CA1068261A - Chromogenic thrombin substrates - Google Patents
Chromogenic thrombin substratesInfo
- Publication number
- CA1068261A CA1068261A CA256,417A CA256417A CA1068261A CA 1068261 A CA1068261 A CA 1068261A CA 256417 A CA256417 A CA 256417A CA 1068261 A CA1068261 A CA 1068261A
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- naphthyl
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
- C07K1/061—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups
- C07K1/064—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups for omega-amino or -guanidino functions
-
- 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
- C07K5/0812—Tripeptides with the first amino acid being neutral and aromatic or cycloaliphatic
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/56—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2337/00—N-linked chromogens for determinations of peptidases and proteinases
- C12Q2337/10—Anilides
- C12Q2337/12—Para-Nitroanilides p-NA
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2337/00—N-linked chromogens for determinations of peptidases and proteinases
- C12Q2337/30—Naphthyl amides, e.g. beta-NA, 2-NA, 4-methoxy-beta-naphthylamine, i.e. 4MNA
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/974—Thrombin
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
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- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
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- Engineering & Computer Science (AREA)
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- General Engineering & Computer Science (AREA)
- Hematology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Neurosurgery (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Abstract of the Disclosure Novel diagnostically active chromogenic substrates with a high specificity for thrombin and thrombin-like enzymes, characterized by the fact that they have the general formula
Description
- 10682~;1 The present invention relates to novel chromogenic substrates for thrombin and thrombin-like enzymes. The substrates according to the invention are especially suitable for the quantitative determination of thrombin or for the study of reactions in which thrombin is formed, inhibited or consumed, or for determination of factors which exert an influence or take part in such -reactions, e.g. for determination of anti-thrombin, prothrombin and heparin.
Synthetic substrates for enzyme determinations have great advan-tages as compared to the natural ones, provided that they fulfil certain conditions, such as a great sensitivity for and specificity for the enzyme, a good solubility in water or the biological test liquid, and easy detectability of some of the splitting products.
One of the hitherto best substrates for thrombin determination is described in our Swedish Patent No. 380,257 and consists of the chromogenic ., ;,.,.~
tripeptide derivative (as regards the abbreviations cf. page 4):
Bz-Phe-Val-Arg-pNA (S-2160) A
This has a great sensitivity for thrombin and gives upon enzymatic hydrolysis the chromophoric product p-nitroaniline which easily can be deter-mined spectrophotometrically. S-2160 has, however, a delimitation due to its relatively low solubility ~1 mg/ml). A low solubility causes the disadvantage , 20 that one has to work very near the saturation limit for the substrate so as to -* achieve a satisfactory substrate concentration. In enzyme determination in - different biological systems a precipitation of the substrate as such can occur or a combined protein/substrate precipitation. The said precipitations ~ ~ will cause erroneous spectrophotometer readings and thus erroneous enzyme '~2 determinatîons. The enzyme substrate S-2160 becomes considerably more soluble -.,2 ~
if the benzo~l group is replaced wi~h H, thus:
'2 ~ H-Phe-Val-Arg-pNA B
The now free protonized amino group on Phe increases the solubility, ~2 but also causes the velocity with which thrombin splits the substrate to de-~ 30 crease heavily, by about 30 times (cf. Table I). Further, the substrate can, , :., - 1 -., ~'~
q~
1Q{;826i in a biological test solution, be decomposed from the N-terminal end by amino peptidases. This is undesirable.
According to the present invention the substrate according to formula B has been modified by exchanging Val for a cyclic imino acid (Aze, Pro or Pip) and L-Phe with D-Phe. As expected the substrates so obtained are still very soluble but quite surprisingly the activity against thrombin is not decreased but instead it is 30-50 times higher than the actlvity for the cor-responding substrate with solely L-amino acids (Table I). Further, the novel substrates are about 400% more active than S-2160. The N-terminal D-amino acid also prevents undesired attack by amino peptidases, since the latter are specific for L-amino acids. The novel chromogenic substrates according to the invention are characterized by the following general formula:
~, .
CH2 CH2-(CH2)n tC,H2)3 NH
., l O l /C~
. Rl or salts thereof, wherein Rl can be hydrogen or hydroxy R2 can be chosen among nitrophenyl, naphthyl, nitronaphthyl, methoxynaphthyl, quinolyl or nitro-quinolyl, n can be 1, 2 or 3, and * indicates the D-configuration.
For the synthesis of the novel chromogenic enzyme substrates con-' ventional protective groups and coupling methods are used all of which are well-known within the peptide chemistry.
As an a-amino protective group carboxybenzoxy or t-butyloxycar-bonyl groups can be used with advantage or any group related thereto, such as, for instance, p-methoxy, p-nitro or p-methoxyphenylazo carbobenzoxy.
As a protection for the ~-quanido group of the arginyl group it is advantageous to use protonization, an N02-group or a p-toluene-sulphonyl group.
Synthetic substrates for enzyme determinations have great advan-tages as compared to the natural ones, provided that they fulfil certain conditions, such as a great sensitivity for and specificity for the enzyme, a good solubility in water or the biological test liquid, and easy detectability of some of the splitting products.
One of the hitherto best substrates for thrombin determination is described in our Swedish Patent No. 380,257 and consists of the chromogenic ., ;,.,.~
tripeptide derivative (as regards the abbreviations cf. page 4):
Bz-Phe-Val-Arg-pNA (S-2160) A
This has a great sensitivity for thrombin and gives upon enzymatic hydrolysis the chromophoric product p-nitroaniline which easily can be deter-mined spectrophotometrically. S-2160 has, however, a delimitation due to its relatively low solubility ~1 mg/ml). A low solubility causes the disadvantage , 20 that one has to work very near the saturation limit for the substrate so as to -* achieve a satisfactory substrate concentration. In enzyme determination in - different biological systems a precipitation of the substrate as such can occur or a combined protein/substrate precipitation. The said precipitations ~ ~ will cause erroneous spectrophotometer readings and thus erroneous enzyme '~2 determinatîons. The enzyme substrate S-2160 becomes considerably more soluble -.,2 ~
if the benzo~l group is replaced wi~h H, thus:
'2 ~ H-Phe-Val-Arg-pNA B
The now free protonized amino group on Phe increases the solubility, ~2 but also causes the velocity with which thrombin splits the substrate to de-~ 30 crease heavily, by about 30 times (cf. Table I). Further, the substrate can, , :., - 1 -., ~'~
q~
1Q{;826i in a biological test solution, be decomposed from the N-terminal end by amino peptidases. This is undesirable.
