Solid-phase substrate containing modified heparin
AssigneeBoard of Regents, The University of Texas System
InventorsGarth L. Nicolson, Motowo Nakajima, Tatsuro Irimura
Life of this patent
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Abstract
A solid phase substrate which yields soluble labeled products upon hydrolysis by a glycosaminoglycan endoglycosidase and methods of producing said substrate are comprised in the present invention. The solid phase substrate comprises glycosaminoglycan bearing labeled substances bound to amino groups. The labeled glycosaminoglycan substrate is reductively aminated at its reducing terminal end to produce an amine-terminus. The substrate is further coupled to an amino-reactive solid matrix through its amine-terminus.
Claims30
A method of producing a solid-phase substrate which yields radioisotopically labeled glycosaminoglycan fragments upon hydrolysis by a glycosaminoglycan endoglycosidase, the method consisting essentially of:
- (a) at least partially N-desulfating or N-deacetylating a quantity of glyucosaminoglycan;
- (b) radiolabeling the at least partially N-desulfated or N-deacetylated glycosaminoglycan with radioisotopically labeled acyl anhydride or acyl halide to produce radioisotopically labeled glycosaminoglycan;
- (c) completely N-acylating the radioisotopically labeled glycosaminoglycan with acyl anhydride or acyl halide;
- (d) reductively aminating a reducing terminal end of said radioisotopically labeled and N-acylated glycosaminoglycan to produce radioisotopically labeled amine-terminal glycosaminoglycan;
- and
- (e) coupling, through its terminal amine, the radioisotopically labeled amine-terminal glycosaminoglycan to an amine-reactive solid-phase support to produce said solid-phase substrate.
A method of producing a solid-phase substrate which yields radioisotopically labeled glycosaminoglycan fragments upon hydrolysis by a heparan sulfate endoglycosidase, the method consisting essentially of the steps of:
- (a) at least partially N-desulfating or N-deacetylating a quantity of heparan sulfate;
- (b) radiolabeling the at least partially N-desulfated or N-deacetylated heparan sulfate with radioisotopically labeled acetic anhydride to produce radioisotopically labeled heparan sulfate;
- (c) completely N-acylating the radioisotopically labeled glycosaminoglycan with acyl anhydride or acyl halide;
- (d) reductively aminating a reducing terminal end of said radioisotopically labeled heparan sulfate to produce radioisotopically labeled amine-terminal heparan sulfate;
- and
- (e) coupling, through its terminal amine, the radioisotopically labeled amine-terminal heparan sulfate to an amino-reactive solid phase support to produce said solid phase substrate.
A solid-phase substrate which yields radio-isotopically labeled glycosaminoglycan fragments upon hydrolysis by a glycosaminoglycan endoglycosidase, said substrate being produced by a process consisting essentially of:
- (a) at least partially N-deacylating or N-desulfating a quantity of glycosaminoglycan;
- (b) radiolabeling the at least partially N-deacylated or N-desulfated glycosaminoglycan with radioisotopically labeled acyl anhydride or acyl halide to produce radioisotopically labeled glycosaminoglycan;
- (c) completely N-acylating the radioisotopically labeled glycosaminoglycan with acyl anhydride or acyl halide;
- (d) reductively aminating a reducing terminal end of said radioisotopically labeled glycosaminoglycan to produce radioisotopically labeled amine-terminal glycosaminoglycan;
- and
- (e) coupling, through its terminal amine, the radioisotopically labeled amine-terminal glycosaminoglycan to an amino-reactive solid-phase support to produce said solid-phase substrate.
A solid-phase substrate which yields radio-isotopically labeled glycosaminoglycan fragments upon hydrolysis by a heparan sulfate endoglycosidase, said substrate being produced by a process consisting essentially of:
- (a) at least partially N-desulfating a quantity of heparan sulfate;
- (b) radiolabeling the at least partially N-desulfated heparan sulfate with radioisotopically labeled acetic anhydride or acetyl chloride to produce radioisotopically labeled heparan sulfate;
- (c) completely N-acylating the radioisotopically labeled heparan sulfate with an acyl anhydride or acyl halide;
- (d) reductively aminating a reducing terminal end of said radioisotopically labeled heparan sulfate to produce radioisotopically labeled amine-terminal heparan sulfate;
- and
- (e) coupling, through its terminal amine, the radioisotopically labeled amine-terminal heparan sulfate to an amine-reactive slid phase support to produce said solid phase substrate.
A solid-phase substrate which yields radio-isotopically labeled glycosaminoglycan fragments upon hydrolysis by a glycosaminoglycan endoglycosidase said substrate consisting essentially of glycosaminoglycan bearing radioisotopically labeled N-acyl groups, said glycosaminoglycan being reductively aminated at its reducing terminal end to produce an amine-terminus and being coupled to an amino-reactive solid matrix through said amine-terminus.
The slid phase substrate of clam 15 wherein the glycosaminoglycan is heparan sulfate and the endoglycosidase is heparanase.
