DK155051B - Tri-peptide derivatives as well as procedure for quantitative analysis of proteolytic enzymes during use thereof - Google Patents
Tri-peptide derivatives as well as procedure for quantitative analysis of proteolytic enzymes during use thereof Download PDFInfo
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DK 15505 1 BDK 15505 1 B
Den foreliggende opfindelse angår hidtil ukendte tripeptidderivater, der er nyttige som substrater til kvantitativ analyse for proteolytiske enzymer fra enzymklassen E.C. 3.4.21, f.eks. organ- eller kirtelkalli-kreiner og plasmin.The present invention relates to novel tripeptide derivatives which are useful as substrates for quantitative analysis for proteolytic enzymes from the enzyme class E.C. 3.4.21, e.g. organ or glandular calluses and plasmin.
5 Såkaldt organkallikrein eller kirtelkallikrein produceres af forskellige organer og kirtler, f.eks. bugspytkirtelen, spytkirtler, nyrer, tarmslimhinde osv. og udskilles i form af et proenzym eller i en aktiv form. Disse organ- eller kirtelkallikreiner afviger kemisk og fysiologisk fra plasmakallikrein. I visse patalogiske tilstande falder organ-10 kallikreinsekretionsniveauet til under eller stiger til over normalværdien. Således falder f.eks. kallikreinsekretionen i urin væsentligt under normalværdien hos patienter, der lider af nyresygdomme. Hos patienter, der lider af nyrecirrose er kallikreinsekretionen næsten fuldstændig undertrykt. Hos patienter, som lider af essentiel hyper-15 tension er kallikreinsekretionen i 24 timers urin signifikant reduceret i gennemsnit med 50% af normalværdien (jfr. f.eks. H.S. Margolius i "Chemistry and Biology of the Kallikrein-Kinin System in Health and Disease", 1974, side 399 - 409). Det er derfor vigtigt at have simple metoder til rådighed til et hurtigt kvantitativt assay for organkalli-20 krein. Det er kendt f.eks. at foretage assay af urinkallikrein ved esteroiyse af visse argininestere, f.eks. tosylargininmethylester (TAME). Ved en forbedret esterolytisk fremgangsmåde anvendes tosylargininmethylester markeret med tritium, og den ved esterolysen frigjorte methanols radioaktivtet måles. Disse esterolytiske fremgangs-25 måder har den ulempe, at de er ubiologiske, for så vidt som kalli-kreinerne er proteolytiske enzymer, der spalter naturlige peptidkæder amidolytisk, men ikke esterolytisk. Endvidere har estersubstrater den ulempe, at de spaltes af adskillige andre enzymer, dvs. spaltes uspecifikt af kallikrein. Fremgangsmåden med anvendelse af TAME marke-30 ret med tritium er complex, for så vidt som methanolen før målingen af det med tritium mærkede methanols radioaktivitet skal ekstraheres fra esterolyseblandingen med en væske, der ikke er blandbar med vand, da det tilbageværende TAME markeret med tritium, som stadig er tilbage i esterolyseblandingen, ellers vil inhibere målingen.5 So-called organ chalice or glandular chalice is produced by various organs and glands, e.g. pancreas, salivary glands, kidneys, intestinal mucosa, etc. and are excreted in the form of a proenzyme or in an active form. These organ or glandular chalice rays differ chemically and physiologically from plasma chalice rays. In certain pathological conditions, the organelle level of secretion is reduced to below or rises above the normal value. Thus, e.g. the uric acid secretion significantly below normal value in patients suffering from kidney disease. In patients suffering from renal cirrhosis, the kallikrein reinsertion is almost completely suppressed. In patients suffering from essential hyper-tension, kallikrein secretion in 24 hours of urine is significantly reduced on average by 50% of normal value (cf., for example, HS Margolius in "Chemistry and Biology of the Kallikrein-Kinin System in Health and Disease" ", 1974, pages 399 - 409). It is therefore important to have simple methods available for a rapid quantitative assay for organ calli-20 cancer. It is known e.g. to carry out assay of urinary alkaline by esteroysis of certain arginine esters, e.g. tosylarginine methyl ester (TAME). In an improved esterolytic process, tosylarginine methyl ester marked with tritium is used and the radioactivity released by the esterolysis is measured. These esterylytic processes have the disadvantage of being unbiological in that the calli-cranes are proteolytic enzymes that cleave natural peptide chains amidolytic but not esterlytic. Furthermore, ester substrates have the disadvantage of being cleaved by several other enzymes, viz. nonspecifically cleaved by kallikrein. The process of using the TAME marker with tritium is complex in that the methanol before the measurement of the radioactivity of the tritium-labeled methanol must be extracted from the esterification mixture with a water immiscible liquid, as the remaining TAME is marked with tritium. which is still left in the esterolysis mixture will otherwise inhibit the measurement.
DK 155051 B i 2 i! .......... ' ji I offentliggjort tysk patentansøgning nr. 25.27.932 beskrives sub- 1 2 1 strater, som har formlen R -Pro-X-Arg-NH-R , hvor R betegner en 2 blokerende gruppe, -NH-R betegner en chromogengruppe og X betegner phenylalanyl, β-cyclohexylalanyl, phenylglycyl eller tyrosyl.DK 155051 B i 2 i! .......... In published German Patent Application No. 25,27,932, substrates are described which have the formula R -Pro-X-Arg-NH-R, where R represents a 2 blocking represents a chromogen group and X represents phenylalanyl, β-cyclohexylalanyl, phenylglycyl or tyrosyl.
5 Disse substrater spaltes særdeles let af plasmakallikrein og giver et 2 . i farvet spaltningsprodukt R -Nl·^, hvis mængde kan måles ved fotometriske, spektrofotometriske eller fluorescensfotometriske metoder.5 These substrates are very readily cleaved by plasma kallikrein and give a 2. in colored cleavage product R -N1 · ^, the amount of which can be measured by photometric, spectrophotometric or fluorescence photometric methods.
Der er gjort forsøg på også at anvende disse substrater til assay for i organ- eller kirtelkallikreiner. Det har imidlertid overraskende vist ! i 10 sig, at disse substrater ikke er følsomme over for organ- eller kirtel- j kallikrein, dvs. ikke spaltes eller kun spaltes i en lille udstrækning i af den sidstnævnte. ·Attempts have also been made to use these substrates as an assay for organ or glandular chalks. However, it has surprisingly proved! in that these substrates are not sensitive to organ or gland kallikrein, ie. not split or only split to a small extent in the latter. ·
Dansk patentansøgning nr. 3126/76 og dansk patentansøgning nr.Danish patent application no. 3126/76 and Danish patent application no.
2400/73 beskriver forskellige tripeptidderivater, som skal anvendes 15 som substrater ved assay for forskellige enzymer. De fra danske patentansøgning nr. 3126/76 kendte tripeptidderivater er imidlertid alle kendetegnet ved, at sidekæden i den N-terminale aminosyre indeholder en 6-leddet aromatisk ring. Tripeptidderivaterne ifølge den foreliggende opfindelse har derimod enten en hydrogeneret 6-leddet 20 ring i den N-terminale aminosyre eller i den midterste aminosyre, og som vist nedenfor har disse strukturforskelle givet årsag til en uventet høj spaltningshastighed hos tripeptidderivaterne.2400/73 describes various tripeptide derivatives to be used as substrates by assay for various enzymes. However, the tripeptide derivatives known from Danish patent application 3126/76 are all characterized in that the side chain of the N-terminal amino acid contains a 6-membered aromatic ring. In contrast, the tripeptide derivatives of the present invention have either a hydrogenated 6-membered ring in the N-terminal amino acid or in the middle amino acid, and as shown below, these structural differences have caused an unexpectedly high cleavage rate of the tripeptide derivatives.
Dansk patentansøgning nr. 2400/73 beskriver blandt andet forbindelser, der indeholder phenylalanin som den N-terminale aminosyre og 25 valin som den midterste aminosyre, medens de foreliggende forbindelser indeholder cyclohexylalanin eller 4-hydroxycyclohexylalanin som den midterste aminosyre, når den N-terminale aminosyre er phenylalanin.Danish Patent Application No. 2400/73 discloses, inter alia, compounds containing phenylalanine as the N-terminal amino acid and valine as the middle amino acid, while the present compounds contain cyclohexylalanine or 4-hydroxycyclohexylalanine as the middle amino acid when the N-terminal amino acid is phenylalanine.
Dansk patentansøgning nr. 3127/76, jfr. DK 146937B angår tripeptid-30 derivater, som er nyttige som substrater til bestemmelse af visse proteolytiske enzymer, især glandulære eller organkallikreiner og plasmin. Forbindelserne ifølge opfindelsen adskiller sig fra de fra dansk patentansøgning nr. 3127/76 kendte forbindelser ved, at den N-terminale aminosyre eller den midterste aminosyre ufravigeligt bærerDanish patent application no. 3127/76, cf. DK 146937B relates to tripeptide derivatives which are useful as substrates for the determination of certain proteolytic enzymes, in particular glandular or organ chalcicles and plasmin. The compounds of the invention differ from the compounds known from Danish Patent Application No. 3127/76 in that the N-terminal amino acid or the middle amino acid inevitably carries
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3 en cyclohexyl-, cyclohexylmethyl-, 4-hydroxy-cyclohexylmethyl- eller phenylradikal på α-carbonatomet, medens de kendte forbindelser enten bærer valin, prolin eller Pip-radikaler i α-stillingen. Bl.a. kan fire forbindelser tages som eksempler, nemlig H-D-valyl-leucyl-arginyl-5 p-nitroaniliddihydrochlorid (forbindelse XVI) (kommercielt tilgængeligt under betegnelsen S-2266), H-D-valyl-leucyl-lysyl-p-nitro-aniliddihy-drochlorid (forbindelse XVIII) (kommercielt tilgængeligt under betegnelsen S-2251), H-D-prolyl-phenylalanyl-arginyl-p-nitroaniliddihydro-chlorid (forbindelse XL) (kommercielt tilgængelig under betegnelsen 10 S-2302) og H-D-pipeco-linyl-phenylalanyl-arginyl-p-nitroanilid-dihy-drochlorid (forbindelse XLI). Forbindelse XVI er et substrat for glandulær kallikrein, forbindelse XVIII er et substrat for plasmin, og forbindelserne XL og XLI er substrater for plasmin glandulær kallikrein og plasmakallikrein. Sammenligningstests har vist, at de fire 15 forbindelser opspaltes enzymatisk ved en given mængde af henholdsvis glandulær kallikrein eller plasmin ved en betydelig lavere hastighed end tripeptidderivaterne ifølge den foreliggende opfindelse.3 is a cyclohexyl, cyclohexylmethyl, 4-hydroxy-cyclohexylmethyl or phenyl radical on the α-carbon atom, while the known compounds either carry valine, proline or Pip radicals in the α-position. Among other things. For example, four compounds may be taken as examples, namely HD-valyl-leucyl-arginyl-5β-nitroanilide dihydrochloride (Compound XVI) (commercially available under the designation S-2266), HD-valyl-leucyl-lysyl-β-nitro-anilide dihydrochloride ( Compound XVIII) (commercially available under the designation S-2251), HD-prolyl-phenylalanyl-arginyl-p-nitroanilide dihydrochloride (Compound XL) (commercially available under the designation 10-S-2302) and HD-pipeclinyl-phenylalanyl-arginyl -p-nitroanilide dihydrochloride (Compound XLI). Compound XVI is a substrate for glandular kallikrein, compound XVIII is a substrate for plasmin, and compounds XL and XLI are substrates for plasmin glandular kallikrein and plasma kallikrein. Comparative tests have shown that the four compounds are enzymatically cleaved at a given amount of glandular kallikrein or plasmin, respectively, at a significantly lower rate than the tripeptide derivatives of the present invention.
