice liver, and that the anti-Xa activity of Compound 5 can be reversed by protamine in an in vitro experiment, while the anti-Xa activity of other compounds, including enoxaparin (brand name LOVENOX), is not reversible by protamine.
• UFH and Compound 5 have similar sensitivity to protamine neutralization.
• the anti-Xa activity of Compound 5 is reversible in an ex vivo experiment. Enoxaparin is only partially reversible, but UFH is fully reversible. And, the anti-Xa activity of Compound 5 can be reversed in an in vivo experiment.
• Heparin and LMWHs are critical for the practice of modern medicine yet their production still depends on a long supply chain, which is vulnerable to contamination and adulteration.
• the US FDA and EMA have implemented a series of new approaches to monitor the purity of heparin drugs.
• Chemoenzymatic synthesis offers an approach for this goal.
• the entire synthesis of homogenous LMWHs requires about 20 synthetic steps and is potentially amenable to large-scale manufacturing.
• We also demonstrate that the synthetic LMWHs have added Attorney Docket No.: 421/330 PCT pharmacological/clinical benefits.
• the chemoenzymatic synthesis of LMWHs provides avenues for the next generation of heparin therapeutics.
• the K d values are the average of two or three determinations.
• the K d value for fondaparinux was previously reported to be 5.9 ⁇ 1 .5 nM (Xu, Y. et al. Science 334, 498-501 , 201 1 ).
• IC 50 represents half maximal inhibitory concentration to inhibit the activity of factor FXa.
• the IC S0 value for fondaparinux was previously reported to be 4.5 ngml "1 (Xu, Y. et al. Science 334, 498-501 , 201 1 ).
• the lower IC 50 value observed for fondaparinux is ascribed to the different protocols used in the two experiments.
• heparin compounds including structurally homogeneous low molecular weight heparin compounds.
• the heparin compounds disclosed herein can be synthesized from a monosaccharide building block using an enzymatic approach as disclosed herein.
• the synthesis of heparin compounds according to the presently disclosed subject matter can include backbone elongation and saccharide modification.
• the presently disclosed subject matter provides a method of synthesizing a Attorney Docket No.: 421/330 PCT heparin compound, comprising providing a saccharide substrate, elongating the saccharide substrate to a saccharide of a desired or predetermined length, performing an epimerization reaction, and performing one or more sulfation reactions, whereby a heparin compound is synthesized.
• the presently disclosed subject matter provides a method of synthesizing a heparin compound, comprising providing a monosaccharide or disaccharide substrate, elongating the monosaccharide or disaccharide substrate to a tetrasaccharide, elongating the tetrasaccharide to a hexasaccharide, wherein the hexasaccharide comprises a N-sulfotransferase substrate residue, converting the N- sulfotransferase substrate residue on the hexasaccharide to a N-sulfo glucosamine (GlcNS) residue performing an epimerization reaction, and performing one or more sulfation reactions selected from the group consisting of a 2-O-sulfation reaction, a 6-O-sulfation reaction, a 3-0- sulfation reaction, and combinations thereof. These steps can be repeated to form a octas
• the elongation step can comprise employing a glycosyl transferase.
• the glycosyl transferase can be /V-acetyl glucosaminyl transferase of E. coli K5 (KfiA) or heparosan synthase-2 (pmHS2) from Pasteurella multocida.
• the elongation step can comprise employing one or more monosaccharides, which can comprise, for example, glucuronic acid (GlcUA), N-acetylated glucosamine (GlcNAc), and N-trifluoroacetyl glucosamine (GlcNTFA).
• the step of converting the N-sulfotransferase substrate residue on the hexasaccharide or heptasaccharide to a N-sulfo glucosamine (GlcNS) residue can comprise employing N-sulfotransferase (NST) and 3'-phosphoadenosine 5'-phosphosulfate (PAPS).
• NST N-sulfotransferase
• PAPS 3'-phosphoadenosine 5'-phosphosulfate
• the epimerization reaction can comprise employing C 5 -epimerase (C 5 -epi)
• the 2-O-sulfation reaction can comprise employing 2-O-sulfotransferase (2-OST)
• the 6-O-sulfation Attorney Docket No.: 421/330 PCT reaction can comprise employing 6-O-sulfotransferase (6-OST)
• the 3- O-sulfation reaction can comprise employing 3-O-sulfotransferase (3-OST).
• the elongation steps can comprise using a glycosyl transferase.
• the glycosyl transferase can be N- acetyl glucosaminyl transferase of E. coli K5 (KfiA).
• a sequence of enzymatic steps are provided for synthesizing a dodecasaccharide as disclosed herein in a highly efficient manner.
• heparin compound synthesis can be initiated from hexasaccharide and continue through several intermediates until the final heparin compound. These intermediates contain multiple - ldoA2S-GlcNS6S- repeating units, posing a synthetic challenge due to the substrate specificity of C 5 -epi.
• a designed sequence of enzymatic steps can be employed for high purity and yields.
• the conversion of GlcA to ldoA2S involves two steps: the C 5 -epi catalyzed epimerization of a GlcA to an IdoA; and 2-OST transferred a sulfo group to the IdoA residue.