According to the present invention the substrate according to formula B has been modified by exchanging Val for a cyclic imino acid (Aze, Pro or Pip) and L-Phe with D-Phe. As expected the substrates so obtained are still very soluble but quite surprisingly the activity against thrombin is not decreased but instead it is 30-50 times higher than the actlvity for the cor-responding substrate with solely L-amino acids (Table I). Further, the novel substrates are about 400% more active than S-2160. The N-terminal D-amino acid also prevents undesired attack by amino peptidases, since the latter are specific for L-amino acids. The novel chromogenic substrates according to the invention are characterized by the following general formula:
~, .
CH2 CH2-(CH2)n tC,H2)3 NH
., l O l /C~
. Rl or salts thereof, wherein Rl can be hydrogen or hydroxy R2 can be chosen among nitrophenyl, naphthyl, nitronaphthyl, methoxynaphthyl, quinolyl or nitro-quinolyl, n can be 1, 2 or 3, and * indicates the D-configuration.
For the synthesis of the novel chromogenic enzyme substrates con-' ventional protective groups and coupling methods are used all of which are well-known within the peptide chemistry.
As an a-amino protective group carboxybenzoxy or t-butyloxycar-bonyl groups can be used with advantage or any group related thereto, such as, for instance, p-methoxy, p-nitro or p-methoxyphenylazo carbobenzoxy.
As a protection for the ~-quanido group of the arginyl group it is advantageous to use protonization, an N02-group or a p-toluene-sulphonyl group.
- 2 -As protection for the hydroxy group in the tyrosine group it is of advantage to use a t-butyl or benzyl group. As a splittable carboxy protective group it is suitable to use methyl, ethyl or benzyl ester.
The coupling between two amino acids or a dipeptide and an amino acid is achieved by activation of the -carboxy group. The activated deri-vative can either by isolated or generated i~ situ and can be for instance p-nitrophenyl, trichlorophenyl, pentachlorophenyl or N-hydroxysuccinimide ester, symmetric or asymmetric anhydride, acid azid, or N-hydroxybenzotriazole ester.
~ The principle for the synthesis can be a stepwise coupling of the ;~ 10 amino acids to the C-terminal arginyl group, either already provided with a coupled chromophoric group which functions as a carboxy protective group or ; provided with a splittable carboxy protective group, and ~he chromophoric group is then coupled to the protected tripeptide derivative, or alternative-` ly it is possible to synthesize the N-terminal dipeptide fragment per se which then is coupled to the arginyl group with or without a chromophoric group $1 according to the statements above.
~j Independently of the chosen principle a purification of inter-mediary and end products by gel filtration chromatography is suitable since this will enable a rapid synthesis work and gives maximal yields.
TLC analysis has been made partly of eluates from GPC and partly i of evaporated and dried end and intermediary products.
The invention is described in more detail in the following non-limiting specific examples.
.~
Abbreviations Amino acids ~if not other~ise stated the L-form is meant):
Arg = Arginine Aze = 2-Azetidine carboxylic acid Phe - Phenylalanine Pip _ Pipecolinic acid Pro ~ Proline ,, .. . .
~068261 Val = Valine AcOH = Acetic acid Bz = Benzoyl Cbo- = Carbobenzoxy-DMF = Dimethylformamide Et3N = Triethylamine EtOAc = Ethylacetate HMPTA = N, N, N', N', N", N"-hexamethylphosphoric acid triamide GPC = Gel filtration chromatography - 10 MeOH = Methanol -OpNP = p-nitrophenoxy . -pNA = p-nitroanilide TLC = Thin-layer chromatography Thin-layer chromatography:
For TLC-analysis preprspared glass plates are used with silica gel F254 ~Merck) as absorption agent. The solvent systems used are the follow-ing:
i! Pl: chloroform: MeOH 9:1 (volume ratio) A : n-butanol : AcOH : water 3:2:1 ~volume ratio) . 20 After finished chromatography the plate is studied in UV-light . ~, .
-~ (254nm) and development is made subsequently with chlorine/o-toluidine reagent according to common practice. When a "homogenous product according to TLC"
~ is stated the analysis is performed on an amount of ~g. The Rf values stated .l are results fTom separate chromatographic procedures.
.,~
~i~ The gel Sephadex(R) G-15 used for the gel filtration is a cross-- linked dextran gel. The gel Sephadex~R) LH-20 is a hydroxypropylated cross-linked dextran gel. The ion exchanger Sephadex~R) QAE-25 used is a cross-linked dextr~n gel with diethyl-(2-hydroxy-propyl)-amino-ethyl as functional ~; group.
'1 These gels are from Pharmacia Fine Chemicals, Uppsala, Sweden.
` :
:
Description of the synthesis I. Cbo-Arg~NO2)pNA
35.3 g ~10 mmol) of dry Cbo-Arg(NO2)-OH are dissolved in 200 ml of dry freshly distilled HMPTA at room temparature, whereupon 10.1 g ~100 mmol) of F.t3N and 24.6 g ~150 mmol) p-nitrophenyl isocyanate is added during stir-ring and moisture-free conditions. After a reaction time of 24 h the solution is poured down in 2 1 of 2% sodiumbicarbonate solution under stirring. The precipi~ation formed is removed by filtration and washed with 2% bicarbonate solution, water, 0.5 N HCl ~aq) and water and finally dried. The raw product is extracted with warm MeOH and difficultly soluble by-products are filtrated.
The filtrate is purified by chromatography on a column of Sephadex~R) LH-20, swelled in and eluated with MeOH.