Opfindelsen angår i overensstemmelse hermed hidtil ukendte chromo-gene substrater, som har høj følsomhed over for visse proteolytiske 20 enzymer af klasse E.C. 3.4.21., især organ- eller kirtelkallikreiner og plasmin, og derfor er nyttige som substrater til kvantitativ analyse for disse enzymer. Tripeptidderivater ifølge opfindelsen har den almene formel IThe invention accordingly relates to novel chromogenic substrates which have high sensitivity to certain proteolytic enzymes of class E.C. 3.4.21., Especially organ or glandular chalcrins and plasmin, and are therefore useful as substrates for quantitative analysis for these enzymes. Tripeptide derivatives of the invention have the general formula I
H - D - X - Y -Z -R IH - D - X - Y -Z -R I
25 hvor a) X betegner alanyl, a-aminobutyryl (But), valyl, norvalyl, leucyl eller norleucyl, og Y betegner cyclohexylalanyl (CHA), cyclohexyltyrosyl (CHT) eller phenylglycyl (Ph'Gly), eller 30 b) X betegner phenylalanyl eller phenylglycyl, og Y betegner cyclohexylalanyl eller cyclohexylty rosy I,Wherein a) X represents alanyl, α-aminobutyryl (But), valyl, norvalyl, leucyl or norleucyl, and Y represents cyclohexylalanyl (CHA), cyclohexyltyrosyl (CHT) or phenylglycyl (Ph'Gly), or b) X represents phenylalanyl or phenylglycyl, and Y represents cyclohexylalanyl or cyclohexylty rosy I,
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JJ
4 i i Z betegner arginyl eller lysyl, og R betegner p-nitrophenylamino, 2-naphthylamino, 4-methoxy-2-naph- j j thylamino, 4-methyl-cumaryl-7-amino, 1,3-di(methoxycarbonyl)phen- j yl-5-amino, quinolyl-5-amino eller 8-nitroquinolyl-5-amino, j 5 og salte deraf med syrer, med det forbehold, at tripeptidderivatet I j ikke kan være | H-D-Val-CHA-Arg-4-methylcumaryl-7-amid, H-D-Val-CHA-Arg-1,3-di(methoxycarbonyI)phenyl-5-amid, H-D-Val-CHA-Arg-2-naphthylamid, H-D-Val-CHA-Arg-4-methoxy-2-naphthylamid, H-D-Val-CHA-Arg-p-10 nitroanilid, H-D-Val-CHA-Lys-p-nitroanilid eller H-D-Ph'-Gly-CHA-Arg-pNA.4 in Z represents arginyl or lysyl, and R represents p-nitrophenylamino, 2-naphthylamino, 4-methoxy-2-naphthylamino, 4-methyl-cumaryl-7-amino, 1,3-di (methoxycarbonyl) phenyl. yl-5-amino, quinolyl-5-amino or 8-nitroquinolyl-5-amino, j5 and its salts with acids, with the proviso that the tripeptide derivative I j cannot be | HD-Val-CHA-Arg-4-methylcumaryl-7-amide, HD-Val-CHA-Arg-1,3-di (methoxycarbonyl) phenyl-5-amide, HD-Val-CHA-Arg-2-naphthylamide, HD-Val-CHA-Arg-4-methoxy-2-naphthylamide, HD-Val-CHA-Arg-p-10 nitroanilide, HD-Val-CHA-Lys-p-nitroanilide or HD-Ph'-Gly-CHA Arg-pNA.
Tripeptidderivaterne med formlen I er tungt opløselige i vandige medier og anvendes derfor fortrinsvis i form af deres salte med syrer, især deres salte med mineralsyrer, f.eks. saltsyre, brom-15 brintesyre, svovlsyre, phosphorsyre osv. eller organiske syrer, f.eks. myresyre, eddikesyre, propionsyre, trimethyleddikesyre, methoxyeddikesyre, halogenerede eddikesyrer såsom trichlor- eller trifluoreddikesyre, aminoeddikesyre, mælkesyre, oxalsyre, malonsyre, citronsyre, benzoesyre, i kernen substituerede aromatiske syrer 20 såsom toluensyrer, chlor- eller brombenzoesyrer, methoxybenzoesyrer eller aminobenzoesyrer eller phthalsyre. Syrens natur er ikke kritisk, da syren ikke deltager i reaktionen mellem substraterne og enzymerne.The tripeptide derivatives of formula I are heavily soluble in aqueous media and are therefore preferably used in the form of their salts with acids, especially their salts with mineral acids, e.g. hydrochloric, hydrobromic, sulfuric, phosphoric, etc. or organic acids, e.g. formic acid, acetic acid, propionic acid, trimethylacetic acid, methoxyacetic acid, halogenated acetic acids such as trichloro or trifluoroacetic acid, aminoacetic acid, lactic acid, oxalic acid, malonic acid, citric acid, benzoic acid, core substituted aromatic acids such as toluene acids, The nature of the acid is not critical as the acid does not participate in the reaction between the substrates and the enzymes.
Forbindelserne med formlen I og deres salte med syrer spaltes hydro-25 lytisk ved indvirkning af visse proteolytiske enzymer af klasse 3.4.21. (jfr. "Enzyme Nomenclature", Elsevier Scientific Publishing Company, Amsterdam 1973, side 238 ff), især organ- eller kirtelkalli-kreiner og plasrrrin. Som et resultat dannes et farvet eller fluorescerende spaltningsprodukt med formlen R-Nf^, hvis mængde kan måles 30 ved fotometriske, spektrofotometriske, fluorescensspektrofotometriske eller el ekt ro kern i ske metoder. De hidtil ukendte forbindelser er derfor nyttige til kvantitativ analyse for proteolytiske enzymer fra enzymklasse E.C. 3.4.21., som spalter naturlige peptidkæder på carboxy-siden af arginin og lysin, især organ- eller kirtelkallikreiner og 35 plasmin samt deres inhibitorer og proenzymer. ' iThe compounds of formula I and their salts with acids are hydrolytically cleaved by the action of certain proteolytic enzymes of class 3.4.21. (cf. "Enzyme Nomenclature", Elsevier Scientific Publishing Company, Amsterdam 1973, page 238 et seq.), especially organ or glandular calli-crins and plasrin. As a result, a colored or fluorescent cleavage product of the formula R-Nf 2, the amount of which can be measured by photometric, spectrophotometric, fluorescence spectrophotometric or el ect ro nuclei, is produced in various methods. Therefore, the novel compounds are useful for quantitative analysis for proteolytic enzymes from enzyme class E.C. 3.4.21., Which cleaves natural peptide chains on the carboxy side of arginine and lysine, especially organ or glandular chalcrins and 35 plasmin, as well as their inhibitors and proenzymes. 'i
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Opfindelsen angår derfor også en fremgangsmåde til kvantitativ analyse for proteolytiske enzymer fra enzymklasse E.C. 3.4.21., som spalter naturlige peptidkæder på caroboxysiden af arginin og lysin, især organ- eller kirtelkallikreiner og plasmin. Fremgangsmåden ifølge 5 opfindelsen er ejendommelig ved, at mediet omsættes med et tripep-tidderivat med den almene formel I eller et salt deraf, og mængden af farvet eller fluorescerende spaltningsprodukt R-Nl·^ dannet ved den hydrolytiske indvirkning af enzymet på tripeptidderivatet måles ved fotometriske, spektrofotometriske, fluorescensspektrofotometriske eller 10 elektrokemiske metoder. Fremgangsmåden ifølge opfindelsen tillader f.eks. at analysere for enzymindholdet i enzympræparater eller enzymniveauet i menneskers eller pattedyrs legemsvæsker, f.eks. urin, bugspyt, tarmslimhinde, mælkekirtelsekretion, svedkirtelsekretion, sputum og blod. Ved hjælp af denne fremgangsmåde kan der udføres 15 assay for frie organkallikreiner og slimhindekallikreiner samt kalli-kreiner dannet ud fra prækallikreiner og endvidere fysiologiske eller ikke-fysiologiske inhibitorer for kallikreiner og fysiologiske eller ikke-fysiologiske aktivatorer for kallikreiner.The invention therefore also relates to a method for quantitative analysis for proteolytic enzymes from enzyme class E.C. 3.4.21., Which cleaves natural peptide chains on the caroboxy side of arginine and lysine, in particular organ or glandular chalice cells and plasmin. The process of the invention is characterized in that the medium is reacted with a tripeptide derivative of the general formula I or a salt thereof and the amount of colored or fluorescent cleavage product R-N1 · formed by the hydrolytic action of the enzyme on the tripeptide derivative is measured by , spectrophotometric, fluorescence spectrophotometric or electrochemical methods. The method according to the invention permits e.g. to analyze for the enzyme content of enzyme preparations or the level of enzymes in human or mammalian body fluids, e.g. urine, pancreas, intestinal mucosa, mammary gland secretion, sweat gland secretion, sputum and blood. By this method, 15 assays can be performed for free organ calli and mucosal calli cleans, as well as callicrins formed from pre-callic clears and further physiological or non-physiological inhibitors of callicrins and physiological or non-physiological activators of callicrins.
Tripeptidderivaterne med formlen I kan fremstilles ved de nedenfor 20 beskrevne fremgangsmåder: 1) Den chromogene gruppe R knyttes til carboxygruppen hos C-ter-minal arginin eller lysin, hvorhos α-aminogruppen beskyttes med en beskyttelsesgruppe, f.eks. en carbobenzoxy- eller tert.butoxycarbo-nylgruppe, idet δ-guanidylgruppen for arginins vedkommende be-25 skyttes ved proton i sering, f.eks. med HCI, nitrering eller tosylering, og ε-aminogruppen for Jysins vedkommende beskyttes med en carbo-benzoxygruppe, eller en p-methyl-, p-methoxy- eller p-chlorbenzyl-oxycarbonylgruppe eller en tert.butoxycarbonylgruppe. Den C-termi-nale R-NH-gruppe tjener også som en beskyttelsesgruppe under den 30 trinvise syntese af peptidkæden. De andre beskyttelsesgrupper kan fjernes selektivt som nødvendigt for at tilknytte yderligere aminosyre-derivater, indtil den ønskede peptidkæde er fuldstændig syntetiseret.The tripeptide derivatives of formula I can be prepared by the methods described below: 1) The chromogenic group R is attached to the carboxy group of C-terminal arginine or lysine, wherein the α-amino group is protected by a protecting group, e.g. a carbobenzoxy or tert-butoxycarbonyl group, the δ-guanidyl group of arginine being protected by proton in-ring, e.g. with HCl, nitration or tosylation, and the ε-amino group for Jysin is protected by a carbenzoxy group, or a p-methyl, p-methoxy or p-chlorobenzyl-oxycarbonyl group or a tert-butoxycarbonyl group. The C-terminal R-NH group also serves as a protecting group during the 30-step synthesis of the peptide chain. The other protecting groups can be selectively removed as necessary to attach additional amino acid derivatives until the desired peptide chain is fully synthesized.
Til sidst spaltes de tilbageværende beskyttelsesgrupper fuldstændig af, uden at R-NH-gruppen påvirkes (jfr. f.eks. Miklos Bodansky et 35 al., "Peptide Synthesis", Interscience Publishers, 1966, side 163 -165).Finally, the remaining protecting groups are completely cleaved without affecting the R-NH group (cf., for example, Miklos Bodansky et al., "Peptide Synthesis", Interscience Publishers, 1966, pp. 163-165).
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6 i i 2) Først syntetiseres peptidkæden (ifølge Bodansky el al., loc.cit.), hvorhos den C-terminale carboxylgruppe i arginin eller lysin midlertidigt beskyttes under anvendelse af en sædvanlig estergruppe, f.eks. en methoxy-, ethoxy- eller benzyloxygruppe for arginins i 5 vedkommende eller en tert.butoxygruppe for lysins vedkommende. j6 in i 2) First, the peptide chain (according to Bodansky et al., Loc. Cit.) Is synthesized, whereby the C-terminal carboxyl group of arginine or lysine is temporarily protected using a conventional ester group, e.g. a methoxy, ethoxy or benzyloxy group for arginine in question or a tert-butoxy group for lysine. j
Estergrupperne kan fjernes ved alkalisk hydrolyse undtagen tert.bu- j toxygruppen, som må fraspaltes selektivt ved hjælp af trifluoreddike- j syre. Hvis δ-guanidylgruppen i arginin protoniseres, fjernes denne j estergruppe enzymatisk ved hjælp af trypsin, idet der ikke foregår 10 nogen racemisering. Derefter tilknyttes den chromogene R-NH-grup- ! pe. Når δ-guanidinogruppen i arginin er beskyttet med en nitro- eller ! tosylgruppe, eller ε-aminogruppe i lysin er beskyttet med en carbo-benzoxy- eller tert.butoxygruppe, og N-terminal-a-aminogruppen i tripeptidderivatet er beskyttet med en carbobenzoxygruppe eller en 15 p-methyl-, p-methoxy- eller p-chlorbenzyloxycarbonylgruppe eller en tert.butoxygruppe, fjernes alle beskyttelsesgrupper samtidigt. Fjernelsen kan udføres ved at behandle det beskyttede tripeptidderivat med vandfrit HF ved stuetemperatur, idet alle de nævnte beskyttelsesgrupper på amino- og δ-guanidinogrupperne således fjernes. Fjer-20 nelsen kan også udføres ved behandling med 2N brombrintesyre i iseddike ved stuetemperatur, hvis det beskyttede tripeptidderivat ikke indeholder nitro- eller tosylbeskyttelsesgrupper.The ester groups can be removed by alkaline hydrolysis except the tertiary butoxy group, which must be selectively cleaved by trifluoroacetic acid. If the δ-guanidyl group in arginine is protonated, this ester group is enzymatically removed by trypsin, with no racemization going on. Then, the chromogenic R-NH group is attached. PE. When the δ-guanidino group in arginine is protected by a nitro or! tosyl group, or ε-amino group in lysine is protected with a carbobenzoxy or tert-butoxy group, and the N-terminal α-amino group in the tripeptide derivative is protected with a carbobenzoxy group or a 15 p-methyl, p-methoxy or p -chlorobenzyloxycarbonyl group or a tert-butoxy group, all protecting groups are removed at the same time. The removal can be carried out by treating the protected tripeptide derivative with anhydrous HF at room temperature, thus removing all the mentioned protecting groups on the amino and δ-guanidino groups. The removal may also be carried out by treatment with 2N hydrochloric acid in glacial acetic acid at room temperature if the protected tripeptide derivative does not contain nitro or tosyl protecting groups.
