US 20090035787A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0035787 A1 Liu et al. (43) Pub. Date: Feb. 5, 2009

(54) ENZYMATIC SYNTHESIS OF SULFATED Publication Classification POLYSACCHARDES WITHOUT DURONIC ACID RESIDUES (51) Int. Cl. GOIN 33/53 (2006.01) (75) Inventors: Jian Liu, Chapel Hill, NC (US); COSB 37/10 (2006.01) Courtney Jones, Durham, NC CI2P 19/26 (2006.01) (US); Jinghua Chen, Wuhan (CN); CI2O 1/02 (2006.01) Yongmei Xu, Chapel Hill, NC (US) C40B 50/06 (2006.01) Correspondence Address: (52) U.S. Cl...... 435/7.1:536/21:536/122; 435/84; JENKINS, WILSON, TAYLOR & HUNT, P. A. 506/26: 435/15; 435/29 Suite 1200 UNIVERSITY TOWER, 3100 TOWER BLVD., (57) ABSTRACT DURHAM, NC 27707 (US) Iduronic acid (Idol JA)-free heparan sulfate (HS)-like com (73) Assignee: The University of North Carolina pounds are provided. Also provided are methods of synthe at Chapel Hill sizing Idol JA-free HS-like compounds. The methods can include providing at least one O-sulfotransferase (OST) (21) Appl. No.: 12/178,434 enzyme and a reaction mixture comprising 3'-phosphoad enosine 5'-phosphosulfate (PAPS); and incubating a polysac (22) Filed: Jul. 23, 2008 charide substrate with the at least one OST and the reaction mixture, whereby the HS-like compounds are synthesized. Related U.S. Application Data Also disclosed are methods of synthesizing a library of HS (60) Provisional application No. 60/961,716, filed on Jul. like compounds and methods of determining the mechanism 23, 2007. of activity of HS-like compounds.

6 HOHC O HCHC HOHC O O Hoog "\,'\ H00 \O \ E \\?ion).0 (09 HO A. YO O ty NDST O HO pi HO OH NHSOH Ho M"HO Ngcl. HO OH (IDURONICACID, Idol)A) (GLCURONICACID, GlcA) (ACETYLCLUCOSAMINE,O GlcNAc) (N-SULFOGLUCOSAMINE, GLcNS) po H03SOHC 0 H03SCHy( 0 (Osc) HOHy( 0 H00C O \ O \ O Ho Y \O O to 1 \, , 6-0ST NHSOH HO-My NHSOHA3' oso NHSOH3 3-0ST HO-1 bso NHSOH3 (6-0, 3-0, YSULFO (6-O-SULFOGLUCOSAMINE, (2-0SULFOIDURONICACID, IdoA2S) GLUCOSAMINE, GlcNS3S6S)

NDS. N-DEACEYLASE/N-SULFOTRANSFERASE 3-0ST. 3-O-SUFOTRANSFERASE 6-0ST. -O-SUFOTRANSFERASE 2-0ST 2-O-SUFORANSFERASE Epi- EPIMERASE US 2009/0035787 A1

HSVHWICH-Id] Patent Application Publication Feb. 5, 2009 Sheet 2 of 8 US 2009/0035787 A1

Patent Application Publication Feb. 5, 2009 Sheet 3 of 8 US 2009/0035787 A1

Figure 2.

A. e--5 - compound 8

4. SO 80 1OO

- Compound 2

40 6O BO 1OO Retention Time (min) Patent Application Publication Feb. 5, 2009 Sheet 4 of 8 US 2009/0035787 A1

Figure 3.

2O Heparir ...... Compound 8 Compound 2 Compound 1 Compound 7 SO

A.

O

0.0 O5 1.O 15 2.O. Concentration of polysaccharide (g/ml) Patent Application Publication Feb. 5, 2009 Sheet 5 of 8 US 2009/0035787 A1

Figure 4.

8000 0.35

-- *s (cpm) O.30 -- O.D. 232 6OOO m 0.25

area 0.20 E E CN SlC 0.15 ()N VO ? g 0.10 C O.05

O.OO

2O 30 40 50 60 70 Fraction Number Patent Application Publication Feb. 5, 2009 Sheet 6 of 8 US 2009/0035787 A1

Figure 5.

s 1OO without epimerase and 2-0ST t E: With epimerase and 2-0ST D 3. 80 s 3 3 60 o 40 is2 20 e s O D- Tetra. Hexa- Octa- Deca- -Dodeca- Full length Size of Substrates Patent Application Publication Feb. 5, 2009 Sheet 7 of 8 US 2009/0035787 A1

Figure 6.

HOHC HOHC HOOC OOC O N-sulfotransferase Yo o / Yo o 1 HO NHSOH O Deacetylated Heparosan PAPS EAF N-sulfo Heparosan (8 mg) (6 mg)

: HQ HOS PAPS O and6-OST-1 6-OST-3 PAPS D PAF (PNP)NO2 HOSOHC HOSOHC O HOOC 3 O O HOOC HOSC O O. o1 os- no OHO b1 Yo NHSOH 7 S. HO NHSOH HO OH PA PAS OH N-sulfo Heparosan 6-O-sulfated Recomparin (2 mg) PAPS Regeneration (4 mg) Patent Application Publication Feb. 5, 2009 Sheet 8 of 8 US 2009/0035787 A1

O OO Hepari (rg/ml)

B 100

2.

1. 100 Recomparin (g/ml) US 2009/0035787 A1 Feb. 5, 2009

ENZYMATC SYNTHESIS OF SULFATED POLYSACCHARDES WITHOUT DURONIC ACID RESIDUES L2 COOH L CHOR3 L2 RELATED APPLICATIONS 1N O 1N O 1N H H 0001. This application claims the benefit of U.S. Provi Y KOR. H. Y KOR, H, Y sional Patent Application Ser. No. 60/961,716, filed Jul. 23, H H H H 2007; the disclosure of which is incorporated herein by ref erence in its entirety. H OR GOVERNMENT INTEREST whereinX is NH or O, R is H, SOH or CH, R is SOH, R 0002 The presently disclosed subject matter was made is Hor SOH, R is H, SOH, CH, or CHCHR is H, SOH, with U.S. Government support under Grant No. AI50050 CH or CHCH R is COOH, COOCH, COOCHs. awarded by National Institutes of Health. Thus, the U.S. CHOSOH, or CHSOH, L is an alpha or beta linkage, L. Government has certain rights in the presently disclosed Sub is an alpha or beta linkage, Y is O or N, and n is 24. In some ject matter. embodiments, the Idol JA-free HS-like compound is provided wherein X is NH, R is SOH, R is SOH, R is SOH, R is TECHNICAL FIELD Hor SOH. Rs is H. L. is an alpha or a beta linkage, L is an 0003. The presently disclosed subject matter relates gen alpha or a beta linkage, and n is 24. In some embodiments, erally to methods of Sulfating polysaccharides. In some the Idol JA-free HS-like compound is provided wherein L is embodiments, the presently disclosed subject matter relates an alpha linkage and L is a beta linkage. to methods of Sulfating polysaccharides using O-sulfotrans 0007. In some embodiments, the Idol JA-free HS-like ferases. compound has a binding affinity to antithrombin ranging from about 20 to about 60 nM. In some embodiments, the BACKGROUND Idol JA-free HS-like compound has an anti-Xa activity rang ing from about 10 to about 500 ng/ml. In some embodiments, 0004 Heparan sulfate (HS) is a ubiquitous component of the Idol JA-free HS-like compound has an anti-IIa activity the cell surface and extracellular matrix. It regulates a wide ranging from about 5 to about 200 ng/ml. In some embodi range of physiologic and pathophysiologic functions, includ ments, the Idol JA-free HS-like compound has substantially ing embryonic development and blood coagulation, and can reduced cell proliferation activity as compared to a HS-like facilitate viral infection (Esko and Selleck (2002) Annu. Rev. compound with iduronic acid residues. Biochem. 71, 435-471; Liu and Thorp (2002) Med. Res. Rev. 0008. In some embodiments, the Idol JA-free HS-like 22, 1-25). HS exerts its biological effects by interacting with compound is provided wherein R is selected from the group the specific proteins involved in a given process (Capila and consisting of CH, CHCH and SOH and Rs is selected Lindhardt (2002) Angew. Chem. Int Ed 41,390-412). HS is a from the group consisting of CH, CHCH and SOH. In highly charged polysaccharide comprising 1->4-linked glu some embodiments, the Idol JA-free HS-like compound is cosamine and glucuronic/iduronic acid units that contain both provided wherein X is O and R is SOH. In some embodi N- and O-sulfo groups. Unique Saccharide sequences within ments, the Idol JA-free HS-like compound is provided HS determine the specificity of the binding of HS to its target wherein L is an alpha linkage and L is an alpha linkage. In proteins (Linhardt (2003) J. Med. Chem. 46, 2551-2564). some embodiments, the Idol JA-free HS-like compound is Heparin, a specialized form of HS, is a commonly used anti provided wherein L is a beta linkage and L is a beta linkage. coagulant drug. Thus, new methods for the synthesis of hep 0009. In some embodiments of the presently disclosed arin and HS attract considerable interest for those developing subject matter, a method of synthesizing an Idol JA-free HS anticoagulant and other HS-related drugs having improved like compound is provided, comprising incubating a polysac pharmacological effects. charide Substrate with a Sulfo donor compound and an enzyme mixture, the enzyme mixture comprising O-sul SUMMARY fotransferase (OST) enzymes 6-OST-1,6-OST-3 and 0005. This Summary lists several embodiments of the 3-OST-1 and substantially no epimerase enzyme, wherein presently disclosed subject matter, and in many cases lists synthesis of the Idol JA-free HS-like compound from the variations and permutations of these embodiments. This polysaccharide Substrate is accomplished. In some embodi Summary is merely exemplary of the numerous and varied ments, the method comprises a polysaccharide Substrate embodiments. Mention of one or more representative features selected from the group consisting of N-sulfo heparosan and of a given embodiment is likewise exemplary. Such an chemically desulfated N-sulfated heparin. In some embodi embodiment can typically exist with or without the feature(s) ments, the OST enzymes are recombinant OST enzymes. In mentioned; likewise, those features can be applied to other some embodiments, the recombinant OST enzymes are pro embodiments of the presently disclosed subject matter, duced in a bacterial expression system. In some embodi whether listed in this Summary or not. To avoid excessive ments, the Sulfo donor compound comprises a compound repetition, this Summary does not list or Suggest all possible capable of donating a Sulfonate or Sulfuryl group. In some combinations of Such features. embodiments, the polysaccharide Substrate is partially Sul 0006. In some embodiments of the presently disclosed fated prior to incubation. subject matter, an iduronic acid (Idol JA)-free heparan sulfate 0010. In some embodiments of the presently disclosed (HS)-like compound comprising the following structure is subject matter, the method of synthesizing an Idol JA-free provided: HS-like compound further comprises (a) providing a reaction US 2009/0035787 A1 Feb. 5, 2009 mixture comprising adenosine 3',5'-diphosphate (PAP), a ride substrate is selected from the group consisting of N-sulfo PAPS regenerating enzyme and a Sulfo donor compound; and heparosan and chemically desulfated N-sulfated heparin. In (b) incubating the reaction mixture for a time period sufficient some embodiments, the O-sulfotransferases are selected to catalyze the production of 3'-phosphoadenosine 5'-phos from the group consisting of 2-OSTs, 3-OSTs and 6-OSTs. In phosulfate (PAPS) from the PAP by the PAPS regenerating Some embodiments, the O-sulfotransferase enzymes are enzyme utilizing the Sulfo donor compound as a Sulfo Sub recombinant OST enzymes. In some embodiments, the strate, whereby the sulfo donor compound is provided. In recombinant OST enzymes are produced in a bacterial Some embodiments, the PAPS regenerating enzyme is an expression system. In some embodiments, the polysaccharide arylsulfotransferase. In some embodiments, the arylsul Substrate is partially Sulfated prior to incubation. In some fotransferase is AST-IV. In some embodiments, the PAPS embodiments, the test is for an activity selected from the regenerating enzyme is an estrogen Sulfotransferase. In some group consisting of anti-thrombin binding activity, anti-Xa embodiments, the Sulfo donor compound is an aryl Sulfate activity, anti-IIa activity, cell proliferation stimulation activ compound. In some embodiments, the aryl Sulfate compound ity, cell growth stimulation activity, activated partial throm is p-nitrophenol sulfate (PNPS). In some embodiments, the boplastin time, prothrombin time and combinations thereof. time period is from about 1 minute to about 30 minutes. In 0014. In some embodiments of the presently disclosed some embodiments, the OST enzymes further comprise Subject matter, the method of determining a mechanism of 2-OST. In some embodiments, the 2-OST enzyme is a recom activity of HS-like compounds further comprises (a) provid binant OST enzyme. In some embodiments, the recombinant ing a reaction mixture comprising adenosine 3',5'-diphos OST enzyme is produced in a bacterial expression system. phate (PAP), a PAPS regenerating enzyme and a sulfo donor 0011. In some embodiments of the presently disclosed compound; and (b) incubating the reaction mixture for a time subject matter, a method of synthesizing a library of HS-like period sufficient to catalyze the production of 3'-phosphoad compounds is provided, comprising incubating polysaccha enosine 5'-phosphosulfate (PAPS), whereby the sulfo donor ride Substrates with a sulfo donor compound and one or more compound is provided. In some embodiments, the PAPS combinations of biosynthetic enzymes, the biosynthetic regenerating enzyme is an arylsulfotransferase. In some enzymes comprising epimerases, N-sulfotransferases and embodiments, the arylsulfotransferase is AST-IV. In some O-sulfotransferases. In some embodiments, the polysaccha embodiments, the PAPS regenerating enzyme is an estrogen ride substrate is N-sulfo heparosan. In some embodiments, Sulfotransferase. In some embodiments, the Sulfo donor com the O-sulfotransferases are selected from the group consist pound is an aryl Sulfate compound. In some embodiments, the ing of 2-OSTs, 3-OSTs and 6-OSTs. In some embodiments, aryl sulfate compound is p-nitrophenol sulfate (PNPS). In the O-sulfotransferase enzymes are recombinant OST Some embodiments, the time period is from about 1 minute to enzymes. In some embodiments, the recombinant OST about 30 minutes. enzymes are produced in a bacterial expression system. In 0015. Accordingly, it is an object of the presently dis Some embodiments, the polysaccharide Substrate is partially closed subject matter to provide iduronic acid-free heparan sulfated prior to incubation. sulfate-like compounds. Further, it is an object of the pres 0012. In some embodiments of the presently disclosed ently disclosed subject matter to provide methods of synthe subject matter, the method of synthesizing a library of HS sizing iduronic acid-free heparan Sulfate-like compounds. like compounds further comprises (a) providing a reaction These objects are achieved in whole or in part by the presently mixture comprising adenosine 3',5'-diphosphate (PAP), a disclosed Subject matter. PAPS regenerating enzyme and a Sulfo donor compound; and 0016. An object of the presently disclosed subject matter (b) incubating the reaction mixture for a time period sufficient having been stated hereinabove, and which is achieved in to catalyze the production of 3'-phosphoadenosine 5'-phos whole or in part by the presently disclosed subject matter, phosulfate (PAPS), whereby the sulfo donor compound is other objects will become evident as the description proceeds provided. In some embodiments, the PAPS regenerating when taken in connection with the accompanying drawings as enzyme is an arylsulfotransferase. In some embodiments, the best described hereinbelow. arylsulfotransferase is AST-IV. In some embodiments, the PAPS regenerating enzyme is an estrogen Sulfotransferase. In BRIEF DESCRIPTION OF THE DRAWINGS Some embodiments, the Sulfo donor compound is an aryl 0017 FIGS. 1A and 1B are schematic drawings illustrat Sulfate compound. In some embodiments, the aryl Sulfate ing the biosynthesis of HS-like compounds. FIG. 1A is a compound is p-nitrophenol sulfate (PNPS). In some embodi schematic drawing of the biosynthetic pathway of HS. FIG. ments, the time period is from about 1 minute to about 30 1B is a schematic drawing of the synthesis of a library of minutes. In some embodiments, the polysaccharide Substrate HS-like compounds using an enzyme-based combinatorial is a chemically desulfated N-sulfated heparin. approach as disclosed herein. Each of compounds 1-8 are 0013. In some embodiments of the presently disclosed non-limiting examples of HS-like compounds prepared using Subject matter, a method of determining a mechanism of the disclosed enzyme-based combinatorial approach. The activity of HS-like compounds is provided, comprising (a) enzyme schemes used for the synthesis of each of compounds synthesizing one or more HS-like compounds by incubating 1-8 are shown in Table 2. one or more polysaccharide Substrates with a Sulfo donor 0018 FIGS. 2A and 2B are reversed-phased ion pairing compound and one or more combinations of biosynthetic (RPIP) HPLC chromatograms of the disaccharide analysis of enzymes, the biosynthetic enzymes comprising epimerases, compounds 8 (FIG. 2A) and 2 (FIG. 2B). The S-labeled N-sulfotransferases and O-sulfotransferases; (b) subjecting polysaccharides were degraded by nitrous acid at pH 1.5 one or more of the HS-like compounds to a test for an activity; followed by sodium borohydride reduction. The resultant and (c) determining a mechanism of activity of the HS-like S-labeled disaccharides were desalted on a BIO-GEL(R) P-2 compounds based on one or more results of the one or more column, and then resolved by RPIP-HPLC chromatography. HS-like compounds. In some embodiments, the polysaccha The elution positions were identified by co-eluting with S US 2009/0035787 A1 Feb. 5, 2009 labeled or H-labeled disaccharide standards, where peak chromogenic Xa substrate. The activity of Xa was determined label 1 represents GlcUA-AnMan3S, 2 represents Idol JA2S by the rate of the increase of the absorbance at 405 nm. The AnMan, 3 represents GlcUA-AnManóS, 4 represents Idol JA activity without polysaccharide was defined as 100%. Each AnManóS. 5 represents Idol JA-AnMan3S6S, 6 represents data point represents the average of two determinations. Error GlcUAAnMan3S6S, and 7 represents Idol JA2S-AnManóS. bars indicate the range. 0019 FIG. 3 is a graph showing the effect of the synthetic HS-like compounds on FGF-2-dependent BaF3 FGFR1c cell proliferation. BaF3 FGFR1c cells were seeded in 96-well DETAILED DESCRIPTION plates with 2 nM FGF2 for control and 2 nM FGF2 plus various concentrations of the following compounds: heparin, 0024. Throughout the specification and claims, a given 1, 2, 7 and 8. Cells were cultured for 40 hrs, followed by chemical formula or name shall encompass all optical iso incubation in the media containing Hthymidine for 4 hrs. mers and stereoisomers, as well as racemic mixtures where The cellular proliferation was determined by IHI thymidine Such isomers and mixtures exist. incorporation into the DNA. Data are meant range of dupli cates. In the graph, heparin is represented by a Solid line and I. General Considerations Solid circle, compound 8 by a dotted line and open circle, compound 2 by a short dashed line and Solid circle, com 0025 Heparan sulfates (HSs) are highly sulfated polysac pound 1 by a dashed and dotted line and open circle, and charides present on the Surface of mammalian cells and in the compound 7 by a broken line and solid circle. extracellular matrix in large quantities. HS is a highly charged 0020 FIG. 4 shows the elution profiles of N-Sisulfo polysaccharide comprising 1->4-linked glucosamine and heparosan oligosaccharides on BIO-GEL(R) P-10 column. glucuronic/iduronic acid units that contain both N- and Low specific Slradioactively labeled N-Sisulfo heparo O-sulfo groups. Heparin, a specialized form of HS, is a com san (1.5x10 cpm/mg) was prepared by incubating deacety monly used anticoagulant drug. Thus, “heparan Sulfate’, as lated heparosan with purified NST and SPAPS. The used herein, includes heparin. polysaccharide (1 mg) was digested with 20 ng of purified 0026. As used herein, the terms “HS-like compound' and heparin lyase Ill. The products were fractionated on a Bio “HS-like molecule' are intended to refer to synthetically Gel RTMP-10 column, which was eluted with a buffer con Sulfated polysaccharides possessing one or more structural taining 25 mM Tris, 1000 mM. NaCl, pH 7.4. The fractions and/or functional properties of HSs. In some embodiments, were monitored by both Sradioactivity and UV 232 nm. In HS-like compounds can contain glucuronic acid or iduronic the graph, Sradioactivity is indicated on the left vertical acid and glucosamine with or without Sulfo groups. As such, axis and represented by the black line and black dots, while HS-like compounds and HS-like molecules include, but are UV optical density (O.D.) at 232 nm is indicated on the right not limited to, synthetic HSs, sulfated polysaccharides and Vertical axis and represented by the gray line and gray dots. heparins. Although exemplary embodiments of particular The fractions containing the oligosaccharides with the HS-like compounds or molecules have been disclosed herein, desired size were pooled for further analysis. the presently disclosed subject matter is not intended to be 0021 FIG. 5 is a bar graph showing the enzymatic synthe limited to the disclosed examples, but rather HS-like com sis of AT-binding oligosaccharides. The oligosaccharide Sub pounds and HS-like molecules include all comparable syn strates (1 lug) or the full length N-sulfo heparosan (1 lug) were thetically sulfated polysaccharides as would be apparent to incubated with a mixture of enzymes, including 6-OST-1 (20 one of ordinary skill. Indeed, one of ordinary skill in the art, ug), 6-OST-3 (20 ug) and 3-OST-1 (10 ug) and SPAPS (30 upon a review of the instant disclosure, is capable of produc uM,285,000 cpm/nmole) at 37°C. for 2 hrs. Alternatively, the ing numerous HS-like compounds based upon the disclosed oligosaccharides (1 lug) or full length N-sulfo heparosan (1 methods and compounds. ug) were preincubated with epimerase (8 g) at 37°C. for 30 0027 HSs play critical roles in a variety of important min followed by incubating with mixture of 6-OST-1 (20 ug), biological processes, including assisting viral infection, regu 6-OST-3 (20 ug), 2-OST (40 ug) and 3-OST-1 (10 ug) in the lating blood coagulation and embryonic development, Sup presence of SIPAPS. The resultant S-labeled oligosac pressing tumor growth, and controlling the eating behavior of charides were incubated with AT, and the complex of AT and test Subjects by interacting with specific regulatory proteins S-labeled oligosaccharides were captured by ConA-agar (Liu, J., and Thorp, S. C. (2002) Med. Res. Rev. 22:1-25; ose column. The amount of S-labeled products using full Rosenberg, R. D., et al., (1997).J. Clin. Invest. 99:2062-2070; length N-sulfo heparosan was defined to be 100%. In the Bernfield, M., et al., (1999) Annu. Rev. Biochem. 68:729-777: graph, the S-labeled oligosaccharides synthesized in the Alexander, C. M., et al., (2000) Nat. Genet. 25:329-332; absence of epimerase and 2-OST are represented by the solid Reizes, O., et al., (2001) Cell 106:105-116). The unique sul black bars while the S-labeled oligosaccharides synthe fated saccharide sequences determine to which specific pro sized with epimerase and 2-OST are represented by the solid teins HSS bind, thereby regulating biological processes. gray bars. 0028. The biosynthesis of HS occurs in the Golgi appara 0022 FIG. 6 is a schematic illustration of the synthetic tus. It is initially synthesized as a copolymer of glucuronic scheme of compound 2 (Recomparin). The components acid and N-acetylated glucosamine by D-glucuronyl and involved in PAPS regeneration system are boxed. N-acetyl-D-glucosaminyltransferase, followed by various 0023 FIGS. 7A and 7B are graphs illustrating the inhibi modifications (Lindahl, U., et al., (1998).J. Biol. Chem. 273: tory effects of heparin (FIG. 1A) and recomparin (compound 24979-24982). These modifications include N-deacetylation 2: FIG. 6), respectively, on Xa activity. Heparin and recom and N-sulfation of glucosamine, Cs epimerization of glucu parin were incubated with AT (20 g/ml), factor Xa (10U/ml) ronic acid to form iduronic acid residues, 2-O-sulfation of and bovine serum albumin (100 ug/ml) in 20 mM sodium iduronic and glucuronic acid, as well as 6-O-sulfation and phosphate and 150 mM. NaCl, pH 7.2, and 1 mM S-2765 3-O-sulfation of glucosamine. Several enzymes that are US 2009/0035787 A1 Feb. 5, 2009 responsible for the biosynthesis of HS have been cloned and heparin, interact with AT, a serine protease inhibitor, to inhibit characterized (Esko, J. D., and Lindahl, U. (2001) J. Clin. the activities of thrombin and factor Xa in the blood coagul Invest 108: 169-173). lation cascade (Rosenberg, R. D., et al., (1997).J. Clin. Invest. 0029. The expression levels of various HS biosynthetic 99:2062-2070). Anticoagulant-active HS(HS") and heparin enzyme isoforms contribute to the synthesis of specific sac contain one or multiple AT-binding sites per polysaccharide charide sequences in specific tissues. HS N-deacetylase/N- chain. This binding site contains a specific pentasaccharide sulfotransferase, 3-O-sulfotransferase, and 6-O-sulfotrans sequence with a structure of -GlcNS(or Ac)6S-GlcUA ferase are present in multiple isoforms. Each isoform is GlcNS3S(+6S)-IdoUA2S-GlcNS6S-. The 3-O-sulfation of believed to recognize a saccharide sequence around the modi glucosamine for generating GlcNS3S(+6S) residue, which is fication site in order to generate a specific sulfated saccharide carried out by 3-OST-1, plays a role in the synthesis of HS' sequence (Liu, J., et al., (1999) J. Biol. Chem. 274:5185 (Liu, J., et al., (1996) J. Biol. Chem. 271:27072-27082: 5192: Aikawa, J.-I., et al., (2001).J. Biol. Chem. 276:5876 Shworak, N. W., et al., (1997) J. Biol. Chem. 272:28008 5882: Habuchi, H., et al., (2000).J. Biol. Chem. 275:2859 28019). 2868). For instance, HS D-glucosaminyl 3-O- 0032. In accordance with some embodiments of the pres sulfotransferase (3-OST) isoforms generate 3-O-sulfated ently disclosed Subject matter, HS-like compounds can have glucosamine residues that are linked to different sulfated a strong binding affinity for AT. By way of non-limiting iduronic acid residues. 3-OST isoform 1 (3-OST-1) transfers example, the binding constant (K) of an HS-like molecule sulfate to the 3-OH position of an N-sulfated glucosamine can range from about 5 to about 100 nM. In some embodi residue that is linked to a glucuronic acid residue at the ments, the binding constant (K) of an HS-like molecule can nonreducing end (GlcUA-GlcNS6S). However, 3-OST iso range from about 20 to about 60 nM. Any suitable approach to form 3 (3-OST-3) transfers sulfate to the 3-OH position of an determine binding affinity can be employed as would be N-unsubstituted glucosamine residue that is linked to a 2-O- appreciated by one of ordinary skill in the art upon review of sulfated iduronic acid at the nonreducing end (IdoUA2S the instant disclosure. GlcNH+6S) (Liu, J., et al., (1999).J. Biol. Chem. 274:38155 0033. In some embodiments the anticoagulantactivity can 38162). The difference in the substrate specificity of 3-OSTs be measured by determining anti-Xa and anti-IIa activities. In results in distinct biological functions. For example, the HS Some embodiments, the anti-Xa and anti-IIa activities can be modified by 3-OST-1 binds to antithrombin (AT) and pos determined in the presence of antithrombin. HS-like com sesses anticoagulant activity (Liu, J., et al., (1996) J. Biol. pounds with a strong binding affinity for AT and/or a high Chem. 271:27072-27082). However, the HS modified by anticoagulant activity can have high anti-Xa and anti-IIa 3-OST-3 (3-OST-3A and 3-OST-3B) binds to glycoprotein D activities. In some embodiments, HS-like compounds with (gD) of herpes simplex virus, type 1 (HSV-1) thus mediating high anticoagulant activity can have ICso values for anti-Xa viral entry (Shukla, D., et al., (1999) Cell 99:13-22). activity ranging from about 10 to about 500 ng/ml. In some 0030 Cell surface HS also assists HSV-1 infection (Wu embodiments, HS-like compounds with high anticoagulant Dunn, D., and Spear, P. G. (1989) J. Virol. 63:52-58). One activity can have ICso values for anti-Xa activity ranging from report (Shukla, D., et al., (1999) Cell 99:13-22) suggests that about 20 to about 100 ng/ml. In some embodiments, HS-like a specific 3-O-sulfated HS is involved in assisting HSV-1 compounds with high anticoagulant activity can have ICso entry. The 3-O-sulfated HS is generated by 3-OST-3 but not values for anti-IIa activity ranging from about 5 to about 200 by 3-OST-1. In addition, the 3-O-sulfated HS provides bind ng/ml. In some embodiments, HS-like compounds with high ing sites for HSV-1 envelope glycoprotein D, which is a key anticoagulant activity can have ICso values for anti-IIa activ viral protein involved in the entry of HSV-1 (Shukla, D., et al., ity ranging from about 5 to about 50 ng/ml. Any suitable (1999) Cell 99:13–22). Because 3-OST-3-modified HS is approach to determine anticoagulant activity can be rarely found in HS from natural Sources, the study Suggests employed as would be appreciated by one of ordinary skill in that HSV-1 recognizes a unique saccharide structure. Indeed, the art upon review of the instant disclosure. the result from the structural characterization of a gld-binding octasaccharide revealed that the octasaccharide possesses a II. Methods of Sulfating Polysaccharides specific saccharide sequence (Liu, J., et al., (2002) J. Biol. 0034. The presently disclosed subject matter provides in Chem. 277:33456-33467). In addition, the binding affinity of some embodiments enzymatic methods for the sulfation of the 3-O-sulfated HS forg) is about 2 uM (Shukla, D., et al., multimilligram amounts of HS-like compounds having par Cell 99:13-22). This affinity is similar to that reported for the ticular functions, using Sulfotransferases coupled with a sys binding of g|D to the protein receptors, suggesting that HSV-1 tem for reducing inhibitory effects from sulfo donor byprod utilizes both protein and HS cell surface receptors to infect ucts. In some embodiments, the system for reducing target cells (Willis, S.H., et al., (1998).J. Virol. 72:5938-5947: inhibitory byproducts comprises a 3'-phosphoadenosine Krummenacher, C., et al., (1999).J. Virol. 73:8127-8137). It is 5'-phosphosulfate (PAPS) regeneration system. In some believed that the interaction between g) and the 3-O-sulfated embodiments, the PAPS regeneration system can utilize aryl the protein entry receptors somehow triggers the fusion Sulotransferase IV, while in Some embodiments estrogen Sul between the virus and the cell in the presence of other viral fotransferase is employed. In some embodiments, the system envelope proteins, including gB, gFI, and gL (Shukla, D., and comprises a phosphatase enzyme. By utilizing the presently Spear, P. G. (2001).J. Clin. Invest. 108:503-510). A study of disclosed sulfation system and selecting appropriate enzy the co-crystal structure of g|D and herpes entry receptor HveA matic modification steps, an inactive precursor polysaccha Suggests that the binding of HveA to g) induces conforma ride can be converted to a HS-like compound having desired tional changes in g|D (Carfi, A., et al., (2001) Mol. Cell. biological properties. 8:169-179). 0035. In some embodiments, the presently disclosed sub 0031 HS-regulated anticoagulation mechanisms have ject matter employs recombinant Sulfotransferases. Because been studied extensively. It is now known that HS, including the recombinant Sulfotransferases can be recombinantly US 2009/0035787 A1 Feb. 5, 2009

