NO142576B - NEW DIAGNOSTIC ACTIVE CHROMOGENIC SUBSTRATES WITH HIGH SPECIFICITY OF THROMBIN AND THROMBIN-LIKE ENZYMES - Google Patents
NEW DIAGNOSTIC ACTIVE CHROMOGENIC SUBSTRATES WITH HIGH SPECIFICITY OF THROMBIN AND THROMBIN-LIKE ENZYMES Download PDFInfo
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- NO142576B NO142576B NO762407A NO762407A NO142576B NO 142576 B NO142576 B NO 142576B NO 762407 A NO762407 A NO 762407A NO 762407 A NO762407 A NO 762407A NO 142576 B NO142576 B NO 142576B
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- Prior art keywords
- thrombin
- arg
- tlc
- pna
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- 108090000190 Thrombin Proteins 0.000 title claims description 15
- 229960004072 thrombin Drugs 0.000 title claims description 15
- 108090000790 Enzymes Proteins 0.000 title claims description 9
- 102000004190 Enzymes Human genes 0.000 title claims description 9
- 229940088598 enzyme Drugs 0.000 title claims description 9
- 239000003593 chromogenic compound Substances 0.000 title claims description 5
- 230000002345 thrombinlike Effects 0.000 title claims description 3
- -1 methoxynaphthyl Chemical group 0.000 claims description 13
- 229910052739 hydrogen Inorganic materials 0.000 claims description 3
- 239000001257 hydrogen Substances 0.000 claims description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 3
- 125000001624 naphthyl group Chemical group 0.000 claims description 2
- 125000006501 nitrophenyl group Chemical group 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 33
- 238000004809 thin layer chromatography Methods 0.000 description 32
- 238000004458 analytical method Methods 0.000 description 27
- 239000000758 substrate Substances 0.000 description 26
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 17
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 15
- 239000000243 solution Substances 0.000 description 15
- 239000000047 product Substances 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 229920005654 Sephadex Polymers 0.000 description 9
- 239000012507 Sephadex™ Substances 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 9
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 8
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 8
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 8
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 7
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 230000007071 enzymatic hydrolysis Effects 0.000 description 4
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- TYMLOMAKGOJONV-UHFFFAOYSA-N 4-nitroaniline Chemical compound NC1=CC=C([N+]([O-])=O)C=C1 TYMLOMAKGOJONV-UHFFFAOYSA-N 0.000 description 3
- 229920002307 Dextran Polymers 0.000 description 3
- CPELXLSAUQHCOX-UHFFFAOYSA-N Hydrogen bromide Chemical compound Br CPELXLSAUQHCOX-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Chemical compound CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 235000019439 ethyl acetate Nutrition 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 3
- 235000017557 sodium bicarbonate Nutrition 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- YDMBNDUHUNWWRP-VJBWXMMDSA-N (2s)-1-[(2r)-2-amino-3-phenylpropanoyl]-n-[(2s)-5-(diaminomethylideneamino)-1-(4-nitroanilino)-1-oxopentan-2-yl]piperidine-2-carboxamide Chemical compound C([C@@H](N)C(=O)N1[C@@H](CCCC1)C(=O)N[C@@H](CCCN=C(N)N)C(=O)NC=1C=CC(=CC=1)[N+]([O-])=O)C1=CC=CC=C1 YDMBNDUHUNWWRP-VJBWXMMDSA-N 0.000 description 2
- 102000004400 Aminopeptidases Human genes 0.000 description 2
- 108090000915 Aminopeptidases Proteins 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 108010016626 Dipeptides Proteins 0.000 description 2
- 150000008575 L-amino acids Chemical class 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- 108010039286 S 2238 Proteins 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- RDOXTESZEPMUJZ-UHFFFAOYSA-N anisole Chemical compound COC1=CC=CC=C1 RDOXTESZEPMUJZ-UHFFFAOYSA-N 0.000 description 2
- 125000003236 benzoyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)=O 0.000 description 2
- 230000008033 biological extinction Effects 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 239000012043 crude product Substances 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000001641 gel filtration chromatography Methods 0.000 description 2
- GNOIPBMMFNIUFM-UHFFFAOYSA-N hexamethylphosphoric triamide Chemical compound CN(C)P(=O)(N(C)C)N(C)C GNOIPBMMFNIUFM-UHFFFAOYSA-N 0.000 description 2
- 125000004435 hydrogen atom Chemical class [H]* 0.000 description 2
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 2
- 238000005342 ion exchange Methods 0.000 description 2
- RNVCVTLRINQCPJ-UHFFFAOYSA-N o-toluidine Chemical compound CC1=CC=CC=C1N RNVCVTLRINQCPJ-UHFFFAOYSA-N 0.000 description 2
- 125000006239 protecting group Chemical group 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 1
- GFNKTLQTQSALEJ-UHFFFAOYSA-N 1-isocyanato-4-nitrobenzene Chemical compound [O-][N+](=O)C1=CC=C(N=C=O)C=C1 GFNKTLQTQSALEJ-UHFFFAOYSA-N 0.000 description 1
- YOETUEMZNOLGDB-UHFFFAOYSA-N 2-methylpropyl carbonochloridate Chemical compound CC(C)COC(Cl)=O YOETUEMZNOLGDB-UHFFFAOYSA-N 0.000 description 1
- JBIJLHTVPXGSAM-UHFFFAOYSA-N 2-naphthylamine Chemical compound C1=CC=CC2=CC(N)=CC=C21 JBIJLHTVPXGSAM-UHFFFAOYSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 150000008574 D-amino acids Chemical group 0.000 description 1
- 125000001711 D-phenylalanine group Chemical group [H]N([H])[C@@]([H])(C(=O)[*])C([H])([H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 1
- HXEACLLIILLPRG-YFKPBYRVSA-N L-pipecolic acid Chemical compound [O-]C(=O)[C@@H]1CCCC[NH2+]1 HXEACLLIILLPRG-YFKPBYRVSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 description 1
- 235000008331 Pinus X rigitaeda Nutrition 0.000 description 1
- 235000011613 Pinus brutia Nutrition 0.000 description 1
- 241000018646 Pinus brutia Species 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 108010094028 Prothrombin Proteins 0.000 description 1
- 102100027378 Prothrombin Human genes 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 150000008064 anhydrides Chemical class 0.000 description 1
