DE3202289A1 - C1-Esterase inhibitor activity determn. e.g. in plasma - by spectrophotometric analysis of chromogenic peptide substrate cleavage prods. (AT 15.04.82) - Google Patents

Abstract

In a new procedure for the determn. of the C1-esterase inhibitor (CEI) activity of CEI-contg. specimens by comparison of the C1-esterase activity of a standardised C1-esterase prepn. with that of the same standardised prepn. in the presence of the CEI-contg. specimen, the C1-esterase activity is determined by spectrophotometric examination of cleavage prods. of chromogenic peptide derivs. R1-A-B-NH-R (I) (where R1 is H, alkyl, a sulphonyl residue (esp. benzenesulphonyl or p-methoxy-benzenesulphonyl) or acyl (esp. alkoxycarbonyl or formyl); A is an amino acid component or di- or tripeptide residue; B is D-Arg, L-Arg, D-Lys, L-Lys, D-Orn, L-Orn, D-Tyr, L-Tyr, D-Ala or L-Ala; and NH-R is a chromophoric or fluorescent gp.). Used for determin. of C1-esterase inhibitor activity in human and animal body fluids.

Description

Procedure for the determination of the C1 esterase inhibitor

Activity of samples containing C1 esterase inhibitor The invention relates to a method for determining the C1 esterase inhibitor activity of C1 - Samples containing esterase inhibitor by comparing the C1 esterase activity of a standardized C1 esterase preparation with the activity of the same standardized Preparation in the presence of the C1 esterase inhibitor-containing sample to be examined, wherein the difference between the values obtained is a measure of the inhibitor activity of the Sample represents.

In human and animal body fluids there is a C1-Esterase ~ Enzyme present, which with an also present inhibitor or inactivator forms an inactive complex. During various examinations or treatments it is important to quantitatively determine the C1 esterase inhibitor content.

The chemical structure of C1 esterase is unknown, but it is white from "Thrombosis Research" 18, no. 6,847-859 (1980) that in the C1 esterase molecule different active centers are present, one of which is through the C1 esterase inhibitor can be blocked.

A well-known method for determining the activity of the C1 esterase inhibitor is based on the ester-splitting effect of C1-esterase. An ester is used as the substrate an aromatic amino acid (e.g. N-acetyl-L-tyrosine ethyl ester ) used, whereby the ester is cleaved and the acid released is determined (Proc. Soc. Exp.

Biol.Med. 101: 608-611 (1959) J. Clin. Pathol. 1980; 33, 167-170). This esterolytic determination method is imprecise and expensive and it is the does not do justice to actual in vivo reactions of the enzyme.

Another method of determination is based on the amidolytic effect the C1 esterase known, u.zw. from "Thrombosis Research", Vol. 18, No. 6, 847-859 (1980).

In this method, the C1-esterase-containing sample is mixed with a chromogenic The tripeptide-based substrate is converted and the extinction of the cleaved chromophore (p-nitroaniline) determined by spectrophotometric means.

With this method, however, the clinically relevant C1 esterase inhibitor content can be determined do not record because the same measurement result was found with and without the addition of an inhibitor will. This is due to the sequence and spatial structure of the chromogenic used there Substrate.

The invention aims to avoid these disadvantages and difficulties the previous determination methods and the task is to determine the activity or the level of C1 esterase inhibitor in human or animal body fluids reliable to determine.

This object is achieved according to the invention by using substrates which specific chromogenic compounds based on di-, tri- or tetrapeptides with chromophoric or fluorescent groups.

The method according to the invention for determining the C1 esterase inhibitor activity is characterized in that the C1 esterase activity to be compared Preparations by spectrophotometric analysis of chromogenic cleavage products Connections of general Formula R1 - A - B - NH - R or their Acid addition salts, in which formula R1 for hydrogen, alkyl, a sulfonyl radical, in particular benzenesulfonyl or p-methoxybenzenesulfonyl (Mbs) or an acyl radical, especially alkoxycarbonyl, such as tert. Butyloxycarbonyl, benzyloxycarbonyl or formyl, A for an amino acid component, a di- or a tripeptide residue, B for one of the following amino acid components: D-Arg, L-Arg, D-Lys, L-Lys, D-Orn, L-Orn, D-Tyr or L-Tyr, D-Ala or L-Ala, and -NH-R for a chromophoric or fluorescent Standing group.

New chromogenic compounds are advantageously used in which R1 represents alkyl having 1 to 4 carbon atoms or represents p-methoxybenzenesulfonyl (Mbs).