expressed in bacteria, and the disclosed methods can use low provided. In some embodiments, the method comprises incu cost Sulfo donors, the presently disclosed Subject matter can bating a polysaccharide Substrate to be sulfated with a reac be readily utilized to synthesize large quantities of biologi tion mixture that comprises at least one Sulfotransferase cally active heparan Sulfates while reducing the production of enzyme, such as for example an O-sulfotransferase (OST) reaction inhibitory byproducts. enzyme, and a sulfo donor, such as for example PAPS. When 0036 Two representative advantages provided by the PAPS is employed, production of the sulfated polysaccharide presently disclosed Subject matter facilitate the large scale from the polysaccharide substrate is catalyzed by the OST synthesis of HS. First, large amounts of all the required HS enzyme with a conversion of the PAPS to adenosine 3',5'- Sulfotransferases can be successfully recombinantly diphosphate (PAP). A reaction condition is further provided expressed in Escherichia coli. Second, the enzymatic sulfa that modifies generated PAP to reduce an inhibitory effect of tion reactions can be coupled with a system for reducing PAP on the polysaccharide sulfation. For example, a PAPS inhibitory effects from sulfo donor byproducts (e.g., PAP) regeneration system can be coupled with the Sulfation reac and reducing costs related to continuously providing a Supply tion to convert PAP into PAPS, orphosphatases can be added of the sulfo donor PAPS. PAPS, a universal sulfate donorand to the reaction mixture to modify PAP such that it does not Source of Sulfate for all Sulfotransferases, is a highly expen compete with PAPS for binding with OSTs. sive and unstable molecule that has been an obstacle to the 0040. In some embodiments, the polysaccharide substrate large-scale production of enzymatically Sulfated products. is a previously N.O-desulfated and subsequently re-N-sul The half-life of PAPS in aqueous solution at pH 8.0 is fated polysaccharide, Such as for example a chemically des approximately 20 hours. Product inhibition by adenosine ulfated N-sulfated (CDSNS) heparin. In other embodiments, 3',5'-diphosphate (PAP) has also been a limiting factor to the polysaccharide Substrate is partially sulfated prior to reac large-scale applications. For example, PAP inhibition of tion mixture incubation. For example, a CDSNS can be hydroxysteroid sulfotransferase was determined to be K = 14 reacted with a particular OST to produce a sulfated polysac uM (Marcus et al. (1980) Anal. Biochem. 107,296). PAP has charide intermediate product that can then be reacted with also been shown to inhibit the sulfotransferase NodST with a one or more different OSTs to further sulfate the polysaccha K=0.1 uM (Lin et al., (1995).J. Am. Chem. Soc. 117, 8031). ride at different locations. This sequential process of reacting In some embodiments of the presently disclosed subject mat the polysaccharide substrate with one or more different OSTs ter, a PAPS regeneration system, such as the system devel can be continued until a final polysaccharide is produced oped by Burkart and colleagues (Burkart et al. (2000).J. Org. exhibiting desired biological activities (based, at least in part, Chem. 65,5565-5574, incorporated herein by reference), has on the sulfation pattern of the polysaccharide). been modified and adapted to be coupled to the enzymatic 0041. In some embodiments the sulfated polysaccharide synthesis reactions. The PAPS regeneration system converts product can be a glycosaminoglycan (GAG). GAGS are the PAP into PAPS, thereby reducing accumulation of inhibitory most abundantheteropolysaccharides in the body. These mol PAP in the reaction mixture and reducing production costs ecules are long unbranched polysaccharides containing a related to providing PAPS to drive the sulfation reaction. In repeating disaccharide unit. The disaccharide units can con other embodiments, phosphatase enzymes can be utilized to tain either of two modified Sugars: N-acetylgalactosamine modify PAP so that it no longer has binding affinity for (GalNAc) or N-acetylglucosamine (GlcNAc) and a uronic Sulfotransferases. acid Such as glucuronate oriduronate. GAGS are highly nega 0037. The presently disclosed sulfation system can be tively charged molecules, with extended conformation that adapted to produce a multitude of HS-like compounds having imparts high viscosity to the solution. Along with the high varied biological activities by selecting appropriate Sul viscosity of GAGs comes low compressibility, which makes fotransferases to include and by controlling (for example, these molecules ideal for a lubricating fluid in the joints. At sequentially controlling) the addition of those Sulfotrans the same time, their rigidity provides structural integrity to ferases to the reaction system to facilitate appropriate timing cells and provides passageways between cells, allowing for of Sulfations of the polysaccharide template. For example, cell migration. The specific GAGs of physiological signifi utilizing the presently disclosed methods, HS-like com cance are hyaluronic acid, dermatan Sulfate, chondroitin Sul pounds can be synthesized with purposeful and specific bio fate, heparin, heparan Sulfate (including heparin), andkeratan logical activities, such as but not limited to: anticoagulant HS, Sulfate. Thus, in some embodiments, the Sulfated polysaccha fibroblast growth factor-2-binding activity, herpes simplex ride product is a HS. In some embodiments, the sulfated virus glycoprotein D (gD)-binding HS, and fibroblast growth polysaccharide product is an anticoagulant-active HS, an factor 2 (FGF2) receptor-binding HS. For example, the enzy antithrombin-binding HS, an FGF-binding HS, and an HSV matic synthesis of ag)-binding octasaccharide can be used to gD-binding HS. inhibit HSV-1 infection (Copeland etal (2008) Biochemistry 0042 FIG. 1B schematically illustrates the enzyme-based 47:5774-5783). combinatorial approach to synthesizing HS-like compounds 0038. By way of example and not limitation, few as two or by selectively reacting a polysaccharide Substrate with one or three enzymatic steps are required for the synthesis of each of more different OSTs and biosynthetic enzymes (Table 2). For these biologically-active HS molecules (See, e.g., FIGS. 1A, example, compound 1 can be produced by incubating a 1B and 7). Thus, the presently disclosed subject matter pro polysaccharide Substrate (e.g., N-sulfo heparosan) with vides for the large scale synthesis of a wide range of HS-like 2-OST, 6-OST-1,6-OST-3 and 3-OST-1, wherein the reaction compounds with specific biological activities. In addition, is substantially free of epimerase. Compound 1 has no idu provided in some embodiments is a model system to better ronic acid residue. Alternatively, incubating a polysaccharide understand the biosynthesis and the functional components of substrate with 6-OST-1,6-OST-3 and 3-OST-1 produces com HS-like compounds. pound 2, which has no iduronic acid residue or 2-O-sulfo 0039. In some embodiments of the presently disclosed group. Further, compound 3 can be synthesized by reacting a Subject matter, a method of Sulfating a polysaccharide is polysaccharide substrate with 3-OST-1 and 2-OST, while US 2009/0035787 A1 Feb. 5, 2009 incubating a polysaccharide Substrate with epimerase in addi more further basic structural units by a beta linkage, repre tion to 3-OST-1 and 2-OST yields compound 4. Compound 3 sented by L, to form the linear chain. Accordingly, an HS has no iduronic acid residue and neither of compounds 3 or 4 like compound of the presently disclosed Subject matter, as have 6-O-sulfation. This enzyme-based combinatorial illustrated in Formula 1, can comprise Lalpha linkages and approach was used to produce compounds 5-8 in a similar Libeta linkages. Alternatively, in some embodiments an HS manner, as illustrated in FIG. 1B and summarized in Table 2. like compound as illustrated in Formula 1 can comprise L. 0043. As illustrated in FIG.1B and summarized in Table 2, beta linkages and L. alpha linkages. Further yet, in some the reactions described above for the production of com embodiments an HS-like compound as illustrated in Formula pounds 1, 2 and 3 are substantially free of epimerase. The 1 can comprise both L and Lalpha linkages, or alternatively, absence of epimerase in these reactions results in the produc both L and L beta linkages. By way of non-limiting tion of Idol JA-free compounds. example, the alpha and beta linkages described herein can 0044) Formula 1 is a structural rendering of the basic struc comprise alpha 1->4 (C.1->4) or beta (B1->4) linkages. The tural unit of HS-like compounds or molecules of the instant L and L. linkages can comprise O or N molecules. In some disclosure, which can be synthesized by the presently dis embodiments both L and L2 can comprise O molecules. In closed methods, including but not limited to the disclosed Some embodiments both L and L2 can comprise N mol enzyme-based combinatorial approach. Formula 1 illustrates ecules. In some embodiments L. can comprise an O molecule the basic structural unit of the HS-like compounds of the and La N molecule. Still yet, in other embodiments, L can instant disclosure as well as non-limiting examples of the comprise a N molecule and L, an O molecule. Further, in variable structural features such as variable R groups. In some Some embodiments, L and L2 can comprise any combination embodiments the basic structural unit of the HS-like com of alpha and beta linkages and O and N molecules. pound illustrated in Formula 1 generally comprises a glucu 0056. There appears to be no known limit to the length of ronic acid residue or an iduronic acid residue linked to a the linear copolymer of basic structural units which form the glucosamine residue. In some embodiments the glucuronic general structure of the HS-like compound. Referring again acid oriduronic acid residue can be linked to the glucosamine to Formula 1, in Some embodiments, n is 21. In some by an alpha linkage, represented by L in Formula 1. In some embodiments, n is 22. In some embodiments, n is 23. In embodiments the glucuronic acid oriduronic acid residue can Some embodiments, n is 24. In some embodiments, n is 25. be linked to the glucosamine by a beta linkage, represented by In some embodiments, n is 210. In some embodiments, n is L in Formula 1. 220. In some embodiments, n is 2100. 0057. In some embodiments, the HS-like molecule illus trated in Formula 1 has as group X an NH or O. In some Formula 1: embodiments, R can be H or SOH. In some embodiments, L2 R6 L1 HORs R can be SOH. In some embodiments, R can be Hor SOH. 1N O 1 N. O 1 N. In some embodiments, R can be H, SOH, CH or CHCH. H H In some embodiments, Rs can be H, SOH, CH or CHCH. Y KoR. H. Y Kor, H. Y - In some embodiments, R can be COOH, COOCH, H H H H COOCH, CHOSOH or CHSOH. 0058. In some embodiments, an HS-like compound of the H OR4 H XR presently disclosed subject matter can comprise an HS-like compound as illustrated in Formula 1, wherein X is NH or O. R is Hor SOH, R is SOH, R is Hor SOH, R is H, SOH, 0045 X-NH or O CH or CHCH. Rs is H, SOH, CH or CHCH R is 0046 R=H, SOH, or CH COOH, COOCH, COOCH, CHOSOH, or CHSOH, 0047 R=SOH L is an alpha or beta linkage, L is an alpha or beta linkage, 0.048 R=H or SOH and n is 24. In some embodiments, an HS-like compound of 0049 Ra-H, SOH, CH, or CHCH the presently disclosed Subject matter can comprise an HS 0050 Rs—H, SOH, CH, or CHCH like compound as illustrated in Formula 1, wherein X is NH, 0051 R-COOH, COOCH COOCH, CHOSOH or R is SOH, R is SOH, R is SOH, R is Hor SOH, Rs is CHSOH H. L. is a beta linkage, L is an alpha linkage, and n is 24. In 0052 Y—O or N some embodiments, an HS-like compound of the presently 0053 L-alpha linkage or beta linkage disclosed Subject matter can comprise an HS-like compound 0054 L-alpha linkage or beta linkage as illustrated in Formula 1, wherein X is NH, R is SOH. R. 0055. The basic structural unit of HS-like compounds is SOH, R is SOH, R is Hor SOH, Rs is H. L. is an alpha illustrated in Formula 1 can be repeated a plurality of times to linkage, L is a beta linkage, and n is 24. In some embodi form a linear copolymer of glucuronic acid or iduronic acid ments, an HS-like compound of the presently disclosed sub and glucosamine residues, resulting in the series of basic ject matter can comprise an HS-like compound as illustrated structural units which form the general structure of the HS in Formula 1, wherein X is NH, R is SOH. R. is SOH. R. like compound. In Formula 1 the brackets with subscript n (I is SOH. R. is Hor SOH. Rs is H. L. is an alpha linkage, La I) represent the repetitive nature of this linear chain of basic is an alpha linkage, and n is 24. In some embodiments, an structural units of glucuronic acid or iduronic acid and glu HS-like compound of the presently disclosed subject matter cosamine residues. In some embodiments, the basic structural can comprise an HS-like compound as illustrated in Formula unit of the HS-like compound is linked to one or more further 1, wherein X is NH, R is SOH, R is SOH, R is SOH. R. basic structural units by an alpha linkage, represented by L. is Hor SOH. Rs is H. L. is a beta linkage, L is a beta linkage, to form the linear chain. In some embodiments, the basic and n is 24. In some embodiments, the HS-like compounds structural unit of the HS-like compound is linked to one or described herein are Idol JA-free. In some embodiments the US 2009/0035787 A1 Feb. 5, 2009

