JPS6257197B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to proteolytic enzymes, particularly enzymes of enzyme class E.
C.3.4.21. Novel tripeptide derivatives useful as substrates for the quantitative analysis of enzymes such as organ or glandular kallikrein, thrombin and plasmin. The so-called organ kallikrein or glandular kallikrein is
It is obtained by various organs and glands, such as the secretory glands, salivary glands, kidneys, gastrointestinal mucosa, etc., and is secreted in the proenzyme form or in the active form. These organ or glandular kallikreins are chemically and physiologically distinct from plasma kallikreins. Under certain pathological conditions, organ kallikrein secretion concentrations either decrease below normal values or increase above normal values. Thus, for example, urinary kallikrein secretion is substantially reduced compared to normal values in patients with kidney disease. Kallikrein secretion is almost completely suppressed in patients with nephrosclerosis. In patients with idiopathic hypertension, 24-hour urinary kallikrein secretion is effectively reduced by an average of 50% of the normal value (see, for example, HS Margolius, “Chemistry and Biology
of the Kallikrein−Kinin System in Health and
Disease", 1974, pp. 399-409). Therefore, it is important to rapidly quantitatively analyze organ kallikreins using simple processing methods. For example, certain arginine esters, e.g. todyl-arginine methyl ester Analyzing urine kallikrein by deesterifying (TAME)
It is publicly known. The improved ester cleavage method uses tritium-labeled tosyl-arginine methyl ester and measures the radioactivity of the methanol released by ester cleavage. However, these esterolysis methods have the disadvantage that they are non-physiological to the extent that kallikrein is a proteolytic enzyme that degrades the natural peptide chain by amidolysis rather than by esterolysis. Furthermore, ester substrates have the disadvantage that they are degraded by a large number of other enzymes, namely in an undefined manner by kallikrein. The method using tritium-labeled TAME is not recommended because the residual tritium-labeled TAME still present in the ester degradation mixture interferes with the measurement prior to the radioactivity measurement of tritium-labeled methanol. It is complex enough that methanol must be extracted from the ester decomposition mixture with a water-immiscible liquid. West German Patent Application No. 2527932, which was published without examination, contains the formula: R ¹ -Pro-X-
Arg-NH-R ² (However, R ¹ represents a protecting group, -
NH-R ² represents a chromophore, and X represents a phenylalanyl group, a β-cyclohexylalanyl group, a phenylglycyl group, or a tyrosyl group). The substrate is very easily degraded by plasma kallikrein to give a colored degradation product R ² -NH ₂ , the amount of which can be determined by photometry, spectrophotometry or fluorometry. can be measured. It has also been attempted to use the substrate to analyze organ or glandular kallikreins. However, it was surprisingly found that the substrate is not sensitive to organ or glandular kallikrein, ie it is not degraded or is only degraded to a small extent by organ or glandular kallikrein. The unexamined published German patent application DE 2629067 describes tripeptide derivatives useful as substrates for the analysis of certain proteolytic enzymes, such as glandular or organ kallikrein and plasmin. . That is, H-D-
Valyl-leucyl-arginyl-p-nitroanilide dihydrochloride and HD-valyl-leucyl-lysyl-p-nitroanilide dihydrochloride are mentioned as two examples. The first tripeptide derivative is a substrate for glandular kallikrein and the second tripeptide derivative is a substrate for plasmin. These two compounds are cleaved by the enzyme to yield p-nitroaniline, the amount of which can be determined photometrically or spectrophotometrically. However, the sensitivity of H-D-valyl-leucyl-arginyl-p-nitroanilide dihydrochloride reaches the limit necessary for accurate analysis of urinary kallikrein in unconcentrated urine. However, when the amide substrate is used in concentrated urine, photometric determination of p-nitroaniline is strongly inhibited by the unique color of the urine. The first in the tripeptide chain of the two tripeptide derivatives described in the West German patent specification mentioned above.
The two amino acids have a water-blocking isopropyl group in the α- or β-position. In order to increase this water-blocking property and subsequently the sensitivity to kallikrein, attempts were made to replace the isopropyl group with an aromatic group, such as a phenyl group, while maintaining the basic structure of the dipeptide chain. However, this attempt failed. The tripeptide substrates obtained by loading aromatic groups are not degraded at all, or in most cases only to a very small extent, by the glandular kallikrein. However, these tripeptide substrates with aromatic groups have surprisingly high sensitivity to plasmin. Moreover, it has surprisingly been found that hydrogenation of aromatic groups in the tripeptide plasmin substrates results in a new class of tripeptide substrates with surprisingly high sensitivity to organ or glandular kallikreins. It has been found that to increase the tripeptide chain only naturally occurring amino acids can be used to obtain substrates that are degraded by proteolytic enzymes. Therefore, by using amino acids that contain a cyclohexyl group and are not naturally occurring, it is possible to obtain chromophoric substrates that are easily degraded by organ or glandular kallikrein and other proteolytic enzymes, such as plasma kallikrein. It was surprising that this was discovered. The present invention relates to specific proteolytic enzymes, particularly enzymes of the enzyme class.
The present invention relates to novel chromophore substrates that have high sensitivity to enzymes of EC3.4.21., such as organ or gland kallikrein, plasmin and thrombin, and are therefore useful as substrates for the quantitative analysis of said enzymes. The substrate has the general formula: H-D-X-Y-Arg-R [wherein, X represents an alanyl group, an α-butyrylamino group, a valyl group, a norvalyl group, a leucyl group, a norleucyl group, or an isoleucyl group, and Y represents a cyclohexylalanyl group, R is p-nitrophenylamino group, 2-naphthylamino group, 4-methoxy-2-naphthylamino group, 4-methyl-coumaryl-7-amino or 1,
3-di(methoxycarbonyl)-phenyl-5-
It is a tripeptide transparent conductor represented by [representing an amino group]. The tripeptide derivatives of the formula are sparingly soluble in aqueous media and can therefore be used in the form of their salts with acids, especially mineral acids such as HCl, HBr, H ₂ SO ₄ ,
H ₃ PO ₄ etc., or organic acids such as formic acid, acetic acid, propionic acid, trimethylacetic acid, methoxyacetic acid, halogenated acetic acid such as trichloro- or trifluoroacetic acid, aminoacetic acid, lactic acid, oxalic acid, malonic acid, citric acid, benzoic acid acids, aromatic acids substituted in the nucleus,
For example, they are advantageously used in the form of salts with toluic acid, chloro- or brobenzoic acid, methoxybenzoic acid and aminobenzoic acid, phthalic acid and the like. The nature of the acid is not important as it does not participate in the reaction between substrate and enzyme. The substrates of the formula and their salts with acids may be used for specific proteolytic enzymes, in particular enzymes of enzyme class EC 3.4.21 (“Enzyme Nomenclature”, Elsevier
Scientific Publishing Company (Amsterdam)
Co., Ltd., 1973, pp. 238 et seq.), e.g. organ kallikrein and glandular kallikrein, hydrolytically degraded by the action of plasmin and thrombin, resulting in chromatic or fluorescent decomposition of the formula R-NH ₂ Products are formed, the amount of which can be determined by photometric, spectrophotometric, fluorescence spectrophotometric or electrochemical methods. Therefore, this new substrate is suitable for proteolytic enzymes,
Particularly enzyme class E that degrades natural peptide chains on the carboxyl side chains of arginine or lysine.