Yield: 29.8 g ~63%) Analysis: Homogenous according to TLC in Pl ~Rf: 0.34) [~]D4 + 20.5 ~c 1.9, DMF) II. Cbo-Pro-Arg~NO2)-pNA
4.8 g (10 mmol) of Cbo-Arg~NO2)-pNA are slurried in 25 ml of dry . AcOH, whereupon 15 ml of 5.6 N HBr in AcOH are added. After a reaction period ;~ of 50 min at room temperature the solution is poured under vigorous stirring into 300 ml of dry ether. The ether phase is sucked from the precipitation obtained and the precipitation is washed with 2 portions of 100 ml of ether each. The HBr-H-Arg(NO2)-pNA thus obtained is dried in vacuum over NaOH at 40C for 16 h. It is subsequently dissolved in 25 ml of DMF and the solution ~1 is coolet to -10C. Now Et3N is added in an amount sufficient for giving a ; moist pH-paper kept immediately above the surface of the solution a weakly basic reaction (1.9 ml). Precipitated Et3N-HBr is removed by filtrat~ion and `~ 4.1 g (11 m~ol) of Cbo-Pro-OpNP are added. After 1 h further 0.7 ml Et3N are ~ added and also after 4 h. The solution is allowed to adjust to room tempera-
The coupling between two amino acids or a dipeptide and an amino acid is achieved by activation of the -carboxy group. The activated deri-vative can either by isolated or generated i~ situ and can be for instance p-nitrophenyl, trichlorophenyl, pentachlorophenyl or N-hydroxysuccinimide ester, symmetric or asymmetric anhydride, acid azid, or N-hydroxybenzotriazole ester.
~ The principle for the synthesis can be a stepwise coupling of the ;~ 10 amino acids to the C-terminal arginyl group, either already provided with a coupled chromophoric group which functions as a carboxy protective group or ; provided with a splittable carboxy protective group, and ~he chromophoric group is then coupled to the protected tripeptide derivative, or alternative-` ly it is possible to synthesize the N-terminal dipeptide fragment per se which then is coupled to the arginyl group with or without a chromophoric group $1 according to the statements above.
~j Independently of the chosen principle a purification of inter-mediary and end products by gel filtration chromatography is suitable since this will enable a rapid synthesis work and gives maximal yields.
TLC analysis has been made partly of eluates from GPC and partly i of evaporated and dried end and intermediary products.
The invention is described in more detail in the following non-limiting specific examples.
.~
Abbreviations Amino acids ~if not other~ise stated the L-form is meant):
Arg = Arginine Aze = 2-Azetidine carboxylic acid Phe - Phenylalanine Pip _ Pipecolinic acid Pro ~ Proline ,, .. . .
~068261 Val = Valine AcOH = Acetic acid Bz = Benzoyl Cbo- = Carbobenzoxy-DMF = Dimethylformamide Et3N = Triethylamine EtOAc = Ethylacetate HMPTA = N, N, N', N', N", N"-hexamethylphosphoric acid triamide GPC = Gel filtration chromatography - 10 MeOH = Methanol -OpNP = p-nitrophenoxy . -pNA = p-nitroanilide TLC = Thin-layer chromatography Thin-layer chromatography:
For TLC-analysis preprspared glass plates are used with silica gel F254 ~Merck) as absorption agent. The solvent systems used are the follow-ing:
i! Pl: chloroform: MeOH 9:1 (volume ratio) A : n-butanol : AcOH : water 3:2:1 ~volume ratio) . 20 After finished chromatography the plate is studied in UV-light . ~, .
-~ (254nm) and development is made subsequently with chlorine/o-toluidine reagent according to common practice. When a "homogenous product according to TLC"
~ is stated the analysis is performed on an amount of ~g. The Rf values stated .l are results fTom separate chromatographic procedures.
.,~
~i~ The gel Sephadex(R) G-15 used for the gel filtration is a cross-- linked dextran gel. The gel Sephadex~R) LH-20 is a hydroxypropylated cross-linked dextran gel. The ion exchanger Sephadex~R) QAE-25 used is a cross-linked dextr~n gel with diethyl-(2-hydroxy-propyl)-amino-ethyl as functional ~; group.
'1 These gels are from Pharmacia Fine Chemicals, Uppsala, Sweden.
` :
:
Description of the synthesis I. Cbo-Arg~NO2)pNA
35.3 g ~10 mmol) of dry Cbo-Arg(NO2)-OH are dissolved in 200 ml of dry freshly distilled HMPTA at room temparature, whereupon 10.1 g ~100 mmol) of F.t3N and 24.6 g ~150 mmol) p-nitrophenyl isocyanate is added during stir-ring and moisture-free conditions. After a reaction time of 24 h the solution is poured down in 2 1 of 2% sodiumbicarbonate solution under stirring. The precipi~ation formed is removed by filtration and washed with 2% bicarbonate solution, water, 0.5 N HCl ~aq) and water and finally dried. The raw product is extracted with warm MeOH and difficultly soluble by-products are filtrated.
The filtrate is purified by chromatography on a column of Sephadex~R) LH-20, swelled in and eluated with MeOH.
Yield: 29.8 g ~63%) Analysis: Homogenous according to TLC in Pl ~Rf: 0.34) [~]D4 + 20.5 ~c 1.9, DMF) II. Cbo-Pro-Arg~NO2)-pNA
4.8 g (10 mmol) of Cbo-Arg~NO2)-pNA are slurried in 25 ml of dry . AcOH, whereupon 15 ml of 5.6 N HBr in AcOH are added. After a reaction period ;~ of 50 min at room temperature the solution is poured under vigorous stirring into 300 ml of dry ether. The ether phase is sucked from the precipitation obtained and the precipitation is washed with 2 portions of 100 ml of ether each. The HBr-H-Arg(NO2)-pNA thus obtained is dried in vacuum over NaOH at 40C for 16 h. It is subsequently dissolved in 25 ml of DMF and the solution ~1 is coolet to -10C. Now Et3N is added in an amount sufficient for giving a ; moist pH-paper kept immediately above the surface of the solution a weakly basic reaction (1.9 ml). Precipitated Et3N-HBr is removed by filtrat~ion and `~ 4.1 g (11 m~ol) of Cbo-Pro-OpNP are added. After 1 h further 0.7 ml Et3N are ~ added and also after 4 h. The solution is allowed to adjust to room tempera-
3, ture over the night. So as to avoid that the excess of Cbo-Pro-OpNP con-, 30 taminates the end product during GPC since they have similar eluation volumes ., .
., ., ~j8~;~
in the chromatographic system used O.5 ml (5 mmol) of n-butyl amine is added.
