EP0000063A1 - Dipeptide derivatives of 7-(N-alpha-substituted or non-substituted X-arginyl)-amino-4-methyl-coumarin - Google Patents
Dipeptide derivatives of 7-(N-alpha-substituted or non-substituted X-arginyl)-amino-4-methyl-coumarin Download PDFInfo
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- EP0000063A1 EP0000063A1 EP78100111A EP78100111A EP0000063A1 EP 0000063 A1 EP0000063 A1 EP 0000063A1 EP 78100111 A EP78100111 A EP 78100111A EP 78100111 A EP78100111 A EP 78100111A EP 0000063 A1 EP0000063 A1 EP 0000063A1
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- methylcoumarin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
- C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/06—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2
- C07D311/08—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring
- C07D311/16—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring substituted in position 7
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
- C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06139—Dipeptides with the first amino acid being heterocyclic
- C07K5/06165—Dipeptides with the first amino acid being heterocyclic and Pro-amino acid; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
- C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0819—Tripeptides with the first amino acid being acidic
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0821—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp
- C07K5/0823—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp and Pro-amino acid; Derivatives thereof
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Definitions
- This invention relates to peptide derivatives, more particularly 7-(Na-substituted or non-substituted-X-arginyl)-amino-4-methyl-coumarin wherein X is a residue of an amino acid selected from the group of glycine, phenylalanine and proline, and to acid salts thereof, which are useful as fluorogenic substrates for determining enzymatic activities.
- a method which comprises reacting a material which is specifically acted by an enzyme with the enzyme and comparing the state of the material before reaction with that after reaction.
- This invention responds to such need and provides synthetic substrates which show specificity to enzymes such as Trypsin and Thrombin.'
- Compounds of the present invention are novel peptide derivatives, which are not described in any reference, more particularly 7-(N ⁇ -substituted or ron-substituted-X-arginyl)amino-4-methyl coumarin wherein X is a residue of an amino acid selected from the group of glycine phenylalanine and proline, and acid salts thereof.
- R 1 When R 1 is a hydrogen atom, the compound is 7-(glycyl- arginyl)-amino-4-methylcoumarin.
- R 1 When R 1 is a prolyl group, the compound is 7-(prolylglycylarginyl)-amino-4-methylcoumarin.
- R 1 When R 1 is a glutamyl group, the compound is 7-(glutamylglycylarginyl)-amino-4-methylcoumarin.
- R 1 When R 1 is a seryl group, the compound is 7-(serylglycylarginyl)-amino-4-methylcoumarin.
- R 1 When R 1 is a leucyl group, the compound is 7-(leucylglycylarginyl)amino-4-methyl-coumarin.
- R 1 When R 1 is a glutamyl group carrying an isoleucyl group in the N ⁇ -position thereof, namely an isoleucylglutamyl group, the compound is 7-(isoleucylglutamylglycylarginyl)-amino-4-methylcoumarin.
- R 1 is a seryl group having a valyl group in the N ⁇ -position thereof, namely a valyl- seryl group
- the compound is 7-(valylserylglycylarginyl)-amino-4-methylcoumarin.
- R 1 is a leucyl group having a valyl group in the N ⁇ -position thereof, namely valyl- leucyl group
- the compound is 7-(valylleucylglycylargin- yl)-amino-4-Methylcoumarin.
- R 3 When R 3 is a hydrogen atom, the compound is 7-(prolyl- arginyl)-amino-4-methylcoumarin. When R 3 is a valyl group, the compound is 7-(valylprolylarginyl)amino-4-methylcoumarin.
- N ⁇ -amino and imino groups in such compounds may be protected by conventional protecting groups for peptides, such as an acyl group (for example, acetyl group and benzoyl group), carbobenzoxy group, tert-alkyloxycarbonyl group, tosyl group and glutaryl group.
- an acyl group for example, acetyl group and benzoyl group
- carbobenzoxy group for example, acetyl group and benzoyl group
- carbobenzoxy group for example, tert-alkyloxycarbonyl group, tosyl group and glutaryl group.
- the hydroxy group thereof may be protected by protecting groups which are used in the conventional method-for synthesizing peptides, such as alkyl groups and benzyl groups.
- the y-carboxyl group thereof may be protected by an ester group with an alcohol such as benzyl alcohol.
- the guanidino group of arginine which constitutes such compounds may be protected by N-guanidino-protecting groups which are used in the conventional method for synthesizing peptides, such as nitro groups, tosyl groups and p-methoxy-benzenesulfonyl groups, and by adding a proton, as in the form of an acid.
- the compounds of the present invention can be produced by conventional methods for synthesizing peptides using 7- arginyl-amino-4-methylcoumarin.
- 7-(glycyl- arginyl)amino-4-methylcoumarin can be produced by reacting glycine whose amino group has been protected, with 7- arginylamino-4-methylcoumarin in the presence of condensing agents such as dicyclohexylcarbodiimide (DCCI), or by reacting an active ester of glycine whose amino group has been protected, with 7-arginylamino-4-methylcoumarin, and then removing the protecting-group of the amino group therein.
- DCCI dicyclohexylcarbodiimide
- 7-(glut- amylglycylarginyl)-amino-4-methylcoumarin By reacting this compound with glutamic acid whose amino group and y-carboxyl group are protected and then removing protecting-groups from both the amino group and y-carboxyl group of thus obtained compound, 7-(glut- amylglycylarginyl)-amino-4-methylcoumarin can be obtained.
- 7-(isoleucyl-glutamylglycylarginyl)amino-4-methylcoumarin can be produced by reacting 7-(glutamylglycylarginyl)-amino-4-methylcoumarin as produced above,with an active ester of isoleucine whose amino group has been protected, an then treating such reacted material in the same manner as above.
- 7-(prolylglycylarginyl)amino-4-methylcoumarin can be produced by reacting 7-(glycylarginyl)amino-4-methylcoumarin obtained above with proline whose imino group has been protected and then removing the protecting group from the thus obtained compound.
- the component hailing the carboxyl group which reacts with component having the amino group is in the active ester form.
- active esters are N-hydroxysuccinimide ester and p-nitrophenyl ester. Reactions using active ester can be operated sufficiently at room temperature, but can be accelerated by heating, if necessary.
- the reaction mixture is concentrated to yield a solid material and the material is purified by column chromatography and then lyophilizel.
- the compounds thus obtained are usually white powders and are decomposed slowly by heating at more than 140°C.
- such protecting-groups can be removed by conventional methods or synthesizing peptides.
- the carbobenzoxy group can be removed by'catalytic hydrogenation in a solvent such as alcohol, and the tert-butyloxycarbonyl group can be removed by reacting the compound in hydrogen fluoride for 30 minutes or by reacting the compound with p-toluene sulfonic acid for 90 minutes.