For the quantitative analysis of enzymes such as organ or glandular kallikrein, plasmin, plasma kallikrein, thrombin or their inhibitors and proenzymes, and other factors involved in the formation or inhibition of C.3.4.21. Useful. An excellent group of tripeptide derivatives consists of compounds of the formula in which Y represents a cyclohexylalanyl group and X represents an alanyl, α-aminobutyryl, valyl, norvalyl, leucyl, norleucyl or isoleucyl group. Following Examples 3, 5, 7, 9, 12, 13, 36, 37,
The tripeptide derivatives described in 38, 40 and 41 are particularly useful as substrates for analyzing urine kallikrein. To analyze kallikrein in human sputum,
Following Examples 3, 5, 7, 9, 12, 13, 36, 37,
The tripeptide derivatives described in 38, 40 and 41 can be used. The tripeptide derivatives described in Examples 36, 37, and 41 below constitute a group of substrates that can be used to analyze plasmin. The present invention relates to a method for the quantitative analysis of proteolytic enzymes, in particular enzymes of the enzyme class EC 3.4.21., which degrade natural peptide chains on the carboxyl side chains of arginine or lysine, such as organ or glandular kallikrein, plasmin and thrombin. Also related. The method according to the invention comprises reacting a substance containing said enzyme or forming or consuming said enzyme with a tripeptide derivative of the formula; It consists of measuring the amount of the fluorescent decomposition product R-NH ₂ by photometry, spectrophotometry, fluorescence spectrophotometry or electrochemical methods. The method of the invention is capable of analyzing, for example, the enzyme content of enzyme specimens or enzyme concentrations in human and mammalian body fluids, such as urine, pancreatic juice, intestinal mucus, mammary gland secretions, sweat gland secretions, sputum and blood. . The method of the invention is particularly useful for the quantitative analysis of organ or glandular kallikrein and plasma kallikrein in the body fluids. According to this method, it is possible to analyze kallikrein formed from free organ kallikrein and mucosal kallikrein or pre-kallikrein, as well as physiological or non-physiological inhibitors of kallikrein and physiological or non-physiological activators of pre-kallikrein. . Tripeptide derivatives of the formula can be prepared by the method described below: (1) The chromophore R is attached to the carboxyl group of the C-terminal arginine, with the α-amino group being protected by a protecting group, e.g. It is protected by a carbobenzoxy group or a t-butoxycarbonyl group, and the δ-guanidyl group is protected by protonation, nitration or tosylation, for example with HCl. Also,
The C-terminal R-NH group serves as a protecting group during the stepwise synthesis of the peptide chain. Other protecting groups can be selectively removed if necessary to attach other amino acid derivatives until the desired peptide chain is fully synthesized. Finally, the remaining protecting groups are completely decomposed without interacting with the R-NH- group (e.g.
Miklos Bodansky et al. “Peptide Synthesis”
Published by Interscience Publishers, 1966, No. 163
~165 pages). (2) First, the peptide chain is synthesized (by the method of Bodansky et al., cited above), in which the C-terminal carboxyl group of arginine is replaced with a conventional ester group, such as methoxy, ethoxy, or benzyloxy. (in the case of arginine) or by a t-butoxy group (in the case of lysine). The ester groups can be removed by alkaline hydrolysis, except for the t-butoxy group, which must be selectively decomposed with trifluoroacetic acid. δ of arginine
- When protonating the guanidyl group, the ester group is removed enzymatically using trypsin, in which case no racemization occurs. next,
Attach the chromophore R-NH-. The δ-guanidino group of arginine is protected by a nitro group or a tosyl group, and the N-terminal α of the tripeptide derivative is
If the -amino group is protected by a carbobenzoxy group or a p-methyl-, p-methoxy- or p-chlorobenzyloxycarbonyl group or a t-butoxy group, all protecting groups are removed at the same time. This removal can be carried out by treating the protected tripeptide derivative with anhydrous HF at room temperature, so that in this case all said protecting groups of the amino and δ-guanidino groups are removed. removed. Also, this removal
If the tripeptide derivative being protected does not contain a protected nitro or tosyl group, this can also be accomplished by treatment with 2N HBr in glacial acetic acid at room temperature. The preparation of tripeptide derivatives according to the invention is detailed in the following examples. Analysis of the eluates and products obtained according to the examples was carried out using glass plates coated with silica gel (Merck
The analysis was carried out by thin layer chromatography using a commercially available commercially available commercially available commercially available product, F254). Thin layer chromatograms were developed with the following solvent system: n-butanol/acetic acid/water (3:1:1) The following abbreviations are used: AcOH = acetic acid AIa = alanine Arg = arginine BOC = t- Butoxycarbonyl But=α-aminobutyric acid Cbo=carbobenzoxy CHA=cyclohexylalanine DMF=dimethylformamide DPA=1,3-di(methoxycarbonyl)-phenyl-(5)-amide TLC=thin layer chromatography Et ₃ N = triethylamine HMPTA = phosphoric acid N,N,N',N',N'',N''-hexamethyltriamide IIe = isoleucine Leu = leucine SS = solvent system Lys = lysine MCA = 4-methyl-coumaryl-(7)- Amide MeOH = methanol 4-MeO-2-NA = 4-methoxy-2-naphthylamide Nleu = norleucine Nval = norvaline OpNP = p-nitrophenoxy pNA = p-nitroanilide THF = tetrahydrofuran Val = valine Peptide chain unless otherwise specified The amino acids in have the L-form. Example 1 H-D-VaI-CHA-Arg-pNA.2HBr 1a Cbo-Arg-pNA.HCl Cbo-Arg-OH.HCl16 (dried over _P2O5 in vacuo) in a ₂₅₀ ml three-necked flask .0g (47.0
mmol) was dissolved in 90 ml of anhydrous HMPTA at 20°C, the atmosphere in the flask being kept moisture-free. To the resulting solution at room temperature was first added a solution of 4.74 g (47.0 mmol) of Et ₃ N in 10 ml of HMPTA, then (100% excess) of p.
- Nitrophenyl isocyanate 16.4g (100
mmol) was added dropwise. After a reaction time of 24 hours at 20°C, most of the HMPTA was removed by distillation in vacuo. This residue was dissolved in 30% AcOH
Extracted several times. This residue was discarded. The combined acetic acid extracts were equilibrated with 30% AcOH and 30%
Further purification was carried out by passage through a column of "Sephadex G-15"®, eluting with AcOH. Fractions of the AcOH eluate, which were degraded by treatment with trypsin to liberate p-nitroaniline, were lyophilized. Thus, 12.6 g of amorphous powder was obtained which was homogeneous in SS as shown by TLC. Elemental analysis and calculation from the empirical formula C ₂₀ H ₂₅ N ₆ O ₅ Cl gave the following values (values from the empirical formula are in parentheses): C = 51.29%
(51.67%), H=5.48% (5.42%), N=17.92%
(18.08%), Cl=7.50% (7.63%). 1b 2HBr.H−Arg−pNA 4.65 g (10 mmol) of compound 1a was dissolved in 40 ml of 2NHBr in glacial acetic acid under stirring at 20 °C in the absence of moisture.