• C 5 -epi catalyzes both the forward and reverse reactions, leading to the incomplete conversion of GlcA to ldoA2S 26 and a complex mixture of products. Such reactions can produce substantial amounts of byproducts thereby rendering the process highly inefficient.
• the conversion of GlcA to ldoA2S can be achieved by C 5 -epi and 2- OST.
• the formation of ldoA2S residues between the elongation steps between octasaccharide, decasaccharide, and dodecasaccharide removes its reactivity towards further C 5 -epi modification. Repeating these steps allows for the elongation up to a dodecasaccharide, and the synthesis of low-molecular weight heparin compounds disclosed herein, while maintaining high efficiency of synthesis.
• using the synthetic process as disclosed herein can heparin compounds, e.g.
• a dodecasaccharide as depicted hererin at efficiency rates of 50%, 60%, 70%, 80%, 90% or more.
• This is in contrast to alternative approaches where a heparin compound is elongated to a desired length, e.g. a Attorney Docket No.: 421/330 PCT dodecasaccharide, and then sulfated, which can yield significantly lower efficiencies, e.g. 2% to 5%.
• a heparin Compound of the presently disclosed subject matter can have an R group that is detectable, for example, has UV or visible light absorbance. Designing and synthesizing a heparin Compound that is detectable, for example has an R group that absorbs UV or visible light, can facilitate product purity detection and isolation. Compounding a heparin compound with a UV or visible light "tag" can facilitate the monitoring of the chemoenzymatic reactions and Compound synthesis. As would be appreciated by one of ordinary skill in the art, detecting a UV or visible light "tag" on a Compound during or after synthesis can be achieved using any number of available spectrophotometric devices. In some embodiments, the "tag" can comprise a para-nitrophenyl.
• the detectable R group can be a hydrophobic R group.
• a hydrophobic R group can allow the product to bind to a C18-column, which can allow for the purification of the synthesized heparins.
• an R group such as an R group that has UV or visible light absorbance, or a hydrophobic R group, can be removable when the synthesis of the heparin is completed.
• removing the R group once its utility is achieved can facilitate the final processing of the heparin Compound.
• the presently disclosed subject matter can utilize sulfotransferases, epimerases, glycosyltransferases, and/or heprosan synthases, among other enzymes as would be appreciated by one of Attorney Docket No.: 421/330 PCT ordinary skill in the art. These enzymes, and others that would be employed by one of ordinary skill in the art, are referred to herein as "the enzymes", “these enzymes", and/or "enzymes”.
• the chemoenzymatic syntheses can employ NST, C 5 -epimerase (C 5 -epi), 2- OST, 6-OST-1 , 6-OST-3, 3-OST-1 , 3-OST-5, /V-acetyl glucosaminyl transferase of E. coli K5 (KfiA), and/or heparosan synthase-2 (pmHS2).
• these and other enzymes employed in the chemoenzymatic syntheses can be expressed in E. coli and purified by appropriate affinity chromatography as described previously (Liu et al., 2010).
• one or more of the converting reactions can employ a base compound or solution as would be appreciated by one of ordinary skill in the art.
• bases can include for example lithium hydroxide or a mixture of triethylamine, CH 3 OH, and/or ⁇ 2 0.
• the chemoenzymatic syntheses employ sulfotransferases, such as O-sulfotransferases (OSTs), to sulfate polysaccharides.
• Sulfotransferases comprise a family of enzymes that catalyze the transfer of a sulfonate or sulfuryl group (S0 3 ) from a sulfo donor compound, i.e. an S0 3 -donor molecule, to an acceptor molecule.
• S0 3 sulfonate or sulfuryl group
• the sulfo donor compound or S0 3 -donor molecule can be the cofactor 3'-phosphoadenosine-5'-phosphosulfate (PAPS).
• sulfation refers to a transfer of a sulfonate or sulfuryl group from one molecule to another.
• Sulfotransferases mediate sulfation of different classes of substrates such as carbohydrates, oligosaccharides, peptides, proteins, flavonoids, and steroids for a variety of biological functions including signaling and modulation of receptor binding (Bowman et al., (1999) Chem. Biol. 6, R9- R22; and Falany (1997) FASEB J. 1 1 , 1 -2).
• Many new sulfotransferases have been identified and cloned (Aikawa et al., (1999) J. Biol. Chem. 274, 2690; Dooley (1998) Chemico-Biol. Interact. 109, 29; Fukuta et al. (1998) Biochim. Biophys. Act. 1399, 57; Habuchi et al., (1998) J. Biol. Chem. 273, Attorney Docket No.: 421/330 PCT
• O-sulfotransferase includes polypeptides and nucleic acids encoding HS O-sulfotransferases, such as for example "2-OST” (e.g. , mouse 2-OST, GENBANK® Accession No. AAC40135; "3-OST-1” (e.g. , human 3-OST-1 , GENBANK® Accession No. NP_005105; "3-OST-3” (e.g. , human 3-OST-3A, GENBANK ® Accession No. NP_006033 and human 3-OST-3B, GENBANK ® Accession No. NP_006032; and "6-OST" (e.g.
• an OST can comprise 3-OST-5.
