Production of defined monodisperse heparosan polymers and unnatural polymers with polysaccharide synthases
View Patent ↗The present invention relates to methodology for polymer grafting by a polysaccharide synthase and, more particularly, polymer grafting using the hyaluronate or chondroitin or heparin/heparosan synthases from Pasteurella , in order to create a variety of glycosaminoglycan oligosaccharides having a natural or chimeric or hybrid sugar structure with a targeted size that are substantially monodisperse in size. The present invention also relates to methodology for polymer grafting by a polysaccharide synthase to form glycosaminoglycan polymers having an unnatural structure.
1. A method for enzymatically producing defined glycosaminoglycan polymers comprising the steps of:
providing at least one functional acceptor, wherein the functional acceptor comprises at least two sugar units, and wherein at least one of the at least two sugar units is selected from the group consisting of uronic acid, a uronic acid analog comprising a substitution at at least one of the C2 and C3 positions thereof, a hexosamine and a hexosamine analog comprising a substitution at at least one of the C2 and C6 positions thereof;
providing a recombinant heparosan synthase having an empty acceptor site and being capable of elongating the at least one functional acceptor in a controlled and/or repetitive fashion to form extended glycosaminoglycan molecules, and wherein the recombinant heparosan synthase is selected from the group consisting of:
(a) a recombinant heparosan synthase having an amino acid sequence as set forth in one of SEQ ID NOS: 6, 8, 66, 70 and 71;
(b) a recombinant heparosan synthase encoded by the nucleotide sequence of one of SEQ ID NOS: 5, 7 and 65;
(c) a recombinant heparosan synthase encoded by a nucleotide sequence that is at least 90% identical to at least one of SEQ ID NOS: 5, 7 and 65;
(d) a recombinant heparosan synthase encoded by a nucleic acid capable of hybridizing to the complement of the polynucleotide of at least one of SEQ ID NOS: 5, 7 and 65 under hybridization conditions comprising hybridization at a temperature of 68° C. in 5×SSC/5× Denhardt's solution/1.0% SDS, followed with washing in 3×SSC at 42° C.;
(e) a recombinant heparosan synthase encoded by a nucleic acid capable of hybridizing to the complement of a polynucleotide encoding an amino acid sequence as set forth in at least one of SEQ ID NOS: 6, 8, 66, 70 and 71 under hybridization conditions comprising hybridization at a temperature of 68° C. in 5×SSC/5× Denhardt's solution/1.0% SDS, followed with washing in 3×SSC at 42° C.; and
providing at least one of a UDP-sugar and a UDP-sugar analog in a stoichiometric ratio to the at least one functional acceptor such that the recombinant heparosan synthase elongates the at least one functional acceptor to provide glycosaminoglycan polymers wherein the glycosaminoglycan polymers have a desired size distribution such that the glycosaminoglycan polymers are substantially monodisperse in size such that the glycosaminoglycan polymers have a polydispersity value in a range of from 1.0 to 1.5, and wherein the desired size distribution is obtained by controlling the stoichiometric ratio of UDP-sugar to functional acceptor.
2. The method of claim 1 wherein uronic acid is further defined as a uronic acid selected from the group consisting of GlcUA, iduronic acid (idoUA) and GalUA; hexosamine is further defined as a hexosamine selected from the group consisting of GlcNAc, GalNAc, GlcN GalN; the uronic acid analog is further defined as a uronic acid analog selected from the group consisting of GlcNAcUA, GlcdiNAcUA, and 2-deoxy-2-fluoro-GlcUA; and the hexosamine analog is further defined as a hexosamine analog selected from the group consisting of GlcN, GlcNAcNAc, GlcN[TFA], GlcNBut, GlcNPro, and 6-F-6-deoxyGlcNAc.
3. The method of claim 1 , further comprising the step of providing a divalent metal ion, wherein the divalent metal ion is selected from the group consisting of manganese, magnesium, cobalt, nickel and combinations thereof, and wherein the method is carried out in a buffer having a pH from about 4 to about 9.
