Patent US5140353A successfully refreshed from Google Patents.
Patent US4897444A successfully refreshed from Google Patents.
US4897444A
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Immobilized fluorogenic substrates for enzymes; and processes for their preparation
Assignee: Research Foundation of the State University of New York
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Filing: 1985-06-03
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Granted: 1990-01-30
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Expires: 2005-06-03
Abstract
Abstract An immobilized fluorogenic substrate useful in processes for identifying and quantifying, intra- and extra-cellularly, and mammalian body fluids, as well as animal cell abstracts is disclosed. The immobilized fluorogenic substrate has the structure R1-NH-R4-R2-R3 wherein R1 represents an enzyme-specific oligopeptide; R2 represents a spacer group which is methylenecarbonyloxy, a methylenecarboxamido, or a methylanesulfonamido group attached to a polymethylene chain which itself has a functional group suitable for coupling with a polymer; R3 represents a biologically inert polymer; and R4 represents a fluorogenic moiety.
Inventors
- 👤 Paul J. Brynes
- 👤 Patricia Andrade-Gordon
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-09 13:17
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/34; C12Q1/37
Claims Analysis
Total Claims: 16
Claim 1
Independent Claim
292 words
An immobilized fluorogenic substrate for identifying and quantifying, intra- and extra-cellularly, the production and secretion of cell-specific enzymes in human and mammalian body fluids as well as in animal extracts, which has the structure: R.sub.1 -NH-R.sub.4 -R.sub.2 -R.sub.3 wherein R 1 represents an oligopeptide which has an amino acid sequence that is enzyme-specific in terms of cleavage of the amino acid sequence at the peptide linkage proximal to a fluorogenic moiety, the oligopeptide comprising naturally occurring amine acids associated with mammalian and bacterial systems, R 1 being joined to NH via a carbonyl group in R 1 ; R 2 represents a spacer group which comprises a group having the structural formula (CH 2 X 1 X 2a (CH 2 ) y X 3 ) wherein X 1 is CO or SO 2 ; X 2 is NH; a is zero or one, provided that when a is zero, X 1 is CO; X 3 is a member selected from the group consisting of NH, NHCO, CONH, OCO, COO or Si(0--) 3 ; and y is an integer from zero to 15; R 3 represent a glass or polymer having a complementary group which forms an ester or amide linkage with the ωsulfonic acid or ω-carboxylic acid function of the spacer group or an ether linkage with the ω-carbon atom and hydroxyl group in the polymer; and NH-R 4 represents a fluorogenic moiety having a second functional group to which is attached the spacer group, said fluorogenic moiety selected from the group consisting of aminoquinolines and their alkyl and alkoxyl derivatives; alkyl, alkoxyl and carboxyl derivatives of aminonaphthalenes; alkyl, akoxyl and carboxyl derivatives of aminocoumarines; alkyl, alkoxyl and carboxyl derivatives of acridines; and alkyl, alkoxyl, amino, nitro and carboxyl derivatives of benzofurazans.
Claim 2
Dependent Claim
292 words
An immobilized fluorogenic substrate for identifying and quantifying, intra- and extra-cellularly, the production and secretion of cell-specific enzymes in human and mammalian body fluids as well as in animal extracts, which has the structure: R.sub.1 -NH-R.sub.4 -R.sub.2 -R.sub.3 wherein R 1 represents an oligopeptide which has an amino acid sequence that is enzyme-specific in terms of cleavage of the amino acid sequence at the peptide linkage proximal to a fluorogenic moiety, the oligopeptide comprising naturally occurring amine acids associated with mammalian and bacterial systems, R 1 being joined to NH via a carbonyl group in R 1 ; R 2 represents a spacer group which comprises a group having the structural formula (CH 2 X 1 X 2a (CH 2 ) y X 3 ) wherein X 1 is CO or SO 2 ; X 2 is NH; a is zero or one, provided that when a is zero, X 1 is CO; X 3 is a member selected from the group consisting of NH, NHCO, CONH, OCO, COO or Si(0--) 3 ; and y is an integer from zero to 15; R 3 represent a glass or polymer having a complementary group which forms an ester or amide linkage with the ωsulfonic acid or ω-carboxylic acid function of the spacer group or an ether linkage with the ω-carbon atom and hydroxyl group in the polymer; and NH-R 4 represents a fluorogenic moiety having a second functional group to which is attached the spacer group, said fluorogenic moiety selected from the group consisting of aminoquinolines and their alkyl and alkoxyl derivatives; alkyl, alkoxyl and carboxyl derivatives of aminonaphthalenes; alkyl, akoxyl and carboxyl derivatives of aminocoumarines; alkyl, alkoxyl and carboxyl derivatives of acridines; and alkyl, alkoxyl, amino, nitro and carboxyl derivatives of benzofurazans.
