Patent US4070245A successfully refreshed from Google Patents.
US4070245A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Substrate for the quantitative determination of proteolytic enzymes
Assignee: DSM Nutritional Products AG
•
Filing: 1976-06-18
•
Granted: 1978-01-24
•
Expires: 1996-06-18
Abstract
Abstract A substrate for the quantitative determination of enzymes in human and mammal body fluids as well as in animal cell extracts and glandular venoms of cold-blooded animals, which has the structure R1 - Gly - Pro - X - NH - R2 wherein R1 represents hydrogen or a blocking acyl or sulfonyl group, R2 represents an aromatic hydrocarbon group which may carry substituents and X represents arginyl or lysyl, -NH-R2 being a chromogenic or fluorescent group capable of yielding a split product NH2-R2 the quantity of which can be measured by photometric, spectrophotometric or fluorescence-photometric methods.
Inventors
- 👤 Lars Gundro Svendsen
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-24 15:17
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/34; C12Q1/37; C07; C07K; C07K5/00; C07K5/04; C07K5/08; C07K5/0802; C07K5/0804; C07K5/0806; C12Q1/56; C12Q2337/00; C12Q2337/10; C12Q2337/12; G; G01; G01N; G01N2333/00; G01N2333/90; G01N2333/914; G01N2333/948; G01N2333/974; G01N2333/976; Y; Y10; Y10S; Y10S435/00; Y10S435/808; Y10S530/00; Y10S530/802
Claims Analysis
Total Claims: 29
Claim 1
Independent Claim
134 words
A chromogenic or fluorescent substrate for the quantitative determination of proteolytic enzymes of class E.C. 3.4.4. which split peptide chains on the carboxyl side of arginine and lysine in human and mammal body fluids in animal cell extracts and in glandular venoms of cold-blooded animals, which has the structure R.sup.1 -- Gly -- Pro -- X -- NH -- R.sup.2 I wherein R 1 represents hydrogen or a blocking acyl or sulfonyl group, R 2 represents an aromatic hydrocarbon group which may carry substituents, and X represents an arginyl or lysyl group, --NH--R 2 being a chromogenic or fluorescent group, and which, when subjected to the proteolytic action of the said enzymes, yields a split product of formula NH 2 --R 2 the quantity of which is measurable by photometric, spectrophotometric or fluoroescence-photometric methods.
Claim 2
Dependent Claim
134 words
A chromogenic or fluorescent substrate for the quantitative determination of proteolytic enzymes of class E.C. 3.4.4. which split peptide chains on the carboxyl side of arginine and lysine in human and mammal body fluids in animal cell extracts and in glandular venoms of cold-blooded animals, which has the structure R.sup.1 -- Gly -- Pro -- X -- NH -- R.sup.2 I wherein R 1 represents hydrogen or a blocking acyl or sulfonyl group, R 2 represents an aromatic hydrocarbon group which may carry substituents, and X represents an arginyl or lysyl group, --NH--R 2 being a chromogenic or fluorescent group, and which, when subjected to the proteolytic action of the said enzymes, yields a split product of formula NH 2 --R 2 the quantity of which is measurable by photometric, spectrophotometric or fluoroescence-photometric methods.
Claim 3
Dependent Claim
17 words
The substrate according to claim 1 which is protonized with a mineral acid or an organic acid.
Claim 4
Dependent Claim
75 words
The substrate according to claim 1 in which the acyl group R 1 has the partial formula R.sup.3 -- CO -- II wherein R 3 represents a. an aliphatic hydrocarbon radical comprising 1 to 17 carbon atoms, b. an araliphatic hydrocarbon radical the aliphatic group of which comprises 1 to 6 carbon atoms, c. a cycloaliphatic hydrocarbon radical, d. an aromatic hydrogen radical, or e. an alkoxy group having 1 to 17 carbon atoms, or
Claim 5
Dependent Claim
4 words
f. a benzyloxy group.
Claim 6
Dependent Claim
4 words
f. a benzyloxy group.
Claim 7
Dependent Claim
43 words
The substrate according to claim 1 in which the sulfonyl group represented by R 1 is an alkanesulfonyl group the alkane radical of which has 1 to 17 carbon atoms or an arylsulfonyl group which can carry one or more lower alkyl substituents.
Claim 8
Dependent Claim
17 words
The substrate according to claim 1 in which R 2 is a p-nitrophenyl, 2-naphthyl or 4-methoxy-2-naphthyl group.