Fremstillingen af tripeptidderivaterne ifølge opfindelsen beskrives detaljeret i de nedenstående eksempler.The preparation of the tripeptide derivatives of the invention is described in detail in the examples below.
25 Analyser af eluater og produkter, der er fremstillet i henhold til eksemplerne, udføres ved tyndtlagschromatografi under anvendelse af glasplader overtrukket med silicagel (Merck, F 254). Tyndtlagschro-matogrammerne udvikles ved hjælp af følgende opløsningsmiddelsystemer: 30 A Chloroform/methanol (9:1).Analyzes of eluates and products prepared according to the Examples are performed by thin-layer chromatography using glass plates coated with silica gel (Merck, F 254). The thin layer chromatograms are developed using the following solvent systems: 30 A Chloroform / methanol (9: 1).
B n-Propanol/ethylacetat/vand (7:1:2).B n-Propanol / ethyl acetate / water (7: 1: 2).
C n-Butanol/eddikesyre/vand (3:1:1).C n -Butanol / acetic acid / water (3: 1: 1).
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Der anvendes følgende forkortelser:The following abbreviations are used:
AcOh = eddikesyreAcOh = acetic acid
Ala = alaninAla = alanine
Arg = arginin 5 BOC = tert.butoxycarbonylArg = arginine BOC = tert.butoxycarbonyl
But = a-aminosmørsyreBut = α-amino butyric acid
Cbo = carbobenzoxy CHA = cyclohexylalanin CHG = cyclohexylglycin 10 CHT = cyclohexyltyrosin = p-hydroxycyclohexylalanin DMF = dimethylformamid TLC = tyndtlagschromatografiCbo = carbobenzoxy CHA = cyclohexylalanine CHG = cyclohexylglycine CHT = cyclohexyl tyrosine = p-hydroxycyclohexylalanine DMF = dimethylformamide TLC = thin layer chromatography
EtgN = triethylamin HMPTA = phosphorsyre-N,N,N',N’,N",N"-hexamethyltriamid 15 Ile = isoleucinEtgN = triethylamine HMPTA = phosphoric acid N, N, N ', N', N ", N" -hexamethyltriamide Ile = isoleucine
Leu = leucin SS = opløsningsmiddelsystem/erLeu = leucine SS = solvent system / s
Lys = lysinLys = lysine
MeOH = methanol 20 Nleu = norleucinMeOH = methanol Nleu = norleucine
Nval = norvalinNval = norvalin
OpNP = p-nitrophenoxyOpNP = p-nitrophenoxy
Phe = phenylalaninPhe = phenylalanine
Ph'Gly = phenylglycin 25 Pip = pipecolinsyre pNA = p-nitroanilid THF = tetrahyd rof uranPh'Gly = phenylglycine Pip = pipecolic acid pNA = p-nitroanilide THF = tetrahydro uranium
Tyr = tyrosinTyr = tyrosine
Val = valin 30 Medmindre andet er angivet, har aminosyrerne i peptidkæderne L-form.Val = valine 30 Unless otherwise indicated, the amino acids in the peptide chains are L-shaped.
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88
Eksempel 1.Example 1.
H-D-But-CHA-Arg-pNA.2HBr.H-D-CHA-But-Arg-pNA.2HBr.
la. Cbo-Arg-pNA.HCI.la. Cbo-Arg-pNA.HCI.
I en 250 ml's trehalset kolbe opløses 16,0 g (47,0 millimol) Cbo-Arg-5 OH.HCI, som er tørret i vakuum over P20^, ' 90 ml absolut HMPTAIn a 250 ml three-neck flask is dissolved 16.0 g (47.0 millimoles) of Cbo-Arg-5 OH.HCI, which is dried in vacuo over P 2 O, 90 ml of absolute HMPTA
ved 20°C, medens atmosfæren i kolben holdes fugtfri. Til den resulterende opløsning sættes ved stuetemperatur først en opløsning af 4,74 g (47,0 millimol) Et^N i 10 ml HMPTA og derefter portionsvis 16,4 g (100 millimol) p-nitrophenylisocyanat (100%'s overskud). Efter en 10 reaktionstid på 24 timer ved 20°C fjernes hoveddelen af HMPTA ved destillation i vakuum. Remanensen ekstraheres adskillige gange med 30%’s AcOH. Remanensen kasseres. De forenede eddikesyreekstrakter renses yderligere ved føre dem gennem en søjle af "Sephadex G-15" ækvilibreret med 30%'s AcOH og elueres med 30%'s AcOH. Den fraktion 15 af AcOH-eluatet, som spaltes ved behandling med trypsin under frigørelse af p-nitroanilin, frysetørres. Således fås 12,6 g af et amorft pulver, som er homogent i opløsningsmiddelsystem C som vist ved TLC.at 20 ° C while keeping the atmosphere in the flask humid. To the resulting solution is added at room temperature first a solution of 4.74 g (47.0 millimoles) of Et 2 N in 10 ml of HMPTA and then portionwise 16.4 g (100 millimoles) of p-nitrophenyl isocyanate (100% excess). After a reaction time of 24 hours at 20 ° C, the majority of HMPTA is removed by distillation in vacuo. The residue is extracted several times with 30% AcOH. The residue is discarded. The combined acetic acid extracts are further purified by passing them through a column of "Sephadex G-15" equilibrated with 30% AcOH and eluted with 30% AcOH. The fraction 15 of the AcOH eluate, which is cleaved by trypsin treatment with release of p-nitroaniline, is lyophilized. Thus, 12.6 g of an amorphous powder is obtained which is homogeneous in solvent system C as shown by TLC.
Analyse: 20 Beregnet for C20H25N6°5CI: C 51,67 H 5,42 N 18,08 Cl 7,63.Calcd for C 20 H 25 N 6 ° 5 Cl: C 51.67 H 5.42 N 18.08 Cl 7.63.
Fundet: C 51,29 H 5,48 N 17,92 Cl 7,50.Found: C 51.29 H 5.48 N 17.92 Cl 7.50.
lb. 2HBr.H-Arg-pNA.lb. 2HBr.H-Arg-pNA.
4,65 g (10 millimol) af forbindelse la behandles under omrøring med 40 ml 2N HBr i iseddike i 45 minutter ved 20°C i fraværelse af fugt.4.65 g (10 millimoles) of compound Ia is treated with stirring with 40 ml of 2N HBr in glacial acetic acid for 45 minutes at 20 ° C in the absence of moisture.
25 Aminosyrederivatet opløses under C02-udvikling. Reaktionsopløsningen sættes dråbevis under kraftig omrøring til 250 ml absolut ether. Dette j resulterer i udfældning af 2HBr. H-Arg-pNA. Den etheriske fase frasuges, hvorefter den faste fase vaskes fire gange med 100 ml af absolut ether til fjernelse af benzylbromid, der er dannet som bipro-30 dukt, samt overskydende HBr og AcOH. Remanensen opløses i 50 ml methanol, pH-værdien indstilles til 4,5 ved tilsætning af Et^N, og j.The amino acid derivative dissolves during CO 2 evolution. The reaction solution is added dropwise with vigorous stirring to 250 ml of absolute ether. This j results in precipitation of 2HBr. H-Arg-pNA. The etheric phase is suctioned off and the solid phase is washed four times with 100 ml of absolute ether to remove benzyl bromide formed as a by-product and excess HBr and AcOH. The residue is dissolved in 50 ml of methanol, the pH is adjusted to 4.5 by the addition of Et 2 N, and j.
i;in;
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9 opløsningen inddampes til tørhed i vakuum ved 30°C. Det resulterende produkt opløses i 75 ml MeOH og føres gennem en søjle af "Sephadex LH-20" (tværbundet dextrangel) ækvilibreret med MeOH. Fra en fraktion af eluatet fås 4,18 g (91,6% af det teoretiske) amorf forbin-5 delse Ib, som er homogen i opløsningsmiddelsystem C som vist ved tyndtlagschromatografi.The solution is evaporated to dryness in vacuo at 30 ° C. The resulting product is dissolved in 75 mL of MeOH and passed through a column of "Sephadex LH-20" (crosslinked dextrangel) equilibrated with MeOH. From a fraction of the eluate, 4.18 g (91.6% of theory) of amorphous Compound Ib is obtained, which is homogeneous in solvent system C as shown by thin layer chromatography.
Analyse:Analysis:
Beregnet for C12H20NgO3Br2: C 31,60 H 4,42 N 18,43 Br 35,03.Calcd for C12 H20 NgO3 Br2: C 31.60 H 4.42 N 18.43 Br 35.03.
Fundet: C31,15 H 4,35 N 18,84 Br 34,81.Found: C31.15 H 4.35 N 18.84 Br 34.81.
10 1c. Cbo-CHA-Arg-pNA.HBr.10 1c. Cbo-CHA-Arg-pNA.HBr.
4,56 g (10 millimol) af forbindelse Ib opløses i 30 ml friskt destilleret DMF, og opløsningen afkøles til -10°C. 1,40 ml (10 millimol) i EtgN sættes til opløsningen under omrøring. Det dannede Et^N.HBr fjernes ved filtrering og vaskes med en lille mængde koldt dimethylformamid.4.56 g (10 millimoles) of compound Ib are dissolved in 30 ml of freshly distilled DMF and the solution is cooled to -10 ° C. Add 1.40 ml (10 millimoles) of EtgN to the solution with stirring. The Et 2 N.HBr formed is removed by filtration and washed with a small amount of cold dimethylformamide.
15 4,69 g (11 millimol) Cbo-CHA-OpNP sættes ved -10°C til filtratet, og reaktionen lades forløbe i 2 - 3 timer i fraværelse af fugt, hvorved reaktionsopløsningens temperatur gradvis når ca. 20°C. Opløsningen afkøles atter til -10°C, puf res med 0,70 ml (5 millimol) EtgN og lades reagere i 2 timer ved -10°C og i ca. 3 timer ved stuetemperatur. Denne 20 procedure gentages med 0,70 ml Et^N, og efter 16 timer inddampes reaktionsopløsningen til tørhed i vakuum ved 50°C. Remanensen opløses i 75 ml 50% AcOH og renses ved gelfiltrering på en søjle af "Sephadex G-15"@ ækvilibreret med 50%'s AcOH. Hovedfraktionen af AcOH-eluatet, der spaltes ved behandling med trypsin under frigørelse af p-nitro-25 anilin, inddampes til tørhed i vakuum ved 40°C. Remanensen opløses i 150 ml MeOH og inddampes atter til tørhed. Den resulterende remanens tørres i en vakuumexsiccator ved 60°C over P205, hvorved fås 5,85 g (88,3% af det teoretiske) af den amorfe forbindelse 1c, som er homogen i opløsningsmiddelsystem C som vist ved TLC.4.69 g (11 millimoles) of Cbo-CHA-OpNP are added at -10 ° C to the filtrate, and the reaction is allowed to proceed for 2 - 3 hours in the absence of moisture, whereby the temperature of the reaction solution gradually reaches approx. 20 ° C. The solution is again cooled to -10 ° C, buffered with 0.70 ml (5 millimoles) of EtgN and allowed to react for 2 hours at -10 ° C and for approx. 3 hours at room temperature. This procedure is repeated with 0.70 ml of Et 2 N and after 16 hours the reaction solution is evaporated to dryness in vacuo at 50 ° C. The residue is dissolved in 75 ml of 50% AcOH and purified by gel filtration on a column of "Sephadex G-15" @ equilibrated with 50% AcOH. The major fraction of the AcOH eluate, which is cleaved by treatment with trypsin to release p-nitroaniline, is evaporated to dryness in vacuo at 40 ° C. The residue is dissolved in 150 ml of MeOH and evaporated again to dryness. The resulting residue is dried in a vacuum desiccator at 60 ° C over P2 O5 to give 5.85 g (88.3% of theory) of the amorphous compound 1c, which is homogeneous in solvent system C as shown by TLC.
30 Analyse:Analysis:
Beregnet for C29H4()N706Br: C 52,57 H 6,09 N 14,80 Br 12,06.Calcd for C 29 H 4 () N 706 Br: C 52.57 H 6.09 N 14.80 Br 12.06.
Fundet: 0 52,28 H 6,16 N 15,09 Br 11,85.Found: 0 52.28 H 6.16 N 15.09 Br 11.85.