- 239000004019 antithrombin Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- IADUEWIQBXOCDZ-UHFFFAOYSA-N azetidine-2-carboxylic acid Chemical group OC(=O)C1CCN1 IADUEWIQBXOCDZ-UHFFFAOYSA-N 0.000 description 1
- 150000001540 azides Chemical class 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- HQABUPZFAYXKJW-UHFFFAOYSA-N butan-1-amine Chemical compound CCCCN HQABUPZFAYXKJW-UHFFFAOYSA-N 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000010908 decantation Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 229910000042 hydrogen bromide Inorganic materials 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- HXEACLLIILLPRG-RXMQYKEDSA-N l-pipecolic acid Natural products OC(=O)[C@H]1CCCCN1 HXEACLLIILLPRG-RXMQYKEDSA-N 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 1
- 125000000636 p-nitrophenyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1*)[N+]([O-])=O 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- NMHMNPHRMNGLLB-UHFFFAOYSA-N phloretic acid Chemical compound OC(=O)CCC1=CC=C(O)C=C1 NMHMNPHRMNGLLB-UHFFFAOYSA-N 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 229940039716 prothrombin Drugs 0.000 description 1
- 230000005588 protonation Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- NRTLTGGGUQIRRT-UHFFFAOYSA-N triethylazanium;bromide Chemical compound [Br-].CC[NH+](CC)CC NRTLTGGGUQIRRT-UHFFFAOYSA-N 0.000 description 1
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 238000002211 ultraviolet spectrum Methods 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
Classifications
-
- 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
-
- 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
-
- 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
-
- 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)
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Neurosurgery (AREA)
- Hematology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (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)
Description
Foreliggende oppfinnelse angår nye kromogene substrater for thrombin og thrombinlignende enzymer. Substratene i henhold til oppfinnelsen er særlig egnet for en kvantitativ bestemmelse av thrombin eller for å studere reaksjoner i hvilke thrombin dannes r inhiberes eller forbrukes, eller for å bestemme fak-torene som utøver en innflytelse eller tar del i slike reaksjoner, f.eks. for bestemmelse av anti-thrombin, prothrombin og heparin. The present invention relates to new chromogenic substrates for thrombin and thrombin-like enzymes. The substrates according to the invention are particularly suitable for a quantitative determination of thrombin or for studying reactions in which thrombin is formed, inhibited or consumed, or for determining the factors that exert an influence or take part in such reactions, e.g. for the determination of anti-thrombin, prothrombin and heparin.
Syntetiske substrater for enzymbestemmelser har store for-deler sammenlignet med de naturlige, forutsatt at de oppfyller visse betingelser, som stor sensitivitet og spesifisitet for enzymet, en god oppløselighet i vann eller den biologiske forsøksvæske og lett påvisbarhet av noen av spaltningsproduktene. Synthetic substrates for enzyme determinations have great advantages compared to natural ones, provided that they meet certain conditions, such as high sensitivity and specificity for the enzyme, a good solubility in water or the biological test fluid and easy detection of some of the cleavage products.
Et av de hittil beste substrater for thrombinbestemmelse er beskrevet i norsk patent 135 245, og består av det kromogene tripeptidderivat: One of the best substrates to date for thrombin determination is described in Norwegian patent 135 245, and consists of the chromogenic tripeptide derivative:
(med hensyn til forkortelsene, se side 4)• (with regard to the abbreviations, see page 4)•
Dette har stor sensitivitet for thrombin og gir ved enzymatisk hydrolyse det kromofore produkt p-nitroanilin som lett kan bestemmes spektrofotometrisk. S-2l6o har imidlertid en begrensning på grunn av dets relativt lave oppløselighet (1 mg/ml). En lav oppløselighet medfører den ulempe at man må arbeide meget nær metningsgrensen for substratet for å oppnå en tilfredsstillende substratkonsentrasjon. I enzymbestemmelser i forskjellige biologiske systemer kan en feining av substratet som sådan inntre, eller en kombinert protein/substratfeining. Disse felninger vil bevirke feilaktige' spektrofotometeravlesninger og således feilaktige enzymbestemmelser. Enzymsubstratet S-2l6o blir betraktelig mere oppløselig hvis benzoylgruppen erstattes med hydrogen, slik: This has great sensitivity to thrombin and upon enzymatic hydrolysis gives the chromophoric product p-nitroaniline which can be easily determined spectrophotometrically. However, S-2l6o has a limitation due to its relatively low solubility (1 mg/ml). A low solubility entails the disadvantage that one must work very close to the saturation limit for the substrate in order to achieve a satisfactory substrate concentration. In enzyme determinations in various biological systems, a screening of the substrate as such can occur, or a combined protein/substrate screening. These folds will cause incorrect spectrophotometer readings and thus incorrect enzyme determinations. The enzyme substrate S-2l6o becomes considerably more soluble if the benzoyl group is replaced by hydrogen, like this:
Den nå frie protoniserte aminogruppe på Phe øker oppløse-ligheten, men bevirker også at hastigheten med hvilken thrombin spalter substratet, øker sterkt, ca. 30 ganger (se tabell I). Videre kan substratet, i en biologisk forsøksoppløsning, spaltes på en uønsket måte fra den N-avsluttede ende av amino-pept idaser. The now free protonated amino group on Phe increases solubility, but also causes the rate at which thrombin cleaves the substrate to increase greatly, approx. 30 times (see Table I). Furthermore, the substrate, in a biological test solution, can be cleaved in an undesired way from the N-terminal end by amino-peptidases.