Treated for 45 minutes. Amino acid derivatives were dissolved under the evolution of _CO2 . This reaction solution was added dropwise to 250 ml of anhydrous ether under vigorous stirring. This solution is
A precipitate of 2HBr.H-Arg-pNA was produced. A-
The solid phase is filtered off with suction, and the solid phase is then freed from the benzyl bromide formed as a by-product as well as the excess
Anhydrous ether to remove HBr and AcOH
Washed 4 times with 100 ml volumes. The residue was dissolved in 50 ml of methanol, the pH was adjusted to 4.5 by adding Et ₃ N, and the solution was concentrated to dryness in vacuo at 30°C. The obtained product
It was dissolved in 75 ml of MeOH and passed through a column of "Sephadex LH-20" (crosslinked dextran gel) equilibrated with MeOH. From this eluate fraction, as shown by TLC
4.18 g (91.6% of theory) of homogeneous amorphous compound 1b in SS were obtained. Elemental analysis and experimental formula
Calculations from C ₁₂ H ₂₀ N ₆ O ₃ Br ₂ gave the following values: C = 31.15% (31.60%), H = 4.35%.
(4.42%), N=18.84% (18.43%) and Br=
34.81% (35.03%). 1c Cbo-CHA-Arg-pNA.HBr 4.56 g (10 mmol) of compound 1b were dissolved in 30 ml of freshly distilled DMF and the solution was cooled to -10<0>C. To this solution was added 1.40 ml of Et ₃ N (10
mmol) was added. The Et ₃ N.HBr formed was removed by filtration and washed with a small amount of cold DMF. Add Cbo− to this solution at −10℃.
Added 4.69 g (11 mmol) of CHA-OpNP,
The reaction was continued for 2-3 hours in the absence of moisture, during which time the temperature of the reaction solution gradually reached about 20°C. The solution was cooled again to −10°C and Et ₃ N0.70
ml (5 mmol) and reacted at -10°C for about 2 hours and at room temperature for about 3 hours. This method was repeated using 0.70 ml of Et ₃ N and the reaction solution was concentrated to dryness in vacuo at 50° C. for 16 hours. This residue was dissolved in 75 ml of 50% AcOH, and “Sephadex G” was equilibrated with 50% AcOH.
AcOH was purified by gel filtration on a −15” column. AcOH was degraded by treatment with trypsin to liberate p-nitroaniline.
The main fraction of the eluate was concentrated to dryness in vacuo at 40°C. This residue was dissolved in 150 ml of MeOH and concentrated again to dryness. The resulting residue was dried over _P2O5 at ₆₀ °C in a vacuum desiccator to yield a homogeneous amorphous compound in SS as shown by TLC.
5.85 g of 1c (88.3% of theory) was obtained. Elemental analysis and calculation from the empirical formula C ₂₉ H ₄₀ N ₇ O ₆ Br gave the following values: C = 52.28% (52.57%), H
= 6.16% (6.09%), N = 15.09% (14.80%) and Br = 11.85% (12.06%). 1d 2HBr.H-CHA-Arg-pNA 5.30 g (8 mmol) of compound 1c were treated with 32 ml of 2NHBr in glacial acetic acid at 20 DEG C. for 40 minutes under stirring. The dipeptide derivative gradually dissolved under the evolution of _CO2 . This reaction solution was added dropwise to 250 ml of anhydrous ether under vigorous stirring. This solution is
A precipitate of 2HBr.H-CHA-Arg-pNA was produced. The ether phase was filtered off with suction and the solid phase was then washed four times with 100 ml portions of anhydrous ether in order to remove the benzyl bromide formed as by-product and excess HBr and AcOH. This residue
Dissolved in 50 ml of MeOH. pH 4.5 using _Et3N
The solution was concentrated to dryness in vacuo at 30°C. Dissolve the obtained residue in 50ml of MeOH,
It was purified using a "Sephadex LH-20" column equilibrated with MeOH.
The fractions of the MeOH eluate, resolved by treatment with trypsin to liberate p-nitroaniline, were concentrated to dryness in vacuo at 30°C. The resulting residue was dried over P ₂ O ₅ at 40 °C in a vacuum desiccator, yielding 4.48 g of homogeneous amorphous compound in SS (91.9 of the theoretical value) as shown by TLC.
%) was obtained. Elemental analysis and experimental formula
Calculations from C ₂₁ H ₃₅ N ₇ O ₄ Br gave the following values: C = 41.80% (41.39%), H = 5.86%
(5.79%), N=16.31% (16.09%) and Br=
25.85% (26.23%). 1e Cbo-D-Val-CHA-Arg-pNA.HBr Compound 1d 3.05 g (5 mmol) was dissolved in 20 ml of freshly distilled DMF and the solution was cooled to -10°C. 0.70 ml (5 mmol) of Et ₃ N was added to this solution under stirring. Formed Et ₃ N.HBr
Remove by washing with cold DMF
Washed with a small amount of Add Cbo− to this solution at −10℃.
2.05 g (5.5 mmol) of D-VaI.OpNP was added under stirring. The reaction mixture was allowed to react for 2-3 hours in the absence of moisture, with the temperature of the reaction solution gradually reaching approximately 20°C. The solution was cooled again to -10°C, buffered with 0.35 ml (2.5 mmol) of Et ₃ N, reacted for about 2 hours at -10°C, and further reacted for 3 hours at room temperature. This method
Repeat with 0.35 ml of Et ₃ N and after 16 hours the reaction solution was concentrated to dryness in vacuo at 50°C. This residue was dissolved in 50 ml of 50% AcOH, and "Sephadex G-" was equilibrated with 50% AcOH.
Purified by gel filtration on a 15" column. The main fraction of the AcOH eluate, which was resolved by treatment with trypsin to liberate p-nitroaniline, was concentrated to dryness in vacuo at 40°C. did.
The residue was dissolved in 100 ml of MeOH and the solution was concentrated again to dryness. The resulting residue was dried over _P2O5 at 60 °C in a vacuum desiccator and analyzed by TLC _.
3.15 g (82.7% of theory) of homogeneous amorphous compound 1e in SS were obtained as shown by . Elemental analysis and calculation from the empirical formula C ₃₄ H ₄₉ N ₈ O ₇ Br gave the following value: C = 52.88% (53.61
%), H=6.54% (6.48%), N=15.08%
(14.71%) and Br=10.25% (10.49%). 1f 2HBr.H-D-VaI-CHA-Arg-pNA Compound 1e2.29 g (3 mmol) was dissolved in 12 ml of 2NHBr in glacial acetic acid under stirring at 20°C in the absence of moisture.
Treated for 40 minutes. The tripeptide derivative gradually dissolved under decarboxylation and generation of _CO2 . This reaction solution was added dropwise to 120 ml of anhydrous ether under vigorous stirring. This solution is 2HBr.H−D
A precipitate of -VaI-CHA-Arg-pNA was produced.