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10 ld. 2HBr.H-CHA-Arg-pNA. j 5,30 g (8 millimol) af forbindelse 1c behandles under omrøring med 32 i j ml 2N HBr i iseddike i 40 minutter ved 20°C. Peptidderivatet opløses i gradvis under (X^-udvikling. Reaktionsopløsningen sættes dråbevis 5 under kraftig omrøring til 250 ml absolut ether. Dette resulterer i j i udfældning af 2HBr.H-CHA-Arg-pNA. Den etheriske fase frasuges, j hvorefter den faste fase vaskes fire gange med 100 ml absolut ether til | fjernelse af benzylbromid, der er dannet som biprodukt, samt overskydende HBr og AcOH. Remanensen opløses i 50 ml MeOH. pH-Værdien 10 indstilles til 4,5 ved hjælp af Et^N, og opløsningen inddampes til tørhed i vakuum ved 30°C. Den resulterende remanens opløses i 50 ml MeOH og renses på en søjle af "Sephadex LH-20" , der er ækvilibreret med10 ld. 2HBr.H-CHA-Arg-pNA. 5.30 g (8 millimoles) of compound 1c is treated with stirring with 32 in j ml 2N HBr in glacial acetic acid for 40 minutes at 20 ° C. The peptide derivative dissolves gradually during (X 3 evolution. The reaction solution is added dropwise 5 with vigorous stirring to 250 ml of absolute ether. This results in precipitation of 2HBr.H-CHA-Arg pNA. The etheric phase is suctioned and the solid phase is washed. four times with 100 ml of absolute ether to remove benzyl bromide formed as a by-product as well as excess HBr and AcOH, the residue is dissolved in 50 ml of MeOH, pH 10 adjusted to 4.5 by Et EtN and the solution evaporate to dryness in vacuo at 30 ° C. The resulting residue is dissolved in 50 ml of MeOH and purified on a column of "Sephadex LH-20" equilibrated with
MeOH. Den fraktion af MeOH-eluatet, der spaltes ved behandling med trypsin under frigørelse af p-nitroanilin, inddampes til tørhed i vakuum 15 ved 30°C. Den resulterende remanens tørres i en vakuumexsiccator ved 40°C over P2O5' hvorved fås 4,48 g (91,9% af det teoretiske) amorf forbindelse Id, som er homogen i opløsningsmiddelsystem C.MeOH. The fraction of the MeOH eluate cleaved by treatment with trypsin under release of p-nitroaniline is evaporated to dryness in vacuo 15 at 30 ° C. The resulting residue is dried in a vacuum desiccator at 40 ° C over P 2 O 5 'to give 4.48 g (91.9% of theory) of amorphous compound Id, which is homogeneous in solvent system C.
Analyse:Analysis:
Beregnet for C^H^N^Br: C 41,39 H 5,79 N 16,09 Br 26,23.Calculated for C C HH HN₂O Br: C, 41.39; H, 5.79; N, 16.09; Br, 26.23.
20 Fundet: C 41,80 H 5,86 N 16,31 Br 25,85.Found: C 41.80 H 5.86 N 16.31 Br 25.85.
le. Cbo-D-But-CHA-Arg-pNA.HBr.scythe. Cbo-D-CHA-But-Arg-pNA.HBr.
3,05 g (5 millimol) forbindelse Id opløses i 20 ml friskt destilleret DMF, og opløsningen afkøles til -10°C. 0,70 ml (5 millimol) EtjN sættes til opløsningen under omrøring. Det dannede EtgN.HBr fjernes 25 ved filtrering og vaskes med en lille mængde koldt DMF. 1,97 g (5,5 millimol) Cbo-D-But.OpNP sættes ved -10°G under omrøring til filtra tet. Reaktronsblandingen lades reagere i 2 - 3 timer i fraværelse af fugt, hvorved reaktionsopløsningstemperaturen gradvis når ca. 20°C.Dissolve 3.05 g (5 millimoles) of compound Id in 20 ml of freshly distilled DMF and cool the solution to -10 ° C. 0.70 ml (5 millimoles) of EtnN is added to the solution with stirring. The EtgN.HBr formed is removed by filtration and washed with a small amount of cold DMF. 1.97 g (5.5 millimoles) of Cbo-D-But.OpNP is added at -10 ° G with stirring to the filtrate. The reaction mixture is allowed to react for 2-3 hours in the absence of moisture, whereby the reaction solution temperature gradually reaches approx. 20 ° C.
Opløsningen afkøles atter til -10°C, pufres med 0,35 ml (2,5 millimol) 30 EtgN og lades reagere i ca. 2 timer ved -10°C og i yderligere 3 timer ved stuetemperatur. Denne procedure gentages med 0,35 ml i Et^N, og efter 16 timer inddampes reaktionsopløsningen til tørhed i vakuum ved 50°C. Remanensen opløses i 50 ml 50%'s AcOH og renses vedThe solution is again cooled to -10 ° C, buffered with 0.35 ml (2.5 millimoles) of EtgN and allowed to react for approx. 2 hours at -10 ° C and for another 3 hours at room temperature. This procedure is repeated with 0.35 ml in Et 2 N and after 16 hours the reaction solution is evaporated to dryness in vacuo at 50 ° C. The residue is dissolved in 50 ml of 50% AcOH and purified
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11 gelfiltrering på en søjle af "Sephadex G-15" ækvilibreret med 50%'s AcOH. Hovedfraktionen af AcOH-eluatet, som spaltes ved behandling med trypsin under frigørelse, af p-nitroanilin, inddampes til tørhed i vakuum ved 40°C. Remanensen opløses i 100 ml MeOH, og opløsningen 5 inddampes atter til tørhed. Den resulterende remanens tørres i en vakuumexsiccator ved 60°C over hvorved fås 3,27 g (87,5% af det teoretiske) af den amorfe forbindelse le, som er homogen i opløsningsmiddelsystem C som vist ved TLC.11 gel filtration on a column of "Sephadex G-15" equilibrated with 50% AcOH. The major fraction of the AcOH eluate, which is cleaved by treatment with trypsin during release, of p-nitroaniline is evaporated to dryness in vacuo at 40 ° C. The residue is dissolved in 100 ml of MeOH and the solution 5 is again evaporated to dryness. The resulting residue is dried in a vacuum desiccator at 60 ° C to give 3.27 g (87.5% of theory) of the amorphous compound 1e, which is homogeneous in solvent system C as shown by TLC.
Analyse: 10 Beregnet for C33H4?N807Br: C 53,01 H 6,34 N 14,99 Br 10,69Calcd for C33H4N807Br: C 53.01 H 6.34 N 14.99 Br 10.69
Fundet: C 52,88 H 6,40 N 15,28 Br 10,53.Found: C 52.88 H 6.40 N 15.28 Br 10.53.
1f. 2HBr.H-D-But-CHA-Arg-pNA.1f. 2HBr.H-D-CHA-But-Arg-pNA.
2,24 g (3 millimol) forbindelse le behandles under omrøring med 12 ml 2N HBr i iseddike i 40 minutter ved 20°C i fraværelse af fugt. Tri-15 peptidderivatet opløses gradvis under decarboxylering og CC^-udvik-ling. Reaktionsopløsningen sættes dråbevis under kraftig omrøring til 120 ml absolut ether. Dette resulterer i udfældning af 2HBr.H-D-But-CHA-Arg-pNA. Den etheriske fase frasuges gennem en filterstav, og derefter vaskes den faste fase fire gange med portioner på 50 ml 20 absolut ether. Den resulterende remanens opløses i 40 ml MeOH. pH-Værdien indstilles til 4,5 ved hjælp af EtgN, og opløsningen inddampes til tørhed i vakuum ved 30°C. Remanensen opløses i 30 ml MeOH og renses på en søjle af "Sephadex LH-20" ækvilibreret med MeOH. Den fraktion af MeOH-eluatet, der spaltes ved behandling med 25 trypsin under frigørelse af p-nitroanilin, inddampes til tørhed i vakuum ved 30°C. Til yderligere rensning opløses den forrensede remanens i 30 ml 33%'s AcOH og renses ved gelfiltrering på en søjle af "Sephadex G-15" ækvilibreret med 33% AcOH. Hovedfraktionen af AcOH-eluatet, som spaltes ved behandling med trypsin under frigø-30 relse af p-nitroanilin, inddampes til tørhed i vakuum ved 40°C. Den resulterende remanens tørres i vakuumexsiccator ved 40°C over Ρ303, hvorved fås 1,71 g (82,1% af det teoretiske) amorf forbindelse 1f, som er homogen i opløsningsmiddelsystem C som vist ved TLC.2.24 g (3 millimoles) of compound le are treated with stirring with 12 ml of 2N HBr in glacial acetic acid for 40 minutes at 20 ° C in the absence of moisture. The tripeptide derivative is gradually dissolved during decarboxylation and CC 2 evolution. The reaction solution is added dropwise with vigorous stirring to 120 ml of absolute ether. This results in the precipitation of 2HBr.H-D-But-CHA-Arg pNA. The etheric phase is aspirated through a filter rod and then the solid phase is washed four times with 50 ml aliquots of absolute ether. The resulting residue is dissolved in 40 ml of MeOH. The pH is adjusted to 4.5 by EtgN and the solution is evaporated to dryness in vacuo at 30 ° C. The residue is dissolved in 30 ml of MeOH and purified on a column of "Sephadex LH-20" equilibrated with MeOH. The fraction of the MeOH eluate cleaved by treatment with 25 trypsin with release of p-nitroaniline is evaporated to dryness in vacuo at 30 ° C. For further purification, the purified residue is dissolved in 30 ml of 33% AcOH and purified by gel filtration on a column of "Sephadex G-15" equilibrated with 33% AcOH. The major fraction of the AcOH eluate, which is cleaved by treatment with trypsin to release p-nitroaniline, is evaporated to dryness in vacuo at 40 ° C. The resulting residue is dried in a vacuum desiccator at 40 ° C above Ρ303 to give 1.71 g (82.1% of theory) of amorphous compound 1f, which is homogeneous in solvent system C as shown by TLC.
i1 12i1 12
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Analyse:Analysis:
Beregnet for C25H47Ng05Br2: C 43,24 H 6,10 N 16,14 Br 23,01Calcd for C25H47NgO5Br2: C 43.24 H 6.10 N 16.14 Br 23.01
Fundet: ~ C 43,18 H 6,16 N 16,27 Br 22,73.Found: ~ C 43.18 H 6.16 N 16.27 Br 22.73.
i ii i
Aminosyreanalysen bekræfter tilstedeværelsen af de forventede amino-5 syrer i de korrekte forhold: Arg:1,01 - CHA:0,98 - D-But:1,00. j i i !The amino acid analysis confirms the presence of the expected amino acids in the correct ratios: Arg: 1.01 - CHA: 0.98 - D-But: 1.00. j i i!
Eksempel 2.Example 2.
2HBr. H-D-But-CHA-Lys-pNA. i 2a. BOC-Lys(e-Cbo)-pNA.2HBr. H-D-CHA-But-Lys-pNA. i 2a. BOC-Lys (e-Cbo) -pNA.
38,05 g (0,1 mol) tørret olie af BOC-Lys(s-Cbo)-OH opløses i en 500 10 ml's trehalset kolbe i 150 ml absolut HMPTA ved 20°C i fraværelse af fugt. Til den resulterende opløsning sættes ved stuetemperatur først en opløsning af 10,12 g (0,1 mol) Et^N i 25 ml HMPTA og derefter portionsvis 24,62 g (0,15 mol) p-nitrophenylisocyanat (50%'s overskud), hvorved der forekommer en kraftig C02-udvikling hver gang.38.05 g (0.1 mole) of dried oil of BOC-Lys (s-Cbo) -OH is dissolved in a 500 10 ml three-neck flask in 150 ml of absolute HMPTA at 20 ° C in the absence of moisture. To the resulting solution is added at room temperature first a solution of 10.12 g (0.1 mole) of Et 3 N in 25 ml of HMPTA and then portionwise 24.62 g (0.15 mole) of p-nitrophenyl isocyanate (50% excess). ), resulting in a strong CO 2 evolution each time.
15 Efter en reaktionstid på 24 timer ved 20°C fjernes hovedandelen af HMPTA ved destillation i vakuum. Remanensen ekstraheres adskillige gange med 2%'s natriumhydrogencarbonatopiøsning og digereres derefter med destilleret vand. Den resulterende remanens tørres i vakuum ved 40°C og ekstraheres derefter adskillige gange med varmt MeOH, 20 indtil remanensen kun indeholder det tungt opløselige biprodukt N,N'-bis(p-nitrophenyl)urea. MeOH-Ekstrakterne inddampes til 300 ml, hvorved nogle urenheder danner et flokkulent bundfald. Efter filtre-ring renses filtratet (330 ml) på en søjle af "Sephadex LH-20" æk-vilibreret med MeOH. Hovedfraktionen af MeOH-eluatet inddampes i 25 vakuum ved 30°C til et lille volumen, hvorved et nålelignende stof krystalliserer. De vundne krystaller frafiltreres og vaskes portionsvis med 50 ml isafkølet MeOH. Efter tørring i en vakuumexsiccator ved 40°C over P20^ ^1,1 g (62,1% af det teoretiske) krystallinsk forbindelse 2a, smeltepunkt ......... hvilket er homogent i opløs- 30 ningsmiddelsystem A og B som vist ved TLC. Moderluden giver enAfter a reaction time of 24 hours at 20 ° C, the majority of HMPTA is removed by distillation in vacuo. The residue is extracted several times with 2% sodium hydrogen carbonate solution and then digested with distilled water. The resulting residue is dried in vacuo at 40 ° C and then extracted several times with hot MeOH until the residue contains only the highly soluble by-product N, N'-bis (p-nitrophenyl) urea. The MeOH extracts are evaporated to 300 ml, whereby some impurities form a flocculent precipitate. After filtration, the filtrate (330 ml) is purified on a column of "Sephadex LH-20" equilibrated with MeOH. The major fraction of the MeOH eluate is evaporated in vacuo at 30 ° C to a small volume, thereby crystallizing a needle-like substance. The obtained crystals are filtered off and washed portionwise with 50 ml of ice-cooled MeOH. After drying in a vacuum desiccator at 40 ° C over P₂O ^ 1.1 g (62.1% of theory) of crystalline compound 2a, melting point ......... which is homogeneous in solvent system A and B as shown by TLC. The mother liquor gives one
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13 yderligere mængde på 5,8 g (11,6% af det teoretiske) af stof 2a, smeltepunkt ........ som er homogent i opløsningsmiddelsystem A og B som vist ved TLC.13 additional amount of 5.8 g (11.6% of theory) of substance 2a, melting point ........ which is homogeneous in solvent systems A and B as shown by TLC.