HS-like compounds described herein are isolated and puri 0065. The terms “OST gene product”, “OST protein', and fied. In some embodiments, the HS-like compounds “OST polypeptide' refer to peptides having amino acid described herein are synthetically synthesized. sequences which are Substantially identical to native amino 0059. In some embodiments, the disclosed HS-like com acid sequences from the organism of interest and which are pounds can be synthesized chemically. Still yet, in some biologically active in that they comprise all or a part of the embodiments the disclosed HS-like compounds can be syn amino acid sequence of a HS O-sulfotransferase isoform, or thesized using a combination of enzymatic and chemical cross-react with antibodies raised against a HS O-sulfotrans methods. ferase isoform polypeptide, or retain all or some of the bio 0060 II.A. Sulfotransferases logical activity of the native amino acid sequence or protein. 0061. As previously noted, the presently disclosed subject Such biological activity can include immunogenicity. matter utilizes sulfotransferases, particularly O-sulfotrans 0066. The terms “OST gene product”, “OST protein', and ferases (OSTs), to sulfate polysaccharides. Sulfotransferases “OST polypeptide' also include analogs of HS O-sulfotrans comprise a family of enzymes that catalyze the transfer of a ferase molecules. By “analog is intended that a DNA or Sulfonate or Sulfuryl group (SOs) from a Sulfo donor com peptide sequence can contain alterations relative to the pound, i.e. an SO-donor molecule, to an acceptor molecule. sequences disclosed herein, yet retain all or some of the By way of non-limiting example, the Sulfo donor compound biological activity of those sequences. Analogs can be derived or SO-donor molecule can be the cofactor 3'-phosphoad from genomic nucleotide sequences as are disclosed herein or enosine-5'-phosphosulfate (PAPS). Even though it is more from other organisms, or can be created synthetically. Those accurate to call these Sulfonation reactions, the term Sulfation skilled in the art will appreciate that other analogs, as yet is still widely used. Therefore, the term "sulfation” as used undisclosed or undiscovered, can be used to design and/or herein refers to a transfer of a sulfonate or sulfuryl group from construct OST analogs. There is no need for a “OST gene one molecule to another. product”, “OST protein', and "OST polypeptide' to com 0062 Sulfotransferases mediate sulfation of different prise all or Substantially all of the amino acid sequence of a classes of substrates Such as carbohydrates, oligosaccharides, native OST gene product. Shorter or longer sequences are peptides, proteins, flavonoids, and steroids for a variety of anticipated to be of use in the presently disclosed subject biological functions including signaling and modulation of matter, shorter sequences are herein referred to as “seg receptor binding (Bowman et al., (1999) Chem. Biol. 6, ments.” Thus, the terms “OST gene product”, “OST protein', R9-R22; and Falany (1997) FASEB.J. 11, 1-2). Within the past and "OST polypeptide' also include fusion or recombinant few years, many new sulfotransferases have been identified HSO-sulfotransferase polypeptides and proteins comprising and cloned (Aikawa et al., (1999).J. Biol. Chem. 274, 2690; sequences of the OST protein. Methods of preparing such Dooley (1998) Chemico-Biol. Interact 109, 29: Fukuta et al. proteins are known in the art. (1998) Biochim. Biophys. Act. 1399, 57: Habuchi et al., 0067. The terms “OST gene”, “OST gene sequence', and (1998).J. Biol. Chem. 273,9208; Mazany et al., (1998) Bio “OST gene segment” refer to any DNA sequence that is chim. Biophys. Act 1407, 92; Nastuk et al. (1998).J. Neuro Substantially identical to a polynucleotide sequence encoding science 18, 7167: Ong et al., (1998).J. Biol. Chem. 273,5190; a HS O-sulfotransferase isoform gene product, protein or Ouyang et al., (1998).J. Biol. Chem. 273,24770; Saeki et al. polypeptide as defined above, and can also comprise any (1998).J. Biochem. 124, 55: Uchimura et al. (1998).J. Biol. combination of associated control sequences. The terms also Chem. 273, 22577; and Yoshinariet al., (1998).J. Biochem. refer to RNA, orantisense sequences, complementary to Such 123, 740). DNA sequences. As used herein, the term “DNA segment 0063. As used herein, the term “O-sulfotransferase refers to a DNA molecule that has been isolated free of total (OST) includes polypeptides and nucleic acids encoding HS genomic DNA of a particular species. Furthermore, a DNA O-sulfotransferases, such as for example “2-OST (e.g., segment encoding a HSO-sulfotransferase polypeptide refers mouse 2-OST, GENBANKR Accession No. AAC40135 to a DNA segment that contains OST coding sequences, yet is (SEQ ID NO:1); “3-OST-1” (e.g., human 3-OST-1, GEN isolated away from, or purified free from, total genomic DNA BANKR Accession No. NP 005105 (SEQ ID NO:2): of a source species, such as for example Homo sapiens. “3-OST-3” (e.g., human 3-OST-3A, GENBANKRAccession Included within the term “DNA segment” are DNA segments No. NP 006033 (SEQ ID NO:3) and human 3-OST-3B, and Smaller fragments of Such segments, and also recombi GENBANKR Accession No. NP 006032 (SEQ ID NO:4); nant vectors, including, for example, plasmids, cosmids, and “6-OST (e.g., mouse 6-OST-1, GENBANKR Accession phages, viruses, and the like. No. NP 056633 (SEQ ID NO:5), mouse 6-OST-2, GEN 0068. The term “substantially identical’, when used to BANKR Accession No. BAA89247 (SEQ ID NO:6), and define eithera OST gene product or amino acid sequence, or mouse 6-OST-3, GENBANKR Accession No. NP 056635 a OST gene or nucleic acid sequence, means that a particular (SEQ ID NO:7)), which are HS 2-O-sulfotransferase, HS sequence varies from the sequence of a natural OST by one or 3-O-sulfotransferase isoform 1, HS 3-O-sulfotransferase iso more deletions, substitutions, or additions, the net effect of form 3, and HS 6-O-sulfotransferase, respectively. which is to retain at least some of biological activity of the 0064. The term “OST includes invertebrate and verte natural gene, gene product, or sequence. Such sequences brate homologs of the O-sulfotransferases (e.g., mammalian include “mutant’ sequences, or sequences in which the bio (such as human and mouse), insect, and avian homologs). As logical activity is altered to some degree but retains at least such, although exemplary embodiments of particular OSTs Some of the original biological activity. have been disclosed herein, the presently disclosed subject 0069. Alternatively, DNA analog sequences are “substan matter is not intended to be limited to the disclosed examples, tially identical to specific DNA sequences disclosed herein but rather “OST, including particular OSTs (e.g., 2-OST, if: (a) the DNA analog sequence is derived from coding 3-OST-1,3-OST-3, and 6-OST), includes all comparable regions of the natural OST gene; or (b) the DNA analog OSTs known to the skilled artisan. sequence is capable of hybridization of DNA sequences of (a) US 2009/0035787 A1 Feb. 5, 2009 under Stringent conditions and which encode biologically active OST gene products; or (c) the DNA sequences are TABLE 1. degenerate as a result of alternative genetic code to the DNA analog sequences defined in (a) and/or (b). Substantially iden Functionally Equivalent Codons tical analog proteins will be greater than about 60%, 70%, Amino Acids Codons 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding sequence of the native protein. Sequences hav Alanine Ala A. GCA GCC GCG GCU ing lesser degrees of identity but comparable biological activ ity are considered to be equivalents. In determining nucleic Cysteine Cys C UGC UGU acid sequences, all Subject nucleic acid sequences capable of Aspartic Acid Asp D GAC GAU encoding Substantially similar amino acid sequences are con sidered to be substantially similar to a reference nucleic acid Glutamic acid Glu E GAA GAG sequence, regardless of differences in codon sequences or Phenylalanine Phe F UUC UUU Substitution of equivalent amino acids to create biologically functional equivalents. Glycine Gly G GGA. GGC GGG GGU 0070 Sequence identity or percent similarity of a DNA or Histidine His H CAC CAU peptide sequence can be determined, for example, by com paring sequence information using the GAP computer pro Isoleucine Ile I AUA AUC AUU gram, available from the University of Wisconsin Geneticist Computer Group. The GAP program utilizes the alignment Lysine Lys K AAA AAG method of Needleman et al. (1970) J Mol Biol 48:443, as Leucine Leu L UUA UUG. CUA CUC CUG. CUU revised by Smith et al. (1981) Adv Appl Math 2:482. Briefly, the GAP program defines similarity as the number of aligned Methionine Met M AUG symbols (i.e., nucleotides or amino acids) that are similar, Asparagine As n N AAC AAU divided by the total number of symbols in the shorter of the two sequences. The preferred parameters for the GAP pro Proline Pro P CCA CCC CCG CCU gram are the default parameters, which do not impose a pen alty for end gaps. See Schwartz et al. (1979) Nuc Acids Res Glutamine Glin Q CAA CAG 6(2):745-755; Gribskov et al. (1986) Nuc Acids Res 14(1): Arginine Arg R. AGA AGG CGA CGC CGG CGU 327-334. 0071. In certain embodiments, the present subject matter Serine Ser S ACG. AGU UCA, UCC UCG UCU concerns the use of OST genes and gene products that include Threonine Thir T ACA ACC ACG ACU within their respective sequences a sequence that is essen tially that of an OST gene, or the corresponding protein. The Waline Wall W. GUA GUC GUG GUU term “a sequence essentially as that of an OST gene, means Tryptophan Trp W UGG that the sequence is Substantially identical or Substantially similar to a portion of an OST gene and contains a minority of Tyrosine Tyr Y UAC UAU bases oramino acids (whether DNA or protein) which are not identical to those of an OST protein oran OST gene, or which (0073. It will also be understood by those of skill in the art are not a biologically functional equivalent. The term “bio that amino acid and nucleic acid sequences can include addi logically functional equivalent” is well understood in the art tional residues, such as additional N- or C-terminal amino and is further defined in detail herein. Nucleotide sequences acids or 5' or 3' nucleic acid sequences, and yet still be encom are “essentially the same' where they have between about passed by the OSTs disclosed herein, so long as the sequence 75% and about 85% or more preferably, between about 86% retains biological protein activity where protein expression is and about 90%, or more preferably greater than 90%, or more concerned. The addition of terminal sequences particularly preferably between about 91% and about 95%, or even more applies to nucleic acid sequences which can, for example, preferably, between about 96% and about 99%; of nucleic include various non-coding sequences flanking either of the 5' acid residues which are identical to the nucleotide sequence or 3' portions of the coding region or can include various of a OST gene. Similarly, peptide sequences which have internal sequences, i.e., introns, which are known to occur within genes. about 60%, 70%, 80%, or 90%, or preferably from 90-95%, 0074 The present subject matter also encompasses the use or more preferably greater than 96%, or more preferably of nucleotide segments that are complementary to the 95-98%, or most preferably 96%, 97%, 98%, or 99% amino sequences of the present Subject matter, in one embodiment, acids which are identical or functionally equivalent or bio segments that are fully complementary, i.e. complementary logically functionally equivalent to the amino acids of an for their entire length. Nucleic acid sequences that are OST polypeptide will be sequences which are “essentially the “complementary are those, which are base-paired according same'. to the standard Watson-Crick complementarity rules. As used 0072 OST gene products and OST encoding nucleic acid herein, the term "complementary sequences' means nucleic sequences, which have functionally equivalent codons, are acid sequences which are substantially complementary, as also covered by the presently disclosed subject matter. The can be assessed by the same nucleotide comparison set forth term “functionally equivalent codon’ is used hereinto refer to above, or is defined as being capable of hybridizing to the codons that encode the same amino acid, Such as the ACG and nucleic acid segment in question under relatively stringent AGU codons for serine. Applicants contemplate Substitution conditions such as those described herein. A particular of functionally equivalent codons of Table 1 into sequences of example of a complementary nucleic acid segment is an anti OSTs disclosed herein as equivalents. sense oligonucleotide. US 2009/0035787 A1 Feb. 5, 2009