After 30 min 10 mmol of diluted HCl are added, the reaction solution is evaporated on a rotavapor, stirred with a couple of portions of water which is removed by decantation. The residue is dissolved in MeOH and chromato-graphed on a column of Sephadex(R) LH-20, swelled in and eluated with MeOH.
The product obtained is homogeneous according to TLC.
Yield: 5.5 g (96%) Analysis: TLC in Pl (Rf: 0.28) [a]D4 - 33.0 (c l.O, DMF) III. Cbo-Pip-Arg(N02)- pNA
Performed according to II.
; Yield: 5.1 g (86%) s Analysis: Homogeneous according to TLC in Pl (Rf: 0.30) [a]D4- 26.2 (c 1.0, DMF) IV. Cbo-Phe-Pip-Arg(N02)-pNa 2.9 g ~5 mmol) Cbo-Pip-Arg(N02)-pNA are dicarbobenzoxylated in HBr ~ in AcOH, precipitated and washed with ether and dried according to II.
!i HBr-H-Pip-Arg(N02)-pNA is then dissolved in 15 ml of DMF. The solution is cooled to -10C, made weakly basic with 0.9 ml of Et3N and filtrated. 3.0 g (7.15 mmol) of Cbo-Phe-OpNP are added and then 0.65 g (5 mmol) of N-hydroxy-' benzotriazole as a catalyst. After 1 h further 0.35 ml of Et3N is added and the same procedure repeated after 4 h. The reaction solution is allowed to .~ increase to room temperature over night. The solution is evaporated to dryness in a rotavapor. The residue is dissolved in EtOAc and treated with 2% sodium bicarbonate solution and water and then evaporated. The residue is now dis-solved in MeOH and chromatographed on Sephadex(R) LH-20 swelled and eluated J~ with MeOH. The product obtained is homogeneous according to TLC.
!
Yield: 2.7 g (73%) l Analysis: TLC in Pl (Rf: 0,35) [a]D4- 32.9 (c 1.0, DMF) . .
~Q6826i V. Cbo-D-Phe-Pip-Arg(N02) -pNA
Performed according to IV.
Yield: 2.6 g ~70%) Analysis: Homogeneous according to TLC in Pl (Rf: 0.44) [a]D4 - 38.4 (c 1.0, DMF) VI. Cbo-Phe-Pro-Arg(N02)-pNA
Performed according to IV.
Yield: 2.9 g (80%) Analysis: Homogeneous according to TLC in Pl (Rf: 0.38) [~]D4- 39.2 (c 1.0, DMF) VII. Cbo-D-Phe-Pro-Arg(N02)-pNA
, Performed according to IV.
Yield: 3.1 g (86%) Analysis: Homogeneous according to TLC in Pl (Rf: 0.46) [a]D4 - 6.2 (c 1.0, DMF) VIII. H-D-Phe-Pro-Arg-pNA 2HCl ~, 175 mg (0.246 mmol) Cbo-D-Phe-Pro-Arg(N02)-pNA is deprotected by reaction with 5 mi of dry HF in the presence of 0.3 ml of anisole in an i apparatus intended for this purpose according to Sakakibara for 60 min at 0C.
i~ 20 After finished reaction and after distillation of all HF the raw product is purified and submitted to ion exchange in two steps:
~ a) GPC on a column of Sephadex(R) G-15, swollen in 33% AcOH in water, j with the same medium as dissolving and eluation medium. The pure product is ~ ~ freeze dried from AcOH (aq).
Z b) Ion exchange on a column of Sephadex(R) QAE-25 in the chloride form, swollen in MeOH:water (95:5) with the same medium as dissolving and eluating medium. The pure product is freeze dried from water.
~; Yield: 120 mg (80%) Analysis: Homogeneous according to TLC in A (Rf: 0.29) Chloride content 11.53% (theoretically 11.6%) ' ~a]D4 - 122 (c 0.5, 50% AcOH (aq)) .~ , ., IX. H-Phe-Pro-Arg-pNA~2HCl Performed according to VIII.
Yield: ~71%) Analysis: Homogeneous according to TLC in A (Rf: 0.22) ; Chloride content 11.0% ~theoretically 11.6%) ~ [a]24 - 73.6 ~c 0.5, 50% AcOH ~aq)) ; X. H-D-Phe-Pip-Arg-pNA 2HCl Performed according to VIII.
Yield: ~72%) Analysis: Homogeneous according to TLC in A ~Rf: 0.44) Chloride content 11.4% ~theoretically 11.3%) []D4 ~ 109 ~c 0.4 50% AcOH ~aq)) XI. H-Phe-Pip-Arg-pNA-2HCl ~;~ Performed according to VIII.
Yield: C55%) Analy~is: Homogeneous accoxd~ng to TLC in A ~Rf: 0.41) Chloride content 11.3% Ctheoretically 11.3%) []24_ 80.3 (c 0.5, 50% AcOH ~aq)) - XII. Cbo-D-Tyr~OBzl)-Pip-Arg~NO2)-pNA
Performed according to IV.
Yield: 3.2 g (75%) ~ Analysis: Homogeneous according to TLC in Pl ~Rf: 0.50) `
,~ XIII. H-D-Tyr-Pip-Arg-pNA
O Performed according to VIII.
~ ~ Yield: C68%) 2~ Analysis: Homogeneous according to TLC in Pl ~Rf: 0.44) .
Chloride content 10.8% (theoretically 11.1%) []D ~ 75.2 ~c 0.5, 50% AcOH ~aq)) ~,~ XIV: Cbo-Aze-Arg~NO2)-pNA
Performed according to II.
.~
:, , :' 1068~6~
Yield: 4.2 g ~75%) Analysis: Homogeneous according to TLC in Pl (Rf: 0.27) XV. Cbo-D-Phe-Aze-ArgCNO2)-pNA
Performed according to IV.
Yield: 2.4 g (69%) Analysis: Homogeneous according to TLC in Pl (Rf: 0 47) XVI. H-D-Phe-Aze-Arg-pNA 2HCl `-Performed according to VIII.