I henhold til foreliggende oppfinnelse er substratet i henhold til formel B blitt modifisert ved å utbytte Val med en cyclisk iminosyre (Aze, Pro eller Pip), og L-Phe er utbyttet med D-Phe. Som ventet er de således erholdte substrater fremdeles meget oppløselige, men helt forbausende har aktiviteten overfor thrombin ikke avtatt, men istedet er den 30 - 50 ganger høyere enn aktiviteten for det tilsvarende substrat med bare L-aminosyrer (tabell I). Videre er de nye substrater ca. 400% mere aktive enn S-2l60. Den N-endeavsluttende D-aminosyre forhindrer også et uønsket angrep av aminopeptidaser da de sistnevnte er spesifikke for L-aminosyrer. De nye kromogene substrater ifølge oppfinnelsen kjennetegnes ved den generelle formel: According to the present invention, the substrate according to formula B has been modified by replacing Val with a cyclic imino acid (Aze, Pro or Pip), and L-Phe has been replaced with D-Phe. As expected, the substrates thus obtained are still very soluble, but surprisingly the activity towards thrombin has not decreased, but instead it is 30 - 50 times higher than the activity for the corresponding substrate with only L-amino acids (table I). Furthermore, the new substrates are approx. 400% more active than S-2l60. The N-terminal D-amino acid also prevents an unwanted attack by aminopeptidases as the latter are specific for L-amino acids. The new chromogenic substrates according to the invention are characterized by the general formula:
eller salter derav, hvor R er hydrogen eller hydroxy, R2 er nitrofenyl, nafthyl eller methoxynafthyl, og n er 1, 2 eller 3. or salts thereof, where R is hydrogen or hydroxy, R 2 is nitrophenyl, naphthyl or methoxynaphthyl, and n is 1, 2 or 3.
Med syntesen av de nye kromogene enzymsubstrater anvendes konvensjonelle beskyttende grupper og koblingsmetoder som alle er velkjente i peptidkjemien. With the synthesis of the new chromogenic enzyme substrates, conventional protecting groups and coupling methods are used, all of which are well known in peptide chemistry.
Som en a-aminobeskyttende gruppe kan med fordel anvendes carboxybenzoxy- eller t-butyloxycarbonylgrupper eller en hvilken som helst dermed beslektet gruppe som f.eks. p-methoxy-, As an α-amino-protecting group, carboxybenzoxy or t-butyloxycarbonyl groups or any related group such as e.g. p-methoxy-,
p-nitro- eller p-methoxyfenylazo-carbobenzoxy. p-nitro- or p-methoxyphenylazo-carbobenzoxy.
Som en beskyttelse for &-guanidogruppen i arginylgruppen er det fordelaktig å anvende protonisering, en nitrogruppe eller en p-toluensulfonylgruppe. As a protection for the &-guanido group in the arginyl group, it is advantageous to use protonation, a nitro group or a p-toluenesulfonyl group.
Som beskyttelse for hydroxygruppen i tyrosingruppen er det fordelaktig å anvende en t-butyl- eller benzylgruppe. Som en avspaltbar carboxy-beskyttende gruppe er det passende å anvende methyl-, ethyl- eller benzylester. As protection for the hydroxy group in the tyrosine group, it is advantageous to use a t-butyl or benzyl group. As a cleavable carboxy-protecting group, it is suitable to use methyl, ethyl or benzyl ester.
Koblingen mellom to aminosyrer eller et dipeptid og en aminosyre oppnåes ved aktivering av a-carboxygruppen. Det aktiverte derivat kan enten isoleres eller dannes in situ, og kan f.eks. være p-nitrofenyl-, triklorfenyl-, pentaklorfenyl-eller N-hydroxysuccinimid-ester, symmetrisk eller asymmetrisk anhydrid, syreazid, eller N-hydroxybenzotriazol-ester. The connection between two amino acids or a dipeptide and an amino acid is achieved by activation of the α-carboxy group. The activated derivative can either be isolated or formed in situ, and can e.g. be p-nitrophenyl, trichlorophenyl, pentachlorophenyl or N-hydroxysuccinimide ester, symmetric or asymmetric anhydride, acid azide, or N-hydroxybenzotriazole ester.
Prinsippet for syntesen kan være en trinnvis kobling av aminosyrene til den carbonavsluttende arginylgruppe, som enten allerede er forsynt med en koblet kromofor gruppe som virker som en carboxybeskyttende gruppe eller forsynt med en avspaltbar carboxy-beskyttende gruppe, og den kromofore gruppe kobles så til det beskyttede tripeptidderivat, eller alternativt er det mulig å syntetisere selve det N-endeavsluttende dipeptidfragment som så kobles til arginylgruppen med eller uten en kromofor gruppe i henhold til det ovenfor anførte. The principle of the synthesis can be a stepwise coupling of the amino acids to the carbon-terminating arginyl group, which is either already provided with a linked chromophoric group that acts as a carboxy-protecting group or provided with a cleavable carboxy-protecting group, and the chromophoric group is then connected to the protected tripeptide derivative, or alternatively it is possible to synthesize the N-terminal dipeptide fragment itself which is then linked to the arginyl group with or without a chromophore group in accordance with the above.
Uavhengig av det valgte prinsipp er en rensning av mellom-og sluttprodukter ved gelfilt reringskromatografi egnet da dette vil muliggjøre et hurtig syntesearbeide og gi maksimale utbytter. Regardless of the chosen principle, a purification of intermediate and final products by gel filtration chromatography is suitable as this will enable rapid synthesis work and give maximum yields.
TLC-analyse har vært utført delvis av eluater fra GPC og delvis av inndampede og tørrede slutt- og mellomprodukter. TLC analysis has been carried out partly on eluates from GPC and partly on evaporated and dried final and intermediate products.
Oppfinnelsen er beskrevet mere detaljert i de følgende, ikke-begrensende, spesifikke eksempler. The invention is described in more detail in the following, non-limiting, specific examples.