- the compound of the present invention can be produced in free form or acid salt form according to the method for production.
- the free form can be easily converted to the acid salt thereof and an acid salt can be easily converted to the free form thereof.
- Examples of the acid salts are salts of inorganic acids such as hydrochloric acid, sulfuric acid, nitric.acid and phosphoric acid, and salts of organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, citric acid and toluene sulfonic acid.
- inorganic acids such as hydrochloric acid, sulfuric acid, nitric.acid and phosphoric acid
- salts of organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, citric acid and toluene sulfonic acid.
- the chemical structure of the compounds of the present invention was identified by elemental analysis, amino acid analysis, ultraviolet spectrum, and comparison of ultraviolet spectrum of material hydrolyzed with trypsin with 7-amino-4-raethylcoumarin.
- the compounds of the present invention are all hydrolyzed specifically by one or more enzyme(s) such as Thrombin, Horseshoe-Crab Blood Coagulating Enzyme, urokinase and/or Blood Coagulating Factor XII a , and therefore are suitable as synthetic substrates of these compounds.
- enzyme(s) such as Thrombin, Horseshoe-Crab Blood Coagulating Enzyme, urokinase and/or Blood Coagulating Factor XII a , and therefore are suitable as synthetic substrates of these compounds.
- Amino acids which compose the compounds of the present invention may have any stereochemical configuration, L, D and DL, if the compounds may be enzymatically hydrolyzed.
- 7-arginylamino-4-methylcoumarin and the salts thereof are also new compounds.
- 7-arginylamino-4-methylcoumarin in the free form can be easily produced by reacting the acid salts thereof with alkaline material.
- N ⁇ -carbobenzoxy-L-arginine hydrochloric acid salt (69.0g) 0.2 M) and 7-amino-4-methylccumarin (17.5g, 0.1 M)
- DMF dimethylformamide
- DCCD Dicyclohexylcarbodiimide
- This product gave a single spot on the thin layer chromatogram (silica gel) with a ternary eluent of n-butyl alcohol, acetic acid and water in 4 : 1 : 1 volume ratio.
- N ⁇ -carbobenzoxy-N G -tosyl-L-arginine (See J. A.C.S. 82, 4576) (6.0 g, 13 mM) was dissolved in DMF (20 ml), and 4-methyl-7-aminocoumarin (1.75 g, 10 mM) was added thereto.
- DCCD (2.47 g, 12 mM) was added with ice-cooling. The mixture was stirred overnight at room temperature and the precipitated dicyclohexyl urea was removed by filtration. DMF was distilled off under reduced pressure, and to the residue chloroform (200 ml) was added for extraction.
- the chloroform solution was washed with 1 N-HCl, 5 % NaHCO 3 and water successively, and then was dried over anhydrous sodium sulfate.
- chloroform was distilled off under reduced pressure, and the residue was purified by column chromatography using silicagel and a binary mixture of chloroform and ethyl acetate in 2 : 1 volume ratio. The main fractions were combined and concentrated.
- the residue was dissolved in chloroform (10 ml) and ether was added thereto to precipitate desired material. This material was separated to give 7-(N ⁇ -carbobenzoxy-N G -tosyl-L-arginyl)amino-4-methylcoumarin (2.31 g, yield: 37 %).
- the precipitate was separated by'filtration and dried.
- the precipitate was dissolved in DMF (8 ml), and N-hydroxysuccinimide ester of tert-butyloxycarbonyl- ⁇ -benzyl-L-glutamic acid (478 mg, 1.1 mM). This mixture was stirred for 2 days at room temperature, and excess ether was added to yield a precipitate.
- This precipitate was separated by filtration and purified by column chromatography (2 x 10 cm) using silica gel as adsorbent and a ternary eluent of chloroform, ethyl acetate and ethyl alcohol in 5 : 5 : 2 volume ratio. The main fractions were were combined and concentrated to yield solid material.
- the residue was purified by column chromatography (2 x 15 cm) using silicagel as adsorbent and ternary mixtures of chloroform, methyl alcohol and acetic acid in 95 : 5 : 3 (adsorption) and then 85 : 20 : 5 (elution) in volume ratio.
- the main fraction was cen- centrated to yield a solid material.
- the material was dissolved in acetic acid (50 ml)and lyophilized to give 7-(N ⁇ -tert-butyloxycarbonyl-L-valyl-L-seryl-glycyl-L-arginyl)amino-4-methylcoumanin hydrochloric acid salt [IX] (320 mg).
- the material was purified by column chromatography (2 x 15 cm) using silica gel as adsorbent and ternary mixtures of chloroform methylalcohol and acetic acid in 95 : 5 : 3 (adsorption) and then 85 : 20 : 5 (elution) in volume ratio.
- the main fraction was concentrated to yield a solid material and dissolved in acetic acid (20 ml). This solution was lyophilized to give 7-(N a - tert-butyloxycarbonyl-0-benzyl-L-serylglycyl-L-arginyl)-amino-4-methyl-coumarin hydrochloric acid salt [XI] (180 mg).
- the material was purified by column chromatography (2 x 15 cm) using silica gel as adsorbent and ternary mixtures of chloroform, methylalcohol and acetic acid in 95 : 5 : 3 (adsorption) and then 85 : 20 : 5 (elution) in volume ratio.
- the main fraction was concentrated to yield solid material, dissolved in acetic acid (20 ml) and lyophilized to give 7-(N ⁇ -tert-butyl- oxycarbonyl-L-valyl-L-leucyl-glycyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt [XII] (350 mg).
- the carbobenzoxy group of the compound thus obtained can be removed by applying a hydrogenation reaction in alcohol thereto.
- the material was purified by column chromatography (2 x 15 cm) using silica gel as adsorbent and ternary mixtures of chloroform, methyl alcohol and acotie acid in 95 : 5 : 3 and then 85 : 20 : 5 volume ratio.
- the main fraction was concentrated to yield solid material, dissolved in acetic acid (10 ml) and lyophilized to give 7-(N ⁇ -tert-butyloxycarbonyl-L-valyl-L-prolyl-L-arginyl)-amino-4-methylcoumarin hydrochloric acid salt [XX] (320 mg).
- the compound produced above (0.1 - 0.2 mM) was dissolved in a mixture of dimethylsulfoxide (5 ml) and water (5 ml) and this solution was diluted with 0.05 M Tris-HCl buffer (pH 8.0) containing both 0.1 M NaCl and 10 mM CaCl 2 to adjust the total volume thereof to 500 ml and thereby a substrate solution was prepared.