• OST includes invertebrate and vertebrate homologs of the O-sulfotransferases (e.g., mammalian (such as human and mouse), insect, and avian homologs).
• O-sulfotransferases e.g., mammalian (such as human and mouse), insect, and avian homologs.
• OST includes invertebrate and vertebrate homologs of the O-sulfotransferases (e.g., mammalian (such as human and mouse), insect, and avian homologs).
• OST including particular OSTs (e.g. , 2-OST, 3-OST-1 , 3-OST-3, and 6- OST), includes all comparable OSTs known to the skilled artisan.
• the disclosed chemoenzymatic syntheses can employ C 5 -epimerase (C 5 -epi).
• C 5 -epi C 5 -epimerase
• the presently disclosed subject matter is not intended to be limited to the disclosed examples, but rather "epimerase", including C 5 -epi, includes all comparable epimerases known to the skilled artisan. Indeed, other epimerases, or compounds having epimerase activity, can be employed without departing from the scope of the presently disclosed subject matter.
• the disclosed chemoenzymatic syntheses can employ W-acetyl glucosaminyl transferase of E. coli K5 (KfiA).
• KfiA W-acetyl glucosaminyl transferase of E. coli K5
• the disclosed chemoenzymatic syntheses can employ heparosan synthase-2 (pmHS2).
• heparosan synthase-2 heparosan synthase-2
• the presently disclosed subject matter is not intended to be limited to the disclosed examples, but rather "heparosan synthase", including pmHS2, includes all comparable heparosan synthases known to the skilled artisan. Indeed, other heparosan synthases, or compounds having similar synthase activity, can be employed without departing from the scope of the presently disclosed subject matter.
• epimerase gene product refers to peptides having amino acid sequences which are substantially identical to native amino acid sequences from the organism of interest and which are biologically active in that they comprise all or a part of the amino acid sequence of an O-sulfotransferase, epimerase, glucosaminyl transferase, or heprosan synthase, or cross-react with antibodies raised against such enzymes, or retain all or some of the biological activity of the native amino acid sequence or protein of such enzymes.
• biological activity can include immunogenicity.
• OST gene product "OST protein”, “OST polypeptide”, “epimerase gene product”, “epimerase protein”, “epimerase polypeptide”, “glucosaminyl transferase gene product”, “glucosaminyl transferase protein”, “glucosaminyl transferase polypeptide”, “heparosan synthase gene product”, “heparosan synthase protein”, and “heparosan synthase polypeptide” also include analogs of the enzymes.
• analog is intended that a DNA or peptide sequence can contain alterations relative to the sequences disclosed herein, yet retain all or some of the biological activity of those sequences.
• Analogs can be derived from genomic nucleotide sequences as are disclosed herein or from other organisms, or can be created synthetically. Those skilled in the art will appreciate that other analogs, as yet undisclosed or undiscovered, can be used to design and/or Compound analogs of these enzymes.
• OST gene product "OST protein”, “OST polypeptide”, “epimerase gene product”, “epimerase protein”, “epimerase polypeptide”, "glucosaminyl transferase gene product”, “glucosaminyl transferase protein”, “glucosaminyl transferase polypeptide”, “heparosan synthase gene product”, “heparosan synthase protein”, and “heparosan synthase polypeptide” to comprise all or substantially all of the amino acid sequence of a native enzyme gene product.
• OST gene product shorter or longer sequences are anticipated to be of use in the presently disclosed subject matter, shorter sequences are herein referred to as “segments.”
• OST gene product "OST protein”, “OST polypeptide”, “epimerase gene product”, “epimerase protein”, “epimerase polypeptide”, “glucosaminyl transferase gene product”, “glucosaminyl transferase protein”, “glucosaminyl transferase polypeptide”, “heparosan synthase gene product”, “heparosan synthase protein”, and “heparosan synthase polypeptide” also include fusion or recombinant polypeptides and proteins comprising sequences of the enzyme protein. Methods of preparing such proteins are known in the art.
• OST gene product refers to any DNA sequence that is substantially identical to a polynucleotide sequence encoding an enzyme isoform gene product, protein or polypeptide as defined above, and can also comprise any combination of associated control sequences.
• DNA segment refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species.
• a DNA segment encoding a polypeptide of one of these enzymes refers to a DNA segment that contains coding sequences for one of these enzymes, yet is isolated away from, or purified free from, total genomic DNA of a source species, such as for example Homo sapiens, included within the term "DNA segment” are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phages, viruses, and the like.
• substantially identical when used to define either a gene product of one of these enzymes or amino acid sequence, or an enzyme gene or nucleic acid sequence, means that a particular sequence varies from the sequence of a natural enzyme by one or more deletions, substitutions, or additions, the net effect of which is to retain at least some of biological activity of the natural gene, gene product, or sequence.
• sequences include "mutant" sequences, or sequences in which the biological activity is altered to some degree but retains at least some of the original biological activity.
• DNA analog sequences are "substantially identical" to specific DNA sequences disclosed herein if: (a) the DNA analog sequence is derived from coding regions of the natural enzyme gene; or (b) the DNA analog sequence is capable of hybridization of DNA sequences of (a) under stringent conditions and which encode biologically active enzyme gene products; or (c) the DNA sequences are degenerate as a result of alternative genetic code to the DNA analog sequences defined in (a) and/or (b).