Forkortelser Abbreviations
Aminosyrer (hvor annet ikke er anført menes L-formen): Amino acids (where not otherwise stated, the L form is meant):
Arg = arginin Arg = arginine
Aze = 2-azetidin-carboxylsyre Aze = 2-azetidine carboxylic acid
Phe = fenylalanin Phe = phenylalanine
Pip = pipecolinsyre Pip = pipecolic acid
Pro = prolin Pro = proline
Val = valin Val = valine
AcOH = eddiksyre AcOH = acetic acid
Bz = benzoyl Cbo- = carbobenzoxy- Bz = benzoyl Cbo- = carbobenzoxy-
DMF = dimethylformamid DMF = dimethylformamide
Et„N = triethylamin Et„N = triethylamine
55
EtOAc = ethylacetat EtOAc = ethyl acetate
HMPTA = N,N,N',N<*>,N",N"-hexamethylfosforsyre-triamid HMPTA = N,N,N',N<*>,N",N"-hexamethylphosphoric acid triamide
GPC = gelfiltreringskromatografi GPC = gel filtration chromatography
MeOH = methanol MeOH = methanol
-OpNP = p-nitrofenoxy -OpNP = p-nitrophenoxy
-pNA = p-nitroanilid -pNA = p-nitroanilide
TLC = tynnskiktskromatografi TLC = thin layer chromatography
Tynnskiktskromatografi Thin layer chromatography
For TLC-analyse anvendes preparerte glassplater med silicagtl F^^^ (Merck) som absorpsjonsmiddel. De anvendte opp-løsningsmiddelsystemer er følgende: For TLC analysis, prepared glass plates with silicagtl F^^^ (Merck) are used as absorbent. The solvent systems used are the following:
P-j^: kloroform/MeOH 9:1 (volumforhold) P-j^: chloroform/MeOH 9:1 (volume ratio)
A : n-butanol/AcOH/vann 3:2:1 (volumforhold) A : n-butanol/AcOH/water 3:2:1 (volume ratio)
Efter avsluttet kromatografi undersøkes platen i UV-lys (254 nm), og fremkallingen utføres derpå med klor/o-toluidin-reagens i henhold til vanlig praksis. Når et "homogent produkt i henhold til TLC" er anført, er analysen utført på en mengde på ^g. De anførte R^-verdier er resultatet av separate kromatografiske metoder. After the chromatography is finished, the plate is examined in UV light (254 nm), and the development is then carried out with chlorine/o-toluidine reagent according to usual practice. When a "homogeneous product according to TLC" is stated, the analysis was carried out on a quantity of ^g. The stated R^ values are the result of separate chromatographic methods.
Gelen Sephadex G-15 anvendt for gelfiltreringen er en tverrbunden dextrangel. Gelen Sephadex<®> LH-20 er en hydroxy-propylert tverrbundet dextrangel. Den anvendte ionebytter The gel Sephadex G-15 used for the gel filtration is a cross-linked dextran. The gel Sephadex<®> LH-20 is a hydroxy-propylated cross-linked dextran. The applied ion exchanger
Sephadex<®> QAE-25 er en tverrbunden dextrangel med diethyl-(2-hydroxypropyl)-aminoethyl som funksjonell gruppe. Disse geler er fra Pharmacia Pine Chemicals, Uppsala, Sverige. Sephadex<®> QAE-25 is a cross-linked dextran with diethyl-(2-hydroxypropyl)-aminoethyl as a functional group. These gels are from Pharmacia Pine Chemicals, Uppsala, Sweden.
Beskrivelse av syntesen Description of the synthesis
I. Cbo-Arg(N02)pNA I. Cbo-Arg(NO2)pNA
35,3 g (IO mmol) tørr Cbo-Arg(N02)-OH oppløses i 200 ml tørt, friskt destillert HMPTA ved værelsetemperatur, hvorpå 10,1 g (100 mmol) Et^N °9 24,6 g (150 mmol) p-nitrofenylisocyanat tilsettes under omrøring og fuktighetsfrie betingelser. Efter en reaksjonstid på 24 timer helles oppløsningen i 2 1 2%-ig 35.3 g (10 mmol) dry Cbo-Arg(NO2)-OH are dissolved in 200 ml dry, freshly distilled HMPTA at room temperature, after which 10.1 g (100 mmol) Et^N °9 24.6 g (150 mmol ) p-nitrophenyl isocyanate is added under stirring and moisture-free conditions. After a reaction time of 24 hours, the solution is poured into 2 1 2%-ig
natriumbicarbonatoppløsning under omrøring. Det dannede bunnfall fjernes ved filtrering og vaskes med 2%-ig bicarbonatopp-løsning, vann, 0,5 N vandig saltsyre og vann, og tørres til slutt. Råproduktet ekstraheres med varm methanol, og vanskelig oppløselige biprodukter frafiltceres, Filtratet renses ved kromatografi på en kolonne av Sephadex<®> LH-20, svellet i og eluert med methanol. sodium bicarbonate solution while stirring. The formed precipitate is removed by filtration and washed with 2% bicarbonate solution, water, 0.5 N aqueous hydrochloric acid and water, and finally dried. The crude product is extracted with hot methanol, and difficult-to-dissolve by-products are filtered off. The filtrate is purified by chromatography on a column of Sephadex<®> LH-20, swollen in and eluted with methanol.
