Reversible heparin molecules and methods of making and using the same

1. A method of synthesizing a heparin compound, comprising: providing a monosaccharide substrate;

elongating the monosaccharide substrate to a hexasaccharide using enzymes N-acetyl glucosaminyl transferase and heparosan synthase-2, and substrates glucuronic acid (GlcUA) and N-trifluoroacetyl glucosamine (GlcNTFA);

converting N-trifluoroacetyl glucosamine (GlcNTFA) residue(s) on the hexasaccharide to N-sulfo glucosamine (GlcNS) residues using N-sulfotransferase (NST), 3′-phosphoadenosine 5′-phosphosulfate (PAPS), and a base to provide an N-sulfated hexasaccharide substrate;

epimerizing the N-sulfated hexasaccharide substrate using C 5 -epimerase (C 5 -epi); sulfating the resultant epimerized hexasaccharide using 2-O-sulfotransferase (2-OST) and 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to provide the following hexasaccharide substrate:

wherein MS is a monosaccharide;

elongating the hexasaccharide substrate to a heptasaccharide using heparosan synthase-2 and glucuronic acid (GlcUA);

elongating the heptasaccharide to an octasaccharide using N-acetyl glucosaminyl transferase and-N-trifluoroacetyl glucosamine (GlcNTFA); epimerizing the resultant octasaccharide using C 5 -epimerase (C 5 -epi); sulfating the resultant epimerized octasaccharide using 2-O-sulfotransferase (2-OST) and 3′-phosphoadenosine 5′-phosphosulfate (PAPS);

converting N-trifluoroacetyl glucosamine (GlcNTFA) residue(s) to N-sulfo glucosamine (GlcNS) residues using N-sulfotransferase (NST), 3′-phosphoadenosine 5′-phosphosulfate (PAPS), and a base to produce an octasaccharide substrate;

elongating the octasaccharide substrate to a decasaccharide using in the following order:

(1) heparosan synthase-2 and glucuronic acid (GlcUA); and

(2) N-acetyl glucosaminyl transferase and N-trifluoroacetyl glucosamine (GlcNTFA);

epimerizing the decasaccharide using C 5 -epimerase (C 5 -epi); sulfating the resultant epimerized decasaccharide using 2-O-sulfotransferase (2-OST) and 3′-phosphoadenosine 5′-phosphosulfate (PAPS);

converting N-trifluoroacetyl glucosamine (GlcNTFA) residue(s)on the resultant sulfated decasaccharide to N-sulfo glucosamine (GlcNS) residues using N-sulfotransferase (NST), 3′-phosphoadenosine 5′-phosphosulfate (PAPS), and a base to produce a decasaccharide substrate;

elongating the decasaccharide substrate to a dodecasaccharide using in the following order:

1) heparosan synthase-2 and glucuronic acid (GlcUA); and

(2) N-acetyl glucosaminyl transferase and N-trifluoroacetyl glucosamine (GlcNTFA) or N-acetylglucosamine (NAcGlc);

epimerizing the dodecasaccharide using C 5 -epimerase (C 5 -epi); sulfating the epimerized dodecasaccharide by the following reactions:

(1) using 2-O-sulfotransferase (2-OST) and 3′-phosphoadenosine 5′-phosphosulfate (PAPS);

(2) using a 6-O-sulfotransferase (6-OST) and 3′-phosphoadenosine 5′-phosphosulfate (PAPS); and

(3) using a 3-O-sulfotransferase (3-OST) and 3′-phosphoadenosine 5′-phosphosulfate (PAPS);

wherein a heparin compound is synthesized.

2. The method of claim 1 , wherein the base is lithium hydroxide.

3. The method of claim 1 , wherein the 6-O-sulfotransferase (6-OST) is 6-O-sulfotransferase 1 and/or 3 (6-OST-1and/or 6-OST-3).

4. The method of claim 1 , wherein the 3-OST is 3-O-sulfotransferase-5 (3-OST-5).

5. The method of claim 1 , wherein the glycosyl transferase is selected from the group consisting of N-acetyl glucosaminyl transferase of E. coli K5 (KfiA) and/or heparosan synthase-2 (pmHS2) from Pasteurella multocida .