4. The method of claim 1 wherein at least one of:
(a) the substantially monodisperse glycosaminoglycan polymers have a molecular weight in a range of from about 3.5 kDa to about 0.5 MDa, and have a polydispersity value in a range of from about 1.0 to about 1.1;
(b) the substantially monodisperse glycosaminoglycan polymers have a molecular weight in a range of from about 0.5 MDa to about 4.5 MDa, and have a polydispersity value in a range of from about 1.0 to about 1.5; and
(c) the substantially monodisperse glycosaminoglycan polymers have a molecular weight in a range of from about 0.5 MDa to about 4.5 MDa, and have a polydispersity value in a range of from about 1.0 to about 1.2.
5. The method of claim 1 wherein the at least one functional acceptor further comprises a moiety selected from the group consisting of a fluorescent tag, a radioactive tag or therapeutic, an affinity tag, a detection probe, a medicant, a biologically active agent, a therapeutic agent, and combinations thereof.
6. The method of claim 1 wherein the at least one of a UDP-sugar and a UDP-sugar analog is radioactive or nuclear magnetic resonance-active.
7. A method for enzymatically producing defined glycosaminoglycan polymers comprising the steps of:
providing at least one functional acceptor, wherein the functional acceptor has at least one sugar unit selected from the group consisting of uronic acid a uronic acid analog comprising a substitution at at least one of the C2 and C3 positions thereof, a hexosamine and a hexosamine analog comprising a substitution at at least one of the C2 and C6 positions thereof;
providing a recombinant heparosan synthase having an empty acceptor site and being capable of elongating the at least one functional acceptor in a controlled and/or repetitive fashion to form extended glycosaminoglycan molecules, and wherein the recombinant heparosan synthase is selected from the group consisting of:
(a) a recombinant heparosan synthase having an amino acid sequence as set forth in one of SEQ ID NOS: 6, 8, 66, 70 and 71;
(b) a recombinant heparosan synthase encoded by the nucleotide sequence of one of SEQ ID NOS: 5, 7 and 65;
(c) a recombinant heparosan synthase encoded by a nucleotide sequence that is at least 90% identical to at least one of SEQ ID NOS: 5, 7 and 65;
(d) a recombinant heparosan synthase encoded by a nucleic acid capable of hybridizing to the complement of the polynucleotide of at least one of SEQ ID NOS: 5, 7 and 65 under hybridization conditions comprising hybridization at a temperature of 68° C. in 5×SSC/5× Denhardt's solution/ 1 . 0 % SDS, followed with washing in 3×SSC at 42° C.;
(e) a recombinant heparosan synthase encoded by a nucleic acid capable of hybridizing to the complement of a polynucleotide encoding an amino acid sequence as set forth in at least one of SEQ ID NOS: 6, 8, 66, 70 and 71 under hybridization conditions comprising hybridization at a temperature of 68° C. in 5×SSC/5× Denhardt's solution/1.0% SDS, followed with washing in 3×SSC at 42° C.; and
providing at least one of a UDP-sugar and a UDP-sugar analog in a stoichiometric ratio to the at least one functional acceptor such that the recombinant heparosan synthase elongates the at least one functional acceptor to provide glycosaminoglycan polymers wherein the glycosaminoglycan polymers have a desired size distribution such that the glycosaminoglycan polymers are substantially monodisperse in size such that the glycosaminoglycan polymers have a polydispersity value in a range of from 1.0 to 1.5, and wherein the desired size distribution is obtained by controlling the stoichiometric ratio of UDP-sugar to functional acceptor.
8. The method of claim 7 wherein the functional acceptor comprises a glycoside of uronic acid.
9. The method of claim 7 wherein the at least one functional acceptor further comprises a moiety selected from the group consisting of a fluorescent tag, a radioactive tag or therapeutic, an affinity tag, a detection probe, a medicant, a biologically active agent, a therapeutic agent, and combinations thereof.
10. The method of claim 1 wherein the functional acceptor is at least one of:
(a) a heparosan oligosaccharide, polysaccharide or polymer;
(b) a heparin oligosaccharide, polysaccharide or polymer;
(c) a heparan oligosaccharide, polysaccharide or polymer;
(d) an acceptor comprising a glycoside of uronic acid; and
(e) a sulfated or modified oligosaccharide, polysaccharide or polymer.