- Tris-HCl buffer pH 8.0
- Each substrate solution (2 ml) was added in a test tube and the obtained test tube was kept at 37°C for 5 minutes. Then, each enzyme solution (20 ⁇ l) was added thereto and incubated at 37°C for 20 minutes with stirring. The incubation was stopped by adding 100 % acetic acid (0.5 ml) thereto.
- the hydrolysis degree was determined by measuring the fluorescence intensity at 460 nm with excitation at 380 mn in the fluorescence spectra. Results are listed in the following table.
- Each value is the fluorescence intensity ratio when the fluorescence intensity of 7-amino-4-methylcoumarim solution (40 ⁇ M, 40 ⁇ mole/l) is 100.
- a value having the mark --.*-- is one with the fluorescence intensity of 7-amino-4-methylcoumarin solution (200 ⁇ M, 200 ⁇ mol/l) being 100.
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Abstract
Description
- This invention relates to peptide derivatives, more particularly 7-(Na-substituted or non-substituted-X-arginyl)-amino-4-methyl-coumarin wherein X is a residue of an amino acid selected from the group of glycine, phenylalanine and proline, and to acid salts thereof, which are useful as fluorogenic substrates for determining enzymatic activities.
- In order to determine enzymatic activities, a method is usually employed which comprises reacting a material which is specifically acted by an enzyme with the enzyme and comparing the state of the material before reaction with that after reaction.
- As materials for the stated purpose, materials occuring in nature can be employed, but synthesized ones are suitably employed in view of mass production and production in pure form. Therefore, compounds which can be substrates for all kinds of enzymes are strongly desired. Such compounds can act as antimetabolites in a living body, and thus it is expected that such compounds can be used as medicines.
- This invention responds to such need and provides synthetic substrates which show specificity to enzymes such as Trypsin and Thrombin.'
- Compounds of the present invention are novel peptide derivatives, which are not described in any reference, more particularly 7-(Nα-substituted or ron-substituted-X-arginyl)amino-4-methyl coumarin wherein X is a residue of an amino acid selected from the group of glycine phenylalanine and proline, and acid salts thereof.
- (1) When X is the residue of glycine, the compounds are represented by the following formula:
- When R1 is a hydrogen atom, the compound is 7-(glycyl- arginyl)-amino-4-methylcoumarin. When R1 is a prolyl group, the compound is 7-(prolylglycylarginyl)-amino-4-methylcoumarin. When R1 is a glutamyl group, the compound is 7-(glutamylglycylarginyl)-amino-4-methylcoumarin. When R1 is a seryl group, the compound is 7-(serylglycylarginyl)-amino-4-methylcoumarin. When R1 is a leucyl group, the compound is 7-(leucylglycylarginyl)amino-4-methyl-coumarin. When R1 is a glutamyl group carrying an isoleucyl group in the Nα-position thereof, namely an isoleucylglutamyl group, the compound is 7-(isoleucylglutamylglycylarginyl)-amino-4-methylcoumarin. When R1 is a seryl group having a valyl group in the Nα-position thereof, namely a valyl- seryl group, the compound is 7-(valylserylglycylarginyl)-amino-4-methylcoumarin. When R1 is a leucyl group having a valyl group in the Nα-position thereof, namely valyl- leucyl group, the compound is 7-(valylleucylglycylargin- yl)-amino-4-Methylcoumarin.
- (2) When X is the residue of phenylalanine, the compounds of the present invention are represented by the following formula:
- (3) When X is the residue of prolin, the compounds of the present invention are represented by the following formula:
- When R3 is a hydrogen atom, the compound is 7-(prolyl- arginyl)-amino-4-methylcoumarin. When R3 is a valyl group, the compound is 7-(valylprolylarginyl)amino-4-methylcoumarin.
- Nα-amino and imino groups in such compounds may be protected by conventional protecting groups for peptides, such as an acyl group (for example, acetyl group and benzoyl group), carbobenzoxy group, tert-alkyloxycarbonyl group, tosyl group and glutaryl group.
- In case of the compounds having a serine residue, the hydroxy group thereof may be protected by protecting groups which are used in the conventional method-for synthesizing peptides, such as alkyl groups and benzyl groups.
- In case of the compunds having a glutamic acid residue, the y-carboxyl group thereof may be protected by an ester group with an alcohol such as benzyl alcohol.
- The guanidino group of arginine which constitutes such compounds may be protected by N-guanidino-protecting groups which are used in the conventional method for synthesizing peptides, such as nitro groups, tosyl groups and p-methoxy-benzenesulfonyl groups, and by adding a proton, as in the form of an acid.
- The compounds of the present invention can be produced by conventional methods for synthesizing peptides using 7- arginyl-amino-4-methylcoumarin. For example, 7-(glycyl- arginyl)amino-4-methylcoumarin can be produced by reacting glycine whose amino group has been protected, with 7- arginylamino-4-methylcoumarin in the presence of condensing agents such as dicyclohexylcarbodiimide (DCCI), or by reacting an active ester of glycine whose amino group has been protected, with 7-arginylamino-4-methylcoumarin, and then removing the protecting-group of the amino group therein. By reacting this compound with glutamic acid whose amino group and y-carboxyl group are protected and then removing protecting-groups from both the amino group and y-carboxyl group of thus obtained compound, 7-(glut- amylglycylarginyl)-amino-4-methylcoumarin can be obtained. 7-(isoleucyl-glutamylglycylarginyl)amino-4-methylcoumarin can be produced by reacting 7-(glutamylglycylarginyl)-amino-4-methylcoumarin as produced above,with an active ester of isoleucine whose amino group has been protected, an then treating such reacted material in the same manner as above. 7-(prolylglycylarginyl)amino-4-methylcoumarin can be produced by reacting 7-(glycylarginyl)amino-4-methylcoumarin obtained above with proline whose imino group has been protected and then removing the protecting group from the thus obtained compound.
- These condensing reactions can desirably be operated in a suitable solvent such as dimethylformamide (DMF), dimethylsulfoxide, water and mixtures thereof.
- It ispreferable that the component hailing the carboxyl group which reacts with component having the amino group, is in the active ester form. Suitable examples of the active esters are N-hydroxysuccinimide ester and p-nitrophenyl ester. Reactions using active ester can be operated sufficiently at room temperature, but can be accelerated by heating, if necessary.
- After completing the reaction, the reaction mixture is concentrated to yield a solid material and the material is purified by column chromatography and then lyophilizel. The compounds thus obtained are usually white powders and are decomposed slowly by heating at more than 140°C.