0075 One technique in the art for assessing complemen 0079 At a biological level, identity is just that, i.e. the tary sequences and/or isolating complementary nucleotide same amino acid at the same relative position in a given sequences is hybridization. Nucleic acid hybridization will be family member of agene family. Homology and similarity are affected by Such conditions as salt concentration, tempera generally viewed as broader terms. For example, biochemi ture, or organic solvents, in addition to the base composition, cally similar amino acids, for example leucine and isoleucine length of the complementary strands, and the number of or glutamate/aspartate, can be present at the same position— nucleotide base mismatches between the hybridizing nucleic these are not identical perse, but are biochemically “similar'. acids, as will be readily appreciated by those skilled in the art. As disclosed herein, these are referred to as conservative Stringent temperature conditions will generally include tem differences or conservative substitutions. This differs from a conservative mutation at the DNA level, which changes the peratures in excess of about 30° C., typically in excess of nucleotide sequence without making a change in the encoded about 37°C., and preferably in excess of about 45° C. Strin amino acid, e.g., TCC to TCA, both of which encode serine. gent salt conditions will ordinarily be less than about 1,000 0080 When percentages are referred to herein, it is meant mM, typically less than about 500 mM, and preferably less to refer to percent identity. The percent identities referenced than about 200 mM. However, the combination of parameters herein can be generated by alignments with the program is much more important than the measure of any single GENEWORKSTM (Oxford Molecular, Inc. of Campbell, parameter. See e.g., Wethmur & Davidson (1968) J Mol Biol Calif., U.S.A.) and/or the BLAST program at the NCBI web 31:349-370. Determining appropriate hybridization condi site. Another commonly used alignment program is entitled tions to identify and/or isolate sequences containing high CLUSTALW and is described in Thompson et al. (1994) levels of homology is well known in the art. See e.g., Sam Nucleic Acids Res 22(22):4673-4680, among other places. brook et al. (2001) Molecular Cloning. A Laboratory I0081. The term “gene' is used for simplicity to refer to a Manual. Third Edition, Cold Spring Harbor Laboratory functional protein, polypeptide or peptide encoding unit. As Press, Cold Spring Harbor, N.Y. will be understood by those in the art, this functional term 0076 For the purposes of specifying conditions of high includes both genomic sequences and cDNA sequences. stringency, preferred conditions are salt concentration of I0082. As noted above, modifications and changes can be about 200 mM and temperature of about 45° C. One example made in the structure of the OST proteins and peptides of such stringent conditions is hybridization at 4xSSC, at 65° described herein and still constitute a molecule having like or C., followed by a washing in 0.1 xSSC at 65° C. for one hour. otherwise desirable characteristics. For example, certain Another exemplary stringent hybridization scheme uses 50% amino acids can be substituted for other amino acids in a formamide, 4xSSC at 42°C. Another example of “stringent protein structure without appreciable loss of interactive conditions' refers to conditions of high Stringency, for capacity with, for example, structures in the nucleus of a cell. example 6xSSC, 0.2% polyvinylpyrrolidone, 0.2% Ficol, Since it is the interactive capacity and nature of a protein that 0.2% bovine serum albumin, 0.1% sodium dodecyl sulfate, defines that protein's biological functional activity, certain 100 g/ml salmon sperm DNA and 15% formamide at 68°C. amino acid sequence Substitutions can be made in a protein Nucleic acids having sequence similarity are detected by sequence (or the nucleic acid sequence encoding it) to obtain hybridization under low stringency conditions, for example, a protein with the same, enhanced, orantagonistic properties. at 50° C. and 10xSSC (0.9 M. NaCl/0.09 M sodium citrate) Such properties can be achieved by interaction with the nor and remain bound when subjected to washing at 55° C. in mal targets of the native protein, but this need not be the case, 1xSSC. Sequence identity can be determined by hybridiza and the biological activity of the presently disclosed subject tion under stringent conditions, for example, at 50° C. or matter is not limited to a particular mechanism of action. It is higher and 0.1xSSC (9 mM NaCl/0.9 mM sodium citrate). thus contemplated in accordance with the present Subject 0077. Nucleic acids that are substantially identical to the matter that various changes can be made in the sequence of provided OSTs, e.g., allelic variants, genetically altered ver the OST proteins and peptides or underlying nucleic acid sions of the gene, etc., bind to the disclosed OSTs under sequence without appreciable loss of their biological utility or stringent hybridization conditions. By using probes, particu activity. larly labeled probes of DNA sequences, one can isolate I0083 Biologically functional equivalent peptides, as used homologous or related genes. The Source of homologous herein, are peptides in which certain, but not most or all, of the genes can be any species, e.g., primate species, particularly amino acids can be substituted. Thus, applicants contemplate human; rodents, such as rats and mice; canines; felines; Substitution of codons that encode biologically equivalent bovines; ovines; equines; insects; yeasts; nematodes; etc. amino acids as described herein into the sequences of the 0078 Between mammalian species, e.g., human, mouse disclosed OSTs, but which are not set forth herein in their and rat, homologs have substantial sequence similarity, i.e. at entirety for convenience. least 75% sequence identity between nucleotide sequences. I0084. Alternatively, functionally equivalent proteins or Sequence similarity is calculated based on a reference peptides can be created via the application of recombinant sequence, which can be a Subset of a larger sequence, such as DNA technology, in which changes in the protein structure a conserved motif coding region, flanking region, etc. A can be engineered, based on considerations of the properties reference sequence will usually be at least about 18 nucle of the amino acids being exchanged. Changes designed by otides long, more usually at least about 30 nucleotides long, man can be introduced through the application of site-di and can extend to the complete sequence that is being com rected mutagenesis techniques, e.g., to introduce improve pared. Algorithms for sequence analysis are known in the art, ments to the antigenicity of the protein or to test OST mutants such as BLAST, described in Altschuletal. (1990).J Mol Biol in order to examine OST sulfotransferase activity, or other 215:403-410. The sequences provided herein are essential for activity at the molecular level. recognizing OST related and homologous proteins in data 0085 Amino acid substitutions, such as those which base searches. might be employed in modifying the OST proteins and pep US 2009/0035787 A1 Feb. 5, 2009

tides described herein, are generally based on the relative will be appreciated that these changes can be effected by similarity of the amino acid side-chain Substituents, for alteration of the encoding DNA, taking into consideration example, their hydrophobicity, hydrophilicity, charge, size, also that the genetic code is degenerate and that two or more and the like. An analysis of the size, shape and type of the codons can code for the same amino acid. amino acid side-chain Substituents reveals that arginine, lysine and histidine are all positively charged residues; that 0092. Thus, it will also be understood that the presently alanine, glycine and serine are all of similar size; and that disclosed subject matter is not limited to the particular nucleic phenylalanine, tryptophan and tyrosine all have a generally acid and amino acid sequences of the OSTs disclosed herein. similar shape. Therefore, based upon these considerations, Recombinant vectors and isolated DNA segments can there arginine, lysine and histidine; alanine, glycine and serine; and fore variously include the O-sulfotransferase polypeptide phenylalanine, tryptophan and tyrosine; are defined herein as encoding region itself, include coding regions bearing biologically functional equivalents. Other biologically func selected alterations or modifications in the basic coding tionally equivalent changes will be appreciated by those of region, or include larger polypeptides which nevertheless skill in the art. comprise the O-sulfotransferase polypeptide-encoding I0086. In making biologically functional equivalent amino regions or can encode biologically functional equivalent pro acid Substitutions, the hydropathic index of amino acids can teins or peptides which have variant amino acid sequences. be considered. Each amino acid has been assigned a hydro Biological activity of an O-sulfotransferase can be deter pathic index on the basis of their hydrophobicity and charge mined using techniques generally known in the art, for characteristics, these are: isoleucine (+4.5); Valine (+4.2): example as disclosed herein in the Examples. leucine (+3.8); phenylalanine (+2.8); cysteine (+2.5); 0093. The nucleic acid segments of the present subject methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine matter, regardless of the length of the coding sequence itself. (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); pro can be combined with other DNA sequences. Such as promot line (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3. ers, enhancers, polyadenylation signals, additional restriction 5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and enzyme sites, multiple cloning sites, other coding segments, arginine (-4.5). and the like. Such that their overall length can vary consider 0087. The importance of the hydropathic amino acid index ably. It is therefore contemplated that a nucleic acid fragment in conferring interactive biological function on a protein is of almost any length can be employed, with the total length generally understood in the art (Kyte et al. (1982) Mol Biol preferably being limited by the ease of preparation and use in 157: 105, incorporated herein by reference). It is known that the intended recombinant DNA protocol. For example, certain amino acids can be substituted for other amino acids nucleic acid fragments can be prepared which include a short having a similar hydropathic index or score and still retain a stretch complementary to a nucleic acid sequence set forth in similar biological activity. In making changes based upon the any of the OSTs disclosed herein, such as about 10 nucle hydropathic index, the Substitution of amino acids whose otides, and which are up to 10,000 or 5,000 base pairs in hydropathic indices are within +2 of the original value is length, with segments of 3,000 being preferred in certain preferred, those, which are within +1 of the original value, are cases. DNA segments with total lengths of about 4,000, particularly preferred, and those within +0.5 of the original 3,000, 2,000, 1,000, 500, 200, 100, and about 50 base pairs in value are even more particularly preferred. length are also contemplated to be useful. 0088. It is also understood in the art that the substitution of 0094. Recombinant vectors form further aspects of the like amino acids can be made effectively on the basis of present subject matter. Particularly useful vectors are those in hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein which the coding portion of the DNA segment is positioned by reference, states that the greatest local average hydrophi under the control of a promoter. The promoter can be that licity of a protein, as governed by the hydrophilicity of its naturally associated with the OST gene, as can be obtained by adjacent amino acids, correlates with its immunogenicity and isolating the 5' non-coding sequences located upstream of the antigenicity, i.e. with a biological property of the protein. It is coding segment or exon, for example, using recombinant understood that an amino acid can be substituted for another cloning and/or polymerase chain reaction (PCR) technology having a similar hydrophilicity value and still obtain a bio and/or other methods known in the art, in conjunction with the logically equivalent protein. compositions disclosed herein. 0089. As detailed in U.S. Pat. No. 4,554,101, the follow 0095. In other embodiments, it is provided that certain ing hydrophilicity values have been assigned to amino acid advantages will be gained by positioning the coding DNA residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0+1): segment under the control of a recombinant, or heterologous, glutamate (+3.0t1); serine (+0.3); asparagine (+0.2): promoter. As used herein, a recombinant or heterologous glutamine (+0.2); glycine (O); threonine (-0.4); proline (-0. promoter is a promoter that is not normally associated with a 5+1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); 3-O-sulfotransferase gene in its natural environment. Such methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine promoters can include promoters isolated from bacterial, (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3. viral, eukaryotic, or mammalian cells. Naturally, it will be 4). important to employ a promoter that effectively directs the 0090. In making changes based upon similar hydrophilic expression of the DNA segment in the cell type chosen for ity values, the Substitution of amino acids whose hydrophi expression. The use of promoter and cell type combinations licity values are within +2 of the original value is preferred, for protein expression is generally known to those of skill in those, which are within it 1 of the original value, are particu the art of molecular biology (See, e.g., Sambrook et al. (2001) larly preferred, and those within +0.5 of the original value are Molecular Cloning: A Laboratory Manual. Third Edition, even more particularly preferred. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 0091. While discussion has focused on functionally N.Y.). The promoters employed can be constitutive or induc equivalent polypeptides arising from amino acid changes, it ible and can be used under the appropriate conditions to direct US 2009/0035787 A1 Feb. 5, 2009

high level expression of the introduced DNA segment, such as embodiments, human and/or mouse estrogen Sulfotransferase is advantageous in the large-scale production of recombinant (EST) can be used in place of or in conjunction with AST-IV. proteins or peptides. 0102 The enzyme AST-IV exists in two oxidative forms 0096. In some embodiments of the method disclosed (Marshallet al., (1997).J. Biol. Chem. 272,9153-9160; Mar herein for sulfating polysaccharides, the OST enzyme is shallet al., (1998) Chem.-Biol. Interact 109, 107-116:Yang et immobilized on a substrate. This provides an advantage in al., (1998) Chem.-Biol. Interact 109, 129-135; Yang et al. that the substrate to which the OSTs are attached can be (1996) Protein Expression Purif 8, 423-429; Guo et al. washed after a sulfation reaction to remove all components of (1994) Chem.-Biol. Interact. 92, 25-31; Chen et al. (1992) the reaction except the bound OSTs. As such, the products of Protein Expression Purif. 3, 421-6; Lin et al. (1998) Anal. the reaction can be more easily separated from the enzymes Biochem. 264, 111-117; and Yang et al., (1997) Protein Eng. catalyzing the reaction and the OSTs can be recycled and 10, 70). These two oxidative forms can be easily resolved utilized again in multiple Sulfation reactions. In some (Yang et al. (1996) Protein Expression Purif 8,423-429), and embodiments, the Substrate is agarose. In particular embodi the resolved physiologically relevant form has been utilized ments, the agarose Substrate is an agarose bead and the OSTs to assay picomole quantities of PAPS and PAP (Lin et al. are linked to the beads. (1998) Anal. Biochem. 264, 111-117). As the bacterial 0097 II.B. Reduction of Inhibitory Effects of PAP expression of rat AST-IV has been demonstrated (Chen et al., 0098. The presently disclosed method for sulfating (1992) Protein Expression Purif. 3, 421-6; and Ozawa et al., polysaccharides can comprise providing a "reaction condi (1990) Nucleic Acids Res. 18,400 1Z.), ASTIV can be cloned tion” that modifies PAP to reduce inhibitory effects of PAP. from a rat liver cDNA library, overexpressed in a recombinant such as competing with PAPS for binding with OSTs, on the bacterial system (e.g., E. coli) and isolated (See, e.g., U.S. polysaccharide Sulfation. In some embodiments, the reaction Pat. No. 6.255,088, herein incorporated by reference in its condition comprises a phosphatase enzyme. The phosphatase entirety). enzyme can remove a phosphate from the PAP, which reduces 0103 Coupling the sulfotransferase catalyzed sulfation its binding affinity for OSTs. In some embodiments, the phos reaction with a PAPS regeneration system can provide a fur phatase is 3'-ribonucleotide phosphohydrolase. ther advantage of generating PAPS utilized in the reaction 0099. In some embodiments, the reaction condition is a directly from PAP. That is, the reaction mixture can be for PAPS regeneration system, which comprises a PAPS regen mulated to combine PAP with a PAPS regenerating enzyme erating enzyme and a sulfo donor compound. The PAPS prior to or simultaneously with addition of a sulfotransferase regenerating enzyme catalyzes regeneration of the PAPS to the reaction mixture. The PAPS regenerating enzyme can from the PAP utilizing the sulfo donor compound as a sub then generate PAPS from the PAP for use by the sulfotrans strate. See, e.g., U.S. Pat. No. 6.255.088; and Burkart et al., ferase, thereby alleviating the need of Supplying any of the (2000).J. Org. Chem. 65,5565-5574, both of which are herein more expensive and unstable PAPS to the reaction mixture. incorporated by reference in their entirety. Thus, the PAPS For example, coupling the PAPS regeneration system to use regeneration system provides the dual advantages of reducing PNPS as a sulfo donor can potentially reduce the cost of the the inhibitory effects of PAP accumulation on sulfotrans synthesis of sulfated polysaccharides by as much as 1,000 ferase activity while also constantly "recharging the reaction fold. As such, in some embodiments of the presently dis mixture with the primary sulfo donor molecule, PAPS. In closed subject matter a method of sulfating a polysaccharide Some embodiments, the PAPS regenerating enzyme is an is provided comprising providing a reaction mixture compris estrogen Sulfotransferase. ing thereinadenosine 3',5'-diphosphate (PAP), a PAPS regen 0100 Thus, an aspect of the presently disclosed subject erating enzyme and a Sulfo donor compound (other than matter is directed to a sulfo donor compound (e.g., PAPS) PAPS, e.g., PNPS) and incubating the reaction mixture for a regeneration process coupled with Sulfation of a polysaccha time period sufficient to catalyze the production of 3'-phos ride Substrate. In particular, the process can be of a type phoadenosine 5'-phosphosulfate (PAPS) from the PAP by the wherein the Sulfation of a polysaccharide Substrate is cata PAPS regenerating enzyme utilizing the Sulfo donor com lyzed by a sulfotransferase, such as one or more OSTs, with a pound as a Substrate. The method further comprises incubat conversion of 3'-phosphoadenosine-5'-phosphosulfate ing a polysaccharide Substrate and at least one O-sulfotrans (PAPS) to adenosine 3',5'-diphosphate (PAP). The sulfation ferase (OST) enzyme with the reaction mixture, wherein process can be coupled with an enzymatic regeneration of the production of a Sulfated polysaccharide from the polysaccha PAPS from the PAP. The enzymatic regeneration can employ ride substrate is catalyzed by the OST enzyme with a conver an arylsulfotransferase as the catalyst and an aryl Sulfate as a sion of the PAPS to PAP and wherein the PAPS regenerating Substrate. In some embodiments, the enzymatic regeneration enzyme then catalyzes regeneration of the PAPS from the can comprise a human or mouse estrogen Sulfotransferase PAP again utilizing the Sulfo donor compound as a Substrate. (EST). As previously disclosed, preferred carbohydrate sub 0104 II.C. Synthesizing Iduronic Acid-Free HS strates can include GAGS, Such as for example heparan Sul 0105. As noted herein, the presently disclosed subject fates, including heparin. matter can be adapted to produce a multitude of sulfated 0101. In some embodiments, the arylsulfotransferase is a polysaccharides, i.e. HS-like compounds, having pre-se recombinant aryl sulfotransferase IV (AST-IV: e.g., rat AST lected biological activities using an enzyme-based combina IV (SEQ ID NO:8)). This enzyme, when coupled to a sul torial approach. In essence, the process of reacting a polysac fotransferase of choice, transfers sulfate from an aryl sulfate charide substrate with specified OSTs can be continued until (e.g., p-nitrophenyl sulfate (PNPS) or any similar compound a final polysaccharide is produced exhibiting the desired bio containing a phenol sulfate moiety) to PAP. This system logical activities, wherein the activities are based, at least in averts product inhibition by PAP while regenerating PAPS in part, on the Sulfation pattern of the polysaccharide. situ and can be monitored quantitatively by measurement of 0106 The presently disclosed subject matter also illus the absorbance of released p-nitrophenol at 400 nm. In some trates that the presence or absence of an iduronic acid (Id US 2009/0035787 A1 Feb. 5, 2009 oUA) residue can also impart biological properties on the 2, 3, 4 or 5% epimerase, and/or to the presence of an amount HS-like compound. In particular, the methods described of epimerase below the limits of a standard detection herein provide for the synthesis of HS-like compounds with approach. out Idol JA residues, i.e. Idol JA-free HS-like compounds. By omitting the inclusion of epimerase in the synthesis of HS III. Methods of Determining the Mechanism of Activity of like compounds, the epimerization of the glucuronic acid HS-Like Compounds residue to an iduronic acid residue is precluded, which results in the synthesis of a HS-like compound that is substantially 0111. The presently disclosed subject matter provides free of Idol JA. As described in more detail in the Examples methods for determining a mechanism of activity of HS-like below, the synthesis of Idol JA-free HS-like compounds can compounds. The enzyme-based combinatorial approach of provide for the synthesis of compounds with the desired synthesizing HS-like compounds provides for the systematic biological properties of HS without untoward biological manipulation of the structural composition of HS-like com effects. In some embodiments, the HS-like compounds pounds. For example, the presently disclosed Subject matter described herein are isolated and/or purified. provides for the synthesis of HS-like compounds with spe 0107. In some embodiments, the terms "Idol JA-free” and cific sulfation patterns. FIG. 1B schematically illustrates the “iduronic acid-free” refer to the substantial absence of idu enzyme-based combinatorial approach to synthesizing HS ronic acid. In some embodiments, the terms "Idol UA-free HS' like compounds by selectively reacting a polysaccharide Sub and “Idol JA-free HS-like compound” refer to HS-like com strate with different OSTs and biosynthetic enzymes (Table pounds Substantially devoid of iduronic acid. In some 2). As such, this method provides a model system to better embodiments, Idol UA-free and iduronic acid-free can refer to understand the biosynthesis of HS-like compounds as well as 0, 0.1, 0.5, 1, 2, 3, 4 or 5% iduronic acid. In some embodi the functional characteristics of each of the components that ments, Idol JA-free can refer to an amount of Idol UA below make up HS-like compounds. limits of detection using 1H-NMR. 0112. In some embodiments, the presently disclosed sub ject matter provides a method of determining the mechanism 0108. In some embodiments, an Idol JA-free HS-like com of activity of HS-like compounds, the method comprising, pound can be synthesized by incubating a polysaccharide synthesizing one or more HS-like compounds by incubating substrate (e.g., N-sulfo heparosan) with 2-OST, 6-OST-1,6- one or more polysaccharide Substrates with a Sulfo donor OST-3 and 3-OST-1 (compound 1, Table 2), wherein the compound and one or more combinations of biosynthetic reaction is substantially free of epimerase. In another embodi enzymes, the biosynthetic enzymes comprising epimerases, ment, a polysaccharide substrate is incubated with 6-OST-1, N-sulfotransferases and O-sulfotransferases; Subjecting one 6-OST-3 and 3-OST-1 to produce compound 2, which also or more of the HS-like compounds to a test for an activity; and has no Idol JA residue due to the substantial absence of epi determining a mechanism of activity of the HS-like com merase. In still yet another embodiment, an Idol JA-free HS pounds based on one or more results of the one or more like compound can be synthesized by reacting a polysaccha HS-like compounds. The method can further comprise pro ride substrate with 3-OST-1 and 2-OST with substantially no viding a reaction mixture comprising adenosine 3',5'-diphos epimerase (compound 3). Although exemplary embodiments phate (PAP), a PAPS regenerating enzyme and a sulfo donor of particular HS-like compounds or molecules have been compound; and incubating the reaction mixture for a time disclosed herein, the presently disclosed Subject matter is not period sufficient to catalyze the production of 3'-phosphoad intended to be limited to the disclosed examples, but rather enosine 5'-phosphosulfate (PAPS), whereby the sulfo donor HS-like compounds include all comparable synthetically sul compound is provided. fated polysaccharides known to one of ordinary skill in the 0113. In some embodiments the test used to elucidate a art. In particular, it is envisioned that one of ordinary skill in mechanism of a component of a HS-like compound can com the art, upon review of the instant disclosure, will be capable prise an anti-thrombin binding activity test. For example, two of producing numerous HS-like compounds based upon the or more HS-like compounds, each synthesized using different disclosed methods. enzyme-based combinatorial reaction schemes and thereby differing in their structural composition, can be analyzed for 0109. In some embodiments, the terms “substantially anti-thrombin binding activity via an anti-thrombin binding free”, “substantial absence' and “substantially no refer to a activity test, to thereby elucidate the one or more structural compound or entity that is predominantly devoid or lacking. components of the HS-like compounds responsible for or In some embodiments, these terms refer to a compound or relevant to the anti-thrombin activity. Likewise, in other entity that is entirely devoid or lacking. By way of non embodiments, the test used to elucidate a mechanism of a limiting example, Substantially free. Substantial absence and component of a HS-like compound can comprise anti-Xa substantially no can refer to the presence of 0, 0.1, 0.5, 1,2,3, activity, anti-IIa activity, cell proliferation stimulation activ 4 or 5% of a compound or entity, and/or to the presence of an ity or cell growth stimulation activity. Still yet, in other amount of compound or entity below the limits of a standard embodiments, the test used to elucidate a mechanism of a detection approach. component of a HS-like compound can comprise activated 0110. In some embodiments, the terms “substantially free partial thromboplastin time (APTT) or prothrombin time of epimerase”, “substantial absence of epimerase' and “sub (PT). stantially no epimerase' refer to reactions for the synthesis of 0114. In some embodiments, the disclosed method of HS-like compounds, wherein the reactions are predominantly determining the mechanism of activity of HS-like com devoid of or lacking in epimerase. In some embodiments, pounds comprises synthesizing one or more HS-like com Substantially free of epimerase, Substantial absence of epime pounds chemically. Still yet, in some embodiments the dis rase and substantially no epimerase can refer to 0, 0.1, 0.5, 1. closed method of determining the mechanism of activity of US 2009/0035787 A1 Feb. 5, 2009