The ether phase was filtered off with suction through a filter bar, and the solid phase was then washed four times with 50 ml portions of absolute ether. The resulting residue was dissolved in 40 ml of MeOH. PH
was adjusted to 4.5 using Et ₃ N and the solution was concentrated to dryness in vacuo at 30°C. This residue is MeOH30
ml and purified with a column of "Sephadex LH-20" equilibrated with MeOH. The fractions of the MeOH eluate, resolved by treatment with trypsin to liberate p-nitroaniline, were concentrated to dryness in vacuo at 30°C. 33% of pre-purified residue for post-purification
It was dissolved in 30 ml of AcOH and purified by gel filtration on a column of "Sephadex G-15" equilibrated with 33% AcOH. p-
The main fraction of the AcOH eluate, resolved by treatment with trypsin to liberate nitroaniline, was concentrated to dryness in vacuo at 40°C. The resulting residue was dried over P ₂ O ₅ at 40 °C in a vacuum desiccator, yielding 1.84 g of homogeneous amorphous compound 1f in SS (86.4% of theory) as shown by TLC.
I got it. Elemental analysis and calculation from the empirical formula C ₂₆ H ₄₄ N ₈ O ₅ Br ₂ gave the following value: C = 43.60
% (44.08%), H=6.41% (6.26%), N=
16.15% (15.82%) and Br=22.21% (22.56
%). Amino acid analysis confirmed the presence of the desired amino acids in the following exact ratios: : Arg:1.00−CHA:0.96−D−CHG:0.98. Rf
Value 0.46. Example 2 2HBr.H−D−VaI−CHA−Arg−MCA 2b 2HBr.H−Arg−MCA 13.0 g (25.9 mmol) of commercially available Cbo−Arg−MCA.HCl was added to 104 ml of a solution of 2NHBr in glacial acetic acid.
(208 mmol) was deblocked according to Example 1b. Dissolve this dry residue in 400 ml of MeOH, and add this solution to “Sephadex LH-
The fractions of the MeOH eluate, which was resolved by treatment with trypsin to liberate 4-methyl-7-amino-coumarin, were concentrated to dryness in vacuo at 30°C. The resulting residue was dried over P ₂ O ₅ at 40° C. in a vacuum desiccator to yield 11.2 g (87.7% of theory) of homogeneous amorphous compound 2b in SS as shown by TLC. Elemental analysis and calculation from the empirical formula C ₁₆ H ₂₃ N ₅ O ₃ Br ₂ gave the following value: C = 39.40
% (38.96%), H=4.61% (4.70%), N=
14.48% (14.20%) and Br=31.90% (32.40
%). 2c Cbo−CHA−Arg−MCA.HBr Compound 2b4.93 g (10 mmol) and Cbo−
4.69 g (11 mmol) of CHA-OpNP was added to 75 ml of freshly distilled DMF. After cooling to −10° C., first 1.40 ml (10 mmol) and then 0.70 ml (5 mmol) of Et ₃ N were added under stirring. This mixture was first heated at -10°C for 30 minutes in the absence of moisture.
The mixture was allowed to react for 4 hours and then for 4 hours at room temperature.
The reaction solution was cooled again to −10°C and Et ₃ N0.70
ml and stirred overnight at 20°C. The reaction mixture was concentrated to dryness in vacuo at 50 DEG C., the residue was dissolved in 200 ml of 50% AcOH, and the solution was purified on a "Sephadex G-15" column. Fractions of the AcOH eluate were digested by treatment with trypsin to liberate 4-methyl-7-amino-coumarin in vacuo.
It was concentrated and dried at 40°C. The residue thus obtained was dried over P ₂ O ₅ at 60 °C in a vacuum desiccator to obtain 5.95 g (85.0% of theory) of homogeneous crystalline compound 2c in SS as shown by TLC. Ta. Elemental analysis and calculation from the empirical formula C ₃₃ H ₄₃ N ₆ O ₆ Br gave the following value: C = 56.33
% (56.65%), H=6.28% (6.19%), N=
12.25% (12.01%) and Br=11.30% (11.42
%). 2d 2HBr.H-CHA-Arg-MCA 5.60 g (8 mmol) of compound 2c in glacial acetic acid
Deblocking was performed according to Example 1d using 32 ml of 2NHBr.
The obtained crude product was dissolved in 100 ml of MeOH,
Add this solution to “Sephadex LH”
-20" column. 4-Methyl-7-
The fractions of the MeOH eluate, resolved by treatment with trypsin to liberate the amino-coumarin, were concentrated to dryness in vacuo at 30°C. The resulting residue was dried over P ₂ O ₅ at 40 °C in a vacuum desiccator, yielding 4.76 g of homogeneous amorphous compound 2d in SS (92.1% of theory) as shown by TLC.
I got it. Elemental analysis and calculations from the empirical formula C ₂₅ H ₃₈ N ₆ O ₄ Br ₂ gave the following values: C=
47.02% (46.45%), H = 6.02% (5.93%), N
= 13.21% (13.00%) and Br = 24.48% (24.72
%). 2e Cbo-D-VaI-CHA-Arg-MCA.HBr 3.23 g (5 mmol) of compound 2d was added according to example 1e.
Cbo-D-VaI-OpNP2.05g (5.5 mmol)
I reacted. 50% of the crude product obtained
It was dissolved in 75 ml of AcOH, and this solution was purified with a "Sephadex G-15" column. The fraction of the AcOH eluate, which was resolved by treatment with trypsin to liberate 4-methyl-7-amino-coumarin, was concentrated to dryness in vacuo at 40°C. This residue was placed in a vacuum desiccator for 60 minutes.
Dry over _P2O5 at _° C and homogeneous amorphous compound in SS as shown by TLC 2e3.21 g
(80.4% of the theoretical value). Elemental analysis and calculation from the empirical formula C ₃₈ H ₅₂ N ₇ O ₇ Br gave the following values: C = 57.05% (57.14%), H = 6.61%.
(6.56%), N=12.49% (12.28%) and Br=
9.82% (10.00%). 2f 2HBr.H-D-VaI-CHA-Arg-MCA 2.40 g (3 mmol) of compound 2d was dissolved in glacial acetic acid.
Deblocking was carried out according to Example 1f using 12 ml of 2NHBr. The obtained crude product was dissolved in 50 ml of MeOH and the solution was purified by “Sephadex LH-
The fractions of the MeOH eluate, which was resolved by treatment with trypsin to liberate 4-methyl-7-amino-coumarin, were concentrated to dryness in vacuo at 30°C. , to purify the pre-purified product by 50%
It was dissolved in 40 ml of AcOH, and the solution was purified by gel filtration through a "Sephadex G-15" column. 4-methyl-7
The main fraction of the AcOH eluate, which was resolved by treatment with trypsin to liberate -amino-coumarin, was concentrated to dryness in vacuo at 40°C. The residue was dried over _P2O5 at 40 °C in a vacuum desiccator, yielding 1.73 g of homogeneous amorphous compound 2f in SS (77.3% _of theory) as shown by TLC.