According to a further preferred embodiment, new chromogenic Compounds used in which A represents an amino acid component A1 with the following Meaning: Sar, L- (Me) Val, D-Leu, L- (Me) Phe, D-Val, D-Phe, L-Phe, L- (Me) Leu, Gly, L-Igln, L-Ala, β-Ala or Gaba, or for a dipeptide A1-A2, wherein A1 is the aforementioned MeaninghatufA2 means Gly, L-Pro, L-Leu, L-Val, L-Ile or a single bond, or for a tripeptide A1-A2-A3, -wherein A1 and A2 have the above meaning and A3 L-Pro or a single bond means stands.

Chromogenic compounds are expediently used in which -NH-R means p-nitroanilide substituted by halogen.

The determination of the inhibitor content in this amidolytic way largely corresponds to in vivo patient conditions.

Preferred compounds that can be used in the inventive Suitable working methods are the following: Mbs-Gly-Gly-L-Pro-L-Arg-o-Cl-pNA Z-Sar-L-Pro-L-Arg-o-Cl-pNA Z-L- (Me) Val-L-Leu-L-Arg-pNA H-D-Leu-L-Leu-L-Arg-o-Cl-pNA Z-L- (Me) Phe-L-Val-L-Arg-pNA Z-D-Val-L-Leu-L-Arg-o-Cl-pNA Z-D-Leu-L-Leu-L-Arg-o-Cl-pNA Z-D-Leu-L-Arg-o-Cl-pNA -HD-Val-L-Leu-L-Arg-o-Cl-pNA ZD-Phe-L-Leu-L'Arg-o-C1-pNA ZL- (Me) Leu-L-Leu-L-Arg- pNA ZL-Phe-L-Val-L-Arg-o-Cl-pNA (Me) 2-Gly-L-Pro-L-Arg-o-Cl-pNA ZL- (Me) Phe-L-Ile-L- Arg-pNA ZD-Tyr-L-Val-L-Arg-pNA ZD-Tyr-L-Leu-L-Arg-o-Cl-pNA Z-'1-Igln-Gly-'1-Arg-pNA Zg-Ala-L -Pro-L-Arg-o-Cl-pNA Mbs-L-Ala-L-Pro-L-Arg-o-Cl-pNA Mbs-Gaba-L-Pro-L-Arg-o-Cl-pNA Mbs-Gly-L-Pro-L-Arg-pNA Mbs-Gly-L-Pro-L-Lys-o-Cl-pNA Mbs-p-Ala-L-Pro-L-Lys-o-Cl-pNA Mbs-Gaba-L-Pro-L-Arg-pNA Have the abbreviations used in the above and in the further description the following meaning: amino acids H-Aib-OH aminoisobutyric acid H-Ala-OH alanine H-P-Ala-OH p-alanine H-Arg-OH arginine H-Gaba-OH γ-aminobutyric acid H-Glu-OH glutamic acid H-Iglu-OH Isoglutamic Acid H-Igln-OH Isoglutamine H-Gly-OH Glycine H-Ile-OH Isoleucine H-Leu-OH Leucine H-Lys-OH Lysine H-Orn-OH Ornithine H-Phe-OH Phenylalanine H-Pro-OH Proline H-Sar-OH Sarcosine H-Tyr-OH Tyrosine H-Val-OH Valine H- (Me) Val-OH N-methylvaline H- (Me) Phe-OH N-methylphenylalanine H- (Me) Leu-OH N-methylleucine (Me) 2Gly-oH N, N-dimethylglycine Other Adoc Adamantyloxycarbonyl Aoc tert. Amyloxycarbonyl Bpoc Z- (p-biphenylyl) isopropyloxycarbonyl Boc tert. Butyloxycarbonyl -o-Cl-pNA o-chloro-p-nitroanilide DCCI dicyclohexylcarbodiimide Ddz o (, - dimethyl-3, 5-dimethoxybenzyloxycarbonyl DMF dimethylformamide EE ethyl acetate Fmoc 9-fluorenylmethoxycarbonyl Foc furfuryloxycarbonyl HAc CH3-COOH HMPT hexamethylphosphoric acid triamide HOBt 1-Hydroxybenzotriazole Iboc Isobornyloxycarbonyl Mbs p-Methoxybenzenesulfonyl MG Molecular weight MeOH methanol pNA p-nitroanilide Nvoc 6-nitroveratryloxycarbonyl PÄ petroleum ether Pipoc Piperidinooxycarbonyl Pz p-Phenylazobenzyloxycarbonyl TFA Trifluoroacetic acid -ONP p-nitrophenyl ester -OPCP pentachlorophenyl ester -OTCP trichlorophenyl ester -OSu hydroxysuccinimide ester Z benzyloxycarbonyl Z (p-Br) - p-bromobenzyloxycarbonyl Z (p-Cl) - p-chlorobenzyloxycarbonyl Z (OMe) - p-Methoxybenzyloxycarbonyl Z (p-NO2) - p-Nitrobenzyloxycarbonyl Z (o-N02) - o-Nitrobenzyloxycarbonyl Z (OMe) 3,5-Dimethoxybenzyloxycarbonyl During manufacture of the chromogenic compounds must pass through the amino functions of the intermediate stages Protecting groups, which are common in peptide chemistry, are blocked.