Claim 3
Dependent Claim
19 words
The substrate of claim 1 wherein R 3 is selected from the group consisting of polyacrylamide, poly(4-aminostyrene) and glass.
Claim 4
Dependent Claim
48 words
The substrate of claim 1 wherein R 3 comprises a copolymer of an N-allyl amide with a polymerizable monomer selected from the group consisting of styrene, butadiene, vinyl acetate, acrylic esters, acrylic amides and mixtures thereof, the N-allyl amide being the non-alkylating terminal group of the spacer group.
Claim 5
Dependent Claim
33 words
The substrate of claim 1 wherein R 2 is selected from the group consisting of CH 2 CONH(CH 2 ) 5 CO; CH 2 CONHCH 2 CH 2 NH; and CH 2 CO.
Claim 6
Dependent Claim
49 words
The substrate of claim 1 wherein R 1 is selected from the group consisting of X-valyl-prolyl-arginyl-Y; X-(D)-phenylalnyl-picolyl-arginyl-Y; X-phenylalanyl-valyl-arginyl-Y; X-glycyl-prolyl-arginyl-Y; X-valyl-leucyl-lysyl-Y; X-(D)-valyl-leucyl-lysyl-Y; X-glytamyl-lysyl-lysyl-Y; X-glycyl-proplyl-lysyl-Y; X-valyl-glycyl-arginyl-Y; X-glutamyl-glycyl-arginyl-Y; X-propyl-phenylalnyl-arginyl-Y; X-(D)-prolyl phenylalanyl-arginyl-Y; X-(D)-valyl-leucyl-arginyl-Y; X-isoleucyl-glutamyl-glycl-arginyl-Y; X-alanyl-propyl-alanyl-Y; X-alanyl-alanyl-prolyl-valyl-Y; and X-alanyl-alanyl-propyl-methionyl-Y; wherein X is a N-terminal blocking agent or H and Y is a fluorescent amine.
Claim 7
Dependent Claim
20 words
The substrate of claim 1 wherein the fluorogenic moiety is selected from the group consisting of 3-amino-quinoline, 2-(N,N-dimethylamino)methyl-6-aminonaphthalene and 4-(N,N-dimethylamino)methyl-6-aminocoumarin.
Claim 8
Dependent Claim
181 words
An immobilized fluorogenic substrate for identifying and quantifying, intra- and extra-cellularly, the production and secretion of cell-specific enxymes in human and mammalian body fluids as well as in animal cell extracts, which has the structure: ##STR16## wherein R 1 represents an oligopeptide which has an amino acid sequence that is enzyme-specific in terms of cleavage of the amino acid sequence at the peptide linkage proximal to the quinoline ring; R 2 represents a spacer group which comprises a group having the structural formula (CH 2 X 1 X 2a (CH 2 ) y X 3 ) wherein X 1 is CO or SO 2 ; X 2 is NH; a is zero or one, provided that when a is zero, X 1 is CO; X 3 a member selected from the group consisting of NH, NHCO, CONH, OCO, COO or Si(O--) 3 ; and y is an integer from zero to 15; and R 3 represents a polymer having a complementary group which forms an ester or amide linkage with the ω-sulfonic acid or ω-carboxylic acid function of the spacer group.
Claim 9
Dependent Claim
181 words
An immobilized fluorogenic substrate for identifying and quantifying, intra- and extra-cellularly, the production and secretion of cell-specific enxymes in human and mammalian body fluids as well as in animal cell extracts, which has the structure: ##STR16## wherein R 1 represents an oligopeptide which has an amino acid sequence that is enzyme-specific in terms of cleavage of the amino acid sequence at the peptide linkage proximal to the quinoline ring; R 2 represents a spacer group which comprises a group having the structural formula (CH 2 X 1 X 2a (CH 2 ) y X 3 ) wherein X 1 is CO or SO 2 ; X 2 is NH; a is zero or one, provided that when a is zero, X 1 is CO; X 3 a member selected from the group consisting of NH, NHCO, CONH, OCO, COO or Si(O--) 3 ; and y is an integer from zero to 15; and R 3 represents a polymer having a complementary group which forms an ester or amide linkage with the ω-sulfonic acid or ω-carboxylic acid function of the spacer group.