Analyse: 5 Beregnet for ^25^32^4^7* ^ ^ 6,44 N 11,19.Analysis: 5 Calculated for ^ 25 ^ 32 ^ 4 ^ 7 * ^ 6.44 N 11.19.
Fundet: C 60,23 H 6,50 N 11,38.Found: C, 60.23; H, 6.50; N, 11.38.
2b. CFgCOOH.H-Lys(s-Cbo)-pNA.2b. CFgCOOH.H-Lys (s-Cbo) -pNA.
25,03 g (50 millimol) forbindelse 2a behandles under kraftig omrøring med 50 ml friskt destilleret vandfrit trifluoreddikesyre ved 20°C i 60 10 minutter i fraværelse af fugtighed. BOC-Gruppen fraspaltes selektivt under CC^-udvikling og frigørelse af isobutylen. Reaktionsopløsningen sættes dråbevis under kraftig omrøring til 750 ml absolut ether, hvorved flokkulent CF^COOH.H-Lys(s-Cbo)-pNA udfældes. Den ethe-riske fase frasuges gennem en filterstav. Den faste fase behandles 15 fire gange med 100 ml's portioner absolut ether. Den resulterende remanens opløses i 200 ml MeOH. pH-Værdien indstilles til 4,5 ved hjælp af EtgN, og opløsningen inddampes til tørhed i vakuum ved25.03 g (50 millimoles) of compound 2a is treated with vigorous stirring with 50 ml of freshly distilled anhydrous trifluoroacetic acid at 20 ° C for 60 minutes in the absence of moisture. The BOC group is selectively cleaved during CC 2 development and isobutylene release. The reaction solution is added dropwise with vigorous stirring to 750 ml of absolute ether to precipitate flocculent CF 2 COOH.H-Lys (s-Cbo) pNA. The etheric phase is aspirated through a filter rod. The solid phase is treated four times with 100 ml portions of absolute ether. The resulting residue is dissolved in 200 ml of MeOH. The pH is adjusted to 4.5 by EtgN and the solution is evaporated to dryness in vacuo at
30°C. Remanensen opløses i 200 ml MeOH og renses på en søjle af "Sephadex LH-20"® ækvilibreret med MeOH. Hovedfraktionen af MeOH-20 eluatet, som er homogen i opløsningsmiddelsystem C som vist ved TLC30 ° C. The residue is dissolved in 200 ml of MeOH and purified on a column of "Sephadex LH-20" ® equilibrated with MeOH. The major fraction of the MeOH-20 eluate, which is homogeneous in solvent system C as shown by TLC
inddampes i vakuum ved 30°C. Den resulterende remanens tørres i en vakuumexsiccator ved 40°C over P2®5 hvorved fås 22,64 g (88,0% af det teoretiske) amorf forbindelse 2b.evaporated in vacuo at 30 ° C. The resulting residue is dried in a vacuum desiccator at 40 ° C over P2®5 to give 22.64 g (88.0% of theory) of amorphous compound 2b.
Analyse: 25 Beregnet for ^22^25^4^7^ C 51,36 H 4,90 N 10,89.Analysis: 25 Calcd for ^ 22 ^ 25 ^ 4 ^ 7 ^ C 51.36 H 4.90 N 10.89.
Fundet: C 51,66 H 4,88 N 11,08.Found: C, 51.66; H, 4.88; N, 11.08.
2c. BOC-CHA-Lys(ε-Cbo)-pNA.2c. BOC-CHA-Lys (ε-Cbo) -pNA.
7,72 (15 millimol) forbindelse 2b opløses i 50 ml frisk destilleret DMF og afkøles til -10°C. Til opløsningen sættes under omrøring 6,48 g 30 (16,5 millimol) BOC-CHA-OpNP og 2,09 ml (15 millimol) EtgN. Blandingen lades reagere i 3 timer i fraværelse af fugtighed, hvorunder DK 155051 Β 14 ί reaktionstemperaturen gradvis når stuetemperatur. Opløsningen afkøles atter til -10°C og pufres med 1,05 mi (7,5 millimol) EtgN. Efter en ! reaktionstid på 5 timer gentages denne procedure med 1,05 ml Et~N. ,7.72 (15 millimoles) of compound 2b is dissolved in 50 ml of freshly distilled DMF and cooled to -10 ° C. To the solution is added stirring 6.48 g of 30 (16.5 millimoles) of BOC-CHA-OpNP and 2.09 ml (15 millimoles) of EtgN. The mixture is allowed to react for 3 hours in the absence of humidity, during which the reaction temperature gradually reaches room temperature. The solution is again cooled to -10 ° C and buffered with 1.05 ml (7.5 millimoles) of EtgN. After one ! reaction time of 5 hours repeats this procedure with 1.05 ml Et ~ N. .
ύ Iύ I
Efter en reaktionstid på 16 timer ved 20°C inddampes reaktionsopløs- j 5 ningen til tørhed i vakuum ved 50°C. Remanensen opløses i 150 mlAfter a reaction time of 16 hours at 20 ° C, the reaction solution is evaporated to dryness in vacuo at 50 ° C. The residue is dissolved in 150 ml
MeOH og renses ved gelfiltrering på en søjle "Sephadex LH-20" ækvilibreret med MeOH. Den første hovedfraktion af MeOH-eluatet, som er homogen i opløsningsmiddelsystem A og B som vist ved tyndt- j lagschromatografi, koncentreres til tørhed i vakuum ved 30°C. Efter j 10 tørring i en vakuumexsiccator ved 40°C over Ρ30^ 8,26 9 (84,2% ! af det teoretiske) amorf forbindelse 2c, som er homogent i opløs ningsmiddelsystem A og B som vist ved TLC.MeOH and purified by gel filtration on a column "Sephadex LH-20" equilibrated with MeOH. The first major fraction of the MeOH eluate, which is homogeneous in solvent systems A and B as shown by thin layer chromatography, is concentrated to dryness in vacuo at 30 ° C. After drying in a vacuum desiccator at 40 ° C over 30 ° 8.26 g (84.2% of theoretical) amorphous compound 2c which is homogeneous in solvent systems A and B as shown by TLC.
Analyse:Analysis:
Beregnet for C^H^yN^Og: C 62,46 H 7,25 N 10,71 15 Fundet: C 63,05 H 7,35 N 10,98.Calculated for C C ^H ^NNN And: C 62.46 H 7.25 N 10.71 Found: C 63.05 H 7.35 N 10.98.
2d. CFgCOOH.H-CHA-Lys(s-Cbo)-pNA.2d. CFgCOOH.H-CHA-Lys (s-Cbo) -pNA.
3,27 g (5 millimol) forbindelse 2c behandles under kraftig omrøring med 10 ml friskt destilleret vandfri trifluoreddikesyre i 60 minutter ved 20°C i fraværelse af fugtighed. Reaktionsopløsningen sættes 20 dråbevis under kraftig omrøring til 100 ml absolut ether, hvorved amorft CFgCOOH. H-CHA-Lys(s-Cbo)-pNA udfældes. Den etheriske fase suges fra. Den faste remanens vaskes tre gange med portioner på 30 ml absolut ether. Den resulterende remanens opløses i 50 ml MeOH. pH-Værdien indstilles til 4,5 ved hjælp af Et^N, og opløsnin- 25 gen inddampes til tørhed i vakuum ved 30°C. Remanensen opløses i 75 ml MeOH og renses på en søjle af "Sephadex LH-20" ækvilibreret med MeOH. Hovedfraktionen af MeOH-eluatet, som er homogen i opløsningsmiddelsystem C som vist ved TLC, inddampes i vakuum ved 30°C. Efter tørring af remanensen i en vakuumexsiccator ved 40°C 30 over P£®5 fås 3,06 g (91,6% af det teoretiske) amorf forbindelse 2d.3.27 g (5 millimoles) of compound 2c are treated with vigorous stirring with 10 ml of freshly distilled anhydrous trifluoroacetic acid for 60 minutes at 20 ° C in the absence of moisture. The reaction solution is added 20 drops with vigorous stirring to 100 ml of absolute ether, thereby amorphous CFgCOOH. H-CHA-Lys (s-Cbo) pNA is precipitated. The etheric phase is sucked off. The solid residue is washed three times with 30 ml portions of absolute ether. The resulting residue is dissolved in 50 ml of MeOH. The pH is adjusted to 4.5 by Et ^N and the solution is evaporated to dryness in vacuo at 30 ° C. The residue is dissolved in 75 ml MeOH and purified on a column of "Sephadex LH-20" equilibrated with MeOH. The major fraction of the MeOH eluate, which is homogeneous in solvent system C as shown by TLC, is evaporated in vacuo at 30 ° C. After drying of the residue in a vacuum desiccator at 40 ° C over P £ 5, 3.06 g (91.6% of theory) of amorphous compound 2d is obtained.
Analyse:Analysis:
Beregnet for C31H40N5O8F3: C 55,77 H 6,04 N 10,49Calcd. For C 31 H 40 N 5 O 8 F 3: C 55.77 H 6.04 N 10.49
Fundet: C 56,08 H 6,15 N 11,01.Found: C 56.08 H 6.15 N 11.01.
DK 155051 BDK 155051 B
15 2e. Cbo-D-But-CHA-Lys(s-Cbo)-pNA.15 2nd. Cbo-D-CHA-But-Lys (s-Cbo) -pNA.
2,00 g (3 mi I limol) forbindelse 2d opløses i 15 ml friskt destilleret DMF og afkøles til -10°C. Til opløsningen sættes under omrøring 1,18 g (3,3 millimol) Cbo-D-But-OpNP og 0,42 ml (3 millimol) EtjN.2.00 g (3 ml in limol) of compound 2d is dissolved in 15 ml of freshly distilled DMF and cooled to -10 ° C. To the solution is added stirred 1.18 g (3.3 millimoles) of Cbo-D-But-OpNP and 0.42 ml (3 millimoles) of EtnN.
5 Blandingen lades reagere i 3 timer i fraværelse af fugtighed, hvorved temperaturen gradvis når 20°C. Reaktionsopløsningen afkøles atter til -10°C og puf res med 0,21 ml (1,5 millimol) Et^N. Efter en reaktionstid på 5 timer ved -10°C når reaktionsblandingen gradvis stuetemperatur.The mixture is allowed to react for 3 hours in the absence of humidity, the temperature gradually reaching 20 ° C. The reaction solution is again cooled to -10 ° C and buffered with 0.21 ml (1.5 millimoles) of Et 2 N. After a reaction time of 5 hours at -10 ° C, the reaction mixture gradually reaches room temperature.
Denne procedure gentages med 0,21 ml Et^N, hvorhos reaktionen 10 tager 16 timer. Reaktionsblandingen inddampes til tørhed i vakuum ved 50°C, og remanensen opløses i 50 ml MeOH og renses ved gelfil-trering på en søjle af "Sephadex LH-20" ækvilibreret med MeOH. Den første hovedfraktion af MeOH-eluatet, som er homogen i opløsningsmiddelsystem A og B som vist ved TLC, inddampes til tørhed i vaku-15 um ved 30°C. Remanensen tørres i en vakuumexsiccator ved 40°C over ^2^5' hvorved fås 2,12 g (91,4% af det teoretiske) af delvis krystallinsk forbindelse 2e.This procedure is repeated with 0.21 ml of Et 2 N, with reaction 10 taking 16 hours. The reaction mixture is evaporated to dryness in vacuo at 50 ° C and the residue is dissolved in 50 ml of MeOH and purified by gel filtration on a column of "Sephadex LH-20" equilibrated with MeOH. The first major fraction of the MeOH eluate, which is homogeneous in solvent systems A and B as shown by TLC, is evaporated to dryness in vacuo at 30 ° C. The residue is dried in a vacuum desiccator at 40 ° C over 2 2 5 'to give 2.12 g (91.4% of theory) of partially crystalline compound 2e.
Analyse:Analysis:
Beregnet for C^H^NgOgt C 63,71 H 6,78 N 10,87 20 Fundet: C 63,48 H 6,82 N 10,99.Calculated for C C ^H ^NgOg C 63.71 H 6.78 N 10.87 Found: C 63.48 H 6.82 N 10.99.
2f. 2HBr.H-D-But-CHA-Lys-pNA.2f. 2HBr.H-But-D-CHA-Lys-pNA.