HS-like compounds comprises synthesizing one or more HS mg/ml glucose, 12.5ug/ml tetracycline, 15ug/mlkanamycin, like compounds using a combination of enzymatic and 35 g/ml chloramphenicol, and 50 ug/ml carbenicillin at 37° chemical methods. C. When the Asoo reached 0.4–0.7, isopropyl-thiogalactopy ranoside (0.15 mM) and L-arabinose (1 mg/ml) were added to IV. Kits induce the expression of 6-OST-3 and chaperonin proteins, 0115 The presently disclosed subject matter further pro respectively. The cells were allowed to shake overnight at 22 vides kits for synthesizing HS-like compounds In some C. Purification was carried out using an amyloseagarose embodiments, the kit comprises at least one Sulfotransferase (New England BioLab) column following the protocols pro enzyme (e.g., at least one OST); and a reagent which modifies vided by the manufacturer. an SO-donor, e.g., adenosine 3',5'-diphosphate (PAP), to I0121 Preparation of enzymatically modified polysaccha reduce an inhibitory effect of the SO-donor on the polysac rides. N-Sulfo Heparosan was prepared by incubating chemi charide sulfation. In some embodiments of the kit the at least cally deacetylated heparosan with purified NST and the com one Sulfotransferase enzyme is contained within a first con ponents for PAPS regeneration system as described below. tainer and the reagent is contained within a second container. N-sulfo heparosan (150 ug) was incubated with SIPAPS The kit can further comprise instructions for Sulfating a (120 uM, 4.5x10 cpm/nmole) and the combination of epi polysaccharide. merase (2.7 mg if included), 2-OST (8 mg if included), 0116. In some embodiments of the kit, the at least one 6-OST-1 (1.2 mg if included), 6-OST-3 (1.4 mg. if included) sulfotransferase enzyme is an OST enzyme selected from the and 3-OST-1 (0.9 mg if included) in 50 mM MES and 1% group consisting of 2-OST, 3-OST-1,3-OST-3, 6-OST, and TritonX-100, pH 7.0. The reactions were incubated at 37° C. combinations thereof. In some embodiments, the OST for 2 hrs with shaking. The resultant S-labeled polysaccha enzyme is a recombinant OST enzyme. Such as for example a rides were purified by DEAE chromatography (Chen et al. recombinant OST enzyme produced in a bacterial expression (2005) Chemistry and Biology 12, 731-756). system. In some embodiments, the OST enzyme is a fusion 0.122 To prepare the enzymatically modified polysaccha protein. Further, in some embodiments, the OST enzyme is rides, a polysaccharide Substrate, in some embodiments an immobilized to a substrate. Such as for example an agarose N-Slsulfo oligosaccharide substrate (1 Jug, 1,500 cpm), bead. was incubated with a mixture of OST enzymes, in some 0117. In some embodiments of the kit, the reagent com embodiments 6-OST-1 (20 ug), 6-OST-3 (20 ug) and 3-OST-1 prises a PAPS regeneration system comprising a PAPS regen (10g), and SPAPS (50 uM, 2.85x10 cpm/nmole) in 100 erating enzyme (e.g., AST-IV) and a sulfo donor compound ul of 50 mM MES and 1% Triton X-100. Alternatively, a (e.g., PNPS). In other embodiments of the kit, the reagent polysaccharide Substrate (1 lug) was incubated with epime comprises a phosphatase enzyme (e.g., 3'-ribonucleotide rase (7 ug) at 37° C. for 30 min before it was incubated with phosphohydrolase). In some embodiments, human and/or a mixture of OST enzymes, in some embodiments 2-OST (40 mouse EST can be provided in place of or in conjunction with ug), 6-OST-1 (20 ug), 6-OST-3 (20 ug) and 3-OST-1 (10 ug). AST-IV. The reactions were terminated by heating at 100° C. for 2 min. 0118. In some embodiments, the kit provides for the syn The resultant sulfated polysaccharides were mixed with AT to thesis of isolated and/or purified HS-like compounds determine the bindings using a ConA-agarose column as described herein. described below. (0123 Disaccharide analysis of polysaccharides. The S EXAMPLES labeled HS was degraded with nitrous acid at pH 1.5, fol lowed by reduction with sodium borohydride (Shively et al. 0119 The following Examples provide illustrative (1976) Biochem. 15, 3932-3942). The resultant S-labeled embodiments. In light of the present disclosure and the gen disaccharides were resolved by a Cs reversed phase column eral level of skill in the art, those of skill will appreciate that (0.46x25 cm) (Vydac) under reversed phase ion pairing the following Examples are intended to be exemplary only (RPIP)HPLC conditions. The identities of the disaccharides and that numerous changes, modifications, and alterations were determined by coeluting with appropriate S-labeled can be employed without departing from the scope of the disaccharide standards (Chen et al. (2003) Glycobiology 13, presently claimed Subject matter. 785-794). 0.124 Determination of the amount of polysaccharides. Materials and Methods for Examples 1-5 The amount of the synthetic polysaccharides was estimated 0120 Expression of HS biosynthetic enzymes. Expres by a colorimetric method using alcian as described (Bjorns sions of HS biosynthetic enzymes, including NST, epimerase, son, S. (1993) Anal. Biochem. 210, 282-291), where the stan 2-OST, 6-OST-1 and 3-OST-1, were carried out in E. coli as dard curve was generated using HS isolated from bovine described previously (Chen et al. (2005) Chemistry and Biol kidney. To measure the concentration of heparosan, N-sulfo ogy 12, 731-756; Kakuta et al. (2003) Biochem. Soc. Trans. heparosan, and N-sulfo heparosan 6-O-sulfate, the polysac 31, 331-334; Edavettal et al. (2004) J. Biol. Chem. 279, charides were depolymerized to disaccharides by a mixture of 25789-25797; Munoz et al. (2006) Biochem. 45,5122-5128). heparin lyase I, II and III. The amount of the resultant disac The expression of 6-OST-3 was also carried out in E. coli. The charides was determined by coeluting with disaccharide stan catalytic domain of mouse 6-OST-3 (Pro'-Pro') was dards (from Seikagaku) on RPIP-HPLC as described above cloned into a pMalc2x vector (New England BioLab) from using UV 232 nm detection. mouse brain Quick clone cDNA library (BD Biosciences). 0.125 Antithrombin-binding. Approximately 1x05 cpm of The expression was carried out in Origami-B cells (Novagen) S-labeled compound was incubated with 5ug of human carrying pGro7 (Takara, Japan) plasmid expressing chapero AT (Cutter Biological) in 50 ul binding buffer containing 10 nin proteins GroEL and GroES of E. coli. Transformed cells mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Mn, 1 mM were grown in 6 liters of LB medium supplemented with 2 Mg", 1 mM Ca", 10 uM dextran sulfate, 0.0004% Triton US 2009/0035787 A1 Feb. 5, 2009

X-100, and 0.02% sodium azide for 30 min at room tempera I0129. PAPS regeneration system. N-terminal (His) ture. Concanavalin A (ConA)-Sepharose (Sigma, 50ll of 1:1 tagged AST-IV was expressed in E. coli and purified as slurry) was then added and the reaction was shaken at room described by Burkart and colleagues (Burkart et al. (2000).J. temperature for 1 h. The beads were then washed by 3x1 ml Org. Chem. 65,5565-5574) at a yields.50 mg/liter of bacterial binding buffer, and the bound polysaccharide was eluted with culture. 1 MNaCl. 0.130 Preparation of N-sulfo heparosan. Heparosan was isolated from E. coli K5 strain using a DEAE column (Vann 0126 Anti-Xa and anti-IIa assays. Assays were based on etal. (1981) Eur: J. Biochem. 116,359-364). The deacetylated two previous methods (Zhanget al. (2001).J. Biol. Chem. 276, hepraosan was achieved under alkaline conditions (Lindahlet 42311-42321 ; Duncan et al. (2004) Biochim Biophys Acta al. (2005) J. Med. Chem. 48, 349-352). The deacetylated 1671, 34-43). Briefly, factor Xa (Enzyme Research Labora heparosan was incubated with purified NST and coupled with tories, South Bend, Ind.) and thrombin (Sigma) were diluted a PAPS regeneration system. Briefly, 2.5 mg purified AST-IV to 1 unit/ml and 8 units/ml with PBS containing 1 mg/ml was incubated with 40 uM PAP and 1 mM p-nitrophenol BSA, respectively. AT was diluted with PBS containing 1 sulfate (PNPS) in 40 ml of 50 mM MES, pH 7.0, 1% Triton mg/ml BSA to give a stock solution at the concentration of 27 X-100, 1 mM MgCl, and 1 mM MnCl at 25° C. for 15 min. uM. The chromogenic substrates, S-2765 (for factor Xa The reaction mixture was mixed with N-sulfotransferase (10 assay) and S-2238 (for thrombin assay), were from Diap mg), 5 mg of deacetylated heparosan added and rotated at 25° harma and prepared at 1 mM with PBS. The synthesized C. for 24h. The N-sulfo heparosan was recovered by a DEAE polysaccharides or heparin was dissolved in a buffer contain column. The resultant product was subjected to a mixture of ing 50 mM Tris-HCl, pH 8.4, 7.5 mM NaEDTA, 175 mM heparin lyases digestion followed by a disaccharide analysis NaCl at various concentrations (1 ng/ml to 10,000 ng/ml). to assess the level of N-sulfation. After two rounds of the The reaction mixture, which consisted of 25 ul of AT stock modifications, the N-sulfation level reached to 80%. Solution and 25ul of the solution containing polysaccharide, I0131 Preparation of N-sulfo oligosaccharides. N-S) was incubated at 37° C. for 2 min. Factor Xa (25 Jul) or sulfoheparosan (1 mg, 1.5x10 cpm) was mixed with 20ng of thrombin (25 ul) was added. After incubating 37° C. for 4 purified heparin lyase III in 1 ml of 50 mM sodium phosphate, min, 25ul of S-2765 or S-2238 was added. The absorbance of pH 7.0. The digestion lasted for 18 hrs and was terminated by the reaction mixture was measured at 405 nm continuously heating at 100° C. for 2 min. The digested polysaccharides for 10 min. The absorbance values were plotted against the were resolved on a BIO-GEL(R) P-10 which was eluted with a reaction time. The initial reaction rates as a function of con buffer containing 25 mM Tris and 1000 mM NaCl, pH 7.4 at centration were used to calculate the ICso values. a flow rate of 2 ml/h. 0127. Affinity coelectrophoresis. The dissociation con stant (K) of each sample and AT was determined using Example 1 affinity co-electrophoresis (Lee etal. (1991) Proc. Natl. Acad. Development of an Enzyme-Based Combinatorial Sci. USA 88,2768-2772). Approximately 4000-5000 cpm of Approach to Synthesize HS-Like Compounds with antithrombin-binding S-labeled polysaccharide was loaded Unique Sulfation Patterns per lane with Zones of AT at concentrations 0, 8, 16, 60 and 120 nM. The gel was run at 400 mA for 2 hours, dried and then (0132) Biosynthesis of HS. The biosynthesis of HS analyzed on a Phosphomager (Amersham BioSciences, involves a series of specialized Sulfotransferases and other Storm 860). The retardation coefficient was calculated at enzymes. Control of the Sulfation pattern mainly depends on R=(Mo-M)/M, where Mo is the mobility of the polysaccha the substrate specificities of the enzymes involved in the ride through the Zone without AT, and M is the mobility of the biosynthetic pathway. HS is initially synthesized as a linear polysaccharide through each separation Zone. The retardation copolymer of glucuronic acid (GlcuA) and N-acetylated glu coefficient was then plotted against the retardation coefficient cosamine (GlcNAc), which then undergoes various modifi divided by its respective concentration of AT. The slope of the cations. These modifications are carried out by a series of line represents -1/K. biosynthetic enzymes, including glucosaminyl N-deacety 0128. FGF2/FGFR1C-mediated proliferation assay. BaF3 lase/N-sulfotransferase, which converts the GlcNAc unit to cells ectopically expressing FGFR1C have been previously an N-sulfoglucosamine (GlcNS) unit (FIG. 1A). After the described (Ornitz et al. (1996) J. Biol. Chem. 271, 15292 N-sulfation, Cs-epimerase converts GlcUA unit to an idu 15297). The BaF3-FGFR1c cells were maintained in RPMI ronic acid (Idol JA) unit. The resultant polysaccharide is fur 1640 media (Sigma, St. Louis, Mo.) supplemented with 10% ther modified by 2-O-sulfotransferase (2-OST), 6-O-sul fetal bovine serum, 0.5 ng/ml IL-3 (PeproTech Inc., Rocky fotransferase (6-OST) and 3-O-sulfotransferase (3-OST) to Hill, N.J.), 2 mM L-glutamine, penicillin (50 IU/ml) and introduce the 2-O-sulfo group to Idol JA/GlcUA, and the 6-O- streptomycin (50 ug/ml), and 50 uM B-mercaptoethanol. For Sulfo/3-O-sulfo groups to the glucosamine unit, respectively mitogenic assays, BaF3 FGFR1c cells were washed three (FIG. 1A). 6-OST is present in three isoforms, and 3-OST is times with RPMI 1640 media to remove IL-3 and resus present in seven isoforms. pended in the growth media lacking IL-3. About 30,000 cells I0133. The presently disclosed subject matter provides were plated per well in a 96-well plate in media containing methods for synthesizing HS-like compounds with unique various concentrations of heparin, compounds 1, 2, 7, and 8 Sulfation patterns by utilizing an enzyme-based combinato and 2 nM of FGF-2 (PeproTech) in a total volume of 200 ul. rial approach. By selectively combining collections of HS The cells were then incubated at 37°C. for 40h. To each well, biosynthetic enzymes the methods disclosed herein provide an additional 50 ul of growth media containing 1 uCi of for the synthesis of a library of HS-like compounds having Hthymidine was added. Cells were harvested after 4-5h by different sulfation patterns (FIG. 1B: Formula 1). Further, this centrifugation. The incorporation of Hthymidine into the enzyme-based combinatorial approach can be used as a gen DNA was determined by scintillation counting. eral method for discovering new HS-like compounds with US 2009/0035787 A1 Feb. 5, 2009

unique biological functions. Additionally, by Systematically cation of the substrate could be demonstrated by the low altering the sulfation pattern the function of specific sulfo Susceptibility of polysaccharide product to undergo addi groups can be elucidated. tional sulfation using SIPAPS with soluble enzymes (Chen 0134. In some embodiments, the disclosed method of syn et al. (2005) J. Biol. Chem. 280, 42817-42825). Under the thesizing of a library of HS-like compounds comprises Syn standard conditions, 2-OST, 6-OST, and 3-OST-1 afforded 98.97, and 98% complete modification, respectively, using thesizing one or more HS-like compounds chemically. Still the PAPS regeneration system (Chen et al. (2005) J. Biol. yet, in some embodiments the disclosed method of synthe Chem. 280, 42817-42825). sizing of a library of HS-like compounds comprises synthe I0137 HS Biosynthetic Enzyme Schemes. The presently sizing one or more HS-like compounds using a combination disclosed subject matter demonstrates that HS-like com of enzymatic and chemical methods. pounds having specific biological activities can be synthe 0135 Expression of HS Sulfotransferases in E. coli. The sized by Subjecting a backbone saccharide polymer to differ enzymes found in synthesizing biologically active HS-like ent enzymatic modifications. N-Sulfo heparosan was used as compounds, including NST, epimerase, and numerous OST a starting material to prepare HS-like compounds with vari enzymes, were expressed and purified as described herein. ous O-sulfation patterns (FIG. 1B). In some embodiments, Four OST enzymes, including 2-OST, 3-OST-1,6-OST-1 and N-sulfation is important for subsequent O-sulfations and/or 6-OST-3, were utilized for the synthesis of HS-like com epimerization. Eight unique HS-like compounds (com pounds. Bacterial expressed 3-OST-1 and 3-OST-3 can pounds 1-8) were prepared using the enzyme-based combi exhibit Substrate specificity and specific enzymatic activity natorial approach, wherein eight different combinations of comparable with those of their counterparts expressed in enzymes were designed. The enzyme combinations and com insect cells (Moon et al. (2004).J. Biol. Chem. 279, 45185 pounds are Summarized in Table 2. Each of compounds 1-8 45193: Edavettal et al. (2004) J. Biol. Chem. 279, 25789 comprise a different sulfation pattern, dictated by the particu 25797). Expression of the catalytic domains of 2-OST and lar OSTs used, e.g., 2-OST, 3-OST and 6-OST, as well as the 6-OST was also achieved in relatively high yield by preparing presence or absence of epimerase which converts glucuronic a fusion protein with MBP and 2-OST or 6-OST in Rosetta acid to iduronic acid (Idol JA). For example, compound 2 was gami B cells. Because 2-OST and 6-OST fusion proteins were synthesized by incubating N-Sisulfo oligosaccharide sub enzymatically active and highly soluble, the MBP domain strates, e.g. N-heparosan, (1 Jug, 1,500 cpm) with a mixture of was retained. 6-OST-1 (20 ug), 6-OST-3 (20 ug) and 3-OST-1 (10 ug) and 0.136 Introduction of PAPS Regeneration System. PAP SPAPS (50 uM, 2.85x10 cpm/nmole) in 100 ul of 50 mM inhibits sulfotransferase-catalyzed reactions. A PAPS regen MES and 1% TritonX-100. Alternatively, for the preparation eration system can be used to convert PAP to PAPS by relying of compound 8, the oligosaccharide Substrates (1 lug) were on AST-IV to catalyze the transfer of the sulfo group from incubated with epimerase (7 ug) at 37° C. for 30 min before PNPS to PAP as illustrated in FIG. 6. The presently disclosed incubation with a mixture of 2-OST (40 ug), 6-OST-1 (20g). subject matter provides for the use of the PAPS regeneration 6-OST-3 (20 ug) and 3-OST-1 (10 ug). The reactions were system coupled with O-sulfotransferases. Complete modifi terminated by heating at 100° C. for 2 min.