I got it. Elemental analysis and calculations from the empirical formula C ₃₀ H ₄₇ N ₇ O ₅ Br ₂ gave the following values: C=
48.12% (48.33%), H = 6.43% (6.35%), N
= 13.38% (13.15%) and Br = 21.18% (21.44
%). Amino acid analysis confirmed the presence of the desired amino acids in the correct ratio: VaI: 1.00 - Arg: 1.02 - D-CHA: 0.97. Rf
Value: 0.42. Example 3 2HBr.H-D-VaI-CHA-Arg-DPA 3a Cbo-Arg-DPA.HCl Anhydrous Cbo-Arg-OH.HCl 34.48g (0.1 mol)
20 to a mixture of 150 ml of freshly distilled anhydrous DMF and 300 ml of anhydrous THF in a 1000 ml three-necked flask.
Dissolved at °C. 10.2 g (0.1 mol) of Et ₃ N were added to the solution cooled to −10° C. under stirring and in the absence of moisture. Furthermore, a solution of 13.65 g (0.1 mol) of isobutyl chloroformate in 50 ml of THF was added dropwise over a period of 20 minutes, after which the reaction temperature never exceeded -5.degree. 10 at temperatures between -10℃ and -5℃
After an additional reaction time of 20.92 g dimethyl 5-amino-isophthalate in 75 ml DMF
(0.1 mol) was added dropwise over 30 minutes, after which the reaction temperature was always kept below -5°C. This reaction mixture was allowed to react for an additional hour at -5°C. The reaction mixture was stirred at 20<0>C overnight and then cooled to -15[deg.]C in order to crystallize _Et3N.HCl . Separate the formed Et ₃ N.HCl and cool
Washed with a small amount of DMF. This solution and the washing solution were concentrated and dried together in vacuo at 50°C. Dissolve this residue in 1000ml of 50% AcOH, and add this solution to 50% AcOH.
It was purified by gel filtration on a column of "Sephadex G-15" equilibrated with AcOH. dimethyl 5-amino-
The main fraction of the AcOH eluate, which was resolved by treatment with trypsin to liberate the isophthalate, was concentrated to dryness in vacuo at 40°C. The residue was dried over P ₂ O ₅ at 50° C. in a vacuum desiccator to yield 24.6 g (45.9% of theory) of homogeneous amorphous compound 3a in SS as shown by TLC. Elemental analysis and calculation from the empirical formula C ₂₄ H ₃₀ N ₅ O ₇ Cl gave the following value: C = 53.21
% (53.78%), H = 5.71% (5.64%), N =
13.20% (13.07%) and Cl=6.52% (6.62%). 3b 2HBr.H-Arg-DPA 21.44 g (40 mmol) of compound 3a were deblocked according to Example 1b. After the usual work-up, the crude product obtained was dissolved in 250 ml of MeOH and the solution was purified by gel filtration on a column of "Sephadex LH-20". Decomposed by treatment with trypsin to liberate dimethyl 5-amino-isophthalate
The MeOH eluate fractions were concentrated to dryness in vacuo.
This residue was purified with P ₂ O ₅ at 40 °C in a vacuum desiccator.
Dry on SS as shown by TLC
Homogeneous amorphous compound 3b19.63% (theoretical value)
93.1%). Elemental analysis and experimental formula
Calculations from C ₁₆ H ₂₅ N ₅ O ₅ Br ₂ gave the following values: C = 36.82% (36.45%), H = 4.67%
(4.78%), N=13.45% (13.28%) and Br=
29.85% (30.31%). 3c Cbo-CHA-Arg-DPA.HBr 5.27 g (10 mmol) of compound 3b was added according to example 1c.
It was reacted with 4.69 g (11 mmol) of Cbo-CHA-OpNP. The crude product obtained after the usual work-up was dissolved in 200 ml of 50% AcOH and this solution was purified on a "Sephadex G-15" column. Fractions of the AcOH eluate were resolved in vacuo by treatment with trypsin to liberate dimethyl 5-amino-isophthalate.
It was concentrated and dried at 40°C. This residue was dried over _P2O5 at 60 °C in a vacuum desiccator to give _a homogeneous amorphous compound in SS as shown by TLC.
6.06 g of 3c (82.6% of theory) was obtained. By elemental analysis and calculation from the empirical formula C ₃₃ H ₄₅ N ₆ O ₈ Br,
The following values were obtained: C = 53.74% (54.02%);
H = 6.28% (6.18%), N = 11.90% (11.46%)
and Br=10.68% (10.89%). 3d 2HBr.H-CHA-Arg-DPA 5.87 g (8 mmol) of compound 3c in glacial acetic acid
Deblocking was performed according to Example 1d using 32 ml of 2NHBr.
The crude product obtained after normal processing
It was dissolved in 100 ml of MeOH, and this solution was purified with a "Sephadex LH-20" column. The fractions of the MeOH eluate, resolved by treatment with trypsin with liberation of dimethyl 5-amino-isophthalate, were concentrated to dryness in vacuo at 30°C. This residue was dried over _P2O5 at 40 °C in a vacuum _desiccator to give a homogeneous amorphous compound in SS as shown by TLC.
g (88.0% of theory). Elemental analysis and calculation from the empirical formula C ₂₅ H ₄₀ N ₆ O ₆ Br gave the following values: C = 43.75% (44.13%), H = 5.88
% (5.93%), N=12.69% (12.35%) and Br
=23.22% (23.49%). 3e Cbo-D-VaI-CHA-Arg-DPA.HBr 3.40 g (5 mmol) of compound 3d was added according to example 1e.
Cbo-D-VaI-OpNP2.05g (5.5 mmol)
I reacted. The crude product obtained after the usual work-up was dissolved in 100 ml of 50% AcOH and this solution was purified on a "Sephadex G-15" column. The fractions of the AcOH eluate, resolved by treatment with trypsin with liberation of dimethyl 5-amino-isophthalate, were concentrated to dryness in vacuo at 40°C. The residue was dried over P ₂ O ₅ at 60° C. in a vacuum desiccator to yield 3.25 g (78.1% of theory) of homogeneous amorphous compound 3e in SS as shown by TLC. Elemental analysis and calculation from the empirical formula C ₃₈ H ₅₄ N ₇ O ₉ Br gave the following values: C = 53.95% (54.80
%), H=6.65% (6.54%), N=12.07%
(11.77%) and Br=9.38% (9.59%). 3f 2HBr.H-D-VaI-CHA-Arg-DPA 2.50 g (3 mmol) of compound 3e in glacial acetic acid
Deblocking was carried out according to Example 1f using 12 ml of 2NHBr. The crude product obtained after normal treatment was mixed with 50 ml of MeOH.
This solution was prepurified using a "Sephadex LH-20" column. The fractions of the MeOH eluate, resolved by treatment with trypsin with liberation of dimethyl 5-amino-isophthalate, were concentrated to dryness in vacuo at 30°C. 50% of this pre-purified product
It was dissolved in 50 ml of AcOH and purified by gel filtration of this solution through a "Sephadex G-15" column. The main fraction of the AcOH eluate, resolved by treatment with trypsin to liberate dimethyl 5-amino-isophthalate, was concentrated to dryness in vacuo at 40°C. This residue was dried in a vacuum desiccator at 40 °C over P ₂ O ₅ to yield 1.93 g of homogeneous amorphous compound 3f in SS (82.6 of theory) as shown by TLC.