Possible protective groups are: Boc, Z, Z (OMe), Z (p-Br), Z (p-Cl), Z (p-N02), Z (o-N02), Z (OMe) 2, Ddz, Nvoc, Bpoc, Aoc, Adoc, Iboc, Fmoc, Foc, Pipoc, Pz, 4 $ «- dimethylbenzyloxycarbonyl, 2-iodo-ethoxycarbonyl, 2- (p-tosyl) -ethoxycarbonyl, 1- (1-Adamantyl) -1-methylethoxycarbonyl (Adpoc), p-toluenesulfonyl, phthalyl, trifluoroacetyl, Triphenylmethyl, o-nitrophenylthio, formyl, benzylsulfonyl, o-hydroxyarylidene, acetoacetyl, 5,5-dimethylcyclohexadione, benzyl, o-nitrophenoxyacetyl and especially Mbs. the Mbs group proves to be specific to many other protective groups according to the invention usable chromogenic substrates as advantageous.

A terminal carboxyl group can either be replaced by the remainder of the Chromophore (-NH-R) or by methyl, ethyl, benzyl, p-nitrobenzyl, benzhydryl, Isopropyl, t-butyl, p-methoxybenzyl, alkylbenzyl, phenacyl, phenyl, 4-picolyl, p-methylthioethyl, 4- (methylthio) phenyl, trimethylsilyl and phthalimidomethyl groups to be protected.

If arginine is used, the side chain of arginine, the guanidino radical, through N -Tosyl-, N -Boc-, N "-Adpoc-, N2-Nitro- and N-Z (p-N02) -, N-Z, N-adamantyloxycarbonyl, N »-isobornyloxycarbonylar groups or by protonation to be protected.

The chromogenic compounds used according to the invention are according to using the methods commonly used in peptide chemistry for making peptide bonds. The carbodiimide, azide, anhydride, isocyanate, ynamine, the phosphorazo, acid chloride or activated amide methods or activated esters (trichlorophenyl ester, pentachlorophenyl ester, p-nitrophenyl ester, Thiophenyl ester, selenophenyl ester, N-hydroxypperidyl ester).

Silica gel plates were used throughout for thin-layer chromatography (TLC prefabricated plates, silica gel 60 F 254 and silica gel 60 without fluorescence indicator) from E. Merck AG., Darmstadt, the following solvent mixtures are used used: A - butanol / glacial acetic acid / water (3: 1: 1) B - chloroform / methanol / ammonia solution (25%) (60:45:20) C - methanol / chloroform (2: 1) D - chloroform / methanol / benzene / water (40: 40: 40: 5).

The method according to the invention is carried out by the following working instructions and examples explained in more detail: Work regulations for production of the chromogenic compounds or their precursors: Working instruction 1-: p-Methoxy-benzenesulfonyl chloride (Mbs-Cl): 80 g of anisole are in 300 ml of absolute Dissolved chloroform, the solution cooled to -100C and dropwise so with a total 172.4 g of chlorosulfonic acid are added so that the temperature does not rise above OOC. To When the reaction has ended, the cold bath is removed and the reaction solution is brought to room temperature warmed up.

After the evolution of gas has ceased, the solution is poured onto ice. the The aqueous phase is separated off in a separating funnel and twice with chloroform shaken out.

The combined chloroform phases are washed with water and washed over Dried sodium sulfate. After stripping off the chloroform, the oil obtained is im Vacuum distilled.

The oil obtained crystallizes on rubbing with a glass rod.