TABLE 2 Summary of the synthetic polysaccharides and the results of their bindings to antithrombin (AT O-Sisulfation level Binding to Binding constant to (nmolestig antithrombin affinity antithrombin (Kod) Compound Modification enzymes Structural features' polysaccharide) column (%)? (nM)

1 2-OST, 6-OST-1, No iduronic acid .4 51% 27nM 6-OST-3 and 3-OST-1 residue 2 6-OST-1, 6-OST-3 and No iduronic acid, .1 52% 57 nM 3-OST-1 no 2-O-sulfation 3 3-OST-1 and 2-OST No iduronic acid, O.9 1.0% Not determined no 6-O-sulfation 4 Epi', 2-OST and No 6-O-sulfation 3 O.7% Not determined 3-OST-1 5 Epi, 2-OST, 6-OST-1 No 3-O-sulfation .5 O.6% Not determined and 6-OST-3 6 Epi and 3-OST-1 No 2-O- and 6-O- 0.4 S.1% Not determined Sulfations 7 Epi, 6-OST-1, 6-OST-3 No 2-O-sulfation 3 29% 25 nM and 3-OST-1 8 Epi, 6-OST-1, 6-OST-3, Very similar to the .5 359 29 nM 3-OST-1 and 2-OST HS isolated from tissues These structural features are compared to heparin and HS isolated from natural sources. °The percentage of the S-labeled polysaccharide bound to AT-affinity column was determined incubating AT and the polysaccha rides. The complex of AT and polysaccharide was captured by using ConA-agarose column. The binding affinity of the polysaccharides and AT was determined using affinity coelectrophoresis. Epi = epimerase US 2009/0035787 A1 Feb. 5, 2009

Example 2 AT were determined to be in the range of 27 to 57 nM (Table 2), which are very similar to that of previously characterized Structural Characterization of Synthesized Polysac anticoagulant HS (Mufioz et al. (2006) Biochem. Biophys. charides Res. Commun. 339,597-602). 0138 Sulfation Characteristics of the Polysaccharides. 0141 Cell Proliferative Activity of the Synthetic Polysac The synthesized HS-like compounds, i.e. compounds 1-8, charides. Heparin and HS form a ternary complex with fibro were degraded and resolved using RPIP-HPLC as described blast growth factors (FGFs) and fibroblast growth factor receptors (FGFR) to stimulate cell proliferation; however, hereinabove. The overall sulfation level among the products this interaction is not related to the anticoagulant activity of was between 0.9 and 1.4 nmoles of O-sulfo groups perug of HS. The instant methods provide for the separation of the polysaccharide, namely 0.39 to 0.61 O-sulfo groups per dis anticoagulant activity and cell proliferation activity of the accharide unit as measured based on the specific Sradio synthetic HS-like compounds using the BaF3 cells model activity (Table 2). Compound 6 was an exception because it system. BaF3 cells overexpressing FGFR1 normally depend contained only 3-O-sulfation, which is an inherently low on IL-3 for growth. In the absence of IL-3, the cell prolifera abundant sulfation type in the HS isolated from a natural tion depends on the addition of both FGF and heparin or HS source (Liu et al. (2002) Med. Res. Rev. 22, 1-25). (Ornitz et al. (1996).J. Biol. Chem. 271, 15292-15297). The 0139 Disaccharide Analysis of the Polysaccharides. cells receiving heparin and compound 8 showed an increase Among the synthesized AT-binding polysaccharides, com in HIthymidine incorporation (FIG. 3). Compound 8 dis pound 2 has the simplest structure because it should contain played a lower extent in stimulating BaF3 cell growth than neither Idol JA nor 2-O-sulfo groups. To further elucidate the that of heparin since its structure is closer to HS than heparin structure of the synthesized polysaccharides, disaccharide (Chen et al. (2005).J. Biol. Chem. 280, 42817-42825). It is analysis of compound 2 was conducted and compared the also noted that compound 1 showed modest activity in stimu disaccharide composition of compound 8. The resultant dis lating cell proliferation. Compounds 2 and 7 had no detect accharides were resolved on reversed-phase ion pairing able activity in stimulating cell proliferation, Suggesting that (RPIP) HPLC (FIG. 2). Indeed, six disaccharides were the presently disclosed methods can provide for the separa observed in compound 8, while only three disaccharides were tion of the anticoagulant activity and cell proliferation activ observed in compound 2. In addition, the ratio of the three ity, such as by removing Idol JA2S and Idol JA residues from disaccharides in compound 2, including GlcUA-AnMan3S, the polysaccharide. GlcUA-AnManéS, and GlcUA-AnMan3S6S, is about 1:2:1 0142. The Anticoagulant Activity of the Synthesized (Table 3), Suggesting that this polysaccharide comprises a Polysaccharides. Heparin achieves its anticoagulant activity repeating tetrasaccharide unit with a structure of -GlcUA by forming a 1:1 complex with AT, which inhibits the activi GlcNS6S-GlcCAGlcNS3S-6S-. The results from disaccha ties of factor Xa and thrombin (Rosenberg et al. (1997) J. ride analysis also suggests that the Idol JA residue might not Clin. Invest 99, 2062-2070). The synthetic HS-like com be essential for the polysaccharide to bind to AT. pounds displayed anticoagulant activity as measured by anti Xa and anti-IIa activities (Table 4). The ICs values of com TABLE 3 pounds 1, 2, 7, and 8 for Xa are 2 to 2.5 fold higher than that of unfractionated heparin, while about 2-fold lower than that Compositional analysis of compounds 2 and 8 of LOVENOX(R), a low molecular weight heparin drug manu Compound 2 Compound 8 factured by Aventis (Aventis, Bridgewater, N.J.). A similar Disaccharides (mol/mol %) (mol/mol %) trend was observed for the anti-la activity of these com pounds. The N-sulfo heparosan 6-O-sulfate was used as a GlcUA-AnManS 24.7% N.D.* negative control showing no inhibitory effect on the activity IdoA2S-AnMan N.D. 35.9% GlcUA-AnManéS 48.8% 23.3% of Xa and IIa. Idol JA-AnManéS N.D. 15.9% Idol JA-AnMan3S6S N.D. 2.2% TABLE 4 GlcUA-AnMan3S6S 26.2% 8.9% IdoA2S-AnManéS N.D. 13.4% Anti-Xa and Anti-IIa activities of the Synthetic polysaccharides *Not detectable Anti-Xa activity, ICso Anti-IIa activity, ICso Compounds (ng ml) (ng ml) Example 3 Heparin 25 8 1 50 18 Determination of the Biological Activities of the 2 50 2O Synthesized HS-Like Compounds 7 45 16 8 40 15 0140. The Binding of the Polysaccharides to AT. Subject N-sulfo heparosan >5,000 >3,000 ing the synthetic polysaccharides to an antithrombin (AT)- 6-O-sulfate affinity column revealed that a significant portion of com LOVENOX (Rb 90 50 pounds 1, 2, 7, and 8 bound to AT, while compounds 3, 4, 5, The synthesis of N-sulfo heparosan 6-O-sulfate is displayed in Supplemen and 6 showed much less or no binding to AT (Table 2). It is tary FIG. 7. noted that 1, 2, 7, and 8 all carry 3-O- and 6-O-sulfo groups, LOVENOX (R) was purchased from a local pharmacy. while 3, 4, 5, and 6 either lacked 3-O-sulfo or 6-O-sulfo groups. Thus, both 3-O-sulfation and 6-O-sulfation of the Example 4 glucosamine unit appear to play a role in binding to AT. In Characterization of the Synthetic HS-Like Com addition, 2-O-sulfation of Idol UA is not essential, as evi pounds without Iduronic Acid denced by the ability of compound 7 to bind to AT. The 0143. As noted above, compound 2 has no Idol JA residue binding constants (KS) of the synthetic polysaccharides and but has high binding affinity to AT and strong anticoagulant US 2009/0035787 A1 Feb. 5, 2009 activity, which appears to be contradictory to previously pub before, compound 2 was synthesized from deacetylated hepa lished results. The Idol JA residue has been implicated to be rosan using sequential modifications with N-sulfotransferase essential for the binding to AT because it provides the con (NST), 6-OST-1,6-OST-3 and 3-OST-1 as illustrated in FIG. formational flexibility (Petitou et al. (2004) Angew. Chem. Int 6. In this synthesis, p-nitrophenol sulfate (PNPS) replaced Ed. 43, 31 18-3133). In the pentasaccharides, the Idol JA resi PAPS as a sulfo donor in the PAPS regeneration system (Chen due exists in either a chair (C) or a skew boat (S) confor et al., (2005).J. Biol. Chem. 280, 42817-42825: Burkartet al. mation (Petitou et al., 2004). Furthermore, the unique chair (2000).J. Org. Chem. 65,5565-5574). Disaccharide analysis conformation (So) of Idol JA2S in a synthetic pentasaccha on the intermediate compounds, including N-sulfo heparosan ride has been proven to be essential for the binding to AT (Das and N-sulfo heparosan 6-O-sulfate, revealed that N-sulfation et al. (2001) Chemistry 7, 4821-4834). However, in light of reached 75.6% and 6-O-sulfation reached 85% (Table 5). The the evidence present herein, it is believed that necessity in the 3-O-sulfation carried out by 3-OST-1 went to about 90% conformational flexibility for AT-binding affinity is size completion, and was estimated to be 0.53-O-sulfo groups/ dependent. To test this hypothesis, the AT-binding efficiency disaccharide based on the modification using SIPAPS. The of HS-like compounds prepared using the mixture of final product, renamed as Recomparin (for recombinant hep enzymes with or without the capability of synthesizing arin), showed anti-Xa activity at the ICso value of 25 ng/ml. Idol JA2S were compared. In one preparation, the oligosac which is very close to the value of heparin at 19 ng/ml (FIG. charides (for the synthesis of oligosaccharides without the 7). Idol JA2S residue) were incubated with the mixture of enzymes containing 6-OST-1,6-OST-3 and 3-OST-1. In TABLE 5 another preparation, the oligosaccharides (for the synthesis of oligosaccharides with the Idol JA2S residue) were incubated Summary of the disaccharide analysis of N-sulfo with a mixture of enzymes containing epimerase and 2-OST heparosan and N-sulfo heparosan 6-O-sulfate as well as 6-OST-1,6-OST-3 and 3-OST-1. Anti-Xa activity, ICso Anti-IIa activity, ICso 0144. The oligosaccharide substrates with structures of Compounds (ng/ml) (ng/ml) AUA-(GlcNS-GlcUA)n-GlcNS-, where n=0, 1, 2, 3, 4 and Heparin 25 8 25, were prepared by Subjecting N-sulfo heparosan to partial 1 50 18 depolymerization with heparin lyase III followed by fraction 2 50 2O ation on gel permeation chromatography (FIG. 4). After the 7 45 16 modifications, the AT-binding S-labeled oligosaccharides were captured by the AT-affinity column, and the data is shown in FIG. 5. Discussion of Examples 1-5 (0145. In the absence of epimerase and 2-OST S-labeled octa- and decasaccharides showed substantial binding to AT, 0148 HS is a major component on the mammalian cell while S-labeled hexasaccharide showed very low yield Surface and in the extracellular matrix with a wide range of (FIG. 5). In contrast, using the same oligosaccharide Sub biological functions. There is considerable interest for devel strates in the presence of epimerase and 2-OST, S-labeled oping HS-based drugs to inhibit tumor growth, bacterial viral hexasaccharides displayed significant binding to AT. As such, infections and bacterial infections and modulate inflamma it appears that the amount of AT-binding hexasaccharides and tory responses, including anticoagulation, antiviral, and anti octasaccharides is about 4 to 5 fold higher than when the cancer activities. Sulfo group-containing saccharide Substrates were modified with a mixture of enzymes contain sequences dominate the specificity of the functions of heparin ing epimerase and 2-OST. These results suggest that the con and HS. Thus, the synthesis of a polysaccharide with the tribution of the Idol JA2S on the AT-binding affinity could be appropriate positioning of these functional groups to carry less essential when the size of the HS-like compound is larger out its unique biological activity is desirable. The presently than an octasaccharide. disclosed subject matter shows that an enzymatic approach is 0146 Synthesis of HS-like compounds using epimerase a viable method for generating the polysaccharides with dif results in compounds containing a mixture of Idol JA and ferent Sulfation patterns and biological functions. The Syn GlcUA because the reaction catalyzed by epimerase is revers thesis can be conducted in parallel to produce a large number ible (Lietal. (1997).J. Biol. Chem. 272,281.58-28163). Con of compounds by varying the levels of individual enzymes. sequently, the product has greater structural heterogeneity, 0149. In summary, this present disclosure provides a novel increasing the possibility of untoward effects of an anticoagu approach to preparing HS-like compounds with unique Sul lant drug. Anticoagulant HS-like compounds without the fation patterns. The presently disclosed Subject matter pro Idol JA residues can decrease the structural heterogeneity and vides methods for enzymatic sulfation and preparation of reduce the complexity in the synthesis of HS-based antico HS-like compounds with distinct biological activities. The agulant drugs. Further, because HS is also involved in tumor presently disclosed methods demonstrate the capability of growth and viral infections, the enzyme-based synthetic using a collection of HS biosynthetic enzymes to synthesize HS-like compounds with selected biological activities. The approach can be used to prepare the HS structures displaying results presented herein also show that the enzyme-based anticancer and antiviral activities. combinatorial approach to synthesizing HS-like compounds Example 5 is fully capable of synthesizing milligram Scales of these anticoagulant polysaccharides. Large-Scale Production of HS-Like Compounds 0150. The generation of a HS library allows for the inves without Iduronic Acid Using an Enzyme-Based tigation of the contribution of each of the sulfo groups to the Combinatorial Approach biological function(s) of HS-like compounds. Utilizing this 0147 Because of its activity and reduced structural het enzyme-based combinatorial HS biosynthetic system, one of erogeneity, compound 2 was synthesized on a larger scale. As ordinary skill in the art can prepare a variety of HS structures US 2009/0035787 A1 Feb. 5, 2009

by Systematically including and/or excluding certain (0162 Bernfield, M., et al., (1999) Annu. Rev. Biochem. 68, enzymes. Unlike chemical Sulfonation approaches, the 729-777. instant methods permit the synthesis of HS-like compounds (0163 Bjornsson, S. (1993) Anal. Biochem. 210, 282-291. that are restricted with certain types of sulfations due to the (0164 Bowman et al., (1999) Chem. Biol. 6, R9-R22. high substrate specificities of sulfotransferases. More par 0.165 Burkart, M. D., Izumi, M., Chapman, E., Lin, C., ticularly, this approach permits the synthesis of biologically and Wong, C. (2000).J. Org. Chem. 65,5565-5574. active HS-like compounds that can mimic the action of HS 0166 Capila, I., and Linhardt, R.J. (2002) Angew. Chem. under physiological conditions. Int Ed. 41,390-412. 0151. As stated hereinabove, employing the enzyme (0167 Carfi, A., et al., (2001) Mol. Cell 8:169-179. based combinatorial approach to synthesize HS-like com (0168 Chen et al., (1992) Protein Expression Purif. 3, 421 pounds revealed that anticoagulant polysaccharides do not 6 require the Idol JA residue. Polysaccharides without an 0169 Chen, J., Duncan, M. B., Carrick, K., Pope, M., and Idol JA residue, i.e. Idol JA-free HS-like compounds, dis Liu, J. (2003) Glycobiology 13,785-794. played strong binding affinity to antithrombin and high anti (0170 Chen et al., (2005) J. Biol. Chem. 280, 42817 Xa and anti-IIa activities. Further, these Idol UA-free HS-like 42825. compounds have Substantially reduced activity in promoting 0171 Conrad, H. (1998) Heparin-binding Proteins, Aca cell proliferation. In some embodiments, a Substantially demic Press, San Diego, Calif. reduced activity in promoting cell proliferation can comprise (0172 Copeland et al., (2008) Biochemistry 47: 5774 a cell proliferation promoting activity of 5% or less of the cell 57.83. proliferation promoting activity of a heparin counterpart. (0173 Das et al. (2001) Chemistry 7, 4821-4834. Stated another way, Idol JA-free HS-like compounds having 0.174 Dementiev, A., Petitou, M., Herbert, J.-M., and Get Substantially reduced activity in promoting cell proliferation tins, P. G. (2004) Nat Struct Biol. 11, 867-863. can have a 95% or greater reduction in cell proliferation promoting activity as compared to a non-Idol JA-free HS-like (0175 Dooley (1998) Chemico-Biol. Interact. 109, 29. compound. As such, the presently disclosed subject matter (0176 Duncan, M. B., Chen, J., Krise, J. P., and Liu, J. provides for the synthesis of functionally specific anticoagul (2004) Biochim. Biophys. Acta 1671, 34-43. lant polysaccharides. 0177 Edavettal, S.C., Lee, K. A., Negishi, M., Linhardt, 0152 Employing the instant methods can also provide for R.J., Liu, J., and Pedersen, L. C. (2004).J. Biol. Chem. 279, the characterization of the precise structure of the anticoagul 25789-25797. lant structure without Idol JA. Characterizing the structure of (0178 Esko, J. D., and Lindahl, U. (2001).J. Clin. Invest. an anticoagulant without Idol JA can simplify the synthesis of 108:169-173. new anticoagulant drugs with reduced untoward effects. Due (0179 Esko, J. D., and Selleck, S. B. (2002) Annu. Rev. to the versatility and flexibility of the enzyme-based combi Biochem. 71, 435-471. natorial synthetic methods disclosed herein, it is believed that 0180 Falany (1997) FASEB.J. 11, 1-2. they area useful tool for identifying the lead structures for the 0181 Feyerabend et al. (2006) Nat. Chem. Biol. 2, 195 development of HS-based therapeutic agents. The presently 196 disclosed methods can aid the exploration of therapeutic 0182 Fukuta et al. (1998) Biochim. Biophys. Act. 1399, applications for HS-like compounds. In addition, enzymatic 57. synthesis of anticoagulant heparin can provide an anticoagul 0183) Fuster et al. (2005) Nat. Rev. Cancer 5,526-542. lant drug with reduced side effects. 0184 Gama et al. (2006) Nat. Chem. Biol. 2, 467-473. 0185 Gribskov et al. (1986) Nuc Acids Res 14(1), 327 REFERENCES 334. 0153. The references listed below as well as all references 0186. Guo et al. (1994) Chem.-Biol. Interact 92, 25-31. cited in the specification are incorporated herein by reference 0187. Habuchi et al., (1998).J. Biol. Chem. 273,9208. to the extent that they supplement, explain, provide a back 0188 Habuchi, H., et al., (2000).J. Biol. Chem. 275,2859 ground for or teach methodology, techniques and/or compo 2868. sitions employed herein. (0189 Kakuta et al. (2003) Biochem. Soc. Trans. 31 (pt2), 0154 Aikawa et al., (1999).J. 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SEQUENCE LISTING

