• 专利附录
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  • 权利要求
  • 类似技术
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    • 专利摘要:
    Serine proteases in a substance such as blood are determined by contacting the substance with a novel chromogenic or fluorgenic substrate for serine proteinases and spectrophotometrically measuring the quantity of a chromophore or fluorescent compound released from the substrate by serine proteases in the substance. The novel substrate has the amino acid sequence-ILe-A-Gly-Arg-wherein A is Asp or Glu substituted in the carboxylic group by esterification or amidation. The novel substrate increases sensitivity and accuracy of the determination.
    公开号:SU736889A3
    申请号:SU772548501
    申请日:1977-11-30
    公开日:1980-05-25
    发明作者:Геран Клаесон Карл；Эрик Аурелл Лейф；Роджер Симонссон Лейф
    申请人:Актиеболагет Каби (Фирма)；
    IPC主号:
    专利说明:

    I.
    Invention relates to biochemistry
    There is a method for determining the activity of serine proteases by affecting the enzyme-containing material on the enzyme substrate I.
    However, the known method does not provide a high accuracy of determining the activity of serine proteases.
    The aim of the invention is to improve the accuracy of the method.
    This is achieved by using a substance of the general formula as a substrate.
     Ilv-A-gki-arg-nn-Y ,,
    where r
    acyl, preferably acetyl or benzoyl; ile - isoleucine ;;
    But aspartic acid or L1taminic acid, which is substituted in the carboxyl group by the formation of an ester with a short alkyl, oxyalkyl, substituted aminoalkyl or cycloshkyl, or guvI, and is doped with a mono- or disubstituted short alkyl, oxyalkyl, and is replaced by an amio-alkyl or heterocyclic group, a non-alkyl group, a non-alkyl group, a non-alkyl group, a non-alkyl group, a non-methyl group, a non-methyl group, a non-methyl group, a non-methyl group, a non-methyl group, a non-methyl group or a non-methyl group can be used. forms a portion of the piperidine morpholine or piperazine ring; glycine;
    gly. arginine; arg nitrophenyl.
    Substrates are obtained from chromogenic or fluorogenic substrates containing amino acids in the sequence, respectively, of il-glu. Ly-gly-arg-and-yl-as-gly-arg, through the formation of complex b of the vfir group or amide free-j-yl, p. carboxyl group in a known manner.
    With thin layer chromatographic analysis of the adsorbent extract
    9L and products take glass plates with silica gel F-254 as the absorption medium. Use the system of solvents: chloroform - methano-acetic acid - water in
    the ratio of 34: 4s9: 2. After thin-layer chromatography, the plates are examined first in ultraviolet rays (254 nm), and then with the help of a hortoluidine reaction. All used amino acids in the absence of
    other indications have an L-configuration,
    Accept the following abbreviations: Apr - arginine, asp - aspartic acid, - to - glutamic acid, gly - glycine, silt -. ioleucine, leu, leucine; Ac. - acetyl, AcOH - acetic acid, B - benzoic, DCCe -. dicyclohexylcarbodiidad, Navy - dimethylformamide, EtjN - triethylamine, HOBT - hydroxybenzotriazole, HOSu N-oxyimide succinic acid, MeOH methanop, OEt - ethyloxy, OMe. -methyloxy, Op NP -. p-nitrophenoxy, OiSopisopropyloxy, rmd - p-nitroanilide GAE - quaternary aminoethyl sepharose, SOCCg thionyl chloride.
    Example G. Benzoyl-yl-glu-arg-p-nitroanilide substrate (5-2222 B2-yl-glu- (OMe) -gly-arg-pNA JASS (mol.v, 748.2).
    Under anhydrous conditions at 0 ° C, 30 ml of distilled is added to O, 5 ml of absolute methanol. After the initial reaction, the solution is left to stand for about 15 minutes at room temperature, then 75 mg of Bx-yl-glu-gly-arg-pN A-it is added () The solution is stirred for 5 hours, then evaporated. The oily product obtained is dissolved in a small amount of methanol and purified by gel chromatography on a column containing Sephadex ZH20 in methanol. Meta- is also used to wash the adsorbent. NOL The pure methyl ester obtained after evaporation of the methanol is dissolved in water and dried in a frozen state. Yield 30 mg (40%). The product is homogeneous. . data, thin-layer chromatography, R 0.33 / ci / n minus 40.3 ° (with 0.5, 50% HAOC / HjO).