Claim 10
Dependent Claim
119 words
A process for preparing an immobilized fluorogenic substrate capable of identifying and quantifying, intra- and extra-cellularly, the production and secretion of cell-specific enzymers in human and mammalian body fluids as well as in animal cell extracts which comprises: (a) coupling an enzyme-specific oligopeptide to the primary amino group of a fluorescent aminoquinoline via an amide linkage; (b) guaternizing the ring nitrogen of the quinoline moiety with an alkylating spacer group selected from haloacetylamino, halo-acetoxy- or halomethylsulfonylamino-polyethylene compounds having an ω-amino (protected), an ω-hydroxyl or ω-carboxylic acid group; (c) coupling the ω-functional group of a spacer moiety with the complementary group of the polymer material to form an ester or an amide linkage, which intrinsically immobilizes the peptidyl quinoline assembly.
Claim 11
Dependent Claim
119 words
A process for preparing an immobilized fluorogenic substrate capable of identifying and quantifying, intra- and extra-cellularly, the production and secretion of cell-specific enzymers in human and mammalian body fluids as well as in animal cell extracts which comprises: (a) coupling an enzyme-specific oligopeptide to the primary amino group of a fluorescent aminoquinoline via an amide linkage; (b) guaternizing the ring nitrogen of the quinoline moiety with an alkylating spacer group selected from haloacetylamino, halo-acetoxy- or halomethylsulfonylamino-polyethylene compounds having an ω-amino (protected), an ω-hydroxyl or ω-carboxylic acid group; (c) coupling the ω-functional group of a spacer moiety with the complementary group of the polymer material to form an ester or an amide linkage, which intrinsically immobilizes the peptidyl quinoline assembly.
Claim 12
Dependent Claim
86 words
A process for preparing an immobilized fluorogenic substrate capable of identifying and quantifying, intra- and extra-cellularly, the production and secretion of cell specific enzymes which comprises: (a) coupling an enzyme-specific oligopeptide to the primary amino group of a fluorescent aminoquinoline via an amide linkage; (b) quaternized the ring nitrogen of the quinoline moiety with an allyl group to produce an allylic monomer; and (c) copolymerizing the allylic monomer with a second polymerizable monomer in the presence of a radial-generating catalyst, to immobilize the enzyme cleavable substrate.
Claim 13
Dependent Claim
86 words
A process for preparing an immobilized fluorogenic substrate capable of identifying and quantifying, intra- and extra-cellularly, the production and secretion of cell specific enzymes which comprises: (a) coupling an enzyme-specific oligopeptide to the primary amino group of a fluorescent aminoquinoline via an amide linkage; (b) quaternized the ring nitrogen of the quinoline moiety with an allyl group to produce an allylic monomer; and (c) copolymerizing the allylic monomer with a second polymerizable monomer in the presence of a radial-generating catalyst, to immobilize the enzyme cleavable substrate.
Claim 14
Dependent Claim
23 words
The process of claim 9 wherein the second monomer is selected from the group consisting of hydroxyethyl methacrylate, vinyl chloride, styrene and butadiene.
Claim 15
Dependent Claim
125 words
A process for preparing an immobilized fluorogenic substrate capable of identifying and quantifying, intra- and extra-cellularly, the production and secretion of cell specific enzymes which comprises: (a) coupling an enzyme-specific oligopeptide to the primary amino group of a fluorescent aminoquinoline via an amide linkage; (b) quaternizing the ring nitrogen of the quinoline moiety with an alkylating spacer group selected from the group consisting of haloacetylamino, haloacetoxy- or halomethylsulfonylamino-polymethylene compounds having an ω-amino (protected), an ω-hydroxyl or ω-carboxylic acid group; (c) reacting the product of step (b) with an allyl amine using a mixed anhydride coupling technique to produce an allylic monomer; and (d) copolymerizing the allylic monomer with a second polymerizable monomer in the presence of a radical-generating catalyst, to immobilize the enzyme cleavable substrate.
Claim 16
Dependent Claim
125 words
A process for preparing an immobilized fluorogenic substrate capable of identifying and quantifying, intra- and extra-cellularly, the production and secretion of cell specific enzymes which comprises: (a) coupling an enzyme-specific oligopeptide to the primary amino group of a fluorescent aminoquinoline via an amide linkage; (b) quaternizing the ring nitrogen of the quinoline moiety with an alkylating spacer group selected from the group consisting of haloacetylamino, haloacetoxy- or halomethylsulfonylamino-polymethylene compounds having an ω-amino (protected), an ω-hydroxyl or ω-carboxylic acid group; (c) reacting the product of step (b) with an allyl amine using a mixed anhydride coupling technique to produce an allylic monomer; and (d) copolymerizing the allylic monomer with a second polymerizable monomer in the presence of a radical-generating catalyst, to immobilize the enzyme cleavable substrate.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1993-01-29 | 1993-07-31 | 1994-01-30 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1997-01-29 | 1997-07-31 | 1998-01-30 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2001-01-29 | 2001-07-31 | 2002-01-30 | $7700.00 | Window Closed / Lapsed |