• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    Easily split enzyme substrates for the quantification of proteases having the general formula wherein R1 = H or a protective group, preferably t-butyl-oxycarbonyl, benzyloxycarbonyl; A, - Gly, Ala, Val, Leu, Ile. Ser, Thr, Pro, Pip, Phe or Tyr; A2 - Arg or Lys; R2 = an aromatic, possibly substituted, hydrocarbon group, wherein -NH-R2 is a physico-chemically determinable group, preferably a chromogenic or fluorogenic group which is split by a present enzyme and then forms a cleavage product of the formula H2N - R2 the amount of which can be quantified. Process for the production of said substrates. Method in the laboratory diagnostics of proteases using said substrates.
    公开号:SU1277904A3
    申请号:SU802893256
    申请日:1980-03-06
    公开日:1986-12-15
    发明作者:Йеран Клаесон Карл；Эрик Аурелл Лейф；Роджер Симонссон Лейф；Ариелли Сало
    申请人:Каби Аб (Фирма)；
    IPC主号:
    专利说明:

     The invention relates to a process for the preparation of tripeptides of the general formula n-Glu-A, -A-NH-R where A is Ala, Gly, Val, Leu, Ser, Thr, Pro, Phe, lie, Pip, Tyr; A j-Arg, Lys; R - p-nitroanilide, new bio-active compounds. containing chromophore troupe, which can be found primitive (biology and medicine). The purpose of the invention is new peptide derivatives with chromophore properties and a higher rate of cleavage. In the analysis of eluates and products obtained by thin-layer chromatography glass plates are used as adsorption medium with silica gel F ,, .. (Megsk) and chromatography graphing is carried out in the following systems: A: n-butanol: acetic acid: water 3: 2: 1 (by volume); P: chloroform: methanol 9: 1 ( about him). After chromatro platings are examined under UV light (254 nm and then sprayed with ninhydrin and treated with a mixture of dicarboxydine /, / chlorine. The following abbreviations are used in the text; HOAc acetic acid; Bz benzoyl; Lo carboxybenzoxy; DCCE; dimethylformamide; EtjN triethylamine; HOBT 1-hydroxy benzotriazole; DSNA dicyclohexylamine; DSI dicyclohexyl urea EtOH ethanol; MeOH methanol; EtOAc ethyl acetate; OpNP p-nitrophenoxy; pNA p-nitroanilide; TFA trifluoroacetic acid. Example 1. n-Glu-Gly-ArgpNAHCl (mp498.9); (ms, 5) 175 ml of concentrated acetic acid and 105 ml (5.6 mol) of HBg in acetic acid are added to 35 g (0.074 mol) of CbO-Arg (NO) pNA and the mixture is stirred for 30 minutes to obtaining a clear solution. Then, with vigorous stirring, the resulting solution is poured into 2 liters of dry ether. The precipitate that forms is filtered off and washed with dry ether, dried in vacuo over NaOH. The resulting hydrobromic H-Arg salt (NO ,,) - pNA is dissolved in 150 ml of dry distilled dimethylformamide and neutralized at low temperature () using triethylamine to obtain an alkaline reaction in a pH-indicator paper. Usually, neutralization consumes Et, N in an amount of approximately 1.5 equivalents of HBg. The resulting HBr is filtered off. While cooling, 1.1 equivalents of 0.81 mol of 26.8 g of Cbo-Gly-OpNP are added to the reaction mixture; after 30 minutes, another 1/2 equivalent of 5.2 mol Et N was added. The mixture was left at room temperature overnight. It is then evaporated in vacuo, the resulting oil is triturated in an aqueous solution of 2% NaHCO, then dissolved in hot methanol and crystallized with stirring and cooling. The crystals formed are filtered and washed with cold methanol. Chromatography in a thin layer shows the presence of only a small amount of impurities, the product is recrystallized from the same solvent. White crystals are obtained, which are a pure product according to the results of thin layer chromatography. Yield: 34 g (86%) (U); , 20 (P,); CoLl -35.4 ° (p. 0.3 Methanol); IbCbo-p-GluOH (m.v. 263.3). 