Yield: ~71%) Analysis: Homogeneous according to TLC in A (Rf: 0.21) -Chloride content 11.6% (theoretically 11.9%) . [a]24 - 130 (c 0.5, 50% AcOH) XVII. Cbo-ArgCN02)-~NA
7.2 g ~20 mmol) of dry Cbo-Arg(N02)-OH are dissolved in 400 ml of . THP. 2.0 g (20 mmol) Et3N are added, whereupon the solution is cooled to -10C
under completely moisture free conditions. 2.7 g (20 mmol) of isobutylchloro-, formate dissolved in 20 ml of THF are added to the cooled solution during 10 min, and after another 10 min 344 g ~20 mmol) of ~-naphthylamine are added.
The reaction mixture i5 allowed to reach room temperature and is left at this temperature for 24 h. The reaction mixture is evaporated in vacuum to dryness, 1~ is treated 3-5 times with distilled water, 3-5 times with a 5% sodium dicar-`~ bonate solution and again 3-5 times with distilled water, after which it is ~; dried in vacuum. The product is dissolved in MeOH and chromatographed on a column of SephadexQR) LH-20, swelled in and eluated with MeOH. The product obtained is homogeneous according to TLC in Pl.
3~
Yield: 8.1 g (84%) AnaIysis: Homogeneous according to TLC in Pl (Rf: 0.40) ~ ] D ~ 7 4 CC 1.0, DMF ) .;, : XVIII. Cbo-Pip-Arg~O2~-~NA
,~ 30 Performed according to II.
. , .:
.. _ g _ ;~ .
1068~
Yield: 4.8 g ~82%) Analysis: Homogeneous according to TLC in Pl (Rf: 0.36) XIX. Cbo-D-Phe-Pip-Arg(N02)-~NA
Performed according to IV.
Yield: 2.6 g ~71%) Analysis: Homogeneous according to TLC in Pl ~Rf: 0.48) XX. H-D-Phe-Pip-Arg-~NA 2HCl Performed according to VIII.
Yield: (68%) Analysis: Homogeneous according to TLC in A (Rf: 0.44) Chloride content 11.2~ (theoretically 11.3%) ~a]D4 - 105 (c 0.5, 50% AcOH (aq)) XXI. Cbo-Arg~NO2)-~NA (4-OMe) Performed according to XVII.
Yield: 7.1 g t70%) Analysis Homogeneous according to TLC in Pl (Rf: 0.42) ,A' ~ XXII. Cbo-Pip-Arg(N02)-~NA (4-OMe) Performed according to II.
Yield: 4.9 g (79%) , 20 Analysis: Homogeneous according to TLC in Pl (Rf: 0.38) XXIII. Cbo-D-Phe-Pip-Arg(NO2)-~NA (4-OMe) Performed according to IV.
Yield: 2.7 g (70%) Analysis: Homogeneous accordîng to TLC in Pl (Rf: 0.51) XXIV: H-D-Phe-Pip-Arg-~NA (4-OMe) 2HCl Performed according to VIII.
,~ ~ Yield: ~64%) Analysis: Homogeneous according to TLC in A ~Rf: 0.44) Chloride content 10.4% ~theoretically 10~7%) ; 30 ~]D4 ~ 102 ~c 0.5, 50% AcOH ~aq)).
... ..... . . .. . .
10682f~
Determination of thrombin by chromogenic substrates:
The substrates prepared according to the examples are used for --determination of thrombin as stated below.
The determination principle is based upon the fact that the split-ting product formed by enzymatic hydrolysis has a UV-spectrum which is essen-tially different from that of the substrate. Thus, the substrate according to Example X, H-D-Phe-Pip-Arg-pNA, has an absorption maximum at 315 nm and the molar extinction coefficient of 12500. At 405 nm the absorption of the sub-strate has almost completely stopped. p-Nitroaniline, split off from the substrate during the enzymatic hydrolysis, has an absorption maximum at 380 nm and a molar extinction coefficient of 13200 which at 405 nm only had decreased to 9620. By spectrophotometrical determination at 405 nm it is thus possible to easily follow the amount of p-nitroaniline formed, which is proportional to the degree of the enzymatic hydrolysis which in its turn is determined by the -active amount o~ thrombin. For other substrates according to the invention rather identical conditions exist and for this reason the determinations have consistently been made at 405 nm.
Table I shows a comparison of the relative reaction velocities be-tween the previously mentioned thrombin substrate S-2160, its non-benzoylated form and substrate according to the invention. This table clearly shows the superiority of the substrates according to the invention. They react 4 times , more rapidly with thrombin than the previously best substratc, S-2160~ and are `; further about 10 times more soluble in water than S-2160.
i , `
..i,, , .
(~
,,, . .
` 10682~i~
SubstrateRel. reaction Solubility in velocity H20 ~mg/ml) A (S-2160) Bz-Phe-Val-Arg-pNA100 0.1 B H-Phe-Val-Arg-pNA 3 IX H-Phe-Pro-Arg-pNA15 VIII H-D-Phe-Pro-Arg-pNA400 3 XI H-Phe-Pip-Arg-pNA 9 X H-D-Phe-Pip-Arg-pNA420 3 Table I
Relative reactions velocity between thrombin (0.4 NIH/ml) and substrate (0.1 ~mol/ml), .
,~ ~
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.
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in the chromatographic system used O.5 ml (5 mmol) of n-butyl amine is added.
After 30 min 10 mmol of diluted HCl are added, the reaction solution is evaporated on a rotavapor, stirred with a couple of portions of water which is removed by decantation. The residue is dissolved in MeOH and chromato-graphed on a column of Sephadex(R) LH-20, swelled in and eluated with MeOH.
The product obtained is homogeneous according to TLC.
Yield: 5.5 g (96%) Analysis: TLC in Pl (Rf: 0.28) [a]D4 - 33.0 (c l.O, DMF) III. Cbo-Pip-Arg(N02)- pNA
Performed according to II.
; Yield: 5.1 g (86%) s Analysis: Homogeneous according to TLC in Pl (Rf: 0.30) [a]D4- 26.2 (c 1.0, DMF) IV. Cbo-Phe-Pip-Arg(N02)-pNa 2.9 g ~5 mmol) Cbo-Pip-Arg(N02)-pNA are dicarbobenzoxylated in HBr ~ in AcOH, precipitated and washed with ether and dried according to II.