Yield: 93.3 g (61% of theory) Molecular formula: C 7H703 SCl MW: 206.65 Elemental analysis: C H S Cl calc .: 40.69% 3.41% 15.52% 17.16% found: 40.32% 3.68 % 15.74% 17.01% M.p .: 430C, Rf: 0.76 D Working instruction 2: Benzyloxycarbonyl-D-phenylalanine (Z-D-Phe-OH): 5 g of H-D-Phe-OH are dissolved in 20 ml of 2N sodium hydroxide solution and 7 ml of benzyl chloroformate are added dropwise at OOC. One relocates the reaction mixture thus obtained to maintain a pH of 9.5 with 2N sodium hydroxide solution using an autotitrator (time required approx 2 hours). The reaction solution is extracted with ether to remove excess To remove benzyl chloroformate. Then you overlay them watery Phase with ethyl acetate, cools to 5 ° C. and acidifies to pH 2.5 with 3N HCl at. The phases are separated in a separating funnel, the aqueous phase two more times shaken out with ethyl acetate. The combined organic phases are then with saturated sodium chloride solution and water and dried over sodium sulfate. After removing the solvent in vacuo, the compound is taken up in ether and excreted with petroleum ether.

Yield: 7.3 g (80.6% of theory) Molecular formula: C1 7H1 7N04 MW: 299.33 Elemental analysis: C H N calc .: 68.21% 5.72% 4.68% found: 68.34% 5.75% 4.73% Melting point: 63 ° C, Rf: 0.31, [α] D20: + 6.98 ° (c = 1, methanol) D Working instruction 3: BenzylOxyCarbonyl-N-methyl-L-phenylalanyl-pentachlorophenyl ester (Z-L- (Me) -Phe-OPCP): 1.56 g of Z-L- (Me) -Phe-OH are combined with 1.48 g of pentachlorophenol in 50 ml of absolute Dissolved methylene chloride and cooled to 0 ° C., then 1.14 is added to the solution g dicyclohexylcarbodiimide, stirred for two hours at this temperature and then overnight at room temperature. After filtering off the precipitated dicyclohexylurea crystallization takes place upon addition of petroleum ether (boiling temperature 30 to 50 ° C.). The crystals are suctioned off, washed with petroleum ether and placed in a desiccator Calcium chloride dried. Recrystallization takes place from methylene chloride / n-hexane.

Yield: 1.5 g (53.6% of theory) Molecular formula: C24H18N04Cl5 MW: 561.68 Elemental analysis: C H N calc .: 51.32% 3.23% 2.49% found: 51.22% 3.14% 2.18% Fp .: 149 ° C, Rf: 0.89, [α] D20: -51.5 ° (c = 1.03, DMF) In an analogous manner, the the following compounds are produced: Benzyloxycarbonyl-p-alanyl-pentachlorophenyl ester (Z-p-Ala - OPCP); M.p .: 1160C, Rf: 0.77 N-Benzyloxycarbonyl-N-methyl-L-leucyl-pentachlorophenyl ester (Z-L- (Me) -Phe-OPCP); M.p .: 65 ° C, [α] D20: -24.2 ° (c = 1; MeOH) benzyloxycarbonyl-D-leucyl-pentachlorophenyl ester (Z-D-Leu - OPCP); Mp .: 122 to 123 ° C, [α] D20: + 23 ° (c = 1; DMF), RfD: 0.79 benzyloxycarbonyl-D-valyl-pentachlorophenyl ester (Z-D - Val-OPCP); M.p .: 139 ° C, [α] D20: + 20.9 ° (c = 1, DMF) Couplable H-Arg (NO2) -o-Cl-pNA Working instruction 4a: o-chloro-p-nitrophenyl isocyanate: In a solution of 20 g o-chloro-p-nitroaniline in absolute dioxane, dry hydrogen chloride gas is passed in until it is saturated. Phosgene is then passed through the solution at 50 ° C. for one hour until it is clear Solution has arisen. The excess phosgene is removed by a stream of dry nitrogen removed. The solution obtained is evaporated to dryness on a rotary evaporator. The residue is recrystallized from petroleum ether.

