Chemoenzymatic synthesis of structurally homogeneous ultra-low molecular weight heparins
View Patent ↗Methods for preparing synthetic heparins are provided. Synthetic heparin compounds, including ultralow molecular weight heparin compounds are provided. Also provided are methods of chemoenzymatically synthesizing structurally homogeneous ultra-low molecular weight heparins. Heparin compounds provided herein can have anticoagulant activity.
1. A method of synthesizing a heparin compound, comprising the following sequential steps while monitoring the steps:
providing a monosaccharide substrate comprising glucuronic acid;
elongating the monosaccharide substrate to a tetrasaccharide using enzymes N-acetyl glucosaminyl transferase and heparosan synthase-2, and substrates glucuronic acid (GlcUA) and N-trifluoroacetyl glucosamine (GIcNTFA), wherein the tetrasaccharide has the following structure:
elongating the tetrasaccharide to a heptasaccharide using enzymes N-acetyl glucosaminyl transferase and heparosan synthase-2, and substrates glucuronic acid (GlcUA), N-trifluoroacetyl glucosamine (GIcNTFA), and N-acetylated glucosamine (GlcNAc), wherein the heptasaccharide has the following structure:
converting N-trifluoroacetyl glucosamine (GIcNTFA) residue(s) on the heptasaccharide to N-sulfo glucosamine (GlcNS) residues using N-sulfotransferase (NST), 3′-phosphoadenosine 5′-phosphosulfate (PAPS), triethylamine, CH 3 OH, and H 2 O;
epimerizing and sulfating the resultant heptasaccharide using C 5 -epimerase (C 5 -epi) and 2-O-sulfotransferase (2-OST) and 3′-phosphoadenosine 5′-phosphosulfate (PAPS);
sulfating the resultant heptasaccharide using 6-O-sulfotransferase-1 (6-OST-1) and 6-O-sulfotransferase-3 (6-OST-3) and 3′-phosphoadenosine 5′-phosphosulfate (PAPS);
sulfating the resultant heptasaccharide using 3-O-sulfotransferase-1 (3-0ST-1) and 3′-phosphoadenosine 5′-phosphosulfate (PAPS); and
purifying the resultant heptasaccharide to give a heparin compound, wherein the heparin compound is synthesized at a yield of greater than 30% and wherein the heparin compound is structurally homogeneous and has the following structure:
2. The method of claim 1 , wherein the enzymes N-acetyl glucosaminyl transferase and heparosan synthase-2 are selected from the group consisting of N-acetyl glucosaminyl transferase of E. coli K5 (KfiA) and heparosan synthase-2 (pmHS2) from Pasteurella multocida.
3. The method of claim 1 , wherein the method has a recovery yield about 400-fold higher than methods of chemically synthesizing heparin.