!i HBr-H-Pip-Arg(N02)-pNA is then dissolved in 15 ml of DMF. The solution is cooled to -10C, made weakly basic with 0.9 ml of Et3N and filtrated. 3.0 g (7.15 mmol) of Cbo-Phe-OpNP are added and then 0.65 g (5 mmol) of N-hydroxy-' benzotriazole as a catalyst. After 1 h further 0.35 ml of Et3N is added and the same procedure repeated after 4 h. The reaction solution is allowed to .~ increase to room temperature over night. The solution is evaporated to dryness in a rotavapor. The residue is dissolved in EtOAc and treated with 2% sodium bicarbonate solution and water and then evaporated. The residue is now dis-solved in MeOH and chromatographed on Sephadex(R) LH-20 swelled and eluated J~ with MeOH. The product obtained is homogeneous according to TLC.
!
Yield: 2.7 g (73%) l Analysis: TLC in Pl (Rf: 0,35) [a]D4- 32.9 (c 1.0, DMF) . .
~Q6826i V. Cbo-D-Phe-Pip-Arg(N02) -pNA
Performed according to IV.
Yield: 2.6 g ~70%) Analysis: Homogeneous according to TLC in Pl (Rf: 0.44) [a]D4 - 38.4 (c 1.0, DMF) VI. Cbo-Phe-Pro-Arg(N02)-pNA
Performed according to IV.
Yield: 2.9 g (80%) Analysis: Homogeneous according to TLC in Pl (Rf: 0.38) [~]D4- 39.2 (c 1.0, DMF) VII. Cbo-D-Phe-Pro-Arg(N02)-pNA
, Performed according to IV.
Yield: 3.1 g (86%) Analysis: Homogeneous according to TLC in Pl (Rf: 0.46) [a]D4 - 6.2 (c 1.0, DMF) VIII. H-D-Phe-Pro-Arg-pNA 2HCl ~, 175 mg (0.246 mmol) Cbo-D-Phe-Pro-Arg(N02)-pNA is deprotected by reaction with 5 mi of dry HF in the presence of 0.3 ml of anisole in an i apparatus intended for this purpose according to Sakakibara for 60 min at 0C.
i~ 20 After finished reaction and after distillation of all HF the raw product is purified and submitted to ion exchange in two steps:
~ a) GPC on a column of Sephadex(R) G-15, swollen in 33% AcOH in water, j with the same medium as dissolving and eluation medium. The pure product is ~ ~ freeze dried from AcOH (aq).
Z b) Ion exchange on a column of Sephadex(R) QAE-25 in the chloride form, swollen in MeOH:water (95:5) with the same medium as dissolving and eluating medium. The pure product is freeze dried from water.
~; Yield: 120 mg (80%) Analysis: Homogeneous according to TLC in A (Rf: 0.29) Chloride content 11.53% (theoretically 11.6%) ' ~a]D4 - 122 (c 0.5, 50% AcOH (aq)) .~ , ., IX. H-Phe-Pro-Arg-pNA~2HCl Performed according to VIII.
Yield: ~71%) Analysis: Homogeneous according to TLC in A (Rf: 0.22) ; Chloride content 11.0% ~theoretically 11.6%) ~ [a]24 - 73.6 ~c 0.5, 50% AcOH ~aq)) ; X. H-D-Phe-Pip-Arg-pNA 2HCl Performed according to VIII.
Yield: ~72%) Analysis: Homogeneous according to TLC in A ~Rf: 0.44) Chloride content 11.4% ~theoretically 11.3%) []D4 ~ 109 ~c 0.4 50% AcOH ~aq)) XI. H-Phe-Pip-Arg-pNA-2HCl ~;~ Performed according to VIII.
Yield: C55%) Analy~is: Homogeneous accoxd~ng to TLC in A ~Rf: 0.41) Chloride content 11.3% Ctheoretically 11.3%) []24_ 80.3 (c 0.5, 50% AcOH ~aq)) - XII. Cbo-D-Tyr~OBzl)-Pip-Arg~NO2)-pNA
Performed according to IV.
Yield: 3.2 g (75%) ~ Analysis: Homogeneous according to TLC in Pl ~Rf: 0.50) `
,~ XIII. H-D-Tyr-Pip-Arg-pNA
O Performed according to VIII.
~ ~ Yield: C68%) 2~ Analysis: Homogeneous according to TLC in Pl ~Rf: 0.44) .
Chloride content 10.8% (theoretically 11.1%) []D ~ 75.2 ~c 0.5, 50% AcOH ~aq)) ~,~ XIV: Cbo-Aze-Arg~NO2)-pNA
Performed according to II.
.~
:, , :' 1068~6~
Yield: 4.2 g ~75%) Analysis: Homogeneous according to TLC in Pl (Rf: 0.27) XV. Cbo-D-Phe-Aze-ArgCNO2)-pNA
Performed according to IV.
Yield: 2.4 g (69%) Analysis: Homogeneous according to TLC in Pl (Rf: 0 47) XVI. H-D-Phe-Aze-Arg-pNA 2HCl `-Performed according to VIII.
Yield: ~71%) Analysis: Homogeneous according to TLC in A (Rf: 0.21) -Chloride content 11.6% (theoretically 11.9%) . [a]24 - 130 (c 0.5, 50% AcOH) XVII. Cbo-ArgCN02)-~NA
7.2 g ~20 mmol) of dry Cbo-Arg(N02)-OH are dissolved in 400 ml of . THP. 2.0 g (20 mmol) Et3N are added, whereupon the solution is cooled to -10C
under completely moisture free conditions. 2.7 g (20 mmol) of isobutylchloro-, formate dissolved in 20 ml of THF are added to the cooled solution during 10 min, and after another 10 min 344 g ~20 mmol) of ~-naphthylamine are added.
The reaction mixture i5 allowed to reach room temperature and is left at this temperature for 24 h. The reaction mixture is evaporated in vacuum to dryness, 1~ is treated 3-5 times with distilled water, 3-5 times with a 5% sodium dicar-`~ bonate solution and again 3-5 times with distilled water, after which it is ~; dried in vacuum. The product is dissolved in MeOH and chromatographed on a column of SephadexQR) LH-20, swelled in and eluated with MeOH. The product obtained is homogeneous according to TLC in Pl.