%) was obtained. Elemental analysis and experimental formula
Calculations from C ₃₀ H ₄₉ N ₇ O ₇ Br ₂ gave the following values: C = 46.84% (46.22%), H = 6.42%
(6.34%), N=12.16% (12.58%) and Br=
20.22% (20.50%). Amino acid analysis confirmed the presence of the desired amino acids in the correct ratio: D-VaI: 1.00-Arg: 0.98-CHA: 1.02. Rf
Value: 0.44. Example 4 2HBr.H-D-VaI-CHA-Arg-2-NA 4b 2HBr.H-Arg-2-NA 9.40 g (20 mmol) of commercially available Cbo-Arg-2-NA.HCl was mixed with glacial acetic acid according to Example 1b. 2NHBr80ml inside
was reacted with a solution of The product obtained after the usual work-up was dissolved in 150 ml of MeOH and this solution was purified on a column of "Sephadex LH-20". The fractions of the MeOH eluate, resolved by treatment with trypsin to liberate 2-naphthylamine, were concentrated to dryness in vacuo at 30°C. This residue was stored in a vacuum desiccator at 40°C.
to give 8.60 g (93.2% of theory) of homogeneous amorphous compound 4b in _SS as shown by _TLC . Elemental analysis and experimental formula
Calculations from C ₁₆ H ₂₃ N ₅ OBr ₂ gave the following values: C = 42.08% (41.67%), H = 5.12%
(5.03%), N=14.68% (15.19%) and Br=
33.96% (34.65%). 4c Cbo-CHA-Arg-2-NA.HBr 4.6 g (10 mmol) of compound 4b was added according to example 1c.
It was reacted with 4.69 g (11 mmol) of Cbo-CHA-OpNP. The crude product obtained after the usual work-up was dissolved in 150 ml of 50% AcOH and this solution was purified on a "Sephadex G-15" column. Decomposed by treatment with trypsin to liberate 2-naphthylamine
The AcOH eluate fractions were concentrated to dryness in vacuo at 40°C. This residue was heated at 60℃ in a vacuum desiccator.
Drying over P ₂ O ₅ gave 5.31 g (79.5% of theory) of amorphous compound 4c, homogeneous in SS as shown by TLC. Elemental analysis and experimental formula
Calculations from C ₃₃ H ₄₃ N ₆ O ₄ Br gave the following values: C = 59.18% (59.37%), H = 6.58%
(6.49%), N=12.87% (12.59%) and Br=
11.55% (11.97%). 4d 2HBr.H-CHA-Arg-2-NA 4.67 g (7 mmol) of compound 4c in glacial acetic acid
Deblocking was carried out according to Example 1d using 28 ml of 2NHBr.
The crude product obtained after normal processing
The solution was purified on a Sephadex LH-20'' column. The fractions of the MeOH eluate, which were digested by treatment with trypsin to liberate 2-naphthylamine, were purified in vacuo. Concentrated to dryness at 30° C. The residue was dried over P ₂ O ₅ at 40° C. in a vacuum desiccator and yielded 3.95 g of homogeneous amorphous compound 4d in SS (91.8 theoretical) as shown by TLC.
%) was obtained. Elemental analysis and experimental formula
Calculations from C ₂₅ H ₃₈ N ₆ O ₂ Br ₂ gave the following values: C = 49.22% (48.87%), H = 6.30%
(6.23%), N=13.61% (13.68%) and Br=
25.84% (26.01%). 4e Cbo-D-VaI-CHA-Arg-2-NA.HBr 3.07 g (5 mmol) of compound 4d was added according to example 1e.
Cbo-D-VaI-OpNP2.05g (5.5 mmol)
I reacted. The crude product obtained after the usual work-up was dissolved in 100 ml of 50% AcOH and this solution was purified on a "Sephadex G-15" column. Decomposed by treatment with trypsin to liberate 2-naphthylamine
The first main fraction of the AcOH eluate was concentrated to dryness at 40 °C in vacuo and at 60 °C in a vacuum dessicator.
Dry over _{P2O5 .} Thus, the homogeneous amorphous compound in SS as shown by TLC
4e3.14 g (81.9% of theory) was obtained. Elemental analysis and calculation from the empirical formula C ₃₈ H ₅₂ N ₇ O ₅ Br gave the following value: C = 58.92% (59.52
%), H=6.93% (6.84%), N=13.02%
(12.79%) and Br=10.18% (10.42%). 4f 2HBr.H-D-Val-CHA.Arg-2-NA Compound 4e1.53 g (2 mmol) in glacial acetic acid
Deblocking was carried out according to Example 1f using 8 ml of 2NHBr. The crude product obtained after normal treatment was mixed with 40 ml of MeOH.
This solution was purified using a "Sephadex LH-20" column.
The main fraction of the MeOH eluate, resolved by treatment with trypsin with the formation of 2-naphthylamine, was concentrated to dryness in vacuo at 30°C. The product was dissolved in 50 ml of 50% AcOH and the solution was purified with a "Sephadex G-15" column. Decomposed by treatment with trypsin with formation of 2-naphthylamine
The main fraction of the AcOH eluate was concentrated to dryness in vacuo at 40°C. This residue was stored in a vacuum desiccator at 40°C.
to give 1.05 g (73.6% of theory) of homogeneous amorphous compound 4f in _SS as shown by _TLC . Elemental analysis and experimental formula
Calculations from C ₃₀ H ₄₇ N ₇ O ₃ Br ₂ gave the following values: C = 50.16% (50.50%), H = 6.71%
(6.64%), N=14.00% (13.74%) and Br=
22.05% (22.40%). Amino acid analysis confirmed the presence of the desired amino acids in the correct ratio: D-VaI: 1.00-Arg: 0.98-CHA: 0.97. Rf
Value: 0.42. Example 5 2HBr.H−D−VaI−CHA−Arg−4−MeO−
2-NA 5b 2HBr.H-Arg-4-MeO-2-NA Commercially available Cbo-Arg-4-MeO-2-NA.
10.0 g (20 mmol) of HCl in glacial acetic acid
Deblocking was performed according to Example 1b using 80 ml of 2NHBr.
The crude product obtained after normal processing
It was dissolved in 150 ml of MeOH, and this solution was purified with a "Sephadex LH-20" column. The main fraction of the MeOH eluate, resolved by treatment with trypsin to liberate 4-methoxy-2-naphthylamine, was concentrated to dryness in vacuo at 30°C. After drying this residue over _P2O5 at 40 °C in a vacuum desiccator _, a homogeneous amorphous compound in SS was obtained as shown by TLC.
8.98 g (91.4% of theory) of 5b was obtained. Elemental analysis and calculation from the empirical formula C ₁₇ H ₂₅ N ₅ O ₂ Br ₂ gave the following values: C = 41.22% (41.57
%), H=5.19% (5.13%), N=14.40%
(14.26%) and Br=32.01% (32.53%). 5c Cbo-CHA-Arg-4-MeO-2-Na.HBr 4.91 g (10 mmol) of compound 5b was added according to Example 1c.