<16 Oc NUMBER OF SEO ID NOS: 8

<210 SEQ ID NO 1 <211 LENGTH: 356 &212> TYPE: PRT <213> ORGANISM: Mus musculus

<4 OO SEQUENCE: 1 Met Gly Lieu. Lieu. Arg Ile Met Met Pro Pro Llys Lieu. Glin Lieu. Lieu Ala 1. 5 1O 15

Wal Wall Ala Phe Ala Wall Ala Met Lieu. Phe Lieu. Glu ASn Glin Ile Glin US 2009/0035787 A1 Feb. 5, 2009 20

- Continued

2O 25 3 O Llys Lieu. Glu Glu Ser Arg Ala Lys Lieu. Glu Arg Ala Ile Ala Arg His 35 4 O 45 Glu Val Arg Glu Ile Glu Glin Arg His Thir Met Asp Gly Pro Arg Glin SO 55 6 O Asp Ala Thir Lieu. Asp Glu Glu Glu Asp Ile Ile Ile Ile Tyr Asn Arg 65 70 7s 8O Val Pro Llys Thr Ala Ser Thr Ser Phe Thr Asn Ile Ala Tyr Asp Leu 85 9 O 95 Cys Ala Lys Asn Arg Tyr His Val Lieu. His Ile Asn. Thir Thir Lys Asn OO OS 1O Asn Pro Wal Met Ser Lieu. Glin Asp Glin Val Arg Phe Wall Lys Asn. Ile 15 2O 25 Thir Thir Trp Asn Glu Met Lys Pro Gly Phe Tyr His Gly His Ile Ser 3O 35 4 O yr Lieu. Asp Phe Ala Lys Phe Gly Val Lys Llys Llys Pro Ile Tyr Ile 45 SO 55 160 Asn Val Ile Arg Asp Pro Ile Glu Arg Lieu Val Ser Tyr Tyr Tyr Phe 65 70 7s Lieu. Arg Phe Gly Asp Asp Tyr Arg Pro Gly Lieu. Arg Arg Arg Lys Glin 8O 85 90 Gly Asp Llys Llys Thr Phe Asp Glu. CyS Val Ala Glu Gly Gly Ser Asp 95 2 OO 2O5 Cys Ala Pro Glu Lys Lieu. Trp Leu Glin Ile Pro Phe Phe Cys Gly His 210 215 22O Ser Ser Glu. Cys Trp Asn Val Gly Ser Arg Trp Ala Met Asp Glin Ala 225 23 O 235 24 O Llys Ser Asn Lieu. Ile Asn. Glu Tyr Phe Lieu Val Gly Val Thr Glu Glu 245 250 255 Lieu. Glu Asp Phe Ile Met Lieu. Lieu. Glu Ala Ala Lieu Pro Arg Val Phe 26 O 265 27 O Arg Gly Ala Thr Asp Lieu. Tyr Arg Thr Gly Lys Llys Ser His Lieu. Arg 27s 28O 285 Llys Thir Thr Glu Lys Llys Lieu Pro Thir Lys Glin Thir Ile Ala Lys Lieu. 290 295 3 OO Gln Glin Ser Asp Ile Trp Llys Met Glu Asn Glu Phe Tyr Glu Phe Ala 3. OS 310 315 32O Lieu. Glu Glin Phe Glin Phe Ile Arg Ala His Ala Val Arg Glu Lys Asp 3.25 330 335 Gly Asp Lieu. Tyr Ile Lieu Ala Glin Asn. Phe Phe Tyr Glu Lys Ile Tyr 34 O 345 350

Pro Llys Ser Asn 355

<210 SEQ ID NO 2 <211 LENGTH: 307 &212> TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO SEQUENCE: 2 Met Ala Ala Lieu Lleu Lieu. Gly Ala Val Lieu. Lieu Val Ala Glin Pro Glin 1. 5 1O 15 US 2009/0035787 A1 Feb. 5, 2009 21

- Continued Lieu Val Pro Ser Arg Pro Ala Glu Lieu. Gly Glin Glin Glu Lieu. Lieu. Arg 2O 25 3 O Lys Ala Gly. Thir Lieu. Glin Asp Asp Val Arg Asp Gly Val Ala Pro Asn 35 4 O 45 Gly Ser Ala Glin Glin Lieu Pro Glin Thir Ile Ile Ile Gly Val Arg Llys SO 55 6 O Gly Gly. Thir Arg Ala Lieu. Lieu. Glu Met Lieu. Ser Lieu. His Pro Asp Wall 65 70 7s 8O Ala Ala Ala Glu Asn. Glu Val His Phe Phe Asp Trp Glu Glu. His Tyr 85 9 O 95 Ser His Gly Lieu. Gly Trp Tyr Lieu Ser Glin Met Pro Phe Ser Trp Pro OO OS 1O His Glin Lieu. Thr Val Glu Lys Thr Pro Ala Tyr Phe Thr Ser Pro Llys 15 2O 25 Val Pro Glu Arg Val Tyr Ser Met Asn Pro Ser Ile Arg Lieu. Lieu. Lieu 3O 35 4 O le Lieu. Arg Asp Pro Ser Glu Arg Val Lieu. Ser Asp Tyr Thr Glin Val 45 SO 55 160 Phe Tyr Asn His Met Gln Lys His Llys Pro Tyr Pro Ser Ile Glu Glu 65 70 7s Phe Lieu Val Arg Asp Gly Arg Lieu. Asn Val Asp Tyr Lys Ala Lieu. Asn 8O 85 90 Arg Ser Leu Tyr His Val His Met Glin Asn Trp Lieu. Arg Phe Phe Pro 95 2 OO 2O5 Lieu. Arg His Ile His Ile Val Asp Gly Asp Arg Lieu. Ile Arg Asp Pro 210 215 22O Phe Pro Glu Ile Gln Lys Val Glu Arg Phe Leu Lys Lieu Ser Pro Glin 225 23 O 235 24 O Ile Asn Ala Ser Asn Phe Tyr Phe Asn Llys Thr Lys Gly Phe Tyr Cys 245 250 255 Lieu. Arg Asp Ser Gly Arg Asp Arg Cys Lieu. His Glu Ser Lys Gly Arg 26 O 265 27 O Ala His Pro Glin Val Asp Pro Llys Lieu. Lieu. Asn Llys Lieu. His Glu Tyr 27s 28O 285 Phe His Glu Pro Asn Lys Llys Phe Phe Glu Lieu Val Gly Arg Thr Phe 290 295 3 OO Asp Trp His 3. OS

<210 SEQ ID NO 3 <211 LENGTH: 406 &212> TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OO SEQUENCE: 3 Met Ala Pro Pro Gly Pro Ala Ser Ala Leu Ser Thr Ser Ala Glu Pro 1. 5 1O 15 Lieu. Ser Arg Ser Ile Phe Arg Llys Phe Lieu. Lieu Met Lieu. Cys Ser Lieu. 2O 25 3 O Lieu. Thir Ser Leu Tyr Val Phe Tyr Cys Lieu Ala Glu Arg Cys Glin Thr 35 4 O 45 Lieu. Ser Gly Pro Val Val Gly Lieu. Ser Gly Gly Gly Glu Glu Ala Gly SO 55 6 O US 2009/0035787 A1 Feb. 5, 2009 22

- Continued

Ala Pro Gly Gly Gly Val Lieu Ala Gly Gly Pro Arg Glu Lieu Ala Val 65 70 7s 8O rp Pro Ala Ala Ala Glin Arg Lys Arg Lieu. Lieu. Glin Lieu Pro Gln Trp 85 9 O 95 Arg Arg Arg Arg Pro Pro Ala Pro Arg Asp Asp Gly Glu Glu Ala Ala OO OS 1O Trp. Glu Glu Glu Ser Pro Gly Lieu Ser Gly Gly Pro Gly Gly Ser Gly 15 2O 25 Ala Gly Ser Thr Val Ala Glu Ala Pro Pro Gly Thr Lieu Ala Lieu. Lieu 3O 35 4 O Lieu. Asp Glu Gly Ser Lys Glin Lieu Pro Glin Ala Ile Ile Ile Gly Val 45 SO 55 160 Llys Lys Gly Gly Thr Arg Ala Lieu. Lieu. Glu Phe Lieu. Arg Val His Pro 65 70 7s Asp Val Arg Ala Val Gly Ala Glu Pro His Phe Phe Asp Arg Ser Tyr 8O 85 90 Asp Llys Gly Lieu Ala Trp Tyr Arg Asp Lieu Met Pro Arg Thr Lieu. Asp 95 2 OO 2O5 Gly Glin Ile Thr Met Glu Lys Thr Pro Ser Tyr Phe Val Thr Arg Glu 210 215 22O Ala Pro Ala Arg Ile Ser Ala Met Ser Lys Asp Thir Lys Lieu. Ile Val 225 23 O 235 24 O Val Val Arg Asp Pro Val Thr Arg Ala Ile Ser Asp Tyr Thr Glin Thr 245 250 255 Lieu. Ser Lys Arg Pro Asp Ile Pro Thr Phe Glu Ser Lieu. Thr Phe Lys 26 O 265 27 O Asn Arg Thir Ala Gly Lieu. Ile Asp Thir Ser Trp Ser Ala Ile Glin Ile 27s 28O 285 Gly Ile Tyr Ala Lys His Lieu. Glu. His Trp Lieu. Arg His Phe Pro Ile 290 295 3 OO Arg Gln Met Lieu. Phe Val Ser Gly Glu Arg Lieu. Ile Ser Asp Pro Ala 3. OS 310 315 32O Gly Glu Lieu. Gly Arg Val Glin Asp Phe Lieu. Gly Lieu Lys Arg Ile Ile 3.25 330 335 Thr Asp Llys His Phe Tyr Phe Asn Llys Thr Lys Gly Phe Pro Cys Lieu. 34 O 345 350 Llys Lys Ala Glu Gly Ser Ser Arg Pro His Cys Lieu. Gly Lys Thir Lys 355 360 365 Gly Arg Thr His Pro Glu Ile Asp Arg Glu Val Val Arg Arg Lieu. Arg 37O 375 38O Glu Phe Tyr Arg Pro Phe Asn Lieu Lys Phe Tyr Gln Met Thr Gly His 385 390 395 4 OO Asp Phe Gly Trp Asp Gly 4 OS

<210 SEQ ID NO 4 <211 LENGTH: 390 &212> TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OO SEQUENCE: 4 Met Gly Glin Arg Lieu. Ser Gly Gly Arg Ser Cys Lieu. Asp Val Pro Gly US 2009/0035787 A1 Feb. 5, 2009 23

- Continued

1. 5 1O 15 Arg Lieu. Leu Pro Gln Pro Pro Pro Pro Pro Pro Pro Val Arg Arg Lys 2O 25 3 O Lieu Ala Lieu. Lieu. Phe Ala Met Lieu. Cys Val Trp Lieu. Tyr Met Phe Lieu. 35 4 O 45 Tyr Ser Cys Ala Gly Ser Cys Ala Ala Ala Pro Gly Lieu. Lieu. Lieu. Lieu. SO 55 6 O Gly Ser Gly Ser Arg Ala Ala His Asp Pro Pro Ala Lieu Ala Thr Ala 65 70 7s 8O Pro Asp Gly Thr Pro Pro Arg Lieu Pro Phe Arg Ala Pro Pro Ala Thr 85 9 O 95 Pro Lieu Ala Ser Gly Lys Glu Met Ala Glu Gly Ala Ala Ser Pro Glu OO OS 1O Glu Glin Ser Pro Glu Val Pro Asp Ser Pro Ser Pro Ile Ser Ser Phe 15 2O 25 Phe Ser Gly Ser Gly Ser Lys Gln Leu Pro Glin Ala Ile Ile Ile Gly 3O 35 4 O Val Llys Lys Gly Gly. Thir Arg Ala Lieu. Lieu. Glu Phe Lieu. Arg Val His 45 SO 55 160 Pro Asp Val Arg Ala Val Gly Ala Glu Pro His Phe Phe Asp Arg Ser 65 70 7s yr Asp Lys Gly Lieu. Ala Trp Tyr Arg Asp Lieu Met Pro Arg Thr Lieu. 8O 85 90 Asp Gly Glin Ile Thr Met Glu Lys Thr Pro Ser Tyr Phe Val Thr Arg 95 2 OO 2O5 Glu Ala Pro Ala Arg Ile Ser Ala Met Ser Lys Asp Thir Lys Lieu. Ile 210 215 22O Val Val Val Arg Asp Pro Val Thr Arg Ala Ile Ser Asp Tyr Thr Glin 225 23 O 235 24 O Thr Lieu Ser Lys Arg Pro Asp Ile Pro Thr Phe Glu Ser Lieu. Thir Phe 245 250 255 Lys Asn Arg Thir Ala Gly Lieu. Ile Asp Thir Ser Trp Ser Ala Ile Glin 26 O 265 27 O Ile Gly Ile Tyr Ala Lys His Lieu. Glu. His Trp Lieu. Arg His Phe Pro 27s 28O 285 Ile Arg Glin Met Lieu. Phe Val Ser Gly Glu Arg Lieu. Ile Ser Asp Pro 290 295 3 OO Ala Gly Glu Lieu. Gly Arg Val Glin Asp Phe Lieu. Gly Lieu Lys Arg Ile 3. OS 310 315 32O Ile Thr Asp Llys His Phe Tyr Phe Asn Llys Thr Lys Gly Phe Pro Cys 3.25 330 335 Lieu Lys Lys Ala Glu Gly Ser Ser Arg Pro His Cys Lieu. Gly Lys Thr 34 O 345 350 Lys Gly Arg Thr His Pro Glu Ile Asp Arg Glu Val Val Arg Arg Lieu. 355 360 365 Arg Glu Phe Tyr Arg Pro Phe Asn Lieu Lys Phe Tyr Gln Met Thr Gly 37O 375 38O His Asp Phe Gly Trp Asp 385 390

<210 SEQ ID NO 5 US 2009/0035787 A1 Feb. 5, 2009 24

- Continued

<211 LENGTH: 4 O1 &212> TYPE: PRT <213> ORGANISM: Mus musculus

<4 OO SEQUENCE: 5 Met Val Glu Arg Ala Ser Llys Phe Val Lieu Val Val Ala Gly Ser Ala 1. 5 1O 15 Cys Phe Met Lieu. Ile Leu Tyr Glin Tyr Ala Gly Pro Gly Lieu. Ser Lieu. 2O 25 3 O Gly Ala Pro Gly Gly Arg Val Pro Pro Asp Asp Lieu. Asp Lieu. Phe Pro 35 4 O 45 Thr Pro Asp Pro His Tyr Glu Lys Llys Tyr Tyr Phe Pro Val Arg Glu SO 55 6 O Lieu. Glu Arg Ser Lieu. Arg Phe Asp Met Lys Gly Asp Asp Val Ile Val 65 70 7s 8O Phe Lieu. His Ile Gln Lys Thr Gly Gly Thr Thr Phe Gly Arg His Leu 85 9 O 95 Val Glin Asn Val Arg Lieu. Glu Val Pro Cys Asp Cys Arg Pro Gly Glin OO OS 1O Llys Lys Cys Thir Cys Tyr Arg Pro Asn Arg Arg Glu Thir Trp Lieu. Phe 15 2O 25 Ser Arg Phe Ser Thr Gly Trp Ser Cys Gly Lieu. His Ala Asp Trp Thr 3O 35 4 O Glu Lieu. Thir Asn. CyS Val Pro Gly Val Lieu. Asp Arg Arg Asp Pro Ala 45 SO 55 160 Gly Lieu. Arg Ser Pro Arg Llys Phe Tyr Tyr Ile Thr Lieu. Lieu. Arg Asp 65 70 7s Pro Val Ser Arg Tyr Lieu. Ser Glu Trp Arg His Val Glin Arg Gly Ala 8O 85 90 Thir Trp Llys Thr Ser Lieu. His Met Cys Asp Gly Arg Thr Pro Thr Pro 95 2 OO 2O5 Glu Glu Lieu Pro Pro Cys Tyr Glu Gly Thr Asp Trp Ser Gly Cys Thr 210 215 22O Lieu. Glin Glu Phe Met Asp Cys Pro Tyr Asn Lieu Ala Asn. Asn Arg Glin 225 23 O 235 24 O Val Arg Met Lieu Ala Asp Lieu. Ser Lieu Val Gly Cys Tyr Asn Lieu. Ser 245 250 255 Phe Ile Pro Glu Ser Lys Arg Ala Glin Lieu. Lieu. Lieu. Glu Ser Ala Lys 26 O 265 27 O Lys Asn Lieu. Arg Gly Met Ala Phe Phe Gly Lieu. Thr Glu Phe Glin Arg 27s 28O 285 Lys Thr Glin Tyr Lieu. Phe Glu Arg Thr Phe Asn Lieu Lys Phe Ile Arg 290 295 3 OO Pro Phe Met Glin Tyr Asn Ser Thr Arg Ala Gly Gly Val Glu Val Asp 3. OS 310 315 32O Glu Asp Thir Ile Arg His Ile Glu Glu Lieu. Asn Asp Lieu. Asp Met Glin 3.25 330 335 Lieu. Tyr Asp Tyr Ala Lys Asp Lieu. Phe Glin Glin Arg Tyr Glin Tyr Lys 34 O 345 350 Arg Glin Lieu. Glu Arg Arg Glu Glin Arg Lieu. Arg Asn Arg Glu Glu Arg 355 360 365 Lieu. Lieu. His Arg Ser Lys Glu Ala Lieu Pro Arg Glu Asp Pro Glu Glu US 2009/0035787 A1 Feb. 5, 2009 25

- Continued

37O 375 38O Pro Gly Arg Val Pro Thr Glu Asp Tyr Met Ser His Ile Ile Glu Lys 385 390 395 4 OO Trp