3~
Yield: 8.1 g (84%) AnaIysis: Homogeneous according to TLC in Pl (Rf: 0.40) ~ ] D ~ 7 4 CC 1.0, DMF ) .;, : XVIII. Cbo-Pip-Arg~O2~-~NA
,~ 30 Performed according to II.
. , .:
.. _ g _ ;~ .
1068~
Yield: 4.8 g ~82%) Analysis: Homogeneous according to TLC in Pl (Rf: 0.36) XIX. Cbo-D-Phe-Pip-Arg(N02)-~NA
Performed according to IV.
Yield: 2.6 g ~71%) Analysis: Homogeneous according to TLC in Pl ~Rf: 0.48) XX. H-D-Phe-Pip-Arg-~NA 2HCl Performed according to VIII.
Yield: (68%) Analysis: Homogeneous according to TLC in A (Rf: 0.44) Chloride content 11.2~ (theoretically 11.3%) ~a]D4 - 105 (c 0.5, 50% AcOH (aq)) XXI. Cbo-Arg~NO2)-~NA (4-OMe) Performed according to XVII.
Yield: 7.1 g t70%) Analysis Homogeneous according to TLC in Pl (Rf: 0.42) ,A' ~ XXII. Cbo-Pip-Arg(N02)-~NA (4-OMe) Performed according to II.
Yield: 4.9 g (79%) , 20 Analysis: Homogeneous according to TLC in Pl (Rf: 0.38) XXIII. Cbo-D-Phe-Pip-Arg(NO2)-~NA (4-OMe) Performed according to IV.
Yield: 2.7 g (70%) Analysis: Homogeneous accordîng to TLC in Pl (Rf: 0.51) XXIV: H-D-Phe-Pip-Arg-~NA (4-OMe) 2HCl Performed according to VIII.
,~ ~ Yield: ~64%) Analysis: Homogeneous according to TLC in A ~Rf: 0.44) Chloride content 10.4% ~theoretically 10~7%) ; 30 ~]D4 ~ 102 ~c 0.5, 50% AcOH ~aq)).
... ..... . . .. . .
10682f~
Determination of thrombin by chromogenic substrates:
The substrates prepared according to the examples are used for --determination of thrombin as stated below.
The determination principle is based upon the fact that the split-ting product formed by enzymatic hydrolysis has a UV-spectrum which is essen-tially different from that of the substrate. Thus, the substrate according to Example X, H-D-Phe-Pip-Arg-pNA, has an absorption maximum at 315 nm and the molar extinction coefficient of 12500. At 405 nm the absorption of the sub-strate has almost completely stopped. p-Nitroaniline, split off from the substrate during the enzymatic hydrolysis, has an absorption maximum at 380 nm and a molar extinction coefficient of 13200 which at 405 nm only had decreased to 9620. By spectrophotometrical determination at 405 nm it is thus possible to easily follow the amount of p-nitroaniline formed, which is proportional to the degree of the enzymatic hydrolysis which in its turn is determined by the -active amount o~ thrombin. For other substrates according to the invention rather identical conditions exist and for this reason the determinations have consistently been made at 405 nm.
Table I shows a comparison of the relative reaction velocities be-tween the previously mentioned thrombin substrate S-2160, its non-benzoylated form and substrate according to the invention. This table clearly shows the superiority of the substrates according to the invention. They react 4 times , more rapidly with thrombin than the previously best substratc, S-2160~ and are `; further about 10 times more soluble in water than S-2160.
i , `
..i,, , .
(~
,,, . .
` 10682~i~
SubstrateRel. reaction Solubility in velocity H20 ~mg/ml) A (S-2160) Bz-Phe-Val-Arg-pNA100 0.1 B H-Phe-Val-Arg-pNA 3 IX H-Phe-Pro-Arg-pNA15 VIII H-D-Phe-Pro-Arg-pNA400 3 XI H-Phe-Pip-Arg-pNA 9 X H-D-Phe-Pip-Arg-pNA420 3 Table I
Relative reactions velocity between thrombin (0.4 NIH/ml) and substrate (0.1 ~mol/ml), .
,~ ~
., ~`
.
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.
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:'`
Claims (8)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A tripeptide derivative of formula and salts thereof, wherein: R1 represents hydrogen or hydroxy; R2 represents nitrophenyl, naphthyl, or methoxy naphthyl; n is an integer chosen from 1, 2 or 3; and * indicates the D-configuration.
2. A tripeptide as claimed in claim 1 of formula
3. A tripeptide as claimed in claim 1 of formula
4. Process for the preparation of a tripeptide derivative of formula and salts thereof, wherein: R1 represents hydrogen or hydroxy; R2 represents nitrophenyl, naphthyl, or methoxy naphthyl; n is an integer chosen from 1, 2, or 3; and * indicates the D-configuration which comprises sequentially coup-ling of the required amino acids to the terminal arginyl moiety, the carboxyl group in the arginyl group carrying a protective group during this reaction, which is then removed and replaced by a group -NHR2.
5. Process according to claim 4, in which the carboxyl group in the arginyl moiety is protected, a group -NHR2, which is left in situ at the end of the process.
6. Process according to claim 4 in which the two amino acids are themselves coupled together prior to being coupled to the arginyl moiety.
7. A method of determining thrombin by enzymatic hydrolysis of a tri-peptide derivative of formula and salts thereof, wherein: R1 represents hydrogen or hydroxy; R2 represents nitrophenyl, naphthyl, or methoxy naphthyl, n is an integer chosen from 1, 2, or 3; and * indicates the D-configuration which comprises determining spectro-scopically either the rate of formation, or the quantity of a chromophore resulting from the hydrolytic splitting of the group -NHR2 from the tripeptide derivative.