It was reacted with 4.69 g (11 mmol) of Cbo-CHA-OpNP. The crude product obtained after the usual work-up was dissolved in 150 ml of 50% AcOH and this solution was purified on a "Sephadex G-15" column. The first main fraction of the AcOH eluate, resolved by treatment with trypsin to liberate 4-methoxy-2-naphthylamine, was concentrated to dryness in vacuo at 40°C. After drying this residue in _a vacuum desiccator at 60 °C over _P2O5 ,
5.36 g (76.8% of theory) of homogeneous amorphous compound 5c in SS as shown by TLC were obtained. Elemental analysis and calculation from the empirical formula C ₃₄ H ₄₅ N ₆ O ₅ Br gave the following value: C = 58.85
% (58.53%), H = 6.59% (6.50%), N =
11.91% (12.05%) and Br=11.32% (11.45
%). 5d 2HBr.H-CHA-Arg-4-MeO-2-NA Compound 5c4.88 g (7 mmol) in glacial acetic acid
Deblocking was carried out according to Example 1d using 28 ml of 2NHBr.
The crude product obtained after normal processing
It was dissolved in 100 ml of MeOH, and this solution was purified with a "Sephadex LH-20" column. The main fraction of the MeOH eluate, resolved by treatment with trypsin with the formation of 4-methoxy-2-naphthylamine, was concentrated to dryness in vacuo at 30°C. After drying this residue over _P2O5 at 40 °C in a vacuum desiccator _, a homogeneous amorphous compound was obtained in SS as shown by TLC.
5d4.12 g (91.3% of theory) was obtained. Elemental analysis and calculation from the empirical formula C ₂₆ H ₄₀ N ₆ O ₃ Br ₂ gave the following values: C = 48.92% (48.46
%), H=6.36% (6.26%), N=12.84%
(13.04%) and Br=24.33% (24.80%). 5e Cbo−D−VaI−CHA−Arg−4−MeO−2
−NA.HBr Compound 5d 3.22 g (5 mmol) according to example 1e
Cbo-D-VaI-OpNP2.05g (5.5 mmol)
react with. The crude product obtained after the usual work-up was dissolved in 125 ml of 50% AcOH and this solution was purified on a "Sephadex G-15" column. The first main fraction of the AcOH eluate, resolved by treatment with trypsin to liberate 4-methoxy-2-naphthylamine, was concentrated to dryness in vacuo at 40°C. After drying this residue in a vacuum desiccator at 60 °C over _P2O5 , 3.15 g (79.1% of theory) of homogeneous amorphous compound _5e in SS was obtained as shown by TLC. . Elemental analysis and empirical formula C ₃₉ H ₅₄ N ₇ O ₆ Br
The calculation from gave the following value: C=
58.35% (58.79%), H = 6.78% (6.83%), N
= 12.68% (12.31%) and Br = 9.82% (10.03
%). 5f 2HBr.H−D−Val−CHA−Arg−4−MeO
-2-NA Compound 5e1.59g (2 mmol) in glacial acetic acid
Deblocking was carried out according to Example 1f using 8 ml of 2NHBr. The crude product obtained after normal treatment was mixed with 40 ml of MeOH.
This solution was prepurified using a "Sephadex LH-20" column. The main fraction of the MeOH eluate, resolved by treatment with trypsin with the formation of 4-methoxy-2-naphthylamine, was concentrated in vacuo at 30°C. 50% of this pre-purified product
It was dissolved in 60 ml of AcOH, and this solution was purified using a "Sephadex G-15" column. The main fraction of the AcOH eluate, resolved by treatment with trypsin with the formation of 4-methoxy-2-naphthylamine, was concentrated to dryness in vacuo at 40°C. This residue is placed in a vacuum desiccator.
Homogeneous amorphous compound 5f1.09 in SS as shown by _TLC after drying over _P2O5 at 40 °C
g (73.3% of theory) was obtained. Elemental analysis and calculation from the empirical formula C ₃₁ H ₄₉ N ₇ O ₄ Br ₂ gave the following values: C = 49.63% (50.07%), H
= 6.70% (6.64%), N = 13.42% (13.19%) and Br = 21.22% (21.49%). Amino acid analysis confirmed the presence of the desired amino acids in the correct ratio: D-VaI: 1.00-Arg: 1.01-D-CHA:
0.98. Rf value 0.47. A series of other tripeptide derivatives were prepared by the methods described in the previous examples. These tripeptide derivatives are summarized in Table 1 below. The tripeptide intermediates used in the preparation of the tripeptide derivatives shown in Table 1 are listed in Table 2.

【table】

【table】

[Table] Example 12 2AcOH.H-D-VaI-CHA-Arg-pNA 2HBr.H-D-VaI-CHA-Arg-pNA (Example 38
7.09 g (10 mmol) of 60% MeOH
Dissolved in 75ml of aqueous solution. This solution is used in the form of acetate as “AmberIite” (registered trademark).
It was packed into a JRA-401 column. 60 this column
% MeOH aqueous solution, followed by replacing HBr with AcOH by ion exchange. The eluate was concentrated to dryness in vacuo at 40°C. After drying the residue over P ₂ O ₅ at 40 °C in a vacuum desiccator, the bromide-free 2AcOH.H−D−VaI−CHA−
6.33 g (98.5% of theory) of Arg-pNA was obtained. According to the above method, other salts with organic acids such as formic acid, propionic acid, oxalic acid, tartaric acid, citric acid, lactic acid, benzoic acid, chlorobenzoic acid, salicylic acid or phthalic acid are prepared from the above-mentioned tripeptide derivatives. be able to. Ion exchangers, e.g. “Amberlite” JRA in the form of hydrochloride
-401, which converts the ion exchanger to the basic OH form by treatment with caustic soda solution and then converts the basic ion exchanger into the basic OH form.
It can be converted to the desired acid salt form by treatment with a solution of a 1:1 mixture of the desired organic acid and its sodium salt in 60% aqueous MeOH. Quantitative analysis of enzymes using the tripeptide substrate according to the invention can be carried out as follows: 1 Urine kallikrein analysis 1 ml of urine and 1 ml of TRIS-imidazole buffer with pH 7.9 and ionic strength 1.0 Incubate at 37°C for 5 minutes, then centrifuge to remove precipitate. 1.4 ml of distilled water heated to 37°C and 0.4 ml of centrifuge
Mix thoroughly in a plastic cuvette. Add to this mixture 0.2 of a 2 x 10 ^-3 mol aqueous substrate solution.
ml and mix the ingredients quickly. This mixture is incubated at 37° C. for exactly 15 minutes. This reaction mixture was then diluted with glacial acetic acid to stop the enzymatic reaction.