6. A method of synthesizing a heparin compound, comprising: providing a hexasaccharide represented by the following structure:

wherein R 2 is H or a detectable tag,

elongating the hexasaccharide to a heptasaccharide using heparosan synthase-2 and glucuronic acid (GlcUA);

elongating the heptasaccharide to an octasaccharide using N-acetyl glucosaminyl transferase and N-trifluoroacetyl glucosamine (GlcNTFA);

epimerizing the octasaccharide using C 5 -epimerase (C 5 -epi); sulfating the resultant epimerized octasaccharide using 2-O-sulfotransferase (2-OST) and 3′-phosphoadenosine 5′-phosphosulfate (PAPS);

converting N-trifluoroacetyl glucosamine (GlcNTFA) residue(s) on the resultant sulfated octasaccharide to N-sulfo glucosamine (GlcNS) residues using N-sulfotransferase (NST), 3′-phosphoadenosine 5′-phosphosulfate (PAPS), and a base to provide an octasaccharide substrate;

elongating the octasaccharide substrate to a decasaccharide using heparosan synthase-2 and N-acetyl glucosaminyl transferase and substrates glucuronic acid (GlcUA) and N-trifluoroacetyl glucosamine (GlcNTFA);

epimerizing the decasaccharide using C 5 -epimerase (C 5 -epi); sulfating the resultant epimerized decasaccharide using 2-O-sulfotransferase (2-OST) and 3′-phosphoadenosine 5′-phosphosulfate (PAPS);

converting N-trifluoroacetyl glucosamine (GlcNTFA) residue(s) on the resultant sulfated decasaccharide to N-sulfo glucosamine (GIcNS) residues using N-sulfotransferase (NST), 3′-phosphoadenosine 5′-phosphosulfate (PAPS), and a base to provide a decasaccharide substrate;

elongating the decasaccharide substrate to a dodecasaccharide using heparosan synthase-2 and N-acetyl glucosaminyl transferase and substrates glucuronic acid (GlcUA) and N-acetyl glucosamine (NAcGlc);

epimerizing the dodecasaccharide using C 5 -epimerase (C 5 -epi); sulfating the resultant epimerized dodecasaccharide using:

(1) 2-O-sulfotransferase (2-OST) and 3′-phosphoadenosine 5′-phosphosulfate PAPS);

(2) 6-O-sulfotransferase 1 and/or 3 (6-OST-1 and/or 6-OST-3), and 3′-phosphoadenosine 5′-phosphosulfate (PAPS); and

(3) 3-O-sulfotransferase 1 (3-OST-1), and 3′-phosphoadenosine 5′-phosphosulfate (PAPS);

wherein a heparin compound represented by the following structure is synthesized:

wherein R 2 is H or a detectable tag.

7. The method of claim 6 , wherein the synthesized heparin compound is a synthetic, low-molecular weight heparin compound with reversible anticoagulant activity, wherein the anticoagulant activity of the heparin compound is reversible by protamine.

8. The method of claim 6 , wherein the base is lithium hydroxide.

9. The method of claim 6 , wherein the glycosyl transferase is selected from the group consisting of N-acetyl glucosaminyl transferase of E. coli K5 (KfiA) and/or heparosan synthase-2 (pmHS2) from Pasteurella multocida .

10. The method of claim 6 , wherein the detectable tag is para-nitrophenyl.

11. A low-molecular weight heparin compound, which is a synthesized material, with reversible anticoagulant activity, represented by the following formula:

wherein R 1 is independently H or —SO 3 H, R 2 is H or a detectable tag, R 3 is —NHSO 3 H or —NHCOCH 3 , and n is an integer of 0-3.

12. The heparin compound of claim 11 , wherein the heparin compound is represented by the following structure:

wherein R 2 is H or a detectable tag.

13. The heparin compound of claim 11 , wherein the heparin compound is represented by the following structure:

wherein R 2 is H or a detectable tag.

14. The heparin compound of claim 11 , wherein the heparin compound comprises at least one 3-O sulfate.

15. The heparin compound of claim 11 , wherein the heparin compound is a dodecamer.

16. The heparin compound of claim 11 , wherein the detectable tag is paranitrophenyl.

17. A method of treating a subject, the method comprising:

providing a subject to be treated;

administering to the subject a heparin compound having anticoagulant activity, wherein the heparin compound is selected from:

wherein R 2 is H or a detectable tag, and wherein the anticoagulant activity of the heparin compound is reversible by protamine.

18. The method of claim 17 , wherein the subject suffers from venous thromboembolism.

19. The method of claim 17 , wherein the subject is renal-impaired.

20. A method of treating a subject in need of anticoagulant therapy, the method comprising:

providing a subject in need of anticoagulant therapy;

administering to the subject a heparin compound having anticoagulant activity; monitoring the subject for heparin-induced thrombocytopenia; and administering to the subject an antidote to reverse the anticoagulant activity of the heparin compound if the subject suffers from heparin-induced thrombocytopenia, wherein the heparin compound is selected from:

wherein R 2 is H or a detectable tag.

21. The method of claim 20 , wherein the antidote to reverse the anticoagulant activity of the heparin compound is protamine.