1,55 g (2 millimol) forbindelse 2e behandles under omrøring med 12 ml 2N HBr i iseddike i 40 minutter ved 20°C i fraværelse af fugtighed. Tripeptidderivatet opløses gradvis under samtidig fraspaltning af de 25 to beskyttelsesgrupper Cbo og BOC og (X^-udvikling. Reaktionsopløsningen sættes dråbevis under kraftig omrøring til 100 ml absolut ether, hvorved flokkulent 2HBr.H-D-But-CHA-Lys-pNA udfældes. Den etheriske fase frasuges efter 30 minutter, og den faste fase vaskes fire gange med portioner på 25 ml absolut ether. Den resulterende 30 remanens opløses i 40 ml MeOH. pH-Værdien indstilles til 4,5 ved hjælp EtgN, og opløsningen inddampes til tørhed i vakuum ved 30°C. Remanensen opløses i 30 ml MeOH og renses på en søjle af "Sephadex LH-20" ækvilibreret med MeOH. Hovedfraktionen af MeOH-eluatet,1.55 g (2 millimoles) of compound 2e is treated with stirring with 12 ml of 2N HBr in glacial acetic acid for 40 minutes at 20 ° C in the absence of moisture. The tripeptide derivative is gradually dissolved with simultaneous cleavage of the two protecting groups Cbo and BOC and (X + evolution. The reaction solution is added dropwise with vigorous stirring to 100 ml of absolute ether, precipitating flocculent 2HBr.HD-But-CHA-Lys pNA. phase is extracted after 30 minutes and the solid phase is washed four times with portions of 25 ml of absolute ether, the resulting residue is dissolved in 40 ml of MeOH, the pH is adjusted to 4.5 by EtgN and the solution is evaporated to dryness in vacuo at 30 DEG C. The residue is dissolved in 30 ml of MeOH and purified on a column of "Sephadex LH-20" equilibrated with MeOH.
DK 155051 BDK 155051 B
16 der spaltes ved behandling med trypsin under frigørelse p-nitroanilin, inddampes til tørhed i vakuum ved 30°C. Til yderligere rensning opløses det forrensede produkt i 25 ml 33%'s AcOH og renses ved gelfiltrering på en søjle af "Sephadex G-15" ækvilibreret med 33%'s 5 AcOH. Den fraktion af AcOH-eluatet, som spaltes ved behandling med trypsin under frigørelse af p-nitroanilin, inddampes til tørhed i vakuum ved 40°C. Den resulterende remanens tørres i en vakuum-exsiccator ved 40°C over hvorved fås 0,99 g (74,3% af det teoretiske) amorft stof 2f, som er homogent i opløsningsmiddelsystem 10 C som vist ved TLC.16 which is cleaved by treatment with trypsin under release p-nitroaniline, evaporated to dryness in vacuo at 30 ° C. For further purification, the purified product is dissolved in 25 ml of 33% AcOH and purified by gel filtration on a column of "Sephadex G-15" equilibrated with 33% 5 AcOH. The fraction of the AcOH eluate which is cleaved by treatment with trypsin to release p-nitroaniline is evaporated to dryness in vacuo at 40 ° C. The resulting residue is dried in a vacuum desiccator at 40 ° C to give 0.99 g (74.3% of theory) of amorphous substance 2f, which is homogeneous in solvent system 10 C as shown by TLC.
Analyse:Analysis:
Beregnet for ^25^42^6^5^r2: ^ 45,05 H 6,35 N 12,61 Br 23,98Calcd for ^ 25 ^ 42 ^ 6 ^ 5 ^ r2: ^ 45.05 H 6.35 N 12.61 Br 23.98
Fundet: C 44,75 H 6,39 N 12,67 Br 23,68.Found: C 44.75 H 6.39 N 12.67 Br 23.68.
Aminosyreanalysen bekræfter tilstedeværelsen af de forventede amino-15 syrer i de korrekte forhold: CHA:0,98 - Lys: 1,00 - D-But:1,02.The amino acid analysis confirms the presence of the expected amino acids in the correct ratios: CHA: 0.98 - Light: 1.00 - D-But: 1.02.
En serie af andre tripeptidderivater fremstilles ved de fremgangsmåder, der beskrives i ovenstående eksempler. Disse tripeptidderivater grupperes i nedenstående tabel I.A series of other tripeptide derivatives are prepared by the methods described in the above examples. These tripeptide derivatives are grouped in Table I below.
Dipeptid- og tripeptidmellemprodukterne, der anvendes til fremstilling af 20 de i tabel I viste derivater, er anført i tabellerne II og III.The dipeptide and tripeptide intermediates used to prepare the derivatives shown in Table I are listed in Tables II and III.
1717
Tabel ITable I
DK 155051 BDK 155051 B
Frem- AnalyseForward Analysis
Udgangspro- gangs- Fundet Beregnet % dukter måde 5 Eksem- (mmol) (eks.) Aminosyreanalyse pel Slutprodukt Udbytte % 3 2AcOH.H-D-Val- 3e (0,75 mmol) (2f) 84,4 C 54,90 55,03 CHT-Lys-pNA 2N HBr/AcOH H 7,78 7,70 10 C30H50N6°10 N 13,05 12,84Initial Found Found Calculated% ducts way 5 Eczema (mmol) (ex) Amino acid analysis final product Yield% 3 2AcOH.HD-Val-3e (0.75 mmol) (2f) 84.4 C 54.90 55, 03 CHT-Lys-pNA 2N HBr / AcOH H 7.78 7.70 C30H50N6 ° 10 N 13.05 12.84
Val:CHT: Lys 1,00:0,98:1,01 4 2HBr.H-D-Leu- 4e (1 mmol) (1f) 85,2 C 44,55 44,88 CHA-Arg-pNA 2N HBr/AcOH H 6,53 6,42 15 C27H46N8°5Br2 N 15,85 15,51Val: CHT: Lys 1.00: 0.98: 1.01 4 2HBr.HD-Leu-4e (1 mmol) (1f) 85.2 C 44.55 44.88 CHA-Arg-pNA 2N HBr / AcOH H 6.53 6.42 C27H46N8 ° 5Br2 N 15.85 15.51
Br 21,92 22,12Br, 21.92; 22.12
Leu:CHA:Arg 1,00:0,98:0,99 5 2HBr.H-D-NIeu- 5e (1 mmol) (1f) 86,0 C 44,75 44,88 20 CHA-Arg-pNA 2N HBr/AcOH H 6,49 6,42 C27H46N8°5Br2 N 15,79 15,51Leu: CHA: Arg 1.00: 0.98: 0.99 5 2HBr.HD-Niu5e (1 mmol) (1f) 86.0 C 44.75 44.88 CHA-Arg pNA 2N HBr / AcOH H 6.49 6.42 C27H46N8 ° 5Br2 N 15.79 15.51
Br 21,82 22,12Br, 21.82; 22.12
Nleu:CHA: Arg 1,00:0,97:0,99 25 6 2HBr.H-D-Nval- 6e (1 mmol) (1f) 87,1 C 44,39 44,08 CHA-Arg-pNA 2N HBr/AcOH H 6,28 6,26 C26H44N8°5Br2 N 16,09 15,82Nleu: CHA: Arg 1.00: 0.97: 0.99 25 2HBr.HD-Nval-6e (1 mmol) (1f) 87.1 C 44.39 44.08 CHA-Arg pNA 2N HBr / AcOH H 6.28 6.26 C26H44N8 ° 5Br2 N 16.09 15.82
Br 22,33 22,56Br, 22.33, 22.56
NvahCHA: Arg 30 1,00:0,98:0,98 18 s 5NvahCHA: Arg 30 1.00: 0.98: 0.98 18 s 5
DK 15505 1 BDK 15505 1 B
7 2HBr. H-D-Phe- 7e (1 mmol) Cif) 82,3 C 47,47 47,63 CHA-Arg-pNA 2N HBr/AcOH H 5,92 5,86 C30H44N8°5Br2 N 15,08 14,817 2HBr. H-D-Phe-7e (1 mmol) Cif) 82.3 C 47.47 47.63 CHA-Arg-pNA 2N HBr / AcOH H 5.92 5.86 C30H44N8 ° 5Br2 N 15.08 14.81
Br 20,85 21,12 5 Phe:CHA:Arg 1,00:0,98:1,01 8 2HBr.H-D-AIa- 8e (1 mmol) (1f) 86,0 C 41,97 42,36 CHA-Arg-pNA 2N HBr/AcOH H 5,92 5,93 C24H40N8°5Br2 N 16,59 16,47 10 Br 23,11 23,49Br 20.85 21.12 Phe: CHA: Arg 1.00: 0.98: 1.01 8 2HBr.HD-Ala8e (1 mmol) (1f) 86.0 C 41.97 42.36 CHA -Arg-pNA 2N HBr / AcOH H 5.92 5.93 C24H40N8 ° 5Br2 N 16.59 16.47 Br 23.11 23.49
Ala:CHA: Arg 1,00:0,97:0,99 9 2HBr. H-D-Nval- 9e (1 mmol) (2f) 76,4 C 45,73 45,89 CHA-Lys-pNA 2N HBr/AcOH H 6,61 6,52 15 C26H44N6°5Br2 N 12,48 12,35Ala: CHA: Arg 1.00: 0.97: 0.99 9 2HBr. H-D-Nval-9e (1 mmol) (2f) 76.4 C 45.73 45.89 CHA-Lys-pNA 2N HBr / AcOH H 6.61 6.52 C26H44N6 ° 5Br2 N 12.48 12.35
Br 23,18 23,49Br 23.18, 23.49
Nval:CHA:Arg 1,00:0,98:0,99 10 2HBr.H-D-Leu- lOe Cl mmol) (2f) 78,0 C 46,39 46,69 20 CHA-Lys-pNA 2N HBr/AcOH H 6,63 6,68 C27H46N6°5Br2 N 12,27 12,10Nval: CHA: Arg 1.00: 0.98: 0.99 (2HBr.HD-Leu10 Cl mmol) (2f) 78.0 C 46.39 46.69 CHA-Lys-pNA 2N HBr / AcOH H 6.63 6.68 C27H46N6 ° 5Br2 N 12.27 12.10
Br 22,81 23,01Br, 22.81, 23.01
Leu: CHA: Lys 1,00:0,97:1,01 25 11 2HBr.H-D-NIeu- Ile (1 mmol) (2f) 77,6 C 46,88 46,69 CHA-Lys-pNA 2N HBr/AcOH H 6,72 6,68 C27H46N6°5Br2 N 12,33 12,10Leu: CHA: Lys 1.00: 0.97: 1.01 25 11 2HBr.HD-NiuIle (1 mmol) (2f) 77.6 C 46.88 46.69 CHA-Lys pNA 2N HBr / AcOH H 6.72 6.68 C27H46N6 ° 5Br2 N 12.33 12.10
Br 22,75 23,01Br, 22.75, 23.01
Nleu:CHA: Lys 30 0,99:0,97:1,00 19Nleu: CHA: Lys 30 0.99: 0.97: 1.00 19
Tabel 11Table 11
DK 155051 BDK 155051 B
Frem-Flow
Udgangspro- gangsmådeInitial procedure
Eksem- Dipeptid- dukter (eks.) Analyse 5 pel precursor (mmol) Udbytte % Fundet % Beregnet % 3c BOC-CHT-Lys- 2b (5 mmol) (2c) 84,3 C 60,48 60,97 (s-Cbo)-pNA BOC-CHT-OpNP H 7,11 7,07 C34H47N5°9 (5'5 mmol) N 10,76 10,46 10 3d CF3COOH.H-CHT- 4c (3 mmol) (2d) 90,8 C 54,08 54,46Eczema Dipeptide Ducts (Ex.) Analysis 5 µl Precursor (mmol) Yield% Found% Calculated% 3c BOC-CHT-Lys-2b (5 mmol) (2c) 84.3 C 60.48 60.97 (s- Cbo) pNA BOC-CHT-OpNP H 7.11 7.07 C34H47N5 ° 9 (5'5 mmol) N 10.76 10.46 3d CF3COOH.H-CHT-4c (3 mmol) (2d) 90, 8 C 54.08 54.46
Lys(s-Cbo)-pNA 6 ml CF3COOH H 6,00 5,90 C31H40N5°9F3 N 10,18 10,24Lys (s-Cbo) pNA 6 ml CF3COOH H 6.00 5.90 C31H40N5 ° 9F3 N 10.18 10.14
Tabel IIITable III
2020
DK 155051 BDK 155051 B
Frem-Flow
Udgangspro- gangsmådeInitial procedure
Eksem- Tripeptid- dukter (eks.) Analyse 5 pel precursor (mmol) Udbytte % Fundet % Beregnet % 3e Cbo-D-Val-CHT- 3d (1,5 mmol) (2e) 77,9 C 62,80 62,83Eczema Tripeptide Products (Ex.) Analysis 5 µl Precursor (mmol) Yield% Found% Calculated% 3e Cbo-D-Val-CHT-3d (1.5 mmol) (2e) 77.9 C 62.80 62, 83
Lys(e-Cbo)-pNA Cbo-D-Val-OpNP H 6,87 6,78 C42H54N6°10 (1,65 mmol) N 10,61 10,47 10 4e Cbo-D-Leu-CHA- Id (2 mmol) (le) 79,8 C 53,95 54,19Lys (e-Cbo) pNA Cbo-D-Val-OpNP H 6.87 6.78 C42H54N6 ° 10 (1.65 mmol) N 10.61 10.47 10th Cbo-D-Leu-CHA-Id ( 2 mmol) (le) 79.8 C 53.95 54.19
Arg-pNA. HBr Cbo-D-Leu- H 6,70 6,63 C35H51N8°7Br 0pNP (2,2 mmo,) N 14,66 14,45Arg-pNA. HBr Cbo-D-Leu-H 6.70 6.63 C35H51N8 ° 7Br 0pNP (2.2 mmo,) N 14.66 14.45
Br 10,14 10,30 5e Cbo-D-Nleu-CHA- Id (2 mmol) (le) 80,4 C 54,08 54,19 15 Arg-pNA.HBr Cbo-D-NIeu- H 6,73 6,63 C35H5lN8°7Br 0pNP (2,2 mmol) N 14,58 14,45Br 10.14 10.30 5th Cbo-D-Nleu-CHA-Id (2 mmol) (le) 80.4 C 54.08 54.19 Arg-pNA.Hbr Cbo-D-NIeu-H 6.73 6.63 C35H5lN8 ° 7Br 0pNP (2.2 mmol) N 14.58 14.45