Utbytte: 29,8 g (63%) Yield: 29.8 g (63%)
Analyse: homogent i henhold til TLC i P.. (Rf: 0,34) Analysis: homogeneous according to TLC in P.. (Rf: 0.34)
II. Cbo-Pro-Arg(N02)-pNA II. Cbo-Pro-Arg(NO2)-pNA
4,8 g (IO mmol) Cbo-Arg(N02)-pNA oppslemmes i 25 ml AcOH, hvorpå 15 ml 5,6 N HBr i AcOH tilsettes. Efter en reaksjonstid på 50 minutter ved værelsetemperatur helles oppløsningen under kraftig omrøring i 300 ml tørr ether. Etherfasen suges fra det erholdte bunnfall, og bunnfallet vaskes med 2 x 100 ml ether. Det således erholdte HBr-H-Arg(NO )-pNA tørres i vakuum over natriumhydroxyd ved 40 C i 16 timer. Det oppløses så i 25 ml DMF, og oppløsningen avkjøles til -10°C. Nå tilsettes Et3<_>N i en mengde tilstrekkelig til å gi et fuktig pH-papir holdt llfC©©Ver overflaten av oppløsningen en svakt basisk reaksjon (1,9 ml). 4.8 g (10 mmol) of Cbo-Arg(NO2)-pNA are suspended in 25 ml of AcOH, after which 15 ml of 5.6 N HBr in AcOH are added. After a reaction time of 50 minutes at room temperature, the solution is poured with vigorous stirring into 300 ml of dry ether. The ether phase is sucked from the precipitate obtained, and the precipitate is washed with 2 x 100 ml of ether. The thus obtained HBr-H-Arg(NO )-pNA is dried in vacuum over sodium hydroxide at 40 C for 16 hours. It is then dissolved in 25 ml of DMF, and the solution is cooled to -10°C. Now Et3<_>N is added in an amount sufficient to give a moist pH paper held llfC©©Ver the surface of the solution a weak basic reaction (1.9 ml).
Utfelt triethylamin-hydrobromid fjernes ved filtrering, og 4,1 g (11 mmol) Cbo-Pro-OpNP tilsettes. Efter 1 time tilsettes ytterligere 0,7 ml triethylamin og likeledes efter 4 timer. Oppløs-ningen får lov til å anta værelsetemperatur over natten. For å unngå at overskuddet av Cbo-Pro-OpNP forurenser sluttproduktet under GPC da de har lignende elueringsvolumer i det anvendte kromatografiske system, tilsettes 0,5 ml (5 mmol) n-butylamin. Efter 30 minutter tilsettes IO mmol fortynnet saltsyre, reak-sjonsoppløsningen inndampes på en roterende inndamper, omrøres med et par porsjoner vann som fjernes ved dekantering. Residuet oppløses i methanol og kromatograferes på en kolonne av "Sephadex" LH-20 svellet i og eluert med methanol. Det erholdte produkt er homogent i henhold til TLC. Precipitated triethylamine hydrobromide is removed by filtration, and 4.1 g (11 mmol) of Cbo-Pro-OpNP are added. After 1 hour, a further 0.7 ml of triethylamine is added and likewise after 4 hours. The solution is allowed to reach room temperature overnight. To avoid the excess of Cbo-Pro-OpNP contaminating the final product during GPC as they have similar elution volumes in the chromatographic system used, 0.5 ml (5 mmol) n-butylamine is added. After 30 minutes, 10 mmol of diluted hydrochloric acid is added, the reaction solution is evaporated on a rotary evaporator, stirred with a couple of portions of water which is removed by decantation. The residue is dissolved in methanol and chromatographed on a column of "Sephadex" LH-20 swollen in and eluted with methanol. The product obtained is homogeneous according to TLC.
Utbytte: 5,5 g (96%) Yield: 5.5 g (96%)
Analyse: TLC i P (Rf: 0,28) Analysis: TLC in P (Rf: 0.28)
III. Cbo-Pip-Arg(N02)-pNA III. Cbo-Pip-Arg(NO2)-pNA
Utført i henhold til II. Carried out in accordance with II.
Utbytte: 5,1 9 (86%) Yield: 5.1 9 (86%)
Analyse: homogent i henhold til TLC i P, (R,: 0,30) Analysis: homogeneous according to TLC in P, (R,: 0.30)
IV. Cbo-Phe-Pip-Arg(N02)-pNA IV. Cbo-Phe-Pip-Arg(NO2)-pNA
2,9 g (5 mmol) Cbo-Pip-Arg(N02)-pNA dicarbobenzoxyleres i hydro-genbromid i eddiksyre, felles og vaskes med ether og tørres i henhold til II. HBr-H-Pip-Arg(N02)-pNA oppløses så i 15 ml DMR. Oppløsningen avkjøles til -10 C, gjøres svakt alkalisk med 0,9 ml triethylamin og filtreres. 3,0 g (7,15 mmol) Cbo-Phe-OpNP tilsettes og derpå 0,65 g (5 mmol) N-hydroxybenzotriazol som kata-lysator. Efter 1 time tilsettes ytterligere 0,35 ml triethylamin, og den samme behandling gjentaes efter 4 timer. Reaksjons-oppløsningen tillates å anta værelsetemperatur over natten. Oppløsningen inndampes til tørrhet på en roterende tørrer. Residuet oppløses i ethylacetat og behandles med 2%-ig natrium-bicarbonatoppløsning og vann og inndampes så. Residuet opp-løses nå i methanol og kromatograferes på Sephadex<®>LH-20 svellet og eluert med methanol. Det erholdte produkt er homogent. i henhold til TLC. 2.9 g (5 mmol) Cbo-Pip-Arg(NO2)-pNA is dicarbobenzoxylated in hydrogen bromide in acetic acid, combined and washed with ether and dried according to II. HBr-H-Pip-Arg(NO 2 )-pNA is then dissolved in 15 ml of DMR. The solution is cooled to -10 C, made slightly alkaline with 0.9 ml of triethylamine and filtered. 3.0 g (7.15 mmol) of Cbo-Phe-OpNP are added and then 0.65 g (5 mmol) of N-hydroxybenzotriazole as catalyst. After 1 hour, a further 0.35 ml of triethylamine is added, and the same treatment is repeated after 4 hours. The reaction solution is allowed to warm to room temperature overnight. The solution is evaporated to dryness on a rotary drier. The residue is dissolved in ethyl acetate and treated with 2% sodium bicarbonate solution and water and then evaporated. The residue is now dissolved in methanol and chromatographed on Sephadex<®>LH-20 swollen and eluted with methanol. The product obtained is homogeneous. according to TLC.