    Example 2. Bz-yl-glu (O.tt) -gly-arg-MAn (mol. 762.2).
    Under anhydrous conditions, at a temperature of minus 60 ml of distilled water, it is added to 1.0 ml of absolute ethanol. After 30 minutes, 150 mg of the substrate S-2222 was added at room temperature. After about 15 hours from the end of the reaction (according to thin layer chromatography), the solution is evaporated. The resulting oil is dissolved in a small amount of 30% HOAc in water and purified by chromatography on a column containing Sephadex-C 15 in 10% -Hejfb aqueous solution. Noaac There is a complex ethylog out. the ether extracted from the adsorbent is dried in a frozen state. Yield 70 mg (46%). The product is homogeneous. data, thin-layer chromatography, R rO, 40 / c (./j3 ° minus 37.7 ° (with 0.50.50 HOAc / HgO),
    Example 3. Br-il-glu (015OR2) -gly-arg-p (mol 776.3).
    . The synthesis is carried out according to Example 2, however, instead of the reference, absolute isopropanol is taken. The reaction is completed after 16 hours. Chromatography and drying in a frozen state is carried out as described in Example 2, Yield 90 mg (58%). The product is homogeneous.
    Q data thin layer chromatography
    , 44 / OC /, minus 36.4 (with 0.5, 50% HOAc / H O).
    Example 4. BZ-yl-glu- (0-cyclohexyl) -gly-arg-p VJ Anea (mol.v. 833.3).
    560 ml SOjCCj added to 1.0 ml
    dry cyclohexanol and, after 1 h, 200. mg 5-2222 are added at room temperature. At the end of the reaction, after about 12 hours, the solution
    0 is evaporated and chromatographed as
    indicated in examples 2 and 3. Then the product is dried in a frozen state. Yield 170 mg (75%). The product is homogeneous according to the thin
    5 chromatography, TJg - O, 50 / i- / r minus 38.6 ° (with 0.5, 50% HOAc / H, O). ; Example 5, B7-yl-glu- (0-CH CH. N (CH,) 2-gly-arg-p N L-HOf (mol, 859.8).
    Q75 g (0.10 mmol) substrate
    S-2222 and 100 mg of dimethyl-aminoethanol-hydrochloride are dissolved in 1, 0 ml of dry distilled, DMF, then 10 MP of pyridine, 10 mg are added,
    - HOBT and finally 25 mg of DCCE is added. The dicyclohexyl carbamide formed is filtered off after about 24 hours and the dimethylformamide solution is evaporated under reduced pressure. The remaining oil is dissolved in a small amount of 95% MeOH 5% H. O and purified on a column containing soAAE-25.
    ion exchanger
    in his
    chloride form. The same solvent mixture is used to wash the adsorbent. According to this procedure, dimethylaminoethyl ester hydrochloride salt is obtained, free from admixture of other peptides; however, the product contains small amounts of some contaminating impurities, for example, dimethylaminoethanol hydrochloride salt. The fraction containing. Complex - dimethylaminoethyl ether,
    evaporated and purified by chromatography on a column containing. Sephadex 15 in 10% HOAc / Hj O. A solution of the pure ester is dried in a frozen state, 60 mg (58%). The product is homogeneous according to
    thin layer chromatography, Rr 0., 50 / oL (1 ° minus 35.0 (with 0.5, 50% HOAc / HjO).
    CONH-CH (CH,) 2
    P. Merper 6. Br-il-pl-gl-arg-pha (mol. 775.3).
    240 mg of substrate 3-2222 HOSu is dissolved in 1 ml of dry distilled OMF. The solution is cooled to minus 5 ° C and 120 mg of tx: CE is added. The temperature is allowed to rise to room temperature and, after 4 hours, the solution is refreshed. cooled to, the dicyclohexyl carbamide precipitated as a precipitate is filtered and washed. The navy solution (about 2 ml) is cooled to 0 ° C and 0.1 MP of pure isopropyl mine is added. After 70 hours at room temperature, the solution is evaporated under reduced pressure, mixed with 5 ml of water and again evaporated. The product is dissolved in approximately 4 ml of 50% SPLA / H2O and purified by chromatography on a column containing Sephadex C-15 in 33% HOAc / H medium. The same solvent mixture is used to wash the adsorbent. The fraction containing pure isopropylamide is evaporated and subjected to ion exchange in a column containing quaternary aminoethyl sepharose AE 25 in its chloride (1 dissolved in 95% MeOH-5% Ho.). The extract from the adsorbent is evaporated, dissolved in water and dried in a frozen state The yield is 120 mg (47%). The product is homogeneous according to thin-layer chromatography, 1 30.3 IdLl minus 30.6 ° (s 0.5, MeOH).