- As regards the compounds having protecting groups in amino or imino groups thereof, such protecting-groups can be removed by conventional methods or synthesizing peptides. For example, the carbobenzoxy group can be removed by'catalytic hydrogenation in a solvent such as alcohol, and the tert-butyloxycarbonyl group can be removed by reacting the compound in hydrogen fluoride for 30 minutes or by reacting the compound with p-toluene sulfonic acid for 90 minutes.
- The compound of the present invention can be produced in free form or acid salt form according to the method for production.The free form can be easily converted to the acid salt thereof and an acid salt can be easily converted to the free form thereof.
- Examples of the acid salts are salts of inorganic acids such as hydrochloric acid, sulfuric acid, nitric.acid and phosphoric acid, and salts of organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, citric acid and toluene sulfonic acid.
- The chemical structure of the compounds of the present invention was identified by elemental analysis, amino acid analysis, ultraviolet spectrum, and comparison of ultraviolet spectrum of material hydrolyzed with trypsin with 7-amino-4-raethylcoumarin.
- The compounds of the present invention are all hydrolyzed specifically by one or more enzyme(s) such as Thrombin, Horseshoe-Crab Blood Coagulating Enzyme, urokinase and/or Blood Coagulating Factor XIIa, and therefore are suitable as synthetic substrates of these compounds.
- Amino acids which compose the compounds of the present invention may have any stereochemical configuration, L, D and DL, if the compounds may be enzymatically hydrolyzed.
- Now, 7-arginylamino-4-methylcoumarin and the salts thereof are also new compounds. 7-arginylamino-4-methylcoumarin in the free form can be easily produced by reacting the acid salts thereof with alkaline material.
- The present invention is illustrated in more detall by the following Examples.
- Nα-carbobenzoxy-L-arginine hydrochloric acid salt (69.0g) 0.2 M) and 7-amino-4-methylccumarin (17.5g, 0.1 M) Were dissolved in dimethylformamide (DMF) (300 ml). Dicyclohexylcarbodiimide (DCCD) (21g, 0.1 M) was added to the mixture with stirring at room temperature. This mixture was stirred overnight at room temperature, and the pro- duced dicyclohexyl urea was removed by filtration. The obtained filtrate was concentrated under reduced pressure and to the obtained residue methyl alcohol (50ml) and ethyl acetate (500ml) were added. The precipitated material was separated by filtration to give crude crystals (19g). These crystals were dissolved in a mixture of heated DMF (30 ml): and heated methyl alcohol (100 ml) and insoluble material was removed by filtration. To the filtrate ethyl acetate (400 ml) was added and thereby precipitated white crystals were separated by filtration and dried to give 7-(Nα-carbobenzoxy-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt (14.5g, yield: 29%).
- m.p. 210-211°C (dec.).
-
- Elemental Analysis:
- m.p. 275°C (dec.)
-
- This product gave a single spot on the thin layer chromatogram (silica gel) with a ternary eluent of n-butyl alcohol, acetic acid and water in 4 : 1 : 1 volume ratio.
- Elemental Analysis:
- m.p. 150°C (dec.)
-
-
- 7-(Nα-carbobenzoxyglycyl-L-arginyl)amino-4-methylocoumarin hydrochloric acid salt (559mg, 1mM) was dissolved in methyl alcohol (50 ml) and palladium-carbon catalyst (50 mg) was added thereto. This mixture was stirred under treating with hydrogen gas for 3 hours. The catalyst was removed by filtration and the thus obtained filtrate was concentrated to yield solid material. To the residue. ether (50 ml) was added to yield a powder. The powder was separated by filtration and dried to give 7-(glycyl-L-arginyl)amino-4-methylcourmarin hydrochloric acid salt [II] (400 mg).
- m.p. 145°C (dec.)
-
-
- 7-(Nα-glycyl-L-arginyl)armino-4-methylcoumarin hydrochloric acid salt (425 mg, 1mM) was dissolved in DMF (10 ml), and N-hydroxysuccinimide ester of carbobenzoxy-L-proline (450 mg, 1.3mM) was added thereto. The mixture was stirred for 15 hours at room temperature and then was concentrated to yield solid material. This material was purified by column chromatography (2 x 10 cm) using silicagel as an adsorbent'an a ternary.eluent of chloroform, methyl alcohol and acetic acid in 95 : 5 : 3 volume ratio. The main fraction was concentrated to yield a solid material. This material was dissolved in acetic acid (20 ml) and lyophilized to give 7-(Nα-carbobenzoxy-L-prolyl-glycyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid as the acetic acid adduct [III] (460 mg).
- m.p. 200°C (dec.)
-
-
- 7-(Nα-carbobenzoxy-L-prolyl-glycyl-L-arginyl)amino-4-methyl-coumarin hydrochloric acid salt (200 mg, 0.27 mM) was dissolved in methyl alcohol (50 ml) and 1 N-HCl (0.27 ml) and palladium-carbon catalyst (20 mg) were added thereto. This mixture was stirred under treating with hydrogen gas for 3 hours. The catalyst was removed by filtration and the filtrate was concentrated to yield a solid material. To the residue ether (50 ml) was added and the thus produced powder was separated by filtration to give 7-(L-prolyl-glycyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt [IV] (150 mg).
- m. p. 185°C (dec.)
-
-
- Nα-carbobenzoxy-NG-tosyl-L-arginine (See J. A.C.S. 82, 4576) (6.0 g, 13 mM) was dissolved in DMF (20 ml), and 4-methyl-7-aminocoumarin (1.75 g, 10 mM) was added thereto. To this mixture DCCD (2.47 g, 12 mM) was added with ice-cooling. The mixture was stirred overnight at room temperature and the precipitated dicyclohexyl urea was removed by filtration. DMF was distilled off under reduced pressure, and to the residue chloroform (200 ml) was added for extraction. The chloroform solution was washed with 1 N-HCl, 5 % NaHCO3 and water successively, and then was dried over anhydrous sodium sulfate. Next, chloroform was distilled off under reduced pressure, and the residue was purified by column chromatography using silicagel and a binary mixture of chloroform and ethyl acetate in 2 : 1 volume ratio. The main fractions were combined and concentrated. The residue was dissolved in chloroform (10 ml) and ether was added thereto to precipitate desired material. This material was separated to give 7-(Nα-carbobenzoxy-NG-tosyl-L-arginyl)amino-4-methylcoumarin (2.31 g, yield: 37 %).
- m. p. 155.0-162.5°C (dec.)
-
- This product gave a single spot (Rf=0.4) on the thin layer chromatogram (silicagel) with a ternary eluent of chloroform, methyl alcohol and acetic acid in 95 : 5 : 3 volume ratio.
- Elemental Analysis:
- m.p. 174.5 - 182.5°C (dec.)