Utbytte: 2,7 9 (73%) Yield: 2.7 9 (73%)
Analyse: TLC i P, (Rf: 0,35) Analysis: TLC in P, (Rf: 0.35)
V. Cbo-D-Phe-Pip-Arg(N02)-pNA V. Cbo-D-Phe-Pip-Arg(NO2)-pNA
Utført i henhold til IV. Carried out in accordance with IV.
Utbytte: 2,6 g (70%) Yield: 2.6 g (70%)
Analyse: homogent i henhold til TLC i P, (Rf: 0,44) Analysis: homogeneous according to TLC in P, (Rf: 0.44)
VI. Cbo-Phe-Pro-Arg(N02)-pNA WE. Cbo-Phe-Pro-Arg(NO2)-pNA
Utført i henhold til IV. Carried out in accordance with IV.
Utbytte: 2,9 g (80%) Yield: 2.9 g (80%)
Analyse: homogent i henhold til TLC i P^ (Rf: 0,38) Analysis: homogeneous according to TLC in P^ (Rf: 0.38)
VII. Cbo-D-Phe-Pro-Arg(N02)-pNA VII. Cbo-D-Phe-Pro-Arg(NO2)-pNA
Utført i henhold til IV. Carried out in accordance with IV.
Utbytte: 3,1 g (86%) Yield: 3.1 g (86%)
Analyse: homogent i henhold til TLC i P 0,46) Analysis: homogeneous according to TLC in P 0.46)
VIII. H-D-Phe-Pro-Arg-pNA-2HCl VIII. H-D-Phe-Pro-Arg-pNA-2HCl
175 mg (0,246 mmol Cbo-D-Phe-Pro-Arg(N02)-pNA befries for beskyttende grupper ved omsetning med 5 ml tørt HF i nærvær av 0,3 ml anisol i et apparat beregnet på dette formål i henhold til Sakakibara i 60 minutter ved 0°C. Efter avsluttet reaksjon og efter avdestillering av alt HF renses råproduktet og under-kastes ionebytte i to trinn: a) GPC på en kolonne av Sephadex G-15, svellet i 33%-ig vandig eddiksyre, med det samme medium som oppløsnings- og 175 mg (0.246 mmol) Cbo-D-Phe-Pro-Arg(NO2)-pNA is freed from protecting groups by reaction with 5 ml of dry HF in the presence of 0.3 ml of anisole in an apparatus designed for this purpose according to Sakakibara in 60 minutes at 0° C. After the reaction has ended and after all HF has been distilled off, the crude product is purified and subjected to ion exchange in two steps: a) GPC on a column of Sephadex G-15, swollen in 33% aqueous acetic acid, with the same medium as dissolution and
elueringsmedium. Det rene produkt frysetørres fra vandig eddiksyre . elution medium. The pure product is freeze-dried from aqueous acetic acid.
b) Ionebytte på en kolonne av Sephadex<®> QAE-25 i klorid-formen, svellet i methanol-.vann (95:5) med det samme medium som b) Ion exchange on a column of Sephadex<®> QAE-25 in the chloride form, swollen in methanol-water (95:5) with the same medium as
oppløsnings- og elueringsmedium. Det rene produkt ble fryse-tørret fra vann.. dissolution and elution medium. The pure product was freeze-dried from water..
Utbytte: 120 mg fflrø) Yield: 120 mg fflrø)
Analyse: homogent i henhold til TLC i A (R^: 0,29) Analysis: homogeneous according to TLC in A (R^: 0.29)
Kloridinnhold 11,53% (teoretisk 11,6%) Chloride content 11.53% (theoretical 11.6%)
IX. H-Phe-Pro-Arg-pNA-2HCl IX. H-Phe-Pro-Arg-pNA-2HCl
Utført i henhold til VIII. Carried out in accordance with VIII.
Utbytte: (71%) Yield: (71%)
Analyse: homogent i henhold til TLC i A (Rf: 0,22) Analysis: homogeneous according to TLC in A (Rf: 0.22)
Kloridinnhold 11,0% (teoretisk 11,6%) Chloride content 11.0% (theoretical 11.6%)
X. H-D-Phe-Pip-Arg-pNA-2HCl X. H-D-Phe-Pip-Arg-pNA-2HCl
Utført i henhold til VIII. Carried out in accordance with VIII.
Utbytte: (72%) Yield: (72%)
Analyse: homogent i henhold til TLC i" A (Rf: 0,44) Analysis: homogeneous according to TLC in" A (Rf: 0.44)
Kloridinnhold 11,4% (teoretisk 11,3%) Chloride content 11.4% (theoretical 11.3%)
XI. H-Phe-Pip-Arg-pNA-2HCl XI. H-Phe-Pip-Arg-pNA-2HCl
Utført i henhold til VIII. ~~"~~ Carried out in accordance with VIII. ~~"~~
Utbytte: (55%) Yield: (55%)
Analyse: homogent i henhold til TLC i A (Rf: 0,4l) Analysis: homogeneous according to TLC in A (Rf: 0.4l)
Kloridinnhold 11,3% (teoretisk 11,3%) Chloride content 11.3% (theoretical 11.3%)
XII. Cbo-D-Tyr(OBzl)-Pip-Arg(N02)-pNA XII. Cbo-D-Tyr(OBzl)-Pip-Arg(NO2)-pNA
Utført i henhold til IV. Carried out in accordance with IV.
Utbytte: 3,2 g (75%) Yield: 3.2 g (75%)
Analyse: homogent i henhold til TLC i P1 (Rf: 0,50) Analysis: homogeneous according to TLC in P1 (Rf: 0.50)
XIII. H-D-Tyr-Pip-Arg-pNA XIII. H-D-Tyr-Pip-Arg-pNA
Utført i henhold til VIII. Carried out in accordance with VIII.
Utbytte: (68%) Yield: (68%)
Analyse: homogent i henhold til TLC i P (Rf: 0,44)) Analysis: homogeneous according to TLC in P (Rf: 0.44))
Kloridinnhold IO,8% (teoretisk 11,1%) Chloride content IO.8% (theoretical 11.1%)
XIV. Cbo-Aze-Arg(N02)-pNA XIV. Cbo-Aze-Arg(NO2)-pNA
Utført i henhold til II. Carried out in accordance with II.