Claim 9
Dependent Claim
148 words
A chromogenic or fluorescent substrate for the quantitative determination of enzymes of glass E.C. 3.4.4. which split peptide chains on the carboxyl side of arginine and of lysine, especially thrombin and thrombin-like enzymes, in human and mammal body fluids, in animal cell extracts and in glandular venoms of cold-blooded animals, which has the structure R.sup.1 -- Gly -- Pro -- Arg -- NH -- R.sup.2 III wherein R 1 is an alkyloxycarbonyl group the alkyl radical of which has 1 to 6 carbon atoms, an aralkyloxycarbonyl group the alkylene group of which has 1 to 6 carbon atoms, an alkanesulfonyl group the alkane radical of which has 1 to 6 carbon atoms, an arylsulfonyl group the aryl radical of which may carry substituents, or an alkanoyl group the alkane radical of which has 1 to 6 carbon atoms, and R 2 is a p-nitrophenyl, 2-naphthyl or 4methoxy-2-naphthyl group.
Claim 10
Dependent Claim
148 words
A chromogenic or fluorescent substrate for the quantitative determination of enzymes of glass E.C. 3.4.4. which split peptide chains on the carboxyl side of arginine and of lysine, especially thrombin and thrombin-like enzymes, in human and mammal body fluids, in animal cell extracts and in glandular venoms of cold-blooded animals, which has the structure R.sup.1 -- Gly -- Pro -- Arg -- NH -- R.sup.2 III wherein R 1 is an alkyloxycarbonyl group the alkyl radical of which has 1 to 6 carbon atoms, an aralkyloxycarbonyl group the alkylene group of which has 1 to 6 carbon atoms, an alkanesulfonyl group the alkane radical of which has 1 to 6 carbon atoms, an arylsulfonyl group the aryl radical of which may carry substituents, or an alkanoyl group the alkane radical of which has 1 to 6 carbon atoms, and R 2 is a p-nitrophenyl, 2-naphthyl or 4methoxy-2-naphthyl group.
Claim 11
Dependent Claim
3 words
N.sup.α -2-Naphthalenesulfonyl-glycyl-prolyl-arginine-p-nitroanilide hydrochloride.
Claim 12
Dependent Claim
3 words
N.sup.α -2-Naphthalenesulfonyl-glycyl-prolyl-arginine-p-nitroanilide hydrochloride.
Claim 13
Dependent Claim
3 words
N.sup.α -Tosyl-glycyl-propyl-arginine-p-nitroanilide hydrochloride.
Claim 14
Dependent Claim
3 words
N.sup.α -Tosyl-glycyl-propyl-arginine-p-nitroanilide hydrochloride.
Claim 15
Dependent Claim
3 words
N.sup.α -Benzenesulfonyl-glycyl-prolyl-arginine-p-nitroanilide hydrochloride.
Claim 16
Dependent Claim
3 words
N.sup.α -Benzenesulfonyl-glycyl-prolyl-arginine-p-nitroanilide hydrochloride.
Claim 17
Dependent Claim
4 words
N 60 -Benzyloxycarbonyl-glycyl-prolyl-arginine-p-nitroanilide hydrochloride.
Claim 18
Dependent Claim
4 words
N 60 -Benzyloxycarbonyl-glycyl-prolyl-arginine-p-nitroanilide hydrochloride.
Claim 19
Dependent Claim
3 words
N.sup.α -Isobutyloxycarbonyl-glycyl-prolyl-arginine-p-nitroanilide hydrochloride.
Claim 20
Dependent Claim
3 words
N.sup.α -Isobutyloxycarbonyl-glycyl-prolyl-arginine-p-nitroanilide hydrochloride.
Claim 21
Dependent Claim
23 words
The substrate according to claim 2 wherein the mineral acid is HCl, HBr, H 2 SO 4 or H 3 PO 4 .
Claim 22
Dependent Claim
18 words
The substrate according to claim 2 wherein the organic acid is formic acid, oxalic acid or tartaric acid.
Claim 23
Dependent Claim
16 words
The substrate according to claim 4 wherein the alkanesulfonyl group is a methane or ethanesulfonyl group.
Claim 24
Dependent Claim
17 words
The substrate according to claim 4 wherein the arylsulfonyl group is a benzene, p-toluene or naphthalene-sulfonyl group.
