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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
• • 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
• • 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
• • 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
• • 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
• • 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
• • 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
• • 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
• • 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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• 1978
  • 1978-06-05 US US05/912,851 patent/US4215047A/en not_active Expired - Lifetime
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