Br 10,08 10,30 6e Cbo-D-Nval-CHA- 1d (2 mmol) (le) 82,6 C 53,28 53,61Br 10.08 10.30 6th Cbo-D-Nval-CHA-1d (2 mmol) (le) 82.6 C 53.28 53.61
Arg-pNA. HBr Cbo-D-Nval- H 6,55 6,48 20 C34H4gNg07Br OpNP (2,2 mmol) N 14,93 14,71Arg-pNA. HBr Cbo-D-Nval-H 6.55 6.48 C34H4gNg07Br OpNP (2.2 mmol) N 14.93 14.71
Br 10,25 10,49 7e Cbo-D-Phe-CHA- 1d (2 mmol) (le) 84,5 C 56,23 56,36Br 10.25 10.49 7th Cbo-D-Phe-CHA-1d (2 mmol) (le) 84.5 C 56.23 56.36
Arg-pNA.HBr Cbo-D-Phe- H 6,18 6,10 C38H49N8°7Br 0pNP (2,2 mmol) N 14,10 13,84 25 Br 9,75 9,87 8e Cbo-D-Ala-CHA- 1d (2 mmol) (le) 88,2 C 52,01 52,39Arg-pNA.HBr Cbo-D-Phe-H 6.18 6.10 C38H49N8 ° 7Br 0pNP (2.2 mmol) N 14.10 13.84 Br 9.75 9.87 8e Cbo-D-Ala CH1-1d (2 mmol) (le) 88.2 C 52.01 52.39
Arg-pNA.HBr Cbo-D-Ala- H 6,19 6,18 ^32^45^8^7Br OpNp (2,2 mmol) N 15,44 15,27Arg-pNA.HBr Cbo-D-Ala-H 6.19 6.18 ^ 32 ^ 45 ^ 8 ^ 7Br OpNp (2.2 mmol) N 15.44 15.27
Br 10,62 10,89Br, 10.62, 10.89
DK 155051 BDK 155051 B
21 9e Cbo-D-Nval- 2d (1,5 mmol) (2e) 90,6 C 63,90 64,10 CHA-Lys(e-Cbo)- Cbo-D-Nval- H 6,98 6,92 pNA OpNP (1,65 mmol) N 10,82 10,68 C42H54N6°9 5 10e Cbo-D-Leu-CHA- 2d (1,5 mmol) (2e) 88,5 C 64,28 64,4821 9th Cbo-D-Nval-2d (1.5 mmol) (2nd) 90.6 C 63.90 64.10 CHA-Lys (e-Cbo) - Cbo-D-Nval-H 6.98 6.92 pNA OpNP (1.65 mmol) N 10.82 10.68 C42H54N6 ° 9 5 10th Cbo-D-Leu-CHA-2d (1.5 mmol) (2nd) 88.5 C 64.28 64.48
Lys(s-Cbo)-pNA Cbo-D-Leu- H 7,11 7,05 C43H56N6°9 OpNP (1,65 mmol) N 10,75 10,49 11e Cbo-D-Nleu-CHA- 2d (1,5 mmol) (2e) 88,9 C 64,18 64,48Lys (s-Cbo) pNA Cbo-D-Leu-H 7.11 7.05 C43H56N6 ° 9 OpNP (1.65 mmol) N 10.75 10.49 11th Cbo-D-Nleu-CHA-2d (1 (5 mmol) (2nd) 88.9 C 64.18 64.48
Lys(e-Cbo)-pNA Cbo-D-Nleu- H 7,08 7,05 10 C43H56N6°9 0pNP (1'65 mmol) N 10,60 10,49Lys (ε-Cbo) pNA Cbo-D-Nleu-H 7.08 7.05 C43H56N6 ° 9 0pNP (1'65 mmol) N 10.60 10.49
Eksempel 12.Example 12.
2AcOH. H-D-Nval-CHA-Arg-pNA.2AcOH. H-D-Nval-CHA-Arg-pNA.
7,09 g (10 millimol) 2HBr. H-D-Nval-CHA-Arg-pNA (fremstillet ifølge 15 eksempel 38) opløses i 75 ml 60%'s vandig MeOH. Opløsningen føres på (i) en søjle af "Amberlite" JRA-401 i acetatform. Søjlen elueres med 60%'s vandig MeOH, hvorved HBr erstattes med AcOH ved ionbytning.7.09 g (10 millimoles) 2HBr. H-D-Nval-CHA-Arg pNA (prepared according to Example 38) is dissolved in 75 ml of 60% aqueous MeOH. The solution is applied to (i) a column of "Amberlite" JRA-401 in acetate form. The column is eluted with 60% aqueous MeOH, thereby replacing HBr with AcOH by ion exchange.
Eluatet inddampes til tørhed i vakuum ved 40°C. Efter tørring i vakuumexsiccatoren ved 40°C over ^2^5 ^,33 9 bromidfrit 20 2AcOH.H-D-Nval-CHA-Arg-pNA (98,5% af det teoretiske).The eluate is evaporated to dryness in vacuo at 40 ° C. After drying in the vacuum desiccator at 40 ° C over ^ 2 ^ 5 ^, 339 bromide-free 20 2AcOH.H-D-Nval-CHA-Arg pNA (98.5% of theory).
De kvantitative enzymassays ved hjælp af tripeptidsubstraterne ifølge opfindelsen kan udføres som følger: 1. Assay for urinkallikrein.The quantitative enzyme assays using the tripeptide substrates of the invention can be carried out as follows: 1. Urine challic acid assay.
1 ml urin og 1 ml TRIS-imidazolpuffer med en pH-værdi på 7,9 og en 25 ionstyrke på 1,0 inkuberes ved 37°C i 5 minutter og centrifugeres derefter til fjernelse af sedimenter.1 ml of urine and 1 ml of TRIS-imidazole buffer with a pH of 7.9 and a 25 ionic strength of 1.0 are incubated at 37 ° C for 5 minutes and then centrifuged to remove sediments.
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22 1,4 ml destilleret vand opvarmet til 37°C og 0,4 ml af centrifugatet blandes godt i en plastcuvette. Til denne blanding sættes 0,2 ml af -3 en 2 x 10 M vandig substratopløsning, og bestanddelene blandes hurtigt. Denne blanding inkuberes i nøjagtig 15 minutter ved 37°C.22 1.4 ml of distilled water heated to 37 ° C and 0.4 ml of the centrifugate are well mixed in a plastic cuvette. To this mixture, 0.2 ml of -3 is added to a 2 x 10 M aqueous substrate solution and the ingredients are quickly mixed. This mixture is incubated for exactly 15 minutes at 37 ° C.
5 Reaktionsblandingen blandes derefter med 0,2 ml iseddike for at stoppe den enzymatiske reaktion. Til farvemåling anvendes en blindprøve, der består af samme bestanddele, men hvortil er sat iseddike før tilsætningen af substratet for at forhindre den enzymatiske reaktion. Den blandede mængde af den farvede forbindelse R-Nl·^ bestem-10 mes fotometrisk eller spektrofotometrisk ved 405 nm ud fra forskellen mellem blindprøven og testprøven. Ud fra den således fundne værdi bestemmes urinkallikreinaktiviteten i urin under anvendelse af nedenstående formel: AOD., c . x V x 1000 x F 15 min.The reaction mixture is then mixed with 0.2 ml of glacial acetic acid to stop the enzymatic reaction. For color measurement, a blank is used which consists of the same constituents, but to which is added glacial acetic acid before the addition of the substrate to prevent the enzymatic reaction. The mixed amount of the colored compound R-N1 · 10 is determined photometrically or spectrophotometrically at 405 nm from the difference between the blank and the test sample. Based on the value thus found, urinary alkaline activity in urine is determined using the formula below: AOD., C. x V x 1000 x F 15 min.
15 = mU/ml urin 15 min. x v x ε.15 = mU / ml urine 15 min. x v x ε.
Δ OD = forøgelsen af den optiske densitet ved 405 nm i løbet af 15 minutter, V = total vol urnenet af testblandingen = 2,2 ml, 20 1000 = omdannelsesfaktor til omdannelse af U til mU, F = fortyndingsfaktor for urin (2), v = volumen af prøve = 0,4 ml, ε = ekstinktionskoefficient divideret med 1000 = 10,4.Δ OD = increase in optical density at 405 nm over 15 minutes, V = total volume of the test mixture = 2.2 ml, 20 1000 = conversion factor for conversion of U to mU, F = dilution factor for urine (2), v = volume of sample = 0.4 ml, ε = extinction coefficient divided by 1000 = 10.4.
Beregningen af urinkallikreinindholdet i urin kan også udføres ved 25 kontinuerlig måling af produktet R-Nl·^ (f.eks. p-nitroanilin) som dannes. Denne metode beskrives nedenfor til anvendelse til assay for kirtel kali i krein i sputum.The calculation of the urinary alkaline content of urine can also be performed by continuously measuring the product R-N1 · (e.g. p-nitroaniline) which is formed. This method is described below for use in assay for glandular potassium in sputum.
Ud over urinkallikrein indeholder urin også urokinase som en pro-teolytisk enzym, der også kan spalte substratet ifølge opfindelsen, om 30 end i mindre udstrækning. I den ovenfor beskrevne assaymetode måles summen af aktiviteterne af urinkallikrein og urokinase. For at få et nøjagtigt tal for urinkallikreinaktiviteten må urokinaseaktiviteten fraregnes. Den sidstnævnte kan bestemmes i et sammenligningsforsøgIn addition to urinary kallic urine, urine also contains urokinase as a proteolytic enzyme that can also cleave the substrate of the invention, albeit to a lesser extent. In the assay method described above, the sum of the activities of urinary kallikrein and urokinase is measured. To obtain an accurate figure for urinary kallicin activity, urokinase activity must be deducted. The latter can be determined in a comparison experiment
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23 ved at tilsætte 0,075 enheder trypsininhibitor (trypsininhibitor fra bovin lunge) pr. ml puffer for fuldstændigt at inhibere urinkallikre-inaktiviteten og kun måle urokinaseaktiviteten.23 by adding 0.075 units of trypsin inhibitor (bovine lung trypsin inhibitor) per ml. ml of buffer to completely inhibit urinary callic inactivity and measure urokinase activity only.
2. Assay for kirtelkallikrein i sputum: 5 0,5 ml sputum blandes med 2 ml TRIS-imidazol puffer (ionstyrke 1,0), og blandingen forinkuberes ved 37°C i 5 minutter. Inkubatet centrifugeres. I en testcuvette anbringes 1,5 ml destilleret vand ved 37°C og 0,25 ml af centrifugatet tilsættes. Bestanddelene blandes godt.2. Sputum Gland Calcium Assay: Mix 0.5 ml of sputum with 2 ml of TRIS-imidazole buffer (ionic strength 1.0) and pre-incubate the mixture at 37 ° C for 5 minutes. The incubate is centrifuged. In a test cuvette, place 1.5 ml of distilled water at 37 ° C and add 0.25 ml of the centrifugate. The ingredients mix well.
_3_3
Derefter tilblandes 0,2 ml af en 2 x 10 M vandig substratopløsning.Then 0.2 ml of a 2 x 10 M aqueous substrate solution is mixed.
10 Ændringen i ekstinktionen ved 405 nm måles derefter kontinuerligt i fra 5 til 10 minutter ved hjælp af en skriver. Ud fra den bestemte værdi for AOD pr. minut beregnes kallikreinaktiviteten pr. ml sputum i mU ved hjælp af nedenstående ligning: AOD . x V x 1000 x F min.10 The change in extinction at 405 nm is then continuously measured for 5 to 10 minutes using a printer. Based on the determined value for AOD per per minute the callikin activity is calculated per minute. ml of sputum in mU using the following equation: AOD. x V x 1000 x F min.
15 = mU/ml sputum v x ε.15 = mU / ml sputum v x ε.