-
- This product gave a singly spot (RF=0.6) on the thin layer chromatogram (silica gel) with a ternary eluent of n-butylalcohol, acetic acid and water in 4 : 1 : 1 volume ratio.
- Elemental Analysis:
- m.p. 150.5 - 135.5°C
-
-
- 7-(Nα-carbobanzoxyglycyl-NG-tosyl-arginyl)amino-4-methylcoumarin (678 mg, 1 mM) was dissolved in a mixture of methyl alcohol (50 ml) and.acetic acid (10 ml), and 5 % palladium-carbon catalyst (50 mg) was added thereto. This mixture was treated with hydrogen gas for 4 hours. The catalyst was removed by filtration and from the filtrate, solvent was distilled off under reduced pressure. The residue was dissolved in methyl alcohol (10 ml) and excess ether was added thereto to yield a precipitate.
- The precipitate was separated by'filtration and dried. The precipitate was dissolved in DMF (8 ml), and N-hydroxysuccinimide ester of tert-butyloxycarbonyl-γ-benzyl-L-glutamic acid (478 mg, 1.1 mM). This mixture was stirred for 2 days at room temperature, and excess ether was added to yield a precipitate. This precipitate was separated by filtration and purified by column chromatography (2 x 10 cm) using silica gel as adsorbent and a ternary eluent of chloroform, ethyl acetate and ethyl alcohol in 5 : 5 : 2 volume ratio. The main fractions were were combined and concentrated to yield solid material. This material was reprecipitated from a binary mixture of methyl alcohol (10 ml) and ether (50 ml) to give 7-(Nα- tert-butyloxycarbonyl-γ-benzyl-L-glutamylglycyl-NG-tosyl-L-arginyl)amino-4-methylcoumarin (705 mg, yield 82.0 %).
- m.p. 118.5 - 123.5°C
- [α]15 -12.7° (C=1, DMF)
-
- To a mixture of 7-(Nα-tert--butyloxycarbonyl-γ-benzyl-L-glutamylglycyl-NG-tosyl-L-arginyl)amino-4-methylcoumarin (432 mg, 0.5 mM) and anisole (0.5 ml), anhydrous hydrogen fluoride (10 ml) was added thereto at -70°C. This mixture was stirred for 60 minutes under cooling in an ice bath. The excess hydrogen fluoride was distilled off under reduced pressure. To the residue water (5 ml) was added and thus obtained water layer was washed with ether (20 ml) and then passed throug a column filled with a ion-exchange resin, Dowex 1 x 2 (acetic acid salt form, 5 ml), which was eluted with water (10 ml). The eluate was lyophilized to give 7-(L-glutamylglycyl-L-arginyl)amiho-4-methylcoumarin [V](225 mg, yield: 74 %), which is amorphous.
-
-
- 7-(L-glutamylglycyl-L-arginyl)amino-4-methyl-coumarin (100 mg, 0.18 mM) was dissolved in DMF (2 ml), and triethylamine (0.03 ml) was added thereto under stirring and cooling. This mixture was adjusted to pH about 7, and then p-nitrophenyl acetate (36.1 mg, 0.23 mM) was added thereto. This mixture was stirred for 2 days at room temperature. Excess ether was added to the mixture and the formed precipitate was separated by filtration and dried. This product was dissolved in 80 % -methyl alcohol (2 ml) and ethyl acetate (30 ml) was added thereto for re-precipitation to give 7-(Nα-acetyl-L-glutamyl- glycyl-L-arginyl)amino-4-methylcoumarin [VI] (99 mg, yield : 84.9 %).
- m.p. 214.0 - 217.5°C (dec.)
-
-
- 7-(L-glutamylglycyl-L-argxnyl)aminc-4-methyl-coumarin (100 mg, 0.18 mM) was dissolved in DMF (2 ml), and triethylamine (0.03 ml) was added thereto with stirring and cooling. This mixture was adjusted to pH about 7, and N-hydroxysuccinimide ester of tert-butyloxycarbonyl-L-iso leucine (75.4 mg, 0.23 mM) was added thereto. This mixture was stirred for 3 days at room temperature. To this mixture excess ether was added and the produced precipitate was separated by filtration and dried. This material was dissolved in methyl alcohol (2 ml), and ether (20 ml) was added thereto for'reprecipitation to give 7-(Nα- tert-butyloxy-carbonyl-L-isaleucyl-L-glutamylglycyl-L-arginyl)amino-4-methylcoumarin [VII] (120 mg, yield: 86.3 %).
- m.p. 208.00- 212.0°C (dec.)
-
-
- 7-(Nα-glycyl-L-arginyl)amino-4-methyl-coumarin hydrochloric acid salt (255 mg, 0.6 mM) was dissolved in a mixture of DMF (1 ml) and water (1 ml), and glutaric acid anhydride (75.2 mg, 0.66 mM) was added thereto. This mixture was stirred for 15 hours at room temperature, and ether (50 ml) was added to yield a precipitate. The precipitate was separated by filtration, dried and purified by column chromatography (2 x 15 cm) using silica gel as adsorbent and a binary eluent of chloroform and methyl alcohol in a 1 : 2 volume ratio. The main fraction was concentrated to yield a solid material. This material was re-recipitated from DMF ( 5 ml)- ether (5 ml) to give 7-(Nα-glutaryl-glycyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt [VIII] (250 mg, yield: 77.4 %).
- m.p. 234.5 - 238.0°C (dec.)
-
-
- 7-L-arginylamino-4-methylcoumarin hydrochloric acid salt 1/2 H20 (377 mg, 1 mM) was dissolved in a mixture of DMF. (10 ml) and water (10 ml), and Na-tert-butyloxycarbonyl-L-valyl-L-seryl-glycine-N-hydroxysuccinimide ester (458 mg, 1 mM) was added thereto. This mixture was stirred for 15 hours at room temperature and concentrated to yield a solid material. The residue was purified by column chromatography (2 x 15 cm) using silicagel as adsorbent and ternary mixtures of chloroform, methyl alcohol and acetic acid in 95 : 5 : 3 (adsorption) and then 85 : 20 : 5 (elution) in volume ratio. The main fraction was cen- centrated to yield a solid material. The material was dissolved in acetic acid (50 ml)and lyophilized to give 7-(Nα-tert-butyloxycarbonyl-L-valyl-L-seryl-glycyl-L-arginyl)amino-4-methylcoumanin hydrochloric acid salt [IX] (320 mg).
- m.p. 160°C (dec.)