Claim 25
Independent Claim
145 words
A method for the quantitative determination of proteolytic enzymes of class E.C. 3.4.4. which split peptide chains on the carboxyl side of arginine and of lysine in human and mammal body fluids, in animal cell extracts and in glandular venoms of cold-blooded animals, which comprises reacting the said body fluids, cell extracts or glandular venoms with a substrate having the following structure R.sup.1 -- Gly -- Pro -- X -- NH -- R.sup.2 I wherein R 1 represents hydrogen or a blocking acyl or sulfonyl group, R 2 , represents an aromatic hydrocarbon group which may carry substituents, and X represents an arginyl or lysyl group, --NH--R 2 being a chromogenic or fluorescent group, and measuring quantitatively by photometric, spectrophotometric or fluorescence-photometric methods the quantity of the split product NH 2 --R 2 formed by the hydrolytic acid of the said enzymes on the substrate.
Claim 26
Independent Claim
145 words
A method for the quantitative determination of proteolytic enzymes of class E.C. 3.4.4. which split peptide chains on the carboxyl side of arginine and of lysine in human and mammal body fluids, in animal cell extracts and in glandular venoms of cold-blooded animals, which comprises reacting the said body fluids, cell extracts or glandular venoms with a substrate having the following structure R.sup.1 -- Gly -- Pro -- X -- NH -- R.sup.2 I wherein R 1 represents hydrogen or a blocking acyl or sulfonyl group, R 2 , represents an aromatic hydrocarbon group which may carry substituents, and X represents an arginyl or lysyl group, --NH--R 2 being a chromogenic or fluorescent group, and measuring quantitatively by photometric, spectrophotometric or fluorescence-photometric methods the quantity of the split product NH 2 --R 2 formed by the hydrolytic acid of the said enzymes on the substrate.
Claim 27
Dependent Claim
29 words
The method according to claim 16 wherein thrombin and thrombin-like enzymes, ecarin thrombin, plasmin and plasminlike enzymes, trypsin and indirectly proenzymes, proenzyme activators and enzyme inhibitors are quantitatively determined.
Claim 28
Independent Claim
170 words
A method for the quantitative determination of thrombin and thrombin-like enzymes in human and mammal body fluids, in animal cell extracts and in glandular venoms of cold-blooded animals, which comprises reacting the said body fluids, cell extracts or glandular venoms with a substrate having the following structure. R.sup.1 --Gly -- Pro -- Arg -- NH -- R.sup.2 wherein R 1 is an alkyloxycarbonyl group the alkyl radical of which has 1 to 6 carbon atoms, an aralkyloxycarbonyl group the alkylene group of which has 1 to 6 carbon atoms, an alkanesulfonyl group the alkane radical of which has 1 to 6 carbon atoms, an arylsulfonyl group the aryl radical of which may carry substituents, or an alkanoyl group the alkane radical of which has 1 to 6 carbon atoms, and R 2 is a p-nitrophenyl, 2-naphthyl or 4-methoxy-2-naphthyl group, and measuring quantitatively by photometric, spectrophotometric or fluorescence-photometric methods the quantity of the split product NH 2 --R 2 formed by the hydrolytic action of the said enzymes on the substrate.
Claim 29
Independent Claim
170 words
A method for the quantitative determination of thrombin and thrombin-like enzymes in human and mammal body fluids, in animal cell extracts and in glandular venoms of cold-blooded animals, which comprises reacting the said body fluids, cell extracts or glandular venoms with a substrate having the following structure. R.sup.1 --Gly -- Pro -- Arg -- NH -- R.sup.2 wherein R 1 is an alkyloxycarbonyl group the alkyl radical of which has 1 to 6 carbon atoms, an aralkyloxycarbonyl group the alkylene group of which has 1 to 6 carbon atoms, an alkanesulfonyl group the alkane radical of which has 1 to 6 carbon atoms, an arylsulfonyl group the aryl radical of which may carry substituents, or an alkanoyl group the alkane radical of which has 1 to 6 carbon atoms, and R 2 is a p-nitrophenyl, 2-naphthyl or 4-methoxy-2-naphthyl group, and measuring quantitatively by photometric, spectrophotometric or fluorescence-photometric methods the quantity of the split product NH 2 --R 2 formed by the hydrolytic action of the said enzymes on the substrate.
References Cited (Backward Citations)
US3884896A
Cited
US3886136A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1981-01-23 | 1981-07-25 | 1982-01-24 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1985-01-23 | 1985-07-25 | 1986-01-24 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1989-01-23 | 1989-07-25 | 1990-01-24 | $7700.00 | Window Closed / Lapsed |