Yield: 20.8 g (90.1% of theory) Molecular formula: C 7H3N203Cl MW: 198.57 Elemental analysis: C H N Cl calc .: 42.34% 1.52% 14.11% 17.85% found: 42.52% 1.74 % 14.33% 17.74% Mp .: 62 C, Rf: 0.77 D Working instruction 4b: N # -nitro-L-arginine: To nitrating acid, consisting of 40 ml Nitric acid, 25 ml of fuming Sulfuric acid and 15 ml of concentrated sulfuric acid are added within 4 hours 30.9 grams of L-arginine. This mixture is stirred until it is completely dissolved and poured these on crushed ice. Then with concentrated ammonia solution to pH 6.8 set. After standing overnight at 40 ° C., the desired product is obtained. To Suction and washing with ice water and ether are dried in a vacuum over P205.

Yield: 30.8 g (81.5% of theory) Molecular formula: C6H1 3N504 MW: 219.20 Elemental analysis: C H N calc .: 32.88% 5.98% 31.95% found: 32.76 9s 5.74% 31.77 % M.p .: 251 C, Rf: 0.26, [α] D20: +24.30 (c = 1.2N HCl) A working instruction 4c: N # -Benzyloxycarbonyl-N # -nitro-L-arginine (Z-Arg (NO2) -OH): 32.8 g -nitroarginine are dissolved in 150 ml of 1N NaOH and then cooled to 0 ° C. Under simultaneous 26.8 ml of carbobenzyloxy chloride and 158 ml of 1N sodium hydroxide solution are added dropwise to the solution added. The mixture is stirred for 2 hours at 5 ° C. and for 2 hours at room temperature. After acidification the reaction mixture at pH = 2 separates the benzyloxycarbonyl compound first as oil, which soon crystallizes through. The precipitated product is dissolved in potassium hydrogen carbonate (4%) added; this solution is extracted three times with ether and then acidified to pH = 2 with 4N hydrochloric acid. The crystalline product is filtered off and dried in vacuo at 35 ° C. over phosphorus pentoxide. Recrystallization takes place from ethanol / water.

Yield: 48 g (90.8% of theory) Molecular formula: C14H19N506 MW: 353.34 Elemental analysis: C H N calc .: 47.59% .5.42% 19.82% found: 47.39% 5.64% 19.66% mp .: 134 to 136 ° C, Rf: 0.38, [α] D20: -3 ° (c = 1, MeOH) D Working instruction 4d: Ne-benzyloxycarbonyl-N-nitro-L-arginyl-o-chloro-p-nitroanilide (Z-Arg (NO2) -o-Cl-pNA): 10.6 g of Z-Arg (NO2) -OH are dissolved in 70 ml of hexamethylphosphoric acid triamide (HMPT) dissolved and treated with 3.04 g (4.22 ml) of triethylamine. Then it goes to 50C cooled and added in portions with 5.96 g of o-chloro-p-nitrophenyl isocyanate and Stirred for 1 hour at 5 ° C. and overnight at room temperature. The reaction solution is then added drop by drop with vigorous stirring into 750 ml of 2.5% NaHCO3 solution, the deposited product is filtered off and with 2% NaHCO3 solution, 0.5N hydrochloric acid and water and then dried over phosphorus pentoxide. Recrystallization takes place from methanol / ether.

Yield: 12.5 g (82.02% of theory) Molecular formula: C20H22N7O7Cl MW: 507.89 Elemental analysis: C H N Cl calc .: 47.30% 4.37% 19.31% 6.98% found. : 47.28% 4.24 % 19.25% 6.77 z Fp .: 142 to 1450C, Rf: 0.78, - 20: -5.2 ° (c = 1, DMF) A working instruction 4e: No-Nitro-L-arginyl-o-chloro-p-nitroanilide hydrobromide (H-Arg (NO2) -o-Cl-pNA.HBr): 11 g of N # -Benzyloxycarbonyl-N # - nitro-L-arginyl-o-chloro-p-nitroanilide with 50 Poured ml of hydrogen bromide / glacial acetic acid and stir for 3 hours at room temperature. After the benzyloxycarbonyl group has been split off, the product is with absolute Ether failed. After the solution has stood at OOC for one hour, the product is filtered off with suction and washed with ether.

Recrystallization takes place from methanol / ether.

Yield: 8 g (80.8% of theory).