The solid phase substrate of clam 15 wherein the amine-reactive solid matrix consists essentially of periodate activated agarose.
A solid phase substrate which yields radioisotopically or fluorescently labeled glycosaminoglycan fragments upon hydrolysis by a glycosaminoglycan endoglycosidase, said substrate consisting essentially of:
- a glycosaminoglycan bearing a fluorescent or radioisotopic label which does not prevent hydrolysis of the labeled glycosaminoglycan by a glycosaminoglycan endoglycosidase, said glycosaminoglycan being linked through its reducing terminal end by a single covalent linkage to a solid matrix.
A method of producing a solid-phase substrate which yields fluorescent or radioisotopic glycosaminoglycan fragments upon hydrolysis by a glycosaminoglycan endoglycosidase, the method consisting essentially of:
- (a) at least partially N-desulfating or N-deacetylating a quantity of glycosaminoglycan;
- (b) labeling amino groups of the at least partially N-desulfated or N-deacetylated glycosaminoglycan with a fluorescent or radioisotopic label to produce labeled glycosaminoglycan which is hydrolyzed by a glyucosaminoglycan endoglycosidase;
- (c) completely N-acylating amino groups of the labeled glycosaminoglycan with acyl anhydride or acyl halide;
- (d) reductively aminating a reducing terminal end of said labeled and N-acylated glycosaminoglycan to produce labeled amine-terminal glycosaminoglycan;
- and
- (e) coupling, through its terminal amine, the labeled amine-terminal glycosaminoglycan to an amine-reactive solid-phase support to produce said solid-phase substrate.
A method of producing a solid-phase substrate which yields fluorescent or radioisotopic labeled glycosaminoglycan fragments upon hydrolysis by a heparan sulfate endoglycosidase, the method consisting essentially of:
- (a) at least partially N-desulfating or N-deacetylating a quantity of heparan sulfate;
- (b) labeling amino groups of the at least partially N-desulfated or N-deacetylated heparan sulfate with a fluorescent or radioisotopic label to produce labeled heparan sulfate which is hydrolyzed by a heparan sulfate endoglycosidase;
- (c) completely N-acylating amino groups of the labeled sulfate with acyl anhydride or acyl halide;
- (d) reductively aminating a reducing terminal end of said labeled heparan sulfate to produce labeled amine-terminal heparan sulfate;
- and
- (e) coupling, through its terminal amine, the labeled amine-terminal heparan sulfate to an amino-reactive solid phase support to produce said solid phase substrate.
A solid-phase substrate which yields fluorescent or radioisotopic glycosaminoglycan fragments upon hydrolysis by a glycosaminoglycan endoglycosidase, said substrate being produced by a process consisting essentially of:
- (a) at least partially N-deacylating or N-desulfating a quantity of glycosaminoglycan;
- (b) labeling amino groups of the at least partially N-deacylated or N-desulfated glycosaminoglycan with a fluorescent or radioisotopic substance to produce labeled glycosaminoglycan which is hydrolyzed by a glycosaminoglycan endoglycosidase;
- (c) completely N-acylating amino groups of the labeled glycosaminoglycan with acyl anhydride or acyl halide;
- (d) reductively aminating a reducing terminal end of said labeled glycosaminoglycan to produce labeled amine-terminal glycosaminoglycan;
- and
- (e) coupling, through its terminal amine, the labeled amine-terminal glycosaminoglycan to an amino-reactive solid-phase support to produce said solid-phase substrate.
A solid-phase substrate which yields fluorescent or radioisotopic glycosaminoglycan fragments upon hydrolysis by a heparan sulfate endoglycosidase, said substrate being produced by a process consisting essentially of:
- (a) at least partially N-deacylating or N-desulfating a quantity of heparan sulfate;
- (b) labeling amino groups of the at least partially N-deacetylated or N-desulfated heparan sulfate with a fluorescent or radioisotopic substance to produce labeled heparan sulfate which is hydrolyzed by a glycosaminoglycan endoglycosidase;
- (c) completely N-acylating amino groups of the labeled heparan sulfate with an acyl anhydride or acyl halide;
- (d) reductively aminating a reducing terminal end of said labeled heparan sulfate to produce labeled amine-terminal heparan sulfate;
- ad
- (e) coupling, through its terminal amine, the labeled amine-terminal heparan sulfate to an amino-reactive solid-phase support to produce said solid-phase substrate.
A solid phase substrate which, upon hydrolysis by a glycosaminoglycan endoglycosidase, yields glycosaminoglycan fragments with a fluorescent or radioisotopic label, the substrate consisting essentially of a glycosaminoglycan having an aminated reducing terminal hexose and bearing a detectable radioisotopic or fluorescent label which does not prevent hydrolysis by a glycosaminoglycan endoglycosidase, said glycosaminoglycan being bound through its aminated reducing terminal hexose to a solid matrix.
Dependencies are inferred from the claim text.