• Substantially identical analog proteins will be equal to or greater than about 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding sequence of the native protein.
• nucleic acid sequences having lesser Attorney Docket No.: 421/330 PCT degrees of identity but comparable biological activity are considered to be equivalents.
• all subject nucleic acid sequences capable of encoding substantially similar amino acid sequences are considered to be substantially similar to a reference nucleic acid sequence, regardless of differences in codon sequences or substitution of equivalent amino acids to create biologically functional equivalents.
• Sequence identity or percent similarity of a DNA or peptide sequence can be determined, for example, by comparing sequence information using the GAP computer program, available from the University of Wisconsin Geneticist Computer Group.
• the GAP program utilizes the alignment method of Needleman et al. (1970) J Moi Biol 48:443, as revised by Smith et al. (1981 ) Adv Appl Math 2:482. Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) that are similar, divided by the total number of symbols in the shorter of the two sequences.
• the preferred parameters for the GAP program are the default parameters, which do not impose a penalty for end gaps. See Schwartz et al. (1979) Nuc Acids Res 6(2):745-755; Gribskov et al. (1986) Nuc Acids Res 14(1 ): 327-334.
• the present subject matter concerns the use of the enzyme genes and gene products that include within their respective sequences a sequence that is essentially that of an enzyme gene, or the corresponding protein.
• a sequence essentially as that of an OST gene means that the sequence is substantially identical or substantially similar to a portion of an OST gene and contains a minority of bases or amino acids (whether DNA or protein) which are not identical to those of an OST protein or an OST gene, or which are not a biologically functional equivalent.
• the terms "a sequence essentially as that of an epimerase gene”, “a sequence essentially as that of a glycosyl transferase gene”, and "a sequence essentially as that of a heparosan synthase gene” have similar meanings.
• nucleotide sequences are "essentially the same” where they have between about 75% and about 85% or more preferably, between about 86% and about 90%, or Attorney Docket No.: 421/330 PCT more preferably greater than 90%, or more preferably between about 91% and about 95%, or even more preferably, between about 96% and about 99%; of nucleic acid residues which are identical to the nucleotide sequence of the enzyme gene.
• peptide sequences which have about 60%, 70%, 80%, or 90%, or preferably from 90-95%, or more preferably greater than 96%, or more preferably 95-98%, or most preferably 96%, 97%, 98%, or 99% amino acids which are identical or functionally equivalent or biologically functionally equivalent to the amino acids of the enzyme polypeptide will be sequences which are "essentially the same".
• amino acid and nucleic acid sequences can include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' nucleic acid sequences, and yet still be encompassed by the enzymes disclosed herein, so long as the sequence retains biological protein activity where protein expression is concerned.
• the addition of terminal sequences particularly applies to nucleic acid sequences which can, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region or can include various internal sequences, i.e., introns, which are known to occur within genes.
• nucleotide segments that are complementary to the sequences of the present subject matter in one embodiment, segments that are fully complementary, i.e. complementary for their entire length.
• Nucleic acid sequences that are "complementary” are those, which are base-paired according to the standard Watson-Crick complementarity rules.
• complementary sequences means nucleic acid sequences which are substantially complementary, as can be assessed by the same nucleotide comparison set forth above, or is defined as being capable of hybridizing to the nucleic acid segment in question under relatively stringent conditions such as those described herein.
• a particular example of a complementary nucleic acid segment is an antisense oligonucleotide.
• Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of Attorney Docket No.: 421/330 PCT nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art.
• Stringent temperature conditions will generally include temperatures in excess of about 30°C, typically in excess of about 37°C, and preferably in excess of about 45°C.
• Stringent salt conditions will ordinarily be less than about 1 ,000 mM, typically less than about 500 mM, and preferably less than about 200 mM.
• preferred conditions are salt concentration of about 200 mM and temperature of about 45°C.
• One example of such stringent conditions is hybridization at 4XSSC, at 65°C, followed by a washing in 0.1XSSC at 65°C for one hour.
• Another exemplary stringent hybridization scheme uses 50% formamide, 4XSSC at 42°C.
• Another example of "stringent conditions” refers to conditions of high stringency, for example 6XSSC, 0.2% polyvinylpyrrolidone, 0.2% Ficoll, 0.2% bovine serum albumin, 0.1 % sodium dodecyl sulfate, 100 ⁇ g/ml salmon sperm DNA and 15% formamide at 68°C.
• Nucleic acids having sequence similarity are detected by hybridization under low stringency conditions, for example, at 50°C and 10XSSC (0.9 M NaCI/0.09 M sodium citrate) and remain bound when subjected to washing at 55°C in 1XSSC. Sequence identity can be determined by hybridization under stringent conditions, for example, at 50°C or higher and 0.1XSSC (9 mM NaCI/0.9 mM sodium citrate).
• Nucleic acids that are substantially identical to the provided enzymes bind to the disclosed OSTs under stringent hybridization conditions.
• probes particularly labeled probes of DNA sequences
• the source of homologous genes can be any species, e.g., primate species, particularly human; rodents, such as rats and mice; canines; felines; bovines; ovines; equines; insects; yeasts; nematodes; etc.