<210 SEQ ID NO 6 <211 LENGTH: 506 &212> TYPE: PRT <213> ORGANISM: Mus musculus

<4 OO SEQUENCE: 6 Met Asp Glu Lys Ser Asn Llys Lieu. Lieu. Lieu Ala Lieu Val Met Lieu. Phe 1. 5 1O 15 Lieu. Phe Ala Val Ile Val Lieu. Glin Tyr Val Cys Pro Gly Thr Glu. Cys 2O 25 3 O Gln Leu Lieu. Arg Lieu. Glin Ala Phe Ser Ser Pro Val Pro Asp Pro Tyr 35 4 O 45 Arg Ser Glu Asp Glu Ser Ser Ala Arg Phe Val Pro Arg Tyr Asn. Phe SO 55 6 O Ser Arg Gly Asp Lieu. Lieu. Arg Llys Val Asp Phe Asp Ile Lys Gly Asp 65 70 7s 8O Asp Lieu. Ile Val Phe Lieu. His Ile Gln Lys Thr Gly Gly Thr Thr Phe 85 9 O 95 Gly Arg His Lieu Val Arg Asn. Ile Glin Lieu. Glu Glin Pro Cys Glu. Cys OO OS 1O Arg Val Gly Glin Llys Lys Cys Thr Cys His Arg Pro Gly Lys Arg Glu 15 2O 25 Thir Trp Leu Phe Ser Arg Phe Ser Thr Gly Trp Ser Cys Gly Lieu. His 3O 35 4 O Ala Asp Trp Thr Glu Lieu. Thir Ser Cys Val Pro Ala Val Val Asp Gly 45 SO 55 160 Lys Arg Asp Ala Arg Lieu. Arg Pro Ser Arg Trp Arg Ile Phe Glin Ile 65 70 7s Lieu. Asp Gly. Thir Ser Lys Asp Arg Trp Gly Ser Ser Asn. Phe Asn. Ser 8O 85 90 Gly Ala Asn Ser Pro Ser Ser Thr Llys Pro Arg Ser Thr Ser Lys Ser 95 2 OO 2O5 Gly Lys Asn. Phe His Tyr Ile Thir Ile Lieu. Arg Asp Pro Val Ser Arg 210 215 22O Tyr Lieu. Ser Glu Trp Arg His Val Glin Arg Gly Ala Thir Trp Lys Ala 225 23 O 235 24 O Ser Lieu. His Val Cys Asp Gly Arg Pro Pro Thr Ser Glu Glu Lieu Pro 245 250 255 Ser Cys Tyr Thr Gly Asp Asp Trp Ser Gly Cys Pro Leu Lys Glu Phe 26 O 265 27 O Met Asp Cys Pro Tyr Asn Lieu Ala Asn. Asn Arg Glin Val Arg Met Lieu. 27s 28O 285 Ser Asp Lieu. Thir Lieu Val Gly Cys Tyr Asn Lieu Ser Val Met Pro Glu 290 295 3 OO Lys Glin Arg Asn Llys Val Lieu. Lieu. Glu Ser Ala Lys Ser Asn Lieu Lys 3. OS 310 315 32O His Met Ala Phe Phe Gly Lieu. Thr Glu Phe Glin Arg Llys Thr Glin Tyr US 2009/0035787 A1 Feb. 5, 2009 26

- Continued

3.25 330 335 Lieu. Phe Glu Lys Thr Phe Asn Met Asin Phe Ile Ser Pro Phe Thr Glin 34 O 345 350 Tyr Asn. Thir Thr Arg Ala Ser Ser Val Glu Ile Asn Glu Glu Ile Glin 355 360 365 Lys Arg Ile Glu Gly Lieu. Asn. Phe Lieu. Asp Met Glu Lieu. Tyr Ser Tyr 37O 375 38O Ala Lys Asp Lieu. Phe Lieu. Glin Arg Tyr Glin Phe Met Arg Glin Lys Glu 385 390 395 4 OO His Glin Asp Ala Arg Arg Lys Arg Glin Glu Glin Arg Llys Phe Lieu Lys 4 OS 410 415 Gly Arg Phe Leu Gln Thr His Phe Glin Ser Glin Ser Glin Gly Glin Ser 42O 425 43 O Gln Ser Glin Ser Pro Gly Glin Asn Lieu Ser Glin Asn Pro Asn Pro Asn 435 4 4 O 445

Pro ASn Glin Asn Lieu. Thir Glin Asn Lieu. Ser His Asn Lieu. Thr Pro Ser 450 45.5 460 Ser Asn Pro Asn. Ser Thr Glin Arg Glu Asn Arg Gly Ser Glin Lys Glin 465 470 47s 48O Gly Ser Gly Glin Gly Glin Gly Asp Ser Gly. Thir Ser Asn Gly Thr Asn 485 490 495 Asp Tyr Ile Gly Ser Val Glu Thir Trp Arg 5 OO 5 OS

<210 SEQ ID NO 7 <211 LENGTH: 470 &212> TYPE: PRT <213> ORGANISM: Mus musculus

<4 OO SEQUENCE: 7 Met Asp Glu Arg Phe Asn Lys Trp Leu Lieu. Thr Pro Val Lieu. Thr Phe 1. 5 1O 15 Lieu. Phe Val Val Ile Met Tyr Glin Tyr Val Ser Pro Ser Cys Thir Ser 2O 25 3 O Ser Cys Thr Asn. Phe Gly Glu Glin Lieu. Arg Ser Gly Glu Ala Arg Pro 35 4 O 45 Pro Ala Val Pro Ser Pro Ala Arg Arg Ala Glin Ala Pro Lieu. Asp Glu SO 55 6 O Trp. Glu Arg Arg Pro Gln Leu Pro Pro Pro Pro Arg Gly Pro Pro Glu 65 70 7s 8O Gly Ser Arg Gly Val Ala Ala Pro Glu Asp Glu Asp Glu Asp Pro Gly 85 9 O 95 Asp Pro Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Pro Asp Pro Glu 1 OO 105 11O Ala Pro Glu Asn Gly Ser Leu Pro Arg Phe Val Pro Arg Phe Asin Phe 115 12O 125 Thir Lieu Lys Asp Lieu. Thir Arg Phe Val Asp Phe Asn. Ile Lys Gly Arg 13 O 135 14 O Asp Val Ile Val Phe Lieu. His Ile Gln Lys Thr Gly Gly Thr Thr Phe 145 150 155 160 Gly Arg His Lieu Val Lys Asn. Ile Arg Lieu. Glu Glin Pro Cys Ser Cys 1.65 17O 17s US 2009/0035787 A1 Feb. 5, 2009 27

- Continued Lys Ala Gly Glin Llys Lys Cys Thr Cys His Arg Pro Gly Lys Lys Glu 18O 185 190 Thir Trp Leu Phe Ser Arg Phe Ser Thr Gly Trp Ser Cys Gly Lieu. His 195 2 OO 2O5 Ala Asp Trp Thr Glu Lieu. Thir Asn. Cys Val Pro Ala Ile Met Glu Lys 210 215 22O Lys Asp Cys Pro Arg Asn His Ser His Thr Arg Asn Phe Tyr Tyr Ile 225 23 O 235 24 O Thir Met Lieu. Arg Asp Pro Val Ser Arg Tyr Lieu. Ser Glu Trp Llys His 245 250 255 Val Glin Arg Gly Ala Thir Trp Llys Thir Ser Lieu. His Met Cys Asp Gly 26 O 265 27 O Arg Ser Pro Thr Pro Asp Glu Lieu Pro Thr Cys Tyr Pro Gly Asp Asp 27s 28O 285 Trp Ser Gly Val Ser Lieu. Arg Glu Phe Met Asp Cys Ser Tyr Asn Lieu. 290 295 3 OO Ala Asn. Asn Arg Glin Val Arg Met Lieu Ala Asp Lieu. Ser Lieu Val Gly 3. OS 310 315 32O Cys Tyr Asn Lieu. Thr Phe Met Asn Glu Ser Glu Arg Asn. Thir Ile Leu 3.25 330 335 Lieu. Glin Ser Ala Lys Asn. Asn Lieu Lys Asn Met Ala Phe Phe Gly Lieu. 34 O 345 350 Thr Glu Phe Glin Arg Llys Thr Glin Phe Leu Phe Glu Arg Thr Phe Asn 355 360 365 Lieu Lys Phe Ile Ser Pro Phe Thr Glin Phe Asn Ile Thr Arg Ala Ser 37O 375 38O Asn Val Asp Ile Asn Asp Gly Ala Arg Glin His Ile Glu Glu Lieu. Asn 385 390 395 4 OO Phe Lieu. Asp Met Glin Lieu. Tyr Glu Tyr Ala Lys Asp Lieu. Phe Glin Glin 4 OS 410 415 Arg Tyr His His Thr Lys Glin Lieu. Glu. His Glin Arg Asp Arg Gln Lys 42O 425 43 O Arg Arg Glu Glu Arg Arg Lieu. Glin Arg Glu. His Arg Ala His Arg Trip 435 4 4 O 445 Pro Lys Glu Asp Arg Ala Met Glu Gly Thr Val Thr Glu Asp Tyr Asn 450 45.5 460 Ser Glin Val Val Arg Trp 465 470

<210 SEQ ID NO 8 <211 LENGTH: 291 &212> TYPE: PRT <213> ORGANISM: Rattus norvegicus

<4 OO SEQUENCE: 8 Met Glu Phe Ser Arg Pro Pro Leu Val His Val Lys Gly Ile Pro Leu 1. 5 1O 15 Ile Llys Tyr Phe Ala Glu Thir Ile Gly Pro Leu Gln Asn Phe Thr Ala 2O 25 3 O Trp Pro Asp Asp Lieu. Lieu. Ile Ser Thr Tyr Pro Llys Ser Gly. Thir Thr 35 4 O 45 Trp Met Ser Glu Ile Lieu. Asp Met Ile Tyr Glin Gly Gly Lys Lieu. Glu SO 55 6 O US 2009/0035787 A1 Feb. 5, 2009 28

- Continued

Cys Gly Arg Ala Ile Ala Arg Pro Phe Lieu. Glu Phe 8O

Cys Pro Gly Val Ser Gly Lieu. Glu T Lieu. Glu Glu Thir Pro

Pro Arg Lieu. Lieu. Thir His Lieu. Pro Ser Luell Luell Pro Glin

Lieu. Lieu. Asp Glin Wall Wall Ile T Ile Ala Arg Asn Ala

Asp Val Val Val Asn. Phe T As yet Ala Luell

Pro Asp Pro Gly T Trp Asp Ser Phe Glu Asn Phe Met Asp 160

Glu Wal Ser Ser Trp Glin Wall Glu Trp Trp

Lieu. Arg His Thr Pro Val Lieu. Tyr Phe Glu Asp Ile

Glu Asn. Pro Glu Ile Lys Lieu. Glu Phe Lieu. Gly

Ser Lieu. Pro Glu Glu Thr Val Asp Ser Wal His His Thir Ser 215

Phe Lys Met Glu Asn Met Thr Thir Thir Ile Pro 225 23 O 24 O

Thir Glu Ile Met His ASn Wal Ser Pro Met Arg Lys Gly Thr 245 250

Thir Gly Asp Trp Asn Thir Phe Thir Wall Ala Glin Asn Glu Arg Phe 26 O 265 27 O

Asp Ala His Ala Lys Thir Met Thr Asp Cys Asp Phe Lys Phe Arg 28O 285 Cys Glu Lieu. 290

What is claimed is: 4. The Idol JA-free HS-like compound of claim 2, having a 1. An iduronic acid (Idol JA)-free heparan sulfate (HS)-like binding affinity to antithrombin ranging from about 20 to compound comprising the following structure: about 60 nM. 5. The Idol JA-free HS-like compound of claim 2, having COOH CHOR L2 L L2 an anti-Xa activity ranging from about 10 to about 500 ng/ml. 1N O 1 N. O 1 N. 6. The Idol JA-free HS-like compound of claim 2, having an anti-IIa activity ranging from about 5 to about 200 ng/ml. -Y or, Y or, -Y- 7. The Idol JA-free HS-like compound of claim 2, wherein the HS-like compound has substantially reduced cell prolif H H H H eration activity as compared to a HS-like compound with H OR iduronic acid residues. 8. The Idol JA-free HS-like compound of claim 1, wherein wherein X is NH or O, R is H, SOH or CH, R is SOH, R R is selected from the group consisting of CH, CHCH and is Hor SOH, R is H, SOH, CH or CHCHRs is H, SOH, SOH and Rs is selected from the group consisting of CH, CH or CHCH R is COOH, COOCH, COOCHs. CHCH and SOH. CHOSOH, or CHSOH, L is an alpha or beta linkage, L. 9. The Idol JA-free HS-like compound of claim 1, wherein is an alpha or beta linkage, Y is O or N, and n is 24. X is O and R is SOH. 2. The Idol JA-free HS-like compound of claim 1, wherein 10. The Idol JA-free HS-like compound of claim 1, wherein X is NH, R is SOH, R is SOH, R is SOH, R is H or L is an alpha linkage and L2 is an alpha linkage. SOH. Rs is H. L. is an alpha or a beta linkage, L is an alpha 11. The Idol JA-free HS-like compound of claim 1, wherein or a beta linkage, and n is 24. L is a beta linkage and L is a beta linkage. 3. The Idol JA-free HS-like compound of claim 2, wherein 12. A method of synthesizing an Idol JA-free HS-like com L is an alpha linkage and L is a beta linkage. pound, comprising incubating a polysaccharide Substrate US 2009/0035787 A1 Feb. 5, 2009 29 with a Sulfo donor compound and an enzyme mixture, the 34. The method of claim 28, further comprising: enzyme mixture comprising O-sulfotransferase (OST) (c) providing a reaction mixture comprising adenosine enzymes 6-OST-1,6-OST-3 and 3-OST-1 and substantially no 3',5'-diphosphate (PAP), a PAPS regenerating enzyme epimerase enzyme, wherein synthesis of the Idol JA-free HS and a sulfo donor compound; and like compound from the polysaccharide Substrate is accom (d) incubating the reaction mixture for a time period suffi plished. cient to catalyze the production of 3'-phosphoadenosine 13. The method of claim 12, wherein the polysaccharide 5'-phosphosulfate (PAPS), Substrate is selected from the group consisting of N-sulfo whereby the sulfo donor compound is provided. heparosan and chemically desulfated N-sulfated heparin. 35. The method of claim 34, wherein the PAPS regenerat 14. The method of claim 12, wherein the OST enzymes are ing enzyme is an arylsulfotransferase. recombinant OST enzymes. 36. The method of claim 35, wherein the arylsulfotrans 15. The method of claim 14, wherein the recombinant OST ferase is AST-IV. enzymes are produced in a bacterial expression system. 37. The method of claim 34, wherein the PAPS regenerat 16. The method of claim 12, wherein the sulfo donor com ing enzyme is an estrogen Sulfotransferase. pound comprises a compound capable of donatinga Sulfonate 38. The method of claim 34, wherein the sulfo donor com or Sulfuryl group. pound is an aryl Sulfate compound. 17. The method of claim 12, wherein the polysaccharide 39. The method of claim 38, wherein the aryl sulfate com substrate is partially sulfated prior to incubation. pound is p-nitrophenol sulfate (PNPS). 18. The method of claim 12, further comprising: 40. The method of claim 34, wherein the time period is (a) providing a reaction mixture comprising adenosine from about 1 minute to about 30 minutes. 3',5'-diphosphate (PAP), a PAPS regenerating enzyme 41. The method of claim 34, wherein the polysaccharide and a sulfo donor compound; and substrate is a chemically desulfated N-sulfated heparin. (b) incubating the reaction mixture for a time period suffi 42. A method of determining a mechanism of activity of cient to catalyze the production of 3'-phosphoadenosine HS-like compounds, the method comprising: 5'-phosphosulfate (PAPS) from the PAP by the PAPS (e) synthesizing one or more HS-like compounds by incu regenerating enzyme utilizing the Sulfo donor com bating one or more polysaccharide Substrates with a pound as a Sulfo Substrate, Sulfo donor compound and one or more combinations of whereby the sulfo donor compound is provided. biosynthetic enzymes, the biosynthetic enzymes com 19. The method of claim 18, wherein the PAPS regenerat prising epimerases, N-sulfotransferases and O-sul ing enzyme is an arylsulfotransferase. fotransferases; 20. The method of claim 19, wherein the arylsulfotrans (f) subjecting one or more of the HS-like compounds to a ferase is AST-IV. test for an activity; and 21. The method of claim 18, wherein the PAPS regenerat (g) determining a mechanism of activity of the HS-like ing enzyme is an estrogen Sulfotransferase. compounds based on one or more results of the one or 22. The method of claim 18, wherein the sulfo donor com more HS-like compounds. pound is an aryl Sulfate compound. 43. The method of claim 42, wherein the polysaccharide 23. The method of claim 22, wherein the aryl sulfate com Substrate is selected from the group consisting of N-sulfo pound is p-nitrophenol sulfate (PNPS). heparosan and chemically desulfated N-sulfated heparin. 24. The method of claim 18, wherein the time period is 44. The method of claim 42, wherein the O-sulfotrans from about 1 minute to about 30 minutes. ferases are selected from the group consisting of 2-OSTs, 25. The method of claim 18, wherein the OST enzymes 3-OSTs and 6-OSTs. further comprise 2-OST. 45. The method of claim 42, wherein the O-sulfotrans 26. The method of claim 25, wherein the 2-OST enzyme is ferase enzymes are recombinant OST enzymes. a recombinant OST enzyme. 46. The method of claim 45, wherein the recombinant OST 27. The method of claim 26, wherein the recombinant OST enzymes are produced in a bacterial expression system. enzyme is produced in a bacterial expression system. 47. The method of claim 42, wherein the polysaccharide 28. A method of synthesizing a library of HS-like com substrate is partially sulfated prior to incubation. pounds, the method comprising incubating polysaccharide 48. The method of claim 42, wherein the test is for an Substrates with a Sulfo donor compound and one or more activity selected from the group consisting of anti-thrombin combinations of biosynthetic enzymes, the biosynthetic binding activity, anti-Xa activity, anti-IIa activity, cell prolif enzymes comprising epimerases, N-sulfotransferases and eration stimulation activity, cell growth stimulation activity, O-sulfotransferases. activated partial thromboplastin time, prothrombin time and 29. The method of claim 28, wherein the polysaccharide combinations thereof. substrate is N-sulfo heparosan. 49. The method of claim 42, further comprising: 30. The method of claim 28, wherein the O-sulfotrans (h) providing a reaction mixture comprising adenosine ferases are selected from the group consisting of 2-OSTs, 3',5'-diphosphate (PAP), a PAPS regenerating enzyme 3-OSTs and 6-OSTs. and a sulfo donor compound; and 31. The method of claim 28, wherein the O-sulfotrans (i) incubating the reaction mixture for a time period suffi ferase enzymes are recombinant OST enzymes. cient to catalyze the production of 3'-phosphoadenosine 32. The method of claim31, wherein the recombinant OST 5'-phosphosulfate (PAPS), enzymes are produced in a bacterial expression system. whereby the sulfo donor compound is provided. 33. The method of claim 28, wherein the polysaccharide 50. The method of claim 49, wherein the PAPS regenerat substrate is partially sulfated prior to incubation. ing enzyme is an arylsulfotransferase. US 2009/0035787 A1 Feb. 5, 2009 30

51. The method of claim 50, wherein the arylsulfotrans- 54. The method of claim 53, wherein the aryl sulfate com ferase is AST-IV. pound is p-nitrophenol sulfate (PNPS). 52. The method of claim 49, wherein the PAPS regenerat- 55. The method of claim 49, wherein the time period is ing enzyme is an estrogen Sulfotransferase. from about 1 minute to about 30 minutes. 53. The method of claim 49, wherein the sulfo donor com pound is an aryl Sulfate compound. ck