Utbytte: 4,2 g(75%) Yield: 4.2 g (75%)
Analyse: homogent i henhold til TLC i P1 ( Rf: 0,27) Analysis: homogeneous according to TLC in P1 ( Rf: 0.27)
XV. Cbo-D-Phe-Aze-Arg(N02)-pNA XV. Cbo-D-Phe-Aze-Arg(NO2)-pNA
Utført i henhold til IV. Carried out in accordance with IV.
Utbytte: 2,4 g (69%) Yield: 2.4 g (69%)
Analyse: homogent i henhold til TLC i P± (Rf: 0,47) Analysis: homogeneous according to TLC in P± (Rf: 0.47)
XVI. H-D-Phe-Aze-Arg-pNA-2HCl XVI. H-D-Phe-Aze-Arg-pNA-2HCl
Utført i henhold til VIII. Carried out in accordance with VIII.
Utbytte: (71%) Yield: (71%)
Analyse: homogent i henhold til TLC i A (R^: 0,21) Analysis: homogeneous according to TLC in A (R^: 0.21)
Kloridinnhold 11,6% ("teoretisk 11,9%) Chloride content 11.6% ("theoretical 11.9%)
XVII. Cbo-Arg(N02)-PNA XVII. Cbo-Arg(NO2)-PNA
7,2 g (20 mmol) tørr Cbo-Arg(N02)-0H oppløses i 400 ml THF. 7.2 g (20 mmol) of dry Cbo-Arg(NO 2 )-OH are dissolved in 400 ml of THF.
2,0 g (20 mmol) triethylamin tilsettes, og derpå avkjøles oppløs-ningen til -10°C under helt fuktighetsfrie betingelser. 2,7 g (20 mmol) isobutylklorformiat oppløst i 20 ml THF tilsettes til den avkjølte oppløsning i løpet av 10 minutter, og efter ytterligere 10 minutter tilsettes 344 g (20 mmol) (3-naf thylamin. Reaksjonsblandingen får lov til å anta værelsetemperatur og hen-settes ved denne temperatur i 24 timer. Reaksjonsblandingen inndampes til tørrhet i vakuum, behandles 3 - 5 ganger med destillert vann, 3 - 5 ganger med 5%-ig natriumbicarbonatoppløs-ning og igjen 3 - 5 ganger med destillert vann, hvorefter den tørres i vakuum. Produktet oppløses i methanol og kromato-graf eres på en kolonne av Sephadex<®> LH-20, svellet i og eluert med methanol. Det erholdte produkt er homogent i henhold til TLC i P . 2.0 g (20 mmol) of triethylamine are added, and then the solution is cooled to -10°C under completely moisture-free conditions. 2.7 g (20 mmol) isobutyl chloroformate dissolved in 20 ml THF is added to the cooled solution over 10 minutes, and after a further 10 minutes 344 g (20 mmol) (3-naphthylamine) is added. The reaction mixture is allowed to reach room temperature and left at this temperature for 24 hours. The reaction mixture is evaporated to dryness in a vacuum, treated 3-5 times with distilled water, 3-5 times with 5% sodium bicarbonate solution and again 3-5 times with distilled water, after which it is dried in vacuo. The product is dissolved in methanol and chromatographed on a column of Sephadex<®> LH-20, swollen in and eluted with methanol. The product obtained is homogeneous according to TLC in P .
Utbytte: 8,1 9 (84%) Yield: 8.1 9 (84%)
Analyse: homogent i henhold til TLC i P^ (Rf: 0,40) Analysis: homogeneous according to TLC in P^ (Rf: 0.40)
XVIII. Cbo-Pip-Arg(N02) -|3NA XVIII. Cbo-Pip-Arg(NO2) -|3NA
Utført i henhold til II. Carried out in accordance with II.
Utbytte: 4,8 9 (82%) Yield: 4.8 9 (82%)
Analyse: homogent i henhold til TLC i P^^ (Rf: 0,36) Analysis: homogeneous according to TLC in P^^ (Rf: 0.36)
XIX. Cbo-D-Phe-Pip-Arg(N02)-PNA XIX. Cbo-D-Phe-Pip-Arg(NO2)-PNA
Utført i henhold til IV. Carried out in accordance with IV.
Utbytte: 2,6 g(7l%) Yield: 2.6 g (7l%)
Analyse: homogent i henhold til TLC i Px (Rf: 0,48) Analysis: homogeneous according to TLC in Px (Rf: 0.48)
XX. H-D-Phe-Pip-Arg-|3NA-2HC1 XX. H-D-Phe-Pip-Arg-|3NA-2HCl
Utført i henhold til VIII. Carried out in accordance with VIII.
Utbytte: (68%) Yield: (68%)
Analyse: homogent i henhold til TLC i A (Rf: 0,44) Analysis: homogeneous according to TLC in A (Rf: 0.44)
Kloridinnhold 11,2% (teoretisk 11,3%) Chloride content 11.2% (theoretical 11.3%)
XXI. Cbo-Arg(N02)-pNA (4-OMe) XXI. Cbo-Arg(NO2)-pNA (4-OMe)
Utført i henhold til XVII. Carried out in accordance with XVII.
Utbytte: 7.1 9 (70%) Yield: 7.1 9 (70%)
Analyse: homogent i henhold til XLC-^i P-^ (Rf: 0,42) Analysis: homogeneous according to XLC-^i P-^ (Rf: 0.42)
XXII. Cbo-Pip-Arg(N02)-pNA (4-OMe) XXII. Cbo-Pip-Arg(NO2)-pNA (4-OMe)
Utført i henhold til II. Carried out in accordance with II.
