Claims
- 1. A method of detecting enzymatic activity, the method comprising:
providing a monomeric substrate comprising the formula X-Y-Z, wherein X comprises a chelator moiety and a chelated paramagnetic or superparamagnetic metal atom or ion, Y comprises a linker moiety, and Z comprises a polymerizing moiety; contacting the substrate with a target tissue, wherein the substrate undergoes polymerization to form a paramagnetic or superparamagnetic polymer, the polymerization being catalyzed by an enzyme in an extracellular matrix or bound to the surfaces of cells of the target tissue; and detecting an increase in relaxivity for the polymer relative to an equivalent amount of unpolymerized substrate, thereby detecting enzymatic activity.
- 2. The method of claim 1, wherein the paramagnetic or superparamagnetic metal atom or ion is a transition metal atom or ion.
- 3. The method of claim 1, wherein the paramagnetic or superparamagnetic metal atom or ion is a lanthanide atom or ion.
- 4. The method of claim 1, wherein the polymerizing moiety comprises any chemical group that can be chemically modified as a result of the catalytic activity of the enzyme to form a covalent chemical bond between either (1) Z and another monomeric substrate or (2) Z and any other polymer or macromolecule present during the reaction, including the enzyme itself.
- 5. The method of claim 1, wherein Z is a moiety that can be accommodated by the catalytic center of the enzyme.
- 6. The method of claim 1, wherein X comprises a structure selected from the group consisting of:
1,4,7,10-tetraazacyclodo-decane-N,N′,N″,N″′-tetraacetic acid; 1,4,7,10-tetraaza-cyclododecane-N,N′,N″-triacetic acid; 1,4,7-tris(carboxymethyl)-10-(2′-hydroxypropyl)-1,4,7,10-tetraazocyclodecane; 1,4,7-triazacyclonane-N,N′,N″-triacetic acid; 1,4,8,11-tetraazacyclotetra-decane-N,N′,N″,N′″-tetraacetic acid; diethylenetriamine-pentaacetic acid (DTPA); ethylenedicysteine; bis(aminoethanethiol)carboxylic acid; triethylenetetraamine-hexaacetic acid; ethylenediamine-tetraacetic acid (EDTA); 1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid; N-(hydroxy-ethyl)ethylenediaminetriacetic acid; nitrilotriacetic acid; and ethylene-bis(oxyethylene-nitrilo)tetraacetic acid.
- 7. The method of claim 1, wherein Z comprises the following structure:
- 8. The method of claim 7, wherein R1, R2, R3, R4 or R5 is at an ortho position relative to the OH substituent, and is selected from the group consisting of OH and OCH3.
- 9. The method of claim 7, wherein R1, R2, R3, R4 or R5 is at a meta position relative to the OH substituent, and is selected from the group consisting of NHC(O)R6 and NR7R8.
- 10. The method of claim 1, wherein Y comprises a structure selected from the group consisting of: an amino acid, an oligopeptide comprising 2-6 amino acid residues, a nucleotide, an oligonucleotide comprising 2-6 nucleotide residues, a C3-C12 alkyl group, a polyethyleneimine, a saccharide, an oligosaccharide, a medium chain fatty acid, a polyamidoamine, a polyacrylic acid, and a polyalcohol.
- 11. The method of claim 1, wherein Y comprises an amino acid or oligopeptide containing 2-6 amino acid residues.
- 12. The method of claim 11, wherein the oligopeptide comprises a glycine residue.
- 13. The method of claim 1, wherein the monomeric substrate comprises the formula:
- 14. The method of claim 1, wherein the enzyme is covalently linked to a targeting moiety, the targeting moiety being bound to a target molecule on the surface of a cell of the target tissue.
- 15. The method of claim 1, wherein the polymer is bound to one or more macromolecules in an extracellular matrix of the target tissue or to the surface of a cell of the target tissue.
- 16. The method of claim 15, wherein the macromolecules are selected from the group consisting of paramagnetic or superparamagnetic polymers, proteins, oligosaccharides, and polynucleotides present in the extracellular matrix or bound to the surface of a cell of the target tissue.
- 17. The method of claim 1, wherein the polymer is not bound to any other macromolecules.
- 18. The method of claim 1, wherein the enzyme is selected from the group consisting of an oxidoreductase, a monophenol oxidase, monophenol monooxygenase, and a catechol oxidase.
- 19. The method of claim 1, wherein the enzyme is selected from the group consisting of tyrosinase, tyrosinase-related protein, lipase, DNA polymerase, thermostable DNA polymerase, RNA polymerase, RNA-dependent DNA polymerase, reverse transcriptase, terminal nucleotide transferase, and polynucleotide phosphorylase.
- 20. The method of claim 18, wherein the enzyme is a monophenol oxidase or catechol oxidase.
- 21. The method of claim 18, wherein the oxidoreductase is selected from the group consisting of a peroxidase and a laccase.
- 22. The method of claim 18, wherein the oxidoreductase is a peroxidase is selected from the group consisting of lactoperoxidase and horseradish peroxidase.