56.3 g (0.20 mol) of Cbo-Glu-OH is dissolved in 400 ml of ethyl acetate, 49.4 g (0.24 mol) of dicyclohexylcarbodiimide, added in 60 MP of ethyl acetate, are added and stirred under cooling on ice bathhouse After 2 hours, the resulting dicyclohexyl urea is filtered off and the remaining solution is evaporated to dryness. The product (Cbo-Glu anhydride) is crystallized from ethyl acetate and petroleum ether. The anhydride is dissolved in a mixture of 20 ml of dioxane and 260 ml of ether, and then 48 ml of dicyclohexylamine (DSNA), dissolved in ether to a volume of 100 ml, are added. After some time, Cbo-p-Glu DSNA precipitates. The mixture is stirred for approximately one hour, and then the dicyclohexyl ammonium salt formed is filtered and washed with ether and ethyl acetate, the dicyclohexylammonium salt is suspended in ethyl acetate and stirred with 25 g (0.18 mol) KHSO, 31 dissolved in water, the resulting Cbo-p- GluOH enters the ethyl acetate phase. The ethyl acetate phase is washed with 10% NaCl in water and dried over,. At this stage there is a danger of the product falling into the pellet. The ethyl acetate phase is evaporated to a low volume and the product is precipitated with petroleum ether, and as a result heavy crystals of Ib are formed, according to TS, which are pure product. Yield: 36.9 g (80%) Ib; , 45 (A); 30.3 (pp. 1, 0 Meta nol); IbCbo-p-Glu-Gly-Arg (NO,) pNA (m..641,6). 10.25 g (18.3 mmol) of H-Gly-Arg (N0 / pNA HBr, prepared by deprotecting Cbo-Gly-Arg / N02) pNa with HBg according to the method described in 1a is dissolved in 35 ml of dry dimethylformamide and neutralized at low temperature () with EtjN to obtain an alkaline reaction on moistened indicator paper. The EtNiHBr formed is filtered off. 2.47 g (18.3 mmol) of oxybenzotriazole and 5.26 g (20 mmol) of CO-p-Glu-OH (Ib) are added to the reaction mixture and then at a low temperature 4.53 g (22 mmol) are added dicyclohexylcarbodiimide, dissolved in a small amount of dimethylformamide. The mixture is left overnight at room temperature, then the reaction solution is evaporated and the resulting oil is triturated with 2% NaHCOj in water and with pure water. The resulting solid compound is triturated in a small amount of acetone, and then hot methanol and a small amount of water are added. Pro 4, the crystallizes upon cooling and stirring, and the resulting crystals are filtered. The resulting lb is a pure product according to the results of chromatography on a thin layer. After thoroughly drying in vacuo, 8.90 g (76%) of Ib are obtained. , 06 (R); 2.5 x O., 8 dimethylformamide). 1.08 g (1.25 mmol) 1b is freed from protecting groups (released) with 30 ml of hydrofluoric acid at 0 ° C for one hour in the presence of 0.6 ml of anisole. After evaporation in vacuum, the product is dissolved in approximately 30 ml 2% acetic acid and washed with a small amount of ether. The aqueous phase is chromatographed on a column of Safadex G-15 (Pharmacia Fain Chemicals) in 2% acetic acid with the same medium for elution. The fraction with pure acetate 1 is lyophilized and passed through a column of ion exchange resin Q AE-25 (Pharmacia Fain Chemicals) in the chloride form in a mixture of ethanol / water (1: 1) and the same medium for elution. The fraction with pure hydrochloride I is lyophilized. 485 ml (78%) 1. Chromatography in a thin layer of Rf shows only one in y — 54.1 (p. 1.0, 50% xagic acid / water). Examples II-XIII, described table. I, carried out by the method of Example 1, physical constants, the outputs are given in Table. 1, the conditions for the coupling reaction and the cleaning methods are given in table. 2. Table 1
    Obzl
    Na0.19R,
    N0;
    Sun 84
    -24.9 95% EtOH
    Prod.pzh15nie tab.
    SPLA
    Continued table. one
    tone + meon
    Continued tabl .. 2
    I 2 HF, anisole, QAE-25 95% MeOH OpNP, crista. EtOAc + HOBT, DCCl, crista. BN + + EtOAc HF, anisole, G-15, 2% on HOAc 5 OpNP, crista. MeOH + H 0 HOBT, DCCl, crista. acetone + MeOH + H 020 HF, anisole, G-15, 2% on HOAc OpNP, crista. MeOH 25 HOBT DCCl, crista. MeOH + HF, anisole, G-15, 2% on jo HOAc OpNP, crista. EtOAc-1-sir HOBT, DCCl, crista. MeOH + HBr / HOAc, QAE 25 50% EtOH
    Tripeptides of general formula (I) obtained by the proposed method and the determination of proteases using substrates with groups subject to cleavage and determination by physicochemical methods have been tested.
    The substrates prepared according to the examples given can be used to determine various enzymes as follows.