Molecular Formula: C12H17N7O5ClBr MW: 454.67 Elemental Analysis: C H N Cl Br calc .: 31.67% 3.77% 21.56% 7.80% 17.58% found: 31.32 * 3.52% 21.71% 7.98 % 17.34% m.p .: 2000C, [α] D20: +17.850 (c = 1, DMF) Working instruction 5: BenzylOxycarbonyl-N-methyl-L-valyl-L-leucyl-L-arginyl-p - nitroanilide- hydroacetate (Z (Me) -Val-Leu-Arg-pNA # HAc): 514 mg of Z (Me) Val-OPCP are added together with 481 mg of H-Leu-Arg - pNA # 2HCl dissolved in 35 ml of absolute dimethylformamide. After adding 0.12 ml of N-methylmorpholine and a spatula tip of hydroxybenzotriazole as a catalyst, the batch is stirred For 28 hours at room temperature. The solution medium is removed in an oil pump vacuum. The residue is taken up in methylene chloride, the substance falls after addition from ether. To remove any remaining impurities, 20% acetic acid is used chromatographed on a Sephadex G 15 column and then freeze-dried.

Yield: 0.65 g (90.9% of theory) Molecular formula: C34H50N8 0 9 MW: 714.82 Elemental analysis: C H N calc .: 57.13% 7.05% 15.68% found: 57.12% 6.89% 15.42 % M.p .: 138 ° C, [α] 54620: -6.4 ° (c = 1, DMF), R: 0.70 In an analogous manner are produced: Benzyloxycarbonyl-sarcosyl-l-alanyl-Nc-benzyloxycarbonyl-L-lysyl-o-chloro-p-nitroanilide (Z-Sar-L-Ala-L-Lys (Z) -o-Cl-pNA; m.p .: 145 ° C, [α] D20: -57.3 ° (c = 1, MeOH) benzyl-oxy-carbonyl-sarcosyl-L -prolyl-L-arginyl-o-chloro-p - nitroanilide hydroacetate (Z-Sar-L-Pro-L-Arg-o-Cl-pNA.HAc); Mp .: 96 to 98 ° C, [α] D20: -1.6 ° (c = 1.03; DMF), RfA: 0.50 benzyloxycarbonyl-L-isoglutamyl-glycyl-L-arginyl-β-nitroanilide hydroacetate (Z-L-Igln-Gly-Arg-pNA.HAc); Mp .: 60 to 63 ° C, [α] D20: -17 ° C (c = 1, DMF), RfA: 0.49 BenzylOxyCarbonyl-P-alanyl-L-prolyl-L-arginyl-o-chloro-p-nitroanilide hydroacetate (Z-P-Ala-L-Pro-L-Arg-o-C1-pNA.HAc); Fp .: 55 to 580C, [α] D20: 440 (c = 1, DMF) BenzylOxyCarbonyl-D-valyl-L-leucyl-L-arginyl-o-chloro-p-nitroanilide-hydroacetate (ZD-Val-L-Leu-L-Arg-o-Cl-pNA.HAc), [α] 54620: -6.80 (c = 0.5, DMF) benzyloxycarbonyl-N-methyl-L-phenylalanyl- L-valyl-L-arginyl-p-nitroanilide hydroacetate (Z-L- (Me) -Phe-L-Val-L-Arg - pNA.HAc); Fp .: 85 to 900C, [α] D20: -21.3 ° (c = 1, DMF), RfA: 0.69 BenzylOxyCarbonyl-N-methyl-L-valyl-L-leucyl-L-arginyl-p-nitroanilide-hydroacetate (Z-L- (Me) -Val-L-Leu-L-Arg-pNA.HAc); Mp .: 1380C, [α] 54620: -6.4 ° (c = 1, DMF), RfA: 0.70 BenzylOxyCarbonyl-N-methyl-L-phenylalanyl-L-isoleucyl-L-arginyl-p-nitroanilide-hydroacetate (Z-L- (Me) -Phe-L-Ile-L-Arg-pNA.HAc); LoC725046: -10.1 ° (c = 1, DMF), RfA: 0.71 benzyloxycarbonyl-N-methyl-L-leucyl-L-leucyl-L-arginyl-p-nitroanilide hydroacetate (Z-L- (Me) -Leu-L-Leu-L-Arg - pNA.HAc); Mp .: 115 to 1200C, [α] D20: +28.10 (C = 1, DMF) Working instruction 6: D-Leucyl-L-leucyl-L-arginyl-o-chloro-p-nitroanilide-dihydrobromide (H-D-Leu-Leu-Arg-o-Cl-pNA.2HBr): 630 mg Z-D-Leu - Leu-Arg-o-Cl-pNA.HAc are with Pour 30 ml of 3N hydrogen bromide / glacial acetic acid over it and leave it at room temperature for 1.5 hours long stirred. The process of splitting off the protective group is determined by thin-layer chromatography tracked. The solution thus obtained is added dropwise with stirring to 250 ml of absolute ether. The precipitated product is filtered off with suction and over solid potassium hydroxide in an oil pump vacuum dried.

