Methods for synthesizing anticoagulant polysaccharides

1. A method of enzymatically synthesizing an N-, 2-O, 3-O, 6-O sulfated, heparan sulfate (N,2,3,6-HS) product, the method comprising the following steps:

(a) providing a starting polysaccharide mixture comprising N-sulfated heparosan;

(b) combining the starting polysaccharide reaction mixture with a reaction mixture comprising a first sulfo group donor and a first sulfotransferase enzyme selected from the group consisting of a hexuronyl 2-O sulfotransferase enzyme (2OST) enzyme and a glucosaminyl 6-O sulfotransferase enzyme (6OST) enzyme, to form a first sulfated polysaccharide product;

(c) combining the first sulfated polysaccharide product with a reaction mixture comprising a second sulfo group donor and a second sulfotransferase enzyme, wherein the second sulfotransferase enzyme is the enzyme that was not selected in step (b), to form a second sulfated polysaccharide product;

(d) combining the second sulfated polysaccharide product with a reaction mixture comprising a third sulfo group donor and a glucosaminyl 3-O sulfotransferase enzyme (3OST) enzyme, to form the N,2,3,6-HS product;

wherein the sulfotransferase enzyme in each of steps (b), (c), and (d) is selected from the group consisting of a natural sulfotransferase enzyme and an engineered sulfotransferase enzyme,

wherein a natural sulfotransferase enzyme has sulfotransferase activity with 3′-phosphoadenosine 5′-phosphosulfate (PAPS) as a sulfo group donor and heparan sulfate as a sulfo group acceptor, and when a natural sulfotransferase enzyme is selected in any of steps (b), (c), or (d), the sulfo group donor is PAPS;

wherein an engineered sulfotransferase enzyme has sulfotransferase activity with an aryl sulfate compound as a sulfo group donor and heparan sulfate as a sulfo group acceptor, and when an engineered sulfotransferase enzyme is selected in any of steps (b), (c), or (d), the sulfo group donor is an aryl sulfate compound; and

wherein at least one of the sulfotransferase enzymes selected in steps (b), (c), and (d) is an engineered sulfotransferase enzyme.

2. The method of claim 1 , wherein the first sulfotransferase enzyme is the 2OST enzyme, and the second sulfotransferase enzyme is the 6OST enzyme.

3. The method of claim 2 , wherein the 3OST enzyme is an engineered sulfotransferase enzyme.

4. The method of claim 3 , wherein the third sulfo group donor is selected from the group consisting of p-nitrophenyl sulfate (PNS) and 4-nitrocatechol sulfate (NCS).

5. The method of claim 4 , wherein the 3OST enzyme comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58.

6. The method of claim 3 , wherein at least one of the 2OST enzyme and the 6OST enzyme is an engineered sulfotransferase enzyme.

7. The method of claim 6 , wherein the 6OST enzyme is an engineered sulfotransferase enzyme.

8. The method of claim 7 , wherein the second sulfo group donor is selected from the group consisting of PNS and NCS.

9. The method of claim 8 , wherein the 6OST enzyme comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61.

10. The method of claim 6 , wherein the 2OST enzyme is an engineered sulfotransferase enzyme.

11. The method of claim 10 , wherein the first sulfo group donor is selected from the group consisting of PNS and NCS.

12. The method of claim 11 , wherein the 2OST enzyme comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 41, and SEQ ID NO: 42.

13. The method of claim 2 , wherein the first sulfotransferase reaction mixture further comprises a glucuronyl C 5 -epimerase enzyme.

14. The method of claim 1 , wherein at least a portion of the glucosamine residues within the N-sulfated heparosan are N-acetylated.

15. The method according to claim 1 , wherein the N,2O,3O,6O-HS product is a heparin product, the heparin product comprising polysaccharides having a sequence motif having the structure of Formula I, below:

wherein X is either a sulfo group or an acetate group and Y is either a sulfo group or a hydroxyl group.

16. The method according to claim 15 , wherein the heparin product has anticoagulant activity and is a polydisperse mixture of polysaccharides having a weight average molecular weight, M w , of at least 1,000 Da.

17. The method according to claim 15 , wherein the heparin product is further fractionated to form a low-molecular weight heparin (LMWH) product, the method further comprising the steps:

(e) providing one or more depolymerization agents; and

(f) treating the heparin product with the one or more depolymerization agents for a time sufficient to depolymerize at least some of the polysaccharides within the heparin product, thereby forming the LMWH product.

18. The method according to claim 17 , wherein one or more depolymerization agents are selected from the group consisting of a β-elimination agent, a deamination agent, and an oxidation agent,

wherein the β-elimination agent is selected from the group consisting of:

a carbon-oxygen lyase reaction mixture comprising at least one carbon-oxygen lyase enzyme, and

one or more basic compounds selected from the group consisting of sodium hydroxide, a quaternary ammonium hydroxide, and a phosphazene base,

wherein the deamination agent is selected from the group consisting of isoamyl nitrate and nitrous acid; and

wherein the oxidizing agent is selected from the group consisting of a peroxide or superoxide compound.

19. The method according to claim 18 , wherein the M w of the LMWH product is in a range from at least about 2,000 Da, and up to about 12,000 Da.

20. The method according to claim 19 , wherein the LMWH product has anticoagulant activity.