Mix with 0.2ml. To measure the color, use a blank sample consisting of the same components but with glacial acetic acid added before the addition of the substrate to inhibit the enzyme reaction. The amount of colored compound R- _NH2 formed is then determined photometrically or spectrophotometrically at 405 nm from the difference between the blank sample and the test sample. From the obtained value, the urinary kallikrein activity in urine is determined using the following formula: △OD = increase in optical density at 405 nm over 15 minutes V = total volume of test mixture = 2.2 ml 1000 = conversion factor to convert U to mU F = dilution factor of urine (2) V = volume of sample = 0.4 ml Extinction coefficient divided by ε = 1000 = 10.4 Calculation of the urinary kallikrein content in urine is carried out by continuously measuring the product R-NH ₂ (e.g. p-nitroaniline) formed. You can also. The application of this method to the analysis of glandular kallikrein in sputum is described below. In addition to urinary kallikrein, urine also contains urokinase, a proteolytic enzyme that can also degrade the substrate according to the invention, albeit in small amounts. In the above analysis method, the total activity of urinary kallikrein and urokinase is measured. To obtain an accurate value for urinary kallikrein activity, urokinase activity must be subtracted. This urokinase activity was determined by the amount of trypsin inhibitor (trypsin inhibitor from bovine lung) per ml of buffer to completely suppress urinary kallikrein activity in a comparative test.
It can be determined by adding 0.075 units and measuring the urokinase activity alone. 2 Glandular kallikrein analysis in sputum: 0.5 ml of sputum is mixed with 2 ml of TRIS-imidazole buffer (ionic strength 1.0) and the mixture is pre-incubated at 37° C. for 5 minutes. This thermostatic material is centrifuged. 1.5 ml of distilled water at 37°C in the test cuvette
to which 0.25 ml of centrifuged material is added.
Mix these ingredients thoroughly. Furthermore, 2×10 ^-3
Add 0.2 ml of molar substrate aqueous solution. next,
The absorbance change at 405 nm is continuously tracked using a recording device for 5-10 minutes. From the measurements of ΔOD per minute, the kallikrein activity per ml of sputum is calculated in mU by the following formula: F=5 V=1.95 v=0.25 IU (unit) = Enzyme capable of decomposing 1 micromole of substrate in 1 minute under optimal conditions of PH, ionic strength, temperature, and substrate concentration or under conditions otherwise specified. amount. In pancreatic juice, pancreatic kallikrein is mainly present in the prekallikrein form and can only be analyzed after activation, for example using trypsin. After activation of prekallikrein, trypsin is inhibited by soybean trypsin inhibitor (SBTI). The kallikrein content in this activated mixture is
It can be determined by one of the methods mentioned above. 3 Plasmin analysis: 1.7 ml of TRIS-imidazole buffer (PH 7.5, ionic strength 0.2) is mixed with 0.1 ml of plasmin solution in 25% glycerol at 37°C and the mixture is incubated at 37°C for 1 minute. Add this mixture 2x to 37°C.
Add 0.2 ml of 10 ^-3 molar substrate aqueous solution and mix the ingredients quickly. Next, the amount of decomposition product R-NH ₂ separated from the substrate per unit time is continuously measured. The plasmin activity per ml of sample is calculated in mU from the values measured every minute using the following formula: ΔE/min. ×V×1000/v×ε=mU of sample/
number of ml △E = amount of degradation products separated per minute V = total volume of test mixture v = volume of sample ε = extinction coefficient divided by 1000 4 Antiplasmin analysis in human plasma: in a ratio of 1:20 0.1 ml of plasma diluted with TRIS-imidazole buffer was added to 1 ml of 25% glycerol.
Human plasmin (AB Kabi (Stockholm,
Mix with 0.02 ml of a solution of 1.25 cU (manufactured by Basle, Switzerland) and 50 ATU of hirudin (manufactured by Pentapharm AG, Basle, Switzerland). This mixture is incubated at 37° C. for 90 seconds. 1.7 ml of TRIS-imidazole buffer (PH 7.5, ionic strength 0.2) at 37° C. is mixed with this thermostatic mixture, and further 0.2 ml of a 2×10 ⁻³ mol substrate aqueous solution is mixed therein. Next, the decomposition product R-NH ₂ separated from the substrate per unit time
Continuously measure the amount of The residual plasmin activity is calculated from the measured values by the method described above. In the blank test, the plasma is replaced by an equivalent amount of buffer, but otherwise the test is performed as described above. The measured plasmin activity corresponds to the starting plasmin amount. Antiplasmin activity is calculated from the difference between the plasmin activity measured in a blank test and the residual plasmin activity measured in a test using plasma using the following formula: F = dilution factor of plasma (20). The substrate according to the invention converts the plasminodiene present in plasma using urokinase or streptokinase in a buffer system and the amount of plasmin formed is determined using one of the substrates according to the invention by the plasmin analysis method described above. It can also be used to analyze plasminodiene in human plasma. The amount of plasminodiene naturally present in plasma is derived from measurements for plasmin since one molecule of plasmin is formed from one molecule of plasminodiene under activation. Table 4 below shows the sensitivity of some substrates according to the invention to organ or glandular kallikrein, plasmin and thrombin.

[Table] The measurement of the degradation product R-NH ₂ formed by enzymatic hydrolysis of a substrate indicates that this degradation product has an ultraviolet spectrum different from that of the substrate and shifts toward higher wavelengths. Based on the forebank condition. Absorption of the substrate at 405 nm is virtually non-existent. p-Nitroaniline as a decomposition product has an absorption maximum at 380 nm and a molar extinction coefficient
Shows 13200. However, the extinction coefficient drops slightly at 405 nm, i.e. to 9650. The amount of substrate enzymatically hydrolyzed, which is proportional to the amount of p-nitroaniline separated, can be determined by spectrophotometric measurements at 405 nm. Measurements at 405 nm are not affected even in the presence of excess substrate. The amount of decomposition product R-NH ₂ is determined by the amount of 2-naphthylamino group, 4-methoxy-2-naphthylamino group, 4
-methyl-coumaryl-(7)-amino group or 1,3
-di(methoxycarbonyl)-phenyl-(5)-
It is measured by fluorescence spectrophotometry using a substrate containing an amino group. In a test system consisting of enzyme, buffer and substrate, radiation of low energy content
The fluorescent decomposition products formed are excited by a high-energy amount of light and then measured continuously at 400-470 nm. The amount of degradation products formed per unit time is measured relative to the enzyme activity present. As mentioned above, one micromole of degradation product per minute corresponds to one enzyme unit based on a given substrate.

Claims (1)

[Claims] 1 General formula: H-D-X-Y-Arg-R [wherein, where Y represents a cyclohexylalanyl group, R represents a p-nitrophenylamino group, a 2-naphthylamino group, a 4-methoxy-2-naphthylamino group, a 4-methyl-coumaryl-7-amino group, or a 1,
3-di(methoxycarbonyl)-phenyl-5-
Tripeptide derivatives represented by [representing an amino group] and salts thereof with acids. 2 Mineral acids, such as HCl, HBr, H ₂ SO ₄ or H ₃ PO ₄
or organic acids such as formic acid, acetic acid, propionic acid,
Halogenated acetic acids such as trimethylacetic acid, methoxyacetic acid, trichloro- or trifluoroacetic acid, aminoacetic acid, lactic acid, oxalic acid, malonic acid, citric acid, benzoic acid, aromatic acids substituted in the nucleus, such as toluic acid, chloro- or the tripeptide derivative according to claim 1, which is protonated with bromobenzoic acid, methoxybenzoic acid and aminobenzoic acid, or phthalic acid.