It is recrystallized from methanol / ether. The substance is through Column chromatography on Sephadex G 15 (Elu- tion agent: 20% Acetic acid) obtained analytically pure.

Yield: 550 mg (91.2% of theory) Molecular formula: C24H41N805ClBr2 MG: 716.91 Elemental analysis: C H N calc .: 40.21% 5.76 * 15.63% found: 40.18% 5.50% 15.80% [α] D20: -62 ° (c = 1, H2O) The following can be produced in an analogous manner: D-Valyl-L-leucyl-L-arginyl-o-chloro-p-nitroanilide-dihydrobromide (H-D-Val-L-Leu-L-Arg-o-Cl-pNA.2HBr); [alpha;] D20: -5.9 ° (c = 1, DMF), RfA: 0.52 in the determination method according to the invention the following reagents were used: Buffer solution: 6.06 g tris (hydroxymethyl) aminomethane from Merck Art.

8382, 10.6 g NaCl from Merck Art. 6404, are distilled in about 900 ml Dissolved water and with about 5 molar hydrochloric acid to a pH value of 6.0 to 9.0 posed, preferably 8.3. Then the volume of the solution is increased to 1000 ml supplemented with distilled water.

Chromogenic compounds are initially used in a concentration of 1 mmol / l dissolved in distilled water.

Preparation of a C1 esterase The preparation is carried out according to the of literature (Journ.

Immunol. 110 p 1010 (1973); Journ. Immunol. 113 p 225 (1974); Protides of biology. Fluids 23rd Collog. 1975 Edit. H. Peeters, Vol 23, p 551 (1975); Biochim. Biophys. Acta 485 p 215 (1977); Proc. Soc. Exp. Biol. Med.

101 p 608 (1959)) known regulations and is based on a concentration of about 500 esterase units per ml analogous to the last-mentioned publication set.

Preparation of a C1 esterase inhibitor (inactivator): The preparation takes place according to the literature (Eur.

J. Biochem. 17 p 254-261 (1970); Vox. Sang. 26 p 118-127 (1974); J. Clin. Invest. 55 p 593 (1975); FEBS-Lett. 79 p 45 (1977)) known regulations and is based on an effectiveness that corresponds to the effectiveness of a fresh plasma pool, set.

The activity of the C1 esterase preparation was determined in the following manner Determined: 1.0 ml of buffer are placed in a plastic tube in a water bath at 370C pipetted. After a warm-up time of about 5 minutes, 0.1 ml of the C1-esterase preparation is added added and mixed. Then 0.1 ml of 0.001 M chromogenic solution is added Link. The solutions are mixed and after a latency of about 10 seconds the solution is placed in a photometer.

The measurement is carried out in single-use plastic cuvettes with one path length of 10 mm at a wavelength of 405 nm and at a temperature of 37 ° C. as The photometer is a Beckman two-beam photometer No. 25 with a wavelength range from 190 to 700 nm and an accuracy of + 0.5%. The increase in absorbance 4 OD / min is being measured.

The C1 esterase activity of a C1 esterase inhibitor (inactivator) -containing Sample was determined in the following way: In parallel tests were 0.1 ml C1 esterase preparation with 0.5 ml each of the sample containing the inhibitor offset. The sample can be a C1 esterase inhibitor preparation prepared like described above, or pooled plasma. After an exposure time of approx 5 minutes 0.5 ml of buffer solution and 0.1 ml of chromogenic compound are added and the0D / min measured.

For comparison, 0.1 ml C1-esterase preparation with 0.5 ml buffer solution instead of a sample containing C1 esterase inhibitor and the enzyme activity of this diluted C1-esterase preparation is determined as described above. By adding of inactivator or inhibitor as human plasma or as a C1 esterase inhibitor preparation obtained therefrom In addition to the enzyme, there is a change in C1-esterase activity. This loss of activity is in direct relation to the activity of the contained in the examined sample Brought inactivator or inhibitor, as can be seen from Table I.