11. The method of claim 1 , wherein the UDP-sugar is selected from the group consisting of UDP-GlcUA, UDP-GalUA UDP-GlcNAc, UDP-Glc, UDP-GalNAc, UDP-GlcN, UDP-GalN, and the UDP-sugar analog is selected from the group consisting of UDP-GlcN, UDP-GlcNAcUA, UDP-GlcNAcNAc, UDP-GlcdiNAcUA, UDP-GlcN[TFA], UDP-GlcNBut, UDP-GlcNPro, UDP-6-F-6-deoxyGlcNAc, and UDP-2-F-2-deoxyGlcUA.
12. The method of claim 7 , wherein the UDP-sugar is selected from the group consisting of UDP-GlcUA, UDP-GalUA UDP-GlcNAc, UDP-Glc, UDP-GalNAc, UDP-GlcN, UDP-GalN, and the UDP-sugar analog is selected from the group consisting of UDP-GlcN, UDP-GlcNAcUA, UDP-GlcNAcNAc, UDP-GlcdiNAcUA, UDP-GlcN[TFA], UDP-GlcNBut, UDP-GlcNPro, UDP-6-F-6-deoxyGlcNAc, and UDP-2-F-2-deoxyGlcUA.
13. A method for enzymatically producing defined glycosaminoglycan polymers comprising the steps of:
providing at least one functional acceptor, wherein the functional acceptor has at least one sugar unit selected from the group consisting of uronic acid, a uronic acid analog comprising a substitution at at least one of the C2 and C3 positions thereof, a hexosamine and a hexosamine analog comprising a substitution at at least one of the C2 and C6 positions thereof;
providing a recombinant heparosan synthase having an empty acceptor site and being capable of elongating the at least one functional acceptor in a controlled and/or repetitive fashion to form extended glycosaminoglycan molecules, and wherein the recombinant heparosan synthase is a single dual-action enzyme possessing GlcUA-transferase and GlcNAc-transferase activities and comprises the motifs of SEQ ID NOS:63 and 64; and
providing at least one of a UDP-sugar and a UDP-sugar analog in a stoichiometric ratio to the at least one functional acceptor such that the recombinant heparosan synthase elongates the at least one functional acceptor to provide glycosaminoglycan polymers wherein the glycosaminoglycan polymers have a desired size distribution such that the glycosaminoglycan polymers are substantially monodisperse in size such that the glycosaminoglycan polymers have a polydispersity value in a range of from 1.0 to 1.5, and wherein the desired size distribution is obtained by controlling the stoichiometric ratio of UDP-sugar to functional acceptor.
14. The method of claim 13 wherein the functional acceptor comprises a glycoside of uronic acid.
15. The method of claim 13 wherein the at least one functional acceptor further comprises a moiety selected from the group consisting of a fluorescent tag, a radioactive tag or therapeutic, an affinity tag, a detection probe, a medicant, a biologically active agent, a therapeutic agent, and combinations thereof.
16. The method of claim 13 wherein the functional acceptor is at least one of:
(a) a heparosan oligosaccharide, polysaccharide or polymer;
(b) a heparin oligosaccharide, polysaccharide or polymer;
(c) a heparan oligosaccharide, polysaccharide or polymer;
(d) an acceptor comprising a glycoside of uronic acid; and
(e) a sulfated or modified oligosaccharide, polysaccharide or polymer.
17. The method of claim 13 , wherein the UDP-sugar is selected from the group consisting of UDP-GlcUA, UDP-GalUA UDP-GlcNAc, UDP-Glc, UDP-GalNAc, UDP-GlcN, UDP-GalN, and the UDP-sugar analog is selected from the group consisting of UDP-GlcN, UDP-GlcNAcUA, UDP-GlcNAcNAc, UDP-GlcdiNAcUA, UDP-GlcN[TFA], UDP-GlcNBut, UDP-GlcNPro, UDP-6-F-6-deoxyGlcNAc, and UDP-2-F-2-deoxyGlcUA.