Utbytte: 4,9 9 (79%) Yield: 4.9 9 (79%)
Analyse: homogent i henhold til TLC i P^ (Rf: 0,38) Analysis: homogeneous according to TLC in P^ (Rf: 0.38)
XXIII. Cbo-D-Phe-Pip-Arg(N02)-|3NA (4-OMe) XXIII. Cbo-D-Phe-Pip-Arg(NO2)-|3NA (4-OMe)
Utført i henhold til IV. Carried out in accordance with IV.
Utbytte: 2, 7 9 (70%) Yield: 2, 7 9 (70%)
Analyse: homogent i henhold til TLC i P± ( Rf: 0,51) Analysis: homogeneous according to TLC in P± ( Rf: 0.51)
XXIV. H-D-Phe-Pip-Arg-pNA (4-OMe)-2HC1 XXIV. H-D-Phe-Pip-Arg-pNA (4-OMe)-2HCl
Utført i henhold til VIII. Carried out in accordance with VIII.
Utbytte: (64%) Yield: (64%)
Analyse: homogent i henhold til TLC i A (Rf: 0,44) Analysis: homogeneous according to TLC in A (Rf: 0.44)
Kloridinnhold IO,4% (teoretisk 10,7%) Chloride content IO.4% (theoretical 10.7%)
Bestemmelse av thrombin med kromogene substrater: Substratene fremstilt i henhold til eksemplene ble anvendt for å bestemme thrombin som anført nedenfor. Bestemmelsesprinsippet bygger på det forhold at spaltnings-produktet dannet ved enzymatisk hydrolyse, har et UV-spektrum som er vesentlig forskjellig;<f>ra det for substratet. Således har substratet i henhold til eksempel X, H-D-Phe-Pip-Arg-pNA, Determination of thrombin with chromogenic substrates: The substrates prepared according to the examples were used to determine thrombin as indicated below. The determination principle is based on the fact that the cleavage product formed by enzymatic hydrolysis has a UV spectrum that is significantly different from that of the substrate. Thus, the substrate according to example X, H-D-Phe-Pip-Arg-pNA, has
et absorpsjonsmaksimum ved 315 nm og den molare ekstinksjons - koeffisient på 12.500. Ved 405 nm er absorpsjonen av substratet nesten fullstendig stanset. p-nitroanilin, avspaltet fra substratet under den enzymatiske hydrolyse, har et absorpsjonsmaksimum ved 380 nm og en molar ekstinksjonskoeffisient på 13.200 som ved 405 nm bare har avtatt til 9.620. Ved spektrofotometrisk bestemmelse ved 405 nm er det således mulig lett å følge mengden av dannet p-nitroanilin, som er porporsjonal med graden av enzymatisk hydrolyse som i sin tur bestemmes av den aktive mengde thrombin. For andre substrater i henhold til oppfinnelsen eksisterer temmelig identiske forhold, og av denne grunn er bestemmelsene konsekvent blitt utført ved 405 nm. an absorption maximum at 315 nm and the molar extinction coefficient of 12,500. At 405 nm, the absorption of the substrate is almost completely stopped. p-nitroaniline, cleaved from the substrate during the enzymatic hydrolysis, has an absorption maximum at 380 nm and a molar extinction coefficient of 13,200 which at 405 nm has only decreased to 9,620. By spectrophotometric determination at 405 nm, it is thus possible to easily follow the amount of p-nitroaniline formed, which is proportional to the degree of enzymatic hydrolysis, which in turn is determined by the active amount of thrombin. For other substrates according to the invention fairly identical conditions exist, and for this reason the determinations have consistently been carried out at 405 nm.
Tabell I viser en sammenligning av de relative reaksjon»;-hastigheter mellom det tidligere nevnte thrombinsubstrat S-2l60, dets ikke-benzoylerte form og substratet i henhold til oppfinnelsen. Denne tabell viser klart overlegenheten av substratene i henhold til oppfinnelsen. De reagerer 4 ganger hurtigere med thrombin enn det beste tidligere kjente substrat, S-2160, og er videre ca. 10 ganger mere oppløselig i vann enn S-2l60. Table I shows a comparison of the relative reaction rates between the previously mentioned thrombin substrate S-2160, its non-benzoylated form and the substrate according to the invention. This table clearly shows the superiority of the substrates according to the invention. They react 4 times faster with thrombin than the best previously known substrate, S-2160, and are further approx. 10 times more soluble in water than S-2l60.
Tabell I Table I
Relative reaksjonshastigheter mellom thrombin (0,4 NIH/ml) og substrat (0,1 umol/ml). Relative reaction rates between thrombin (0.4 NIH/ml) and substrate (0.1 umol/ml).
Claims (1)
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 (3)
| Publication Number | Publication Date |
|---|---|
| NO762407L NO762407L (en) | 1977-01-12 |
| NO142576B true NO142576B (en) | 1980-06-02 |
| NO142576C NO142576C (en) | 1980-09-10 |
Family
ID=20325116
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| NO762407A NO142576C (en) | 1975-07-11 | 1976-07-09 | NEW DIAGNOSTIC ACTIVE CHROMOGENIC SUBSTRATES WITH HIGH SPECIFICITY FOR THROMBIN AND THROMBIN-LIKE ENZYMES |
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 |
| US20090075298A1 (en) | 2005-05-20 | 2009-03-19 | 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 AU AU15305/76A patent/AU497547B2/en not_active Expired
- 1976-06-25 AT AT463976A patent/AT348686B/en not_active IP Right Cessation
- 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 BE BE168774A patent/BE843970A/en not_active IP Right Cessation
- 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 ES ES449739A patent/ES449739A1/en not_active Expired
- 1976-07-09 NO NO762407A patent/NO142576C/en unknown
- 1976-07-09 DK DK312676A patent/DK145799C/en not_active IP Right Cessation
- 1976-07-09 FR FR7621196A patent/FR2317280A1/en active Granted
- 1976-07-09 PL PL1976191068A patent/PL103003B1/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 DD DD7600193785A patent/DD128691A5/en unknown
- 1976-07-10 JP JP51082442A patent/JPS5224590A/en active Granted
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