Table I Chromogenic Compound Activity of C1 Esterase Preparations Human plasma C1 esterase inhibition tor preparation [(# OD / min) # 1000] [(# OD / min) # 1000] [(# OD / min) # 1000] Mbs-Gly-L-Pro-L-Arg-pNA 59 6 7 Z-L- (Me) Phe-L-Val-L-Arg - pNA 55 5 5 ZD-Leu-L-Arg-o-Cl-pNA 43 5 4 ZD-Leu-L-Leu-L-Arg-o - Cl-pNA 41 6 4 ZL- (Me) Val-L-Leu- L-Arg - pNA 108 10 10 ZD-Phe-L-Leu-L-Arg-o - Cl-pNA 27 2 1 ZL-Igln-Gly-L-Arg-pNA 70 8 7 ZD-Val-L-Leu-L-Arg- o - Cl-pNA 44 4 5 H-D-Leu-L-Leu-L-Arg-o - Cl-pNA 56 5 6 Mbs-Gly-Gly-L-Pro-L - Arg-o-Cl-pNA 27 3 3 HD-Val-L-Leu-L-Arg-o-Cl-pNA 35 3 4 ZD-Tyr-L-Val-L-Arg-pNA 40 1 0 ZD-Tyr-L-Leu-L-Arg- o - Cl-pNA 36 2 2 Other preferred substrates cleavable by C1 esterase, which can detect the C1 esterase activity changed by the addition of inhibitors, are listed in Table II.

Table II Chromogenic compound activity according to C1 activity Esterase pre-addition of C1 ready in Esterase-In - [# OD # 1000 / min] hibitor preparation [(# OD / min) # 100g] Z-Sar-L-Pro-L-Arg-o-CI-pNA 10 1 Z-L- (Me) Leu-L-Leu-L-Arg-pNA 26 0 ZL-Phe-L-Val-L-Arg-o-Cl-pNA 24 1 (Me) 2Gly-L-Pro-L-Arg-o-Cl-pNA 21 2 ZL- (Me) Phe-L-Ile -L-Arg-pNA 20 2 Zp-Ala-L-Pro-L-Arg-o-Cl-pNA 22 1 Mbs-L-Ala-L-Pro-L-Arg-o-Cl-pNA 37 1 Mbs-Gaba-L-Pro- L-Arg-o-Cl-pNA 37 0 Mbs-Gly-L-Pro-L-Lys-o-Cl-pNA 17 0 Mbs-P-Ala-L-Pro-L-Lys-o-C1-pNA 11 0 Mbs-Gaba-L-Pro- L-Arg-pNA 44 3

Claims (4)

Claims: 1. Method for determining the C1 esterase inhibitor activity of samples containing C C1 esterase inhibitor by comparing the C1 esterase activity a standardized C1-esterase preparation with the activity of the same standardized Preparation in the presence of the C1 esterase inhibitor-containing sample to be examined, wherein the difference between the values obtained is a measure of the inhibitor activity of the Sample represents, characterized in that the C1-esterase activity to be compared Preparations by spectrophotometric analysis of chromogenic cleavage products Compounds of the general formula R1 - A - B - NH - R or their acid addition salts it is determined in which formula R1 for hydrogen, alkyl, a sulfonyl radical, in particular benzenesulfonyl or p-methoxybenzenesulfonyl (Mbs) or an acyl radical, especially alkoxycarbonyl, such as tert. Butyloxycarbonyl, benzyloxycarbonyl or Formyl, A for an amino acid component, a di- or a tripeptide residue, B for one of the following amino acid components: D-Arg, L-Arg, D-Lys, L-Lys, D-Orn, L-Orn, D-Tyr or L-Tyr, D-Ala or L-Ala, and -NH-R for a chromophoric or fluorescent Standing group. 2. The method according to claim 1, characterized in that new chromogenic Compounds can be used where R1 is alkyl of 1 to 4 carbon atoms or is p-methoxybenzenesulfonyl (Mbs). 3. The method according to claim 1 or 2, characterized in that new chromogenic compounds can be used in which A is an amino acid component A1 with the following meaning: Sar, L- (Me) Val, D-Leu, L- (Me) Phe, D-Val, D-Phe, L-Phe, L- (Me) Leu, Gly, L-Igln, L-Ala, ß-Ala or Gaba, or for a dipeptide A1-A2, where A1 has the aforementioned meaning and A2 Gly, L-Pro, L-Leu, L-Val, L-Ile or means a single bond, or for a tripeptide A1-A2-A3, wherein A1 and A2 above Have meaning and A3 means L-Pro or a single bond, is. 4. The method according to claims 1 to 3, characterized in that that chromogenic compounds are used in which -NH-R substituted by halogen means p-nitroanilide.