• homologs have substantial sequence similarity, i.e. at least 75% sequence identity between nucleotide sequences. Sequence similarity is calculated based on a reference sequence, which can be a subset of a larger sequence, such as a conserved motif, coding region, flanking region, etc. A reference sequence will usually be at least about 18 nucleotides long, more usually at least about 30 nucleotides long, and can extend to the complete sequence that is being compared. Algorithms for sequence analysis are known in the art, such as BLAST, described in Altschul et al. (1990) J Mol Biol 215:403-410. The sequences provided herein are essential for recognizing enzymes related and homologous proteins in database searches.
• identity is just that, i.e. the same amino acid at the same relative position in a given family member of a gene family.
• Homology and similarity are generally viewed as broader terms.
• biochemically similar amino acids for example leucine and isoleucine or glutamate/aspartate, can be present at the same position - these are not identical per se, but are biochemically "similar". As disclosed herein, these are referred to as conservative differences or conservative substitutions. This differs from a conservative mutation at the DNA level, which changes the nucleotide sequence without making a change in the encoded amino acid, e.g., TCC to TCA, both of which encode serine.
• percent identities can be generated by alignments with the program GENEWORKSTM (Oxford Molecular, Inc. of Campbell, California, U.S.A.) and/or the BLAST program at the NCBI website. Another commonly used alignment program is entitled CLUSTAL W and is described in Thompson et al. (1994) Nucleic Acids Res 22(22):4673-4680, among other places.
• gene is used for simplicity to refer to a functional protein, polypeptide or peptide encoding unit. As will be understood by those in the Attorney Docket No.: 421/330 PCT art, this functional term includes both genomic sequences and cDNA sequences.
• Biologically functional equivalent peptides are peptides in which certain, but not most or all, of the amino acids can be substituted. Thus, applicants contemplate substitution of codons that encode biologically equivalent amino acids as described herein into the sequences of the disclosed enzymes, but which are not set forth herein in their entirety for convenience.
• functionally equivalent proteins or peptides can be created via the application of recombinant DNA technology, in which changes in the protein structure can be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man can be introduced through the application of site-directed mutagenesis techniques, e.g., to introduce improvements to the antigenicity of the protein or to test enzyme mutants in order to examine enzyme activity, or other activity at the molecular level.
• Site-directed mutagenesis techniques e.g., to introduce improvements to the antigenicity of the protein or to test enzyme mutants in order to examine enzyme activity, or other activity at the molecular level.
• Amino acid substitutions such as those which might be employed in modifying enzyme proteins and peptides described herein, are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
• An analysis of the size, shape and type of the amino acid side-chain substituents reveals that arginine, lysine and histidine are all positively charged residues; that alanine, glycine and serine are all of similar size; and that phenylalanine, tryptophan and tyrosine all have a generally similar shape.
• arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine are defined herein as biologically functional equivalents.
• Other biologically functionally equivalent changes will be appreciated by those of skill in the art.
• hydropathic index of amino acids can be considered.
• Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+ 4.5); valine (+ 4.2); leucine (+ 3.8); phenylalanine (+ 2.8); cysteine (+ 2.5); methionine (+ 1.9); alanine (+ 1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (- 0.9); tyrosine (-1.3); proline (-1 .6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
• hydrophilicity values have been assigned to amino acid residues: arginine (+ 3.0); lysine (+ 3.0); aspartate (+ 3.0 ⁇ 1 ); glutamate (+ 3.0 ⁇ 1); serine (+ 0.3); asparagine (+ 0.2); glutamine (+ 0.2); glycine (0); threonine (-0.4); proline (- 0.5 ⁇ 1 ); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (- 2.5); tryptophan (-3.4).
• the presently disclosed subject matter is not limited to the particular nucleic acid and amino acid sequences of the enzymes disclosed herein, including sulfotransferases, epimerases, glycosyltransferases, and/or heprosan synthases.
• Recombinant vectors and isolated DNA segments can therefore variously include the enzyme polypeptide-encoding region itself, include coding regions bearing selected alterations or modifications in the basic coding region, or include larger polypeptides which nevertheless comprise the enzyme polypeptide-encoding Attorney Docket No.: 421/330 PCT regions or can encode biologically functional equivalent proteins or peptides which have variant amino acid sequences.
• Biological activity of an enzyme can be determined using techniques generally known in the art, for example as disclosed herein in the Examples.
• nucleic acid segments of the present subject matter can be combined with other DNA sequences, such as promoters, enhancers, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length can vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length can be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.
• nucleic acid fragments can be prepared which include a short stretch complementary to a nucleic acid sequence set forth in any of the enzymes disclosed herein, such as about 10 nucleotides, and which are up to 10,000 or 5,000 base pairs in length, with segments of 3,000 being preferred in certain cases. DNA segments with total lengths of about 4,000, 3,000, 2,000, 1 ,000, 500, 200, 100, and about 50 base pairs in length are also contemplated to be useful.
• Recombinant vectors form further aspects of the present subject matter.
• Particularly useful vectors are those in which the coding portion of the DNA segment is positioned under the control of a promoter.