8. Method according to claim 7 wherein the group -NHR2 is a para-nitroanilyl group, the released chromophore is para-nitroaniline, and the spectroscopic determinations are carried out at 405 nm.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SE7507975-6A SE407405B (en) | 1975-07-11 | 1975-07-11 | NEW CHROMOGENATE THROMBIN SUBSTRATE |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1068261A true CA1068261A (en) | 1979-12-18 |
Family
ID=20325116
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA256,417A Expired CA1068261A (en) | 1975-07-11 | 1976-07-06 | Chromogenic thrombin substrates |
Country Status (22)
| Country | Link |
|---|---|
| JP (1) | JPS5224590A (en) |
| AT (1) | AT348686B (en) |
| AU (1) | AU497547B2 (en) |
| BE (1) | BE843970A (en) |
| CA (1) | CA1068261A (en) |
| CH (1) | CH622287A5 (en) |
| CS (1) | CS196314B2 (en) |
| DD (1) | DD128691A5 (en) |
| DE (1) | DE2629195C3 (en) |
| DK (1) | DK145799C (en) |
| ES (1) | ES449739A1 (en) |
| FI (1) | FI56525C (en) |
| FR (1) | FR2317280A1 (en) |
| GB (1) | GB1510926A (en) |
| IL (1) | IL49829A (en) |
| IT (1) | IT1062605B (en) |
| NL (1) | NL188355C (en) |
| NO (1) | NO142576C (en) |
| PL (1) | PL103003B1 (en) |
| SE (1) | SE407405B (en) |
| SU (1) | SU719492A3 (en) |
| ZA (1) | ZA763586B (en) |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5255593A (en) * | 1975-10-30 | 1977-05-07 | Ajinomoto Kk | Measuring method of enzyme activity |
| DE2757992A1 (en) * | 1977-12-24 | 1979-06-28 | Boehringer Mannheim Gmbh | METHOD FOR PROTHROMBIN DETERMINATION |
| DE2812943C3 (en) * | 1978-03-23 | 1981-05-14 | Boehringer Mannheim Gmbh, 6800 Mannheim | Method and reagent for determining the biological activity of heparin in plasma |
| US4289498A (en) * | 1979-01-08 | 1981-09-15 | Ortho Diagnostics, Inc. | One-stage prothrombin assay and compositions useful therein |
| FR2471410A2 (en) * | 1979-12-17 | 1981-06-19 | Jozefonvicz Marcel | Electrochemical protease or anti-protease determn. - by amperometric determn. of amine released from peptide amide substrate |
| EP0018002B1 (en) * | 1979-04-24 | 1983-02-09 | Marcel Jozefonvicz | Process for the determination of proteases and antiproteases |
| DE3005540A1 (en) * | 1980-02-14 | 1981-08-20 | Boehringer Mannheim Gmbh, 6800 Mannheim | METHOD AND REAGENT FOR DETERMINING THE BIOLOGICAL ACTIVITY OF HEPARIN IN PLASMA |
| DK155051C (en) * | 1980-05-06 | 1989-07-03 | Pentapharm Ag | TRIPEPTIDE DERIVATIVES AND PROCEDURES FOR QUANTITATIVE ANALYSIS OF PROTEOLYTIC ENZYMES UNDER USE thereof |
| JPS5856695A (en) * | 1981-09-28 | 1983-04-04 | Nitto Boseki Co Ltd | Novel substrate for assay of thrombin |
| DE3211254A1 (en) * | 1982-03-26 | 1983-09-29 | Boehringer Mannheim Gmbh, 6800 Mannheim | METHOD FOR DETECTING THE PRESENCE OF AN ALLERGY AND FOR SPECIFIC DETECTING THE ALLERGY RESPONSIBLE FOR THE ALLERGY |
| DK201084A (en) * | 1983-04-28 | 1984-10-29 | Kimberly Clark Co | PROCEDURE FOR DETERMINING CATHEPSIN B IN THE PRESENCE OF OTHER PROTEOLYTIC ENZYMES AND RELATIONSHIPS FOR USING THE PROCEDURE |
| CA2609113A1 (en) | 2005-05-20 | 2006-11-23 | Mitsubishi Kagaku Iatron, Inc. | Method of analyzing enzyme |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SE380257B (en) * | 1972-05-02 | 1975-11-03 | Bofors Ab | NEW DIAGNOSTIC OPERATING SUBSTRATES WITH HIGH SPECIFICITY FOR THROMBIN AND OTHER PROTEOLYTIC ENZYMES OF THE PEPTIDYL-PEPTIDE HYDROLASES |
| SE380258B (en) * | 1972-05-02 | 1975-11-03 | Bofors Ab | NEW DIAGNOSTIC OPERATING SUBSIDIES WITH HIGH SPECIFICITY FOR TRYPSIN AND OTHER PEPTIDYL-PEPTIDE HYDROLASES OF THE PEPTIDYL TYPE |
-
1975
- 1975-07-11 SE SE7507975-6A patent/SE407405B/en not_active IP Right Cessation
-
1976
- 1976-06-16 ZA ZA763586A patent/ZA763586B/en unknown
- 1976-06-17 IL IL49829A patent/IL49829A/en unknown
- 1976-06-21 GB GB25580/76A patent/GB1510926A/en not_active Expired
- 1976-06-25 AT AT463976A patent/AT348686B/en not_active IP Right Cessation
- 1976-06-25 AU AU15305/76A patent/AU497547B2/en not_active Expired
- 1976-06-29 DE DE2629195A patent/DE2629195C3/en not_active Expired
- 1976-06-29 NL NLAANVRAGE7607104,A patent/NL188355C/en not_active IP Right Cessation
- 1976-07-06 CA CA256,417A patent/CA1068261A/en not_active Expired
- 1976-07-08 CS CS764544A patent/CS196314B2/en unknown
- 1976-07-09 DD DD7600193785A patent/DD128691A5/en unknown
- 1976-07-09 BE BE168774A patent/BE843970A/en not_active IP Right Cessation
- 1976-07-09 FR FR7621196A patent/FR2317280A1/en active Granted
- 1976-07-09 CH CH887476A patent/CH622287A5/en not_active IP Right Cessation
- 1976-07-09 SU SU762379659A patent/SU719492A3/en active
- 1976-07-09 PL PL1976191068A patent/PL103003B1/en unknown
- 1976-07-09 DK DK312676A patent/DK145799C/en not_active IP Right Cessation
- 1976-07-09 NO NO762407A patent/NO142576C/en unknown
- 1976-07-09 IT IT50364/76A patent/IT1062605B/en active
- 1976-07-09 FI FI762010A patent/FI56525C/en not_active IP Right Cessation
- 1976-07-09 ES ES449739A patent/ES449739A1/en not_active Expired
- 1976-07-10 JP JP51082442A patent/JPS5224590A/en active Granted
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