-
-
- 7-(Nα-tert-butyloxycarbonyl-O-benzyl-L-serylglycyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt (120 mg, 0.28 mM) was dissolved in methyl alcohol (50 ml), and palladium-carbon catalyst (20 mg) was added. Through this mixture hydrogen gas was passed for 4 hours with stirring. The catalyst was removed by filtration and the filtrate was concentrated to yield a solid material. The residue was dissolved in acetic acid (10 ml) and lyophilized to give 7-(Nα-tert-butyloxycarbonyl-L-seryl- glycyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt [X] (70 mg).
- m.p. 180°C (dec.)
-
-
- 7-(Nα-glycyl-L-arginyl)amino-4-methyl-coumarin hydrochloric acid salt (230 mg, 0.54 mM) was dissolved in DMF (5 ml), and Nα-ter t-butyloxycarbonyl-O-benzyl-L-serine-N-hydroxysuccinimide ester (253 mg, 0.65 mM) was added thereto. This mixture was stirred for 15 hours at room temperature and concentrated to yield a solid material. The material was purified by column chromatography (2 x 15 cm) using silica gel as adsorbent and ternary mixtures of chloroform methylalcohol and acetic acid in 95 : 5 : 3 (adsorption) and then 85 : 20 : 5 (elution) in volume ratio. The main fraction was concentrated to yield a solid material and dissolved in acetic acid (20 ml). This solution was lyophilized to give 7-(Na - tert-butyloxycarbonyl-0-benzyl-L-serylglycyl-L-arginyl)-amino-4-methyl-coumarin hydrochloric acid salt [XI] (180 mg).
- m.p. 160°C (dec.)
-
-
- 7-L-arginylamino-4-methylcoumarin hydrochloric acid salt 1/2 H20 (377 mg, 1 mM). was dissolved in a mixture of DMF (5 ml) and water (5 ml), Nα-tert-butyloxycarbonyl-L-valyl-L-leucylglycine-N-hydroxysuccinimide ester (484 mg, 1 mM) was added thereto. This mixture was stirred for 15 hours .at room temperature and concentrated to yield a solid material. The material was purified by column chromatography (2 x 15 cm) using silica gel as adsorbent and ternary mixtures of chloroform, methylalcohol and acetic acid in 95 : 5 : 3 (adsorption) and then 85 : 20 : 5 (elution) in volume ratio. The main fraction was concentrated to yield solid material, dissolved in acetic acid (20 ml) and lyophilized to give 7-(Nα-tert-butyl- oxycarbonyl-L-valyl-L-leucyl-glycyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt [XII] (350 mg).
- m.p. 150°C (dec.)
-
-
- 7-(Nα-glycyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt (91.4 mg, 0.215 mM). was dissolved in DMF ( 5 ml) and N-tert-butyloxycarbonyl-L'-lsucins-N-hydroxysuccinimide ester (85 mg, 0.26 mM) was added thereto. This mixture was stirred for 15 hours at room temperature and concentrated to yield a solid material. The material was purified by column chromatography (1 x 15 cm) using silica gel as adsorbent and ternary mixtures of chloroform, methyl alcohol and acetic acid in 95 : 5 : 3 and then 85 : 20.: 5 volume ratio. The main fraction was concentrated to solid material, then dissolved in acetic acid (20 ml) and lyophilized to give 7-(Nα-tert-butyloxycarbonyl-L-leucylglycyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt [XIII] (120 mg).
- m.p. 160°C (dec.)
-
-
- 7-(Nα-glycyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt (136 mg, 0.32 mM) was dissolved in DMF (5 ml), and Nα-carbobenzoxy-L-leucine-N-hydroxysuccinimide ester (140 mg, 0.38 mM) was added thereto. This mixture was stirred for 15 hours at room temperature and concentrated to yield a solid material. The material was purified by column chromatography (1 x 15 cm) using silica gel and ternary mixtures of chloroform, methyl alcohol and acetic acid in 95 : 5 : 3 and then 85 : 20 :5 volume ratio. The main fraction was concentrated to yield solid material, dissolved in acetic acid (20 ml) and lyophilized to give 7-(Na -carbobenzoxy-L-leucyl-glycyl-L-. argaxiyl)amino-4-methylcoumarin hydrochloric acid salt [XIV] (150 mg).
- m.p. 150°C (dec.)
-
-
- 7-L-arginylamino-4-mathylcoumarin hydrochloric acid salt 1/2 H20 (754 mg, 2 mM) was dissolved in a mixture of DMF (10 ml) and water (10 ml), and carbobenzoxy-L-phenylalanine-N-hydroxysuccinimide ester (1.03 g, 2.6 mM) was added thereto. This mixture was stirred for 15 hours at room temperature and concentrated to yield solid material. The material was purified by column chromatography (2 x 15 cm) using silica gel as adsorbent and.ternary mixture of chloroform, methylalcohol and acetic acid in 95 : 5 :3 volume ratio. The main fraction was concentrated to yield a solid material, dissolved in acetic acid (30 ml) and lyophilized to give 7-(Na -carbobenzoxy-L-phenylalanyl-L-arginyl)amino-4-methylcoumiarin hydrochloric acid salt [XV] (920 mg)
- m.p. 150°C (dec.)
-
-
- 7-(N α-carbobenzoxy-L-phenylalanyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt (1.3 g, 2 mM) was dissolved in methyl alcohol (100 ml), and palladium-carbon catalyst (100 mg) was added. Through this mixture hydrogen gas was passed for 3 hours with stirring. The catalyst was removed by filtration and the filtrate was concentrated to yield a.solid material. To the material ether (100 ml) was added and thus produced powder was separated by filtration to give 7-(L-phenylalanyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt [XVI] (1.0 g).
- m.p. 140°C (dec.)
-
-
- 7-(L-phenylalanyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt (515 mg, 1 mM) was dissolved in DMF (5 ml), and carbobenzoxy-L-proline-N-hydroxysuccinimide ester (450 mg, 1.3 mM) was added thereto. This mixture was stirred for 15 hours and concentrated to yield a solid material. The material was purified by column chromatography (2 x 15 cm) using silica gel as an adsorbent and a ternary mixture of chloroform, methyl alcohol and acetic acid in 95 : 5 : 3 volume ratlo. The main fraction was concentrated to yield solid material. The solid material was dissolved in acetic acid and lyophilized to give 7-(Nα-carbobenzoxy-L-prolyl-L-phenylalanyl-L-arginyl)amino-4-methylcoumarin hydrochloric acid salt as adduct of acetic acid [XVII] (510 mg).
- m.p. 160°C (dec.)