- 23. The method of claim 14, wherein the targeting moiety is selected from the group consisting of a primary antibody, a secondary antibody, a cell adhesion molecule, a cytokine, a cell surface receptor molecule, or a fragment thereof that recognizes a preselected binding partner.
- 24. The method of claim 14, wherein the enzyme is a peroxidase and the targeting moiety is selected from the group consisting of a primary antibody and a secondary antibody.
- 25. The method of claim 1, wherein the target tissue comprises human or animal neovasculature.
- 26. The method of claim 1, wherein the target tissue comprises diseased or developmental human or animal tissue.
- 27. A method of detecting enzymatic activity using magnetic resonance imaging, the method comprising:
providing a monomeric substrate polymerizable in the presence of an enzyme or as a result of an enzyme-catalyzed reaction, the substrate comprising the formula X-Y-Z, wherein X comprises a chelator moiety and a chelated paramagnetic or superparamagnetic metal atom or ion, Y comprises a linker moiety, and Z comprises a polymerizing moiety; contacting the substrate with a target tissue, wherein the substrate undergoes polymerization to form a paramagnetic or superparamagnetic polymer, the polymerization being catalyzed by an enzyme in an extracellular matrix or bound to the surfaces of cells of the target tissue; and detecting an increase in relaxivity for the polymer relative to an equivalent amount of unpolymerized substrate, thereby detecting enzymatic activity.
- 28. A composition comprising a compound of formula X-Y-Z, wherein X comprises a chelator moiety, Y comprises a linker moiety, and Z comprises a polymerizing moiety.
- 29. The composition of claim 28, wherein the compound further comprises a paramagnetic or superparamagnetic metal atom or ion.
- 30. The composition of claim 29, wherein the paramagnetic or superparamagnetic metal atom or ion is a transition metal atom or ion.
- 31. The composition of claim 29, wherein the paramagnetic or superparamagnetic metal atom or ion is a lanthanide atom or ion.
- 32. The composition of claim 29, wherein the metal ion is selected from the group consisting of an iron ion, a dysprosium ion, a europium ion and a manganese ion.
- 33. The composition of claim 29, wherein the metal ion is a gadolinium ion.
- 34. The composition of claim 28, wherein the polymerizing moiety comprises any chemical group that can be chemically modified as a result of the catalytic activity of an enzyme to form a covalent chemical bond between either (1) Z and another compound of formula X-Y-Z or (2) Z and any polymer or macromolecule.
- 35. The composition of claim 28, wherein Z is a moiety that can be accommodated by the catalytic center of an enzyme.
- 36. The composition of claim 28, wherein X comprises a structure selected from the group consisting of:
1,4,7,10-tetraazacyclodo-decane-N,N′,N″,N′″-tetraacetic acid; 1,4,7,10-tetraaza-cyclododecane-N,N′,N″-triacetic acid; 1,4,7-tris(carboxymethyl)-10-(2′-hydroxypropyl)-1,4,7,10-tetraazocyclodecane; 1,4,7-triazacyclonane-N,N′,N″-triacetic acid; 1,4,8,11-tetraazacyclotetra-decane-N,N′,N″,N″′-tetraacetic acid; diethylenetriamine-pentaacetic acid (DTPA); ethylenedicysteine; bis(aminoethanethiol)carboxylic acid; triethylenetetraamine-hexaacetic acid; ethylenediamine-tetraacetic acid (EDTA); 1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid; N-(hydroxy-ethyl)ethylenediaminetriacetic acid; nitrilotriacetic acid; and ethylene-bis(oxyethylene-nitrilo)tetraacetic acid.
- 37. The composition of claim 28, wherein Z comprises the following structure:
- 38. The composition of claim 37, wherein R1, R2, R3, R4 or R5 is at an ortho position relative to the OH substituent, and is selected from the group consisting of OH and OCH3.
- 39. The composition of claim 37, wherein R1, R2, R3, R4 or R5 is at a meta position relative to the OH substituent, and is selected from the group consisting of NHC(O)R6 and NR7R8.
- 40. The composition of claim 28, wherein Y comprises a structure selected from the group consisting of: an amino acid, an oligopeptide comprising 2-6 amino acid residues, a nucleotide, an oligonucleotide comprising 2-6 nucleotide residues, a C3-C12 alkyl group, a polyethyleneimine, a saccharide, an oligosaccharide, a medium chain fatty acid, a polyamidoamine, a polyacrylic acid, and a polyalcohol.
- 41. The composition of claim 28, wherein Y comprises an amino acid or oligopeptide containing 2-6 amino acid residues.
- 42. The composition of claim 41, wherein the oligopeptide comprises a glycine residue.
- 43. The composition of claim 29, wherein the compound comprises the formula:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application No. 60/241,566, filed Oct. 19, 2000, and U.S. Provisional Application No. 60/310,335, filed Aug. 6, 2001, both of which are incorporated herein by reference in their entirety.
Provisional Applications (2)
|
Number |
Date |
Country |
|
60241566 |
Oct 2000 |
US |
|
60310335 |
Aug 2001 |
US |