    Prm and meer 7. Br-il-glu-gly-,
     CONH) -apr-pNA (mol 802.1)
    Synthesis Carried out according to Example B, however, piperidine is used instead of isopropylamine. Yield 105 mg (40%). The product is homogeneous according to thin layer chromatography, R.j 0,50 / Ы / 2 minus 34 ° - (с 0.5, MeOH). Example 8. V1.-IL-GLYU-GLI-:
    .c6NlCH,; CHj-OM) -apr-pNA (mol 822.3)
    The synthesis is carried out as in example b, but diethanolamine is used instead of isoprpylamine. Yield 120 J45%). The product is homogeneous according to thin layer chromatography, TJf-O, 25 / oL (minus 31 ° (with 0.5 MeOH).
    Example E-Br-il-asp (oiSoPv-) -gli-arg-r And A-HCt I lo b. 762.3).
    30 ml of distilled BFB are added to O, 5 MP of dry isopropanol, and after 30 minutes, 73 mg (0.10 mmol) of Bz-yl-as-gly-arg-pN A-HCE (mol. 720.2). At the end of the reaction, after about 18 hours, the solution is evaporated, chromatographed and dried in a frozen state, as indicated in Examples 2, 3 and 4.
    Yolkhod 32 mg (42%). Product odnoren on. . thin layer chromatography, 46 / o (/ |, minus 22.7 (s - 0.5, 50% HOAc / H-jO).
    Lrimer 10. Eg-il-asp5 (OEt) .- rJiH-apr-p I A-HSE (mol. 1b.74 8.2) 1 The synthesis is carried out as in Example 9, but instead of isopropanol stadium. The output of 28 mg (37%). The product is homogeneous according to thin layer Q chromatography, 40 / ot / f minus 23, (with 0.5, 50% HOAc / H O). Example 11. Br-il-asp-gly coNH-cHtoiOj
    -ar-p NAHCC (mol. c. 761.3). , Synthesis is carried out as in Example 6. Bg.-IL-as-Gli-Arg-R NA-HCt is used as the starting material instead of 5-2222, Yield 100 mg (40%). The product is homogeneous according to the data, thin-layer chromatography, R - 0.40 / Oil
    0 minus 20.1 (s - 0.5, MeOH).
    Example 12. B2.-Il-as-gly0
    CON
    -apr-pNA (mol-, c. 818.3).
    The synthesis is carried out according to example 11,. but morpholine is used instead of isopropylamine. Yield 95 mg (35%). The product is homogeneous. according to thin-layer chromatography, R — 0.46 / s1 / minus 24.2 ° (s — 0.5 MeOH).
    The definitions of K and (Ktp Michaelis constant Tsdaks maxgsh-, on speed), get, using the equation of Linevever-Burka:
    o waw; so
    Then c - "is mixed with the substrate in a buffer solution and the hydrolysis rate is determined spectrophotometrically. Substrate concentration (Zo) is measured, and the enzyme concentration is kept constant. Then, the values of the values, inverse speeds -, are plotted on the graph relative to the values of the inverse concentration -, According to the obtained graph of Linewever - Burka, 3 is determined (see table)
    The extract is dissolved in water up to 2 mmol / l, the buffer, the enzyme and the substrate are mixed and the change in absorbability (O1Umin) is counted at
    405 nm at. 37 ° C
    The proposed method, using chromogenic substrates, ensures high sensitivity and accuracy of determination for a short time.
    time, which allows for the analysis of a large number of samples.
    736889
    权利要求:
    Claims (1)
    [1]
    1. Teyen A, P., Lee M., Ayild & ard U. Research in the field of rhombus. 1976, 8, 413.
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    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    SE7613463A|SE437153B|1976-12-01|1976-12-01|SPECIFIC CHROMOGENA ENZYM SUBSTATE FOR SERINE PROTEASES|
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