• the promoter can be that naturally associated with an enzyme gene, as can be obtained by isolating the 5' non-coding sequences located upstream of the coding segment or exon, for example, using recombinant cloning and/or polymerase chain reaction (PCR) technology and/or other methods known in the art, in conjunction with the compositions disclosed herein.
• PCR polymerase chain reaction
• a recombinant or heterologous promoter is a promoter that is not normally associated with an enzyme gene in its natural environment.
• Such promoters can include promoters isolated from bacterial, viral, eukaryotic, or mammalian cells.
• promoter that effectively directs the expression of the DNA segment in the cell type chosen for expression.
• the use of promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology (See, e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).
• the promoters employed can be constitutive or inducible and can be used under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins or peptides.
• an enzyme of the presently disclosed subject matter e.g. sulfotransferases, epimerases, glycosyltransferases, and/or heprosan synthases
• a substrate is agarose.
• the agarose substrate is an agarose bead and the enzymes are linked to the beads.
• the presently disclosed methods for synthesizing heparin compounds can comprise the use of a PAPS regenerating enzyme and a sulfo donor compound.
• the PAPS regenerating enzyme catalyzes regeneration of the PAPS from the PAP utilizing the sulfo donor compound as a substrate. See, e.g., U.S. Patent No. 6,255,088; and Burkart et al., (2000) J. Org. Chem. 65, 5565-5574, both of which are herein incorporated by reference in their entirety.
• the PAPS regeneration system provides the dual advantages of reducing the inhibitory effects of PAP accumulation on sulfotransferase activity while also constantly "recharging" the reaction mixture with the Attorney Docket No.: 421/330 PCT primary sulfo donor molecule, PAPS.
• the PAPS regenerating enzyme is an estrogen sulfotransferase.
• an aspect of the presently disclosed subject matter is directed to a sulfo donor compound (e.g., PAPS) regeneration process coupled with sulfation of a polysaccharide substrate.
• the process can be of a type wherein the sulfation of a polysaccharide substrate is catalyzed by a sulfotransferase, such as one or more OSTs, with a conversion of 3'- phosphoadenosine-5'-phosphosulfate (PAPS) to adenosine 3', 5'- diphosphate (PAP).
• PAPS 3'- phosphoadenosine-5'-phosphosulfate
• PAP adenosine 3', 5'- diphosphate
• the sulfation process can be coupled with an enzymatic regeneration of the PAPS from the PAP.
• the enzymatic regeneration can employ an arylsulfotransferase as the catalyst and an aryl sulfate as a substrate.
• the enzymatic regeneration can comprise a human or mouse estrogen sulfotransferase (EST).
• EST estrogen sulfotransferase
• preferred carbohydrate substrates can include GAGs, such as for example heparan sulfates, including heparin.
• Coupling the sulfotransferase catalyzed sulfation reaction with a PAPS regeneration system can provide a further advantage of generating PAPS utilized in the reaction directly from PAP. That is, the reaction mixture can be formulated to combine PAP with a PAPS regenerating enzyme prior to or simultaneously with addition of a sulfotransferase to the reaction mixture. The PAPS regenerating enzyme can then generate PAPS from the PAP for use by the sulfotransferase, thereby alleviating the need of supplying any of the more expensive and unstable PAPS to the reaction mixture.
• a method of sulfating a polysaccharide comprising providing a reaction mixture comprising therein adenosine 3',5'-diphosphate (PAP), a PAPS regenerating enzyme and a sulfo donor compound (other than PAPS, e.g., PNPS) and incubating the reaction mixture for a time period sufficient to catalyze the production of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) from the PAP by the PAPS regenerating enzyme utilizing the sulfo donor compound as a Attorney Docket No.: 421/330 PCT substrate.
• PAP adenosine 3',5'-diphosphate
• PAPS regenerating enzyme e.g., PNPS
• the method further comprises incubating a polysaccharide substrate and at least one O-sulfotransferase (OST) enzyme with the reaction mixture, wherein production of a sulfated polysaccharide from the polysaccharide substrate is catalyzed by the OST enzyme with a conversion of the PAPS to PAP and wherein the PAPS regenerating enzyme then catalyzes regeneration of the PAPS from the PAP, again utilizing the sulfo donor compound as a substrate.
• OST O-sulfotransferase
• heparin compound is one of the following:
• the heparin compound used in such treatments is a heparin compound comprising a synthetic, low-molecular weight heparin compound with reversible anticoagulant activity, wherein the anticoagulant activity of the heparin compound is reversible by protamine, wherein the anticoagulant activity is reversed by about 50% or more in the presence of 1 ug/ml of protamine, as disclosed herein.
• heparin compound can comprise for example a heparin compound comprising a structure selected from:
• R 2 is present or absent, wherein when R 2 is present it comprises a detectable tag, optionally para-nitrophenyl.
• the antidote to reverse the anticoagulant activity of the heparin compound is protamine.
• a subject to be treated in any of the methods of treatment disclosed herein can be a subject suffering from venous thromboembolism.
• a subject to be treated can be a subject that is renal-impaired.
• the subject is a human subject.