-
-
- 7-(Nα-carbobenzoxy-L-prolyl-L-phenylalanyl-L-arginyl) amino-4-methylcoumarin hydrochloric acid salt (200 mg, 0 0 27 mM) was dissolved in methyl alcohol (50 ml), and 1N-HCl (0.27 ml) and palladium-carbon catalyst '(20 mg) were added thereto. Through this mixture hydrogen gas was passed with stirring for 3 hours. The catalyst was removed by filtration and the filtrate was concentrated to yield a solid residue. To the residue ether (50 ml) was added and thus produced powder was separated by filtration to give 7-(L-prolyl-L-phenylalanyl-L-arginyl)-amino-4-methylcoumarin hydrochloric acid salt [XVIII] (150 mg).
- m.p. 185°C (dec.)
- [
-
- 7-L-arginylamino-4-methylcoumarin hydrochloric acid salt 1/2 H20 (377 mg, 1 mM) was dissolved in a mixture of DMF (10 ml) and water (10 ml), and carbobenzoxy-L-proline-N-hydroxysuccinimide ester (450 mg, 1.3 mM) was added thereto. This mixture was stirred for 15 hours at room temperature and concentrated to yield a solid material. The solid material was purified by column chromatography (2 x 15 cm) using silica gel as adsorbent and ternary mixtures of chloroform, methyl alcohol and acetic acid in 95 : 5 : 3 and then 85 : 20 : 5 volume ratio. The main fraction was concentrated to yield a solid material, dissolved in acetic acid (10 ml), and lyophilized to give 7-(Nα-carbobenzoxy-L-prolyl-L-arginyl)araino-4-methylcoumarin hydrochloric acid salt [XIX] (530 mg).
- m.p. 198°C (dec.)
- [
-
- The carbobenzoxy group of the compound thus obtained can be removed by applying a hydrogenation reaction in alcohol thereto.
- 7-L-arginylamino-4-methylcoumarin hydrochloric acid salt 1/2 H20 (377 mg, 1 mM) was dissolved in a mixture of DMF (5 ml) and water (5 ml), and -tert-butyloxycarbonyl-L-valyl-L-proline-p-nitrophenyl ester (566 mg, 1.3 mM) was added thereto. The mixture was stirred for 15 hours at room temperature and concentrated to yield a solid material. The material was purified by column chromatography (2 x 15 cm) using silica gel as adsorbent and ternary mixtures of chloroform, methyl alcohol and acotie acid in 95 : 5 : 3 and then 85 : 20 : 5 volume ratio. The main fraction was concentrated to yield solid material, dissolved in acetic acid (10 ml) and lyophilized to give 7-(Nα-tert-butyloxycarbonyl-L-valyl-L-prolyl-L-arginyl)-amino-4-methylcoumarin hydrochloric acid salt [XX] (320 mg).
- m. p. 155°C (dec.)
-
-
- The compound produced above (0.1 - 0.2 mM) was dissolved in a mixture of dimethylsulfoxide (5 ml) and water (5 ml) and this solution was diluted with 0.05 M Tris-HCl buffer (pH 8.0) containing both 0.1 M NaCl and 10 mM CaCl2to adjust the total volume thereof to 500 ml and thereby a substrate solution was prepared.
- Each substrate solution (2 ml) was added in a test tube and the obtained test tube was kept at 37°C for 5 minutes. Then, each enzyme solution (20 µl) was added thereto and incubated at 37°C for 20 minutes with stirring. The incubation was stopped by adding 100 % acetic acid (0.5 ml) thereto.
- The hydrolysis degree was determined by measuring the fluorescence intensity at 460 nm with excitation at 380 mn in the fluorescence spectra. Results are listed in the following table.
- Each value is the fluorescence intensity ratio when the fluorescence intensity of 7-amino-4-methylcoumarim solution (40 µM, 40 µmole/l) is 100.
- A value having the mark --.*-- is one with the fluorescence intensity of 7-amino-4-methylcoumarin solution (200 µM, 200 µmol/l) being 100.
-
Claims (1)
Applications Claiming Priority (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP52066517A JPS5842862B2 (en) | 1977-06-06 | 1977-06-06 | peptide derivative |
JP66517/77 | 1977-06-06 | ||
JP52067718A JPS5811424B2 (en) | 1977-06-08 | 1977-06-08 | Novel polypeptide |
JP67719/77 | 1977-06-08 | ||
JP67718/77 | 1977-06-08 | ||
JP6771977A JPS543074A (en) | 1977-06-08 | 1977-06-08 | Novel peptide derivative |
Publications (2)
Publication Number | Publication Date |
---|---|
EP0000063A1 true EP0000063A1 (en) | 1978-12-20 |
EP0000063B1 EP0000063B1 (en) | 1981-06-10 |
Family
ID=27299158
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP78100111A Expired EP0000063B1 (en) | 1977-06-06 | 1978-06-06 | Dipeptide derivatives of 7-(n-alpha-substituted or non-substituted x-arginyl)-amino-4-methyl-coumarin |
Country Status (3)
Country | Link |
---|---|
US (1) | US4215047A (en) |
EP (1) | EP0000063B1 (en) |
DE (1) | DE2860749D1 (en) |
Cited By (19)
Publication number | Priority date | Publication date | Assignee | Title |
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EP0004256A1 (en) * | 1978-02-07 | 1979-09-19 | Kabi AB | Easily split substrates for the quantification of proteases, a process for their production and a method for the quantification of proteases |
EP0016800A1 (en) * | 1978-08-03 | 1980-10-15 | American Hospital Supply Corp | Determination of proteolytic enzymes in body fluids and fluorogenic substrates. |
EP0018112A1 (en) * | 1979-04-23 | 1980-10-29 | Robert Eugene Smith | Substrates and method for determining proteolytic enzymes |
EP0025190A2 (en) * | 1979-09-10 | 1981-03-18 | BEHRINGWERKE Aktiengesellschaft | Chromogenic compounds and their use as enzymatic substrates |
FR2497798A1 (en) * | 1981-01-09 | 1982-07-16 | Pharmindustrie | NOVEL PEPTIDES CARRYING A FLUOROPHORE, PROCESS FOR THEIR PREPARATION AND THEIR APPLICATION TO FLUORIMETRIC DETERMINATION OF ENDOTOXINS |
US4675289A (en) * | 1983-04-12 | 1987-06-23 | Ajinomoto Company, Incorporated | Method of measuring the number of eumycete cells |
US5064756A (en) * | 1982-04-14 | 1991-11-12 | Carr Anthony H | Microbial susceptibility assay using 7-n-(aminoacyl)-7-amido-4-methylcoumarins |
US5073488A (en) * | 1988-11-29 | 1991-12-17 | Minnesota Mining And Manufacturing Company | Rapid method for determining efficacy of a sterilization cycle and rapid read-out biological indicator |
US5079144A (en) * | 1982-04-14 | 1992-01-07 | Radiometer Corporate Development Ltd. | Microorganism testing with a hydrolyzable fluorogenic substrate |