F = 5 V = 1,95 v = 0,25 20 1 U (enhed) = enzymmængde, der er i stand til at spalte 1ymol sub strat på 1 minut under optimale eller på anden måde definerede pH-betingelser, ionstyrke, temperatur og substratkoncentration.F = 5 V = 1.95 v = 0.25 20 1 U (unit) = amount of enzyme capable of cleaving 1ymol of substrate in 1 minute under optimal or otherwise defined pH conditions, ionic strength, temperature and substrate concentration.
I bugspyt er pancreatisk kallikrein til stede hovedsageligt i form af prækallikrein, som der kun kan udføres assay for efter aktivering, 25 f.eks. ved hjælp af trypsin. Efter aktivering af prækallikrein inhibe-res trypsin ved hjælp af sojabønnetrypsininhibitor (SBTI). Kallikre-inindholdet i denne aktiverede blanding kan bestemmes ved en af de ovenfor beskrevne fremgangsmåder.In pancreas, pancreatic kallikrein is present mainly in the form of precallikrein, for which assay can only be performed after activation, e.g. using trypsin. Following activation of precallikrein, trypsin is inhibited by soybean trypsin inhibitor (SBTI). The potassium content of this activated mixture can be determined by one of the methods described above.
3. Assay for plasmin: 30 1,7 ml TRIS-imidazolpuffer (pH-værdi 7,5, ionstyrke 0,2) blandes med 0,1 ml af en opløsning af plasmin i 25%'s glycerol ved 37°C, og bian- ! 243. Plasmin Assay: Mix 1.7 ml of TRIS-imidazole buffer (pH 7.5, ionic strength 0.2) with 0.1 ml of a solution of plasmin in 25% glycerol at 37 ° C, and bian-! 24
DK 155051 BDK 155051 B
_2 dingen inkuberes ved 37°C i 1 minut. 0,2 ml af en vandig 2 x 10 M substratopløsning ved 37°C sættes til blandingen, og bestanddelene blandes hurtigt. Mængden af spaltningsproduktet R-Nl·^, som frigøres fra substratet pr. tidsenhed, måles derefter kontinuerligt. Ud fra 5 værdien bestemt pr. minut beregnes plasminaktiviteten pr. ml prøve i mU ud fra følgende ligning: ΔΕ/min. x V x 1000 - = mU/ml prøve v x ε.The things are incubated at 37 ° C for 1 minute. 0.2 ml of an aqueous 2 x 10 M substrate solution at 37 ° C is added to the mixture and the ingredients are quickly mixed. The amount of cleavage product R-N1 · 4 released from the substrate per unit of time is then continuously measured. From the 5 value determined per Plasma activity per minute is calculated per minute. ml sample in mU based on the following equation: ΔΕ / min. x V x 1000 - = mU / ml sample v x ε.
10 ΔΕ = mængden af spaltningsprodukt frigjort pr. minut, V = totalvolumen af testblanding, v = volumen af prøve, ε = ekstinktionskoefficient divideret med 1000.10 ΔΕ = amount of cleavage product released per minute, V = total volume of test mix, v = volume of sample, ε = extinction coefficient divided by 1000.
4. Assay for antiplasmin i humant plasma: 15 0,1 ml plasma fortyndet med TRIS-imidazolpuffer i forholdet 1:20 blandes med 0,02 ml af en opløsning af 1,25 CU humant plasmin (præparat fra firmaet AB Kabi, Stockholm, Sverige) og 50 ATU hirudin (præparat fra firmaet Pentapharm A.G., Basel, Schweiz) pr. ml i 25%'s glycerol. Blandingen inkuberes i 90 sekunder ved 37°C. Inku-20 batet blandes med 1,7 ml TRIS-imidazolpuffer (pH-værdi 7,5, ionstyrke 0,2) ved 37°C og derefter med 0,2 ml af en 2 x 10 ^ M vandig substratopløsning. Mængden af spaltningsproduktet R-Nl·^ frigjort fra substratet pr. tidsenhed måles derefter kontinuerligt. Ud fra den bestemte værdi beregnes den tilbageværende plasminaktivitet på den 25 ovenfor beskrevne måde.4. Human plasma antiplasmin assay: 0.1 ml of plasma diluted with 1:20 TRIS-imidazole buffer is mixed with 0.02 ml of a solution of 1.25 CU of human plasmin (preparation from AB Kabi, Stockholm, Sweden) and 50 ATU hirudin (preparation from Pentapharm AG, Basel, Switzerland) per ml in 25% glycerol. The mixture is incubated for 90 seconds at 37 ° C. The incubate is mixed with 1.7 ml of TRIS-imidazole buffer (pH 7.5, ionic strength 0.2) at 37 ° C and then with 0.2 ml of a 2 x 10 µM aqueous substrate solution. The amount of cleavage product R-N1 · 4 released from the substrate per time unit is then continuously measured. From the determined value, the residual plasmin activity is calculated in the manner described above.
I et blindforsøg erstattes plasmaet med den tilsvarende mængde puffer, men ellers udføres testen på den ovenfor beskrevne måde. Den bestemte plasminaktivitet svarer til udgangsplasminaktiviteten. Anti-plasminaktiviteten beregnes ud fra forskellen mellem plasminaktiviteten 30 bestemt i blindprøven og den tilbageværende plasminaktivitet bestemt i testen under anvendelse af plasma ifølge nedenstående formel: . 25In a blank test, the plasma is replaced by the corresponding amount of buffer, but otherwise the test is performed in the manner described above. The particular plasmin activity corresponds to the starting plasmin activity. The anti-plasmin activity is calculated from the difference between the plasmin activity 30 determined in the blank and the remaining plasmin activity determined in the test using the plasma of the following formula:. 25
DK 15505TBDK 15505TB
(4Eblindprøve - 4Eplasmaprøve)/min· x V x F x 1000 = mlU/ml v x ε plasma F = fortyndingsfaktor for plasma (20).(4Eblind sample - 4Eplasm sample) / min · x V x F x 1000 = mlU / ml v x ε plasma F = plasma dilution factor (20).
5 Substraterne ifølge opfindelsen kan også anvendes til assay for plasminogen i humant plasma ved at omdanne plasminogen, der er til stede i plasmaet, ved hjælp af urokinase eller streptokinase i et puffersystem og bestemme mængden af det dannede plasmin ved hjælp af et af substraterne ifølge opfindelsen i overensstemmelse med den ovenfor 10 beskrevne plasmin-assay-metode. Mængden af plasminogen, der oprindeligt er til stede i plasmaet, afledes af den værdi, der bestemmes for plasmin, da der efter aktivering dannes ét molekylplasmin ud fra ét molekyleplasminogen.The substrates of the invention can also be used to assay for plasminogen in human plasma by converting plasminogen present in the plasma by urokinase or streptokinase into a buffer system and determining the amount of plasmin formed by one of the substrates of the invention according to the plasmin assay method described above. The amount of plasminogen initially present in the plasma is derived from the value determined for plasmin, as after activation one molecular plasmin is formed from one molecular plasminogen.
I nedenstående tabel IV er følsomheden for nogle af substraterne 15 ifølge opfindelsen over for organ- eller kirtelkallikrein og plasmin angivet.In Table IV below, the sensitivity of some of the substrates 15 according to the invention to organ or glandular chalice and plasmin is indicated.
Tabel IVTable IV
Urinkallikreinaktivitet i 1 ml human urin0, submandibularis kallikrein i 1 ml sputum, humant plasmin og human NIH-thrombin, målt ved hjælp 20 af substraterne ifølge opfindelsen ved konstante substrat- og enzymkoncentrationer. Til sammenligning er de tilsvarende værdier for de kendte substrater 2HCI.H-D-Val-Leu-Arg-pNA (A), 2HCI.H-D-Val-Leu-Lys-pNA (B), H-D-Pro-Phe-Arg-pNA (C) og H-D-Pip-Phe-Arg- pNA (D) (jfr. dansk patentansøgning nr. 3127/76) også angivet.Urinary kallikrein activity in 1 ml of human urine 0, submandibular kallikrein in 1 ml of sputum, human plasmin and human NIH thrombin, as measured by the substrates of the invention at constant substrate and enzyme concentrations. In comparison, the corresponding values for the known substrates are 2HCI.HD-Val-Leu-Arg-pNA (A), 2HCI.HD-Val-Leu-Lys-pNA (B), HD-Pro-Phe-Arg-pNA ( C) and HD-Pip-Phe-Arg-pNA (D) (cf. Danish Patent Application No. 3127/76) are also indicated.
-4 25 Substratkoncentration: 2 x 10 M.-4 25 Substrate Concentration: 2 x 10 M.
DK 155051 BDK 155051 B
26 Mængde i nanomol af spaftningsproduktet R-Nl·^ frigjort på 1 minut af henholdsvis 1 ml human urin, 1 ml human sputum og 1 CU-enhed humant plasmin.26 Quantity in nanomoles of the R-N1 · 1 releasing product released in 1 minute of 1 ml of human urine, 1 ml of human sputum and 1 CU unit of human plasmin, respectively.
5 Urin- Submandibula- Humant kallikrein ris kallikrein plasmin A 0,90 12,2 83 B 0,30 5,7 347 10 C 0,81 - 451 D 1,08 - 4015 Urinary Submandibula- Human kallikrein rice kallikrein plasmin A 0.90 12.2 83 B 0.30 5.7 347 10 C 0.81 - 451 D 1.08 - 401
Substrater ifølge 15 eksempel 1 2,99 31,2 623 2 2,22 21,0 532 3 1,40 16,5 532 20 4 3,11 35,9 1069 5 2,08 28,1 1179 6 3,12 41,3 969 7 2,14 28,5 814 8 1,73 22,4 433 25 9 1,87 18,04 655 10 1,73 19,3 778 11 0,77 8,4 695 ° Urinprøve fremkommet ved at blande 100 ml portioner morgenurin fra raske personer.Substrates of Example 1 2.99 31.2 623 2 2.22 21.0 532 3 1.40 16.5 532 20 4 3.11 35.9 1069 5 2.08 28.1 1179 6 3.12 41 , 3 969 7 2.14 28.5 814 8 1.73 22.4 433 25 9 1.87 18.04 655 10 1.73 19.3 778 11 0.77 8.4 695 ° Urine sample obtained by mixing 100 ml portions of morning urine from healthy people.
30 _ Målingen af spaltningsproduktet R-N^ dannet ved den enzymatiske hydrolyse af substratet bygger på den forudsætning, at spaltnings-The measurement of the cleavage product R-N 2 formed by the enzymatic hydrolysis of the substrate is based on the assumption that
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CH441279A (en) * | 1960-01-26 | 1967-08-15 | Hoechst Ag | Process for the preparation of benzenesulfonyl-cyclohexylureas |
WO1980002559A1 (en) * | 1979-05-11 | 1980-11-27 | Pentapharm Ag | Tripeptidic derivatives |
DK145799B (en) * | 1975-07-11 | 1983-03-07 | Kabi Ab | TRIPEPTIDES OR SALTS THEREOF USED AS DIAGNOSTIC CHROMOGENT SUBSTRATE WITH HIGH SPECIFICITY ABOVE THROMBIN AND THROMBIN SIMILAR ENZYMES |
DK146937B (en) * | 1975-07-11 | 1984-02-20 | Kabi Ab | TRIPEPTIDES OR SALTS THEREOF USED AS DIAGNOSTIC CHROMOGENT SUBSTRATE FOR SERINE PROTEASES |
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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 |
JPS4942396A (en) * | 1973-05-01 | 1974-04-20 | ||
CH634662A5 (en) * | 1976-05-28 | 1983-02-15 | Pentapharm Ag | USE OF TRIPEPTIDE DERIVATIVES FOR THE QUANTITATIVE DETERMINATION OF PLASMINOGEN ACTIVATORS. |
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1987
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CH441279A (en) * | 1960-01-26 | 1967-08-15 | Hoechst Ag | Process for the preparation of benzenesulfonyl-cyclohexylureas |
DK145799B (en) * | 1975-07-11 | 1983-03-07 | Kabi Ab | TRIPEPTIDES OR SALTS THEREOF USED AS DIAGNOSTIC CHROMOGENT SUBSTRATE WITH HIGH SPECIFICITY ABOVE THROMBIN AND THROMBIN SIMILAR ENZYMES |
DK146937B (en) * | 1975-07-11 | 1984-02-20 | Kabi Ab | TRIPEPTIDES OR SALTS THEREOF USED AS DIAGNOSTIC CHROMOGENT SUBSTRATE FOR SERINE PROTEASES |
WO1980002559A1 (en) * | 1979-05-11 | 1980-11-27 | Pentapharm Ag | Tripeptidic derivatives |
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DK155781A (en) | 1981-11-07 |
JPS62294695A (en) | 1987-12-22 |
JPS62294696A (en) | 1987-12-22 |
JPS62296899A (en) | 1987-12-24 |
DK159458C (en) | 1991-03-04 |
NO811510L (en) | 1981-11-09 |
JPH0244518B2 (en) | 1990-10-04 |
JPH0244840B2 (en) | 1990-10-05 |
JPH0244839B2 (en) | 1990-10-05 |
DK509788A (en) | 1988-09-13 |
NO155540B (en) | 1987-01-05 |
DK155051C (en) | 1989-07-03 |
JPS572253A (en) | 1982-01-07 |
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