• HS biosynthetic enzymes A total of nine enzymes were used for the synthesis, including NST, C 5 -epi, 2-OST, 6-OST-1 , 6-OST- 3, 3-OST-1 , 3-OST-5, KfiA, and pmHS2. All enzymes were expressed in E. coli and purified by appropriate affinity chromatography as described Attorney Docket No.: 421/330 PCT previously (Liu, R. et al., J Biol Chem 285, 34240-34249, 2010; Xu, D., Moon, A., Song, D., Pedersen, L.C. & Liu, J. Nat Chem Biol 4, 200-202, 2008).
• UDP-GlcNTFA The preparation of UDP-GlcNTFA was started from glucosamine (Sigma-Aldrich), which was first converted to GlcNTFA by reacting with S- ethyl trifluorothioacetate (Sigma-Aldrich) followed the protocol described previously (Liu, R. et al., J Biol Chem 285, 34240-34249, 2010). The resultant GlcNTFA was converted to GlcNTFA-1 -phosphate using N- acetylhexosamine 1 -kinase (Zhao, G., Guan, W., Cai, L. & Wang, P.G., Nat. Protoc. 5, 636-646, 2010).
• the plasmid expressing /V-acetylhexoamine 1- kinase was a generous gift from Prof. Peng Wang (Georgia State University), and the expression of the enzyme was carried out in E. coli as reported (Zhao, G., Guan, W., Cai, L. & Wang, P.G., Nat. Protoc. 5, 636- 646, 2010).
• the UDP-GlcNTFA synthesis was completed by transforming GlcNTFA-1 -phosphate using glucosamine- -phosphate acetyltransferase/ - acetylglucosamine-1 -phosphate uridyltransferase (GlmU) as described (Liu, R. et al., J Biol Chem 285, 34240-34249, 2010).
• the resultant UDP- GlcNTFA was ready for the elongation reaction involved in using KfiA as described below.
• GlcA-pnp 1.2 mM was incubated with KfiA (20 ⁇ gm ⁇ 1 ) in a buffer containing Tris (25 mM, pH 7.5), MnCI 2 (15 mM) and UDP-GlcNTFA (1.5 mM), at room temperature Attorney Docket No.: 421/330 PCT overnight.
• disaccharide substrate GlcNTFA- GlcA-pnp (1.2 mM)
• pmHS2 20 ⁇ gm ⁇ 1
• a buffer containing Tris 25 mM, pH 7.5
• MnCI 2 15 mM
• UDP-GIcA 1.5 mM
• the product after each elongation step was purified using a C 8 column (0.75 * 20 cm; Biotage), which was eluted with a linear gradient of 0-100% acetonitrile in H 2 0 and 0.1 % TFA in 60 min at a flow rate of 2 mlmin "1 .
• the pentasaccharide backbone was further subjected to detrifluoroacetylation, followed by /V-sulfation with /V-sulfotransferase (NST).
• the pentasaccharide backbones (280 mg) were dried and resuspended in 30 ml of 0.1 M LiOH on ice for 2h.
• the degree of completion of the detrifluoroacetylation reaction was monitored by PAMN-HPLC analysis and ESI-MS.
• the pH of the reaction mixture decreased to 7.0 and incubated of MES 50 mM pH 7.0, N- sulfotransferase 10 ⁇ 1 and 0.5 mM PAPS in 780 ml at 37°C overnight, where the amount of PAPS was about 1.5-times molar amount of NH 2 groups in the pentasaccharide.
• /V-sulfated product was purified by Q column, and the purified product with the structure of GlcA-GlcNS-GlcA- GlcNS-GicA-PNP was dialyzed.
• the conversion of compound 6 to 1 , compound 7 to 2 and compound 8 to 3 followed same reactions, including detrifluoroacetylation/A/-sulfation, 6-O-sulfation and 3-O-sulfation by 3-OST-1 enzyme.
• the conversion of compound 9 to 4 involved 2 steps, including 6-O-sulfation and 3-OST-1 sulfation.
• the conversion of compound 4 to 5 was completed by 3-O- sulfation using 3-OST-5 enzyme.
• the 6-O-sulfation reaction contained 50 mM MES, pH 7.0, 1.5 mM PAPS and 0.3 mM substrate, 0.2 mgmr 1 6-OST-1 and 0.2 mgmr 1 6-OST-3 in 20 to 40 ml overnight at 37°C.
• LMWH Compounds 1-5 was carried out by a Q-Sepharose column, which was eluted with a linear gradient of 30-100% 2M NaCI in 20mM NaOAc at pH 5.0 in 2 h at a flow rate of 2 mlmin "1 .
• UFH from US Pharmacopea
• LOVENOX ® from local pharmacy
• LMWH Compounds 1 to 5 was dissolved in PBS at various concentrations (3 to 600 ⁇ gm ⁇ 1 ).
• the reaction mixture which consisted of 20 ⁇ of human plasma (Sigma-Aldrich, St. Louis, Missouri, United States of America) and 8 ⁇ of the solution containing the sample, was incubated at room temperature for 5 min. Factor Xa (100 ⁇ ) was then added. After incubating at room temperature for 4 min, 30 ⁇ of S-2765 substrate was added. The absorbance of the reaction mixture was measured at 405 nm continuously for 5 min. The absorbance values were plotted against the reaction time. The initial reaction rates as a function of concentration were used to calculate the IC 50 values.