US5223401A (en) * | 1988-11-29 | 1993-06-29 | Minnesota Mining And Manufacturing Company | Rapid read-out sterility indicator |
US5252484A (en) * | 1988-11-29 | 1993-10-12 | Minnesota Mining And Manufacturing Company | Rapid read-out biological indicator |
US5342970A (en) * | 1991-05-30 | 1994-08-30 | Laboratoires Eurobio | Hydrosoluble coumarin derivatives, their preparation and their use as an enzyme substrate or for the preparation of such substrates |
US8043845B2 (en) | 2006-09-20 | 2011-10-25 | American Sterilizer Company | Sterilization indicator |
US8173388B2 (en) | 2008-09-30 | 2012-05-08 | American Sterilizer Company | Self-contained biological indicator |
WO2012061213A1 (en) | 2010-11-01 | 2012-05-10 | 3M Innovative Properties Company | Method of detecting a biological activity |
WO2012071131A1 (en) | 2010-11-24 | 2012-05-31 | 3M Innovative Properties Company | Methods and compositions to detect a biological activity |
US8372624B2 (en) | 2006-09-20 | 2013-02-12 | American Sterilizer Company | Genetically engineered biological indicator |
US8840837B2 (en) | 2010-11-01 | 2014-09-23 | 3M Innovative Properties Company | Biological sterilization indicator and method of using same |
EP3366315A1 (en) | 2017-02-23 | 2018-08-29 | Ethicon, Inc. | Method to read biological indicator |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4388233A (en) * | 1981-05-15 | 1983-06-14 | The Regents Of The University Of California | Synthetic substrates for enzyme analysis |
US4448715A (en) * | 1981-11-02 | 1984-05-15 | University Of Miami | Tagged pyroglu-L-Phe-L-Arg derivatives, substrates and assays for kallikrein |
US5055594A (en) * | 1990-07-19 | 1991-10-08 | Becton, Dickinson And Company | Fluorogenic trypotophanase substrates |
DE19834374B4 (en) * | 1998-07-30 | 2004-03-04 | Preh-Werke Gmbh & Co. Kg | Knob of a control unit |
CN109265373A (en) * | 2018-11-03 | 2019-01-25 | 郑州安图生物工程股份有限公司 | A kind of amino acids substrate and its preparation method and application |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2150146A5 (en) * | 1971-08-16 | 1973-03-30 | Smith Robert |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US2929822A (en) * | 1955-10-31 | 1960-03-22 | Geigy Ag J R | 3-substituted 7-carbalkoxyamino-coumarins |
DE1293160B (en) * | 1964-03-07 | 1969-04-24 | Bayer Ag | Process for the preparation of coumarin compounds |
-
1978
- 1978-06-05 US US05/912,851 patent/US4215047A/en not_active Expired - Lifetime
- 1978-06-06 DE DE7878100111T patent/DE2860749D1/en not_active Expired
- 1978-06-06 EP EP78100111A patent/EP0000063B1/en not_active Expired
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2150146A5 (en) * | 1971-08-16 | 1973-03-30 | Smith Robert |
Cited By (40)
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EP0004256A1 (en) * | 1978-02-07 | 1979-09-19 | Kabi AB | Easily split substrates for the quantification of proteases, a process for their production and a method for the quantification of proteases |
EP0016800A1 (en) * | 1978-08-03 | 1980-10-15 | American Hospital Supply Corp | Determination of proteolytic enzymes in body fluids and fluorogenic substrates. |
EP0016800A4 (en) * | 1978-08-03 | 1981-01-08 | American Hospital Supply Corp | Determination of proteolytic enzymes in body fluids and fluorogenic substrates. |
EP0034586B1 (en) * | 1979-04-23 | 1983-08-31 | Robert Eugene Smith | 4-trifluoromethylcoumarin peptide derivatives and their use in proteinase assays |
EP0018112A1 (en) * | 1979-04-23 | 1980-10-29 | Robert Eugene Smith | Substrates and method for determining proteolytic enzymes |
EP0025190A2 (en) * | 1979-09-10 | 1981-03-18 | BEHRINGWERKE Aktiengesellschaft | Chromogenic compounds and their use as enzymatic substrates |
EP0025190B1 (en) * | 1979-09-10 | 1984-06-13 | BEHRINGWERKE Aktiengesellschaft | Chromogenic compounds and their use as enzymatic substrates |
FR2497798A1 (en) * | 1981-01-09 | 1982-07-16 | Pharmindustrie | NOVEL PEPTIDES CARRYING A FLUOROPHORE, PROCESS FOR THEIR PREPARATION AND THEIR APPLICATION TO FLUORIMETRIC DETERMINATION OF ENDOTOXINS |
EP0056210A3 (en) * | 1981-01-09 | 1982-08-18 | Pharmuka Laboratoires | Method for the fluorimetric determination of endotoxins, peptides containing a fluorophore used in the said method and their method of preparation |
WO1982002382A1 (en) * | 1981-01-09 | 1982-07-22 | Monsigny Michel | Method for the fluorometric determination of endotoxins,new peptides carrying a fluorophorous substance usable in said method and method for its preparation |
EP0056210A2 (en) * | 1981-01-09 | 1982-07-21 | Centre National De La Recherche Scientifique (Cnrs) | Method for the fluorimetric determination of endotoxins, peptides containing a fluorophore used in the said method and their method of preparation |
US5064756A (en) * | 1982-04-14 | 1991-11-12 | Carr Anthony H | Microbial susceptibility assay using 7-n-(aminoacyl)-7-amido-4-methylcoumarins |
US5079144A (en) * | 1982-04-14 | 1992-01-07 | Radiometer Corporate Development Ltd. | Microorganism testing with a hydrolyzable fluorogenic substrate |
US4675289A (en) * | 1983-04-12 | 1987-06-23 | Ajinomoto Company, Incorporated | Method of measuring the number of eumycete cells |
US5073488A (en) * | 1988-11-29 | 1991-12-17 | Minnesota Mining And Manufacturing Company | Rapid method for determining efficacy of a sterilization cycle and rapid read-out biological indicator |
US5223401A (en) * | 1988-11-29 | 1993-06-29 | Minnesota Mining And Manufacturing Company | Rapid read-out sterility indicator |
US5252484A (en) * | 1988-11-29 | 1993-10-12 | Minnesota Mining And Manufacturing Company | Rapid read-out biological indicator |
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Also Published As
Publication number | Publication date |
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US4215047A (en) | 1980-07-29 |
EP0000063B1 (en) | 1981-06-10 |
DE2860749D1 (en) | 1981-09-17 |
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