• Reaction consisted of MES 50 mM pH 7.0, 10 mM MnCI 2 , 5 mM MgCI 2 , 3- OST-5 O. l mgmr 1 and 0.5 nmol [ 35 S] PAPS (specific activity of [ 35 S]PAPS was 2.2 10 4 cpm/pmol), oligosaccharide 5 ⁇ g in total 500 ⁇ at 37°C overnight.
• the 35 S-labeled compound 5 was purified by a DEAE-HPLC column.
• the dissociation constant (K d ) of each sample and AT was determined using affinity co-electrophoresis (Lee, M.K. & Lander, A. D., Proc. Natl. Acad. Sci. USA 88, 2768-2772, 1991 ). Approximately 1500-2500 cpm of 35 S-labeled LMWH Compounds 1 to 5 was loaded per lane with zones of AT at concentrations 0, 8, 16, 32, 60, 120, 250, 500 and 1000 nM. The gel was performed at 300 mA for 2 h, dried and analyzed on a Phospholmager (Amersham Biosciences, Wauwatosa, Wisconsin, United States of America Storm 860).
• the retardation coefficient was then plotted against the retardation coefficient divided by its respective concentration of AT. The slope of the line represents -1/K d .
• Protamine (15 mgkg “1 ) or phosphate buffer saline (PBS) was administered intravenously via retro-orbital plexus injection, and 5 min later blood samples were drawn from the inferior vena cava into syringes preloaded with 3.2% solution of sodium citrate (final volume ratio 9:1 ). To obtain mouse plasma, blood samples were centrifuged at 4,000 g for 15 min at 4°C. Mouse plasma was then used to determine anti-Xa activity. Ex vivo analysis of anti-Xa activity was done similar to the in vitro study described above.
• PBS phosphate buffer saline
• LC binary pump After injection of 8.0 pL oligosaccharide mixture (1.0 pgpL "1 ) through an Agilent 1200 autosampler, HPLC binary pump was used to deliver the gradient from 10% A to 35% A over 40 min at a flow rate of 150 ⁇ "1 .
• the LC column was directly connected online to the standard electrospray ionization source of LTQ-Orbitrap XL Fourier transform (FT) mass spectrometer (MS) (Thermo Fisher Scientific, San-Jose, California, United States of America).
• the source parameters for FT-MS detection were optimized using Arixtra ® (purchased at a pharmacy) to minimize the insource fragmentation and sulfate loss and maximize the signal/ noise in the negative-ion mode.
• LMWH LMWH Compounds and intermediates were analyzed by 1 D H-NMR and 2D NMR ( H- 1 H COSY, 1 H- 13 C HMQC). All NMR experiments were Attorney Docket No.: 421/330 PCT performed at 298 K on Bruker Avance II 800 MHz spectrometer with Topsin 2.1 software. Samples (3.0 to 6.0 mg) were each dissolved in 0.5 ml D 2 0 (99.996%, Sigma-Aldrich) and lyophilized three times to remove the exchangeable protons. The samples were re-dissolved in 0.4 ml D 2 0 and transferred to NMR microtubes (OD 5 mm, Norrell NMR tubes from Sigma Aldrich).
• the LMWH Compounds and intermediates were also analyzed by 1 D 1 H-NMR, 1 D 13 C-NMR and 2D NMR ( 1 H- 1 H COSY, 1 H- 13 C HSQC) on Varian Inova 500 MHz spectrometer with VnmrJ 2.2D software.
• Samples (2.0 to 5.0 mg) were dissolved in 0.5 ml D 2 0 (99.994%, Sigma-Aldrich) and lyophilized three times to remove the exchangeable protons. The samples were re-dissolved in 0.5 ml D 2 0 and transferred to NMR microtubes (OD 5 mm, Norrell).
• 1 D 1 H-NMR experiments were performed with 256 scans and an acquisition time of 768 msec.
• mice were anesthetized in a small 34°C chamber with a flow of oxygen containing 4% isoflurane. Once the mice were unconscious, they were individually placed on a heated pad with a nose cone fitted over their snout with a constant flow of oxygen containing 2% isoflurane.
• a specific amount of 35 S-labeled LMWH Compound, LMWH, or UFH was injected via the lateral tail vein using a 27G1/2 needle mounted on a 1 ml syringe.
• the labeled material was allowed to circulate in the blood for 12 min while the mouse lay unconscious.
• the abdominal cavity was exposed by incision and the liver was collected, washed, and weighed.
• Approximately 100 mg from each of the lobes was homogenized in 0.75 ml 1% NP-40 and then centrifuged at 12,000 x g for 2 min to pellet insoluble material.
• the supernatant was then added to 4 ml scintillation fluid, mixed for 30 min by Attorney Docket No. : 421/330 PCT rocking and then radioactivity was assessed by a Beckman Coulter LS6500 scintillation counter.
• the data is presented as the percentage of CPM in total liver divided by total CPM injected ⁇ standard error of 3-5 mice per ligand.

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