    .. 3 the definition is based on the fact that the cleavage product, formed as a result of enzymatic hydrolysis, has a UV spectrum that is essentially different from the UV spectrum of the substrate. Thus, all p-nitroanilide substrates of the invention, for example, are characterized by a maximum
    277904
    10 Continued table. 2
    one
    absorption at 310 with a molar extinction coefficient of about 12,000. At 405 nm, the absorption of these substrates almost completely disappears. p-Nitroanilide, which is released from substrates during enzymatic hydrolysis, has an absorption maximum at 380 nm and a molar extinction coefficient of 13,200, which at 405 nm is reduced only to 9620. Therefore, by means of spectrophotometric data at 406 nm, it is easy to monitor the amount of p-nitroanilide formed, which is proportional to the rate of znimamaticheskogo hydrolysis, which, in turn, It is determined by the amount of active enzyme. OpNP, crista. MeOH + H 0 HOBT, BCCl, crista. MeOH + HBr / HOAc, QAE-25 50% EtOH OpNP, crista. MeOH + H O HOBT, DCCl, crista. acetone + MeOH Hbr / HOAc, QAE-25, 50% EtOH OpNP, MeOH + H ,, 0 HOBT, DCCl, acetone + MeOH HF, anisole, QAE-25, 95% MeOH OpNP, ethyl acetate + HOBT ether , DCCl DMF-ethyl ethyl acetate HF, anisole, QAE 25 50% EtOH OpNP MeOH + H 0 EtOH + H 0
    1) 127790412
    In tab. Figure 3 shows the values of each enzyme, the reaction rate pz, the rates of reaction of the synthetic substrate relative to the substrate of comparison of strata with the enzymes. For (reaction rate 1).
    T a b l and c a 3
    权利要求:
    Claims (1)
    [1]
    SRI of serine and SH proteases. Invention Formula
    Table 4
    Plasmin
    Trypsin
    Fxa
    Urokinase I
    where A is Ala, Gly, Val, Leu, Ser, Thr, Pro, Phe, He, Pip, Tyr; A, - Arg, Lys;
    R is p-nitroanilide, characterized in that the amino acid derivative of formula 11,
    where R have the indicated meanings,
    interact with the corresponding amino acid and then the required peptide structure is gradually increased by combining the remaining amino acids, the R group being used. As can be seen from the tables above, it is used as a protective
    The proposed substrates are hydrolyzed by the C-terminal carboxyl group with perraction enzymes faster than the resulting amino acid.
    Tissue activator
    one
    0.040
    55
    known, and can be used as reagents in determining
    SRI of serine and SH proteases. Invention Formula
    The method of obtaining tripeptides of General formula 1
    n-Glu-A-A-NH-R
    where A is Ala, Gly, Val, Leu, Ser, Thr, Pro, Phe, He, Pip, Tyr; A, - Arg, Lys;
    R is p-nitroanilide, characterized in that the amino acid derivative of formula 11
    55
    类似技术:
    公开号 | 公开日 | 专利标题
    SU1277904A3|1986-12-15|Method of producing tripeptides
    US4061625A|1977-12-06|Novel chromogenic thrombin substrates
    FI56829C|1980-04-10|NYA DIAGNOSTIC VERKSAMMA SUBSTRAT MED HOEG SPECIFICATION FOR TRYPSIN OR ANDRA ENZYMER AV TYPE PEPTIDYLPEPTIDHYDROLASER
    CA1161432A|1984-01-31|Tripeptide derivatives and their application inassaying enzymes
    EP0046742B1|1984-06-27|Peptide substrates for determination of protease activity
    US4214049A|1980-07-22|Assay of serine proteases using novel chromogenic enzyme substrates
    PL90746B1|1977-01-31|
    EP0170797B1|1987-09-16|Novel substrate for determining the activity of blood coagulation factor xa |
    SU786853A3|1980-12-07|Method of detecting activity of x-prolyldipeptidylaminopeptidane
    US4221706A|1980-09-09|Chromogenic substrates
    US4428874A|1984-01-31|Tripeptide derivatives
    US4247454A|1981-01-27|Novel chromogenic thrombin substrates
    US4216142A|1980-08-05|Chromogenic substrates for the proteolytic enzymes
    US4568636A|1986-02-04|Tripeptide derivatives
    SE448167B|1987-01-26|PEPTIDE DERIVATIVES TO USE AS SUBSTRATE FOR DETERMINATION OF ENZYMATIC ACTIVITY
    DK145799B|1983-03-07|TRIPEPTIDES OR SALTS THEREOF USED AS DIAGNOSTIC CHROMOGENT SUBSTRATE WITH HIGH SPECIFICITY ABOVE THROMBIN AND THROMBIN SIMILAR ENZYMES
    US4406832A|1983-09-27|Peptide-type substrates useful in the quantitative determination of endotoxin
    GB2126720A|1984-03-28|A method for determination of enzyme activity
    US4169015A|1979-09-25|Novel chromogenic thrombin substrates
    US4443367A|1984-04-17|Peptide and peptolide substrates for mammalian collagenase
    Fridkin et al.1977|Thiolysis of O-2, 4-dinitrophenyltyrosines: Spectrophotometric monitoring of the reaction and its use in peptide synthesis
    US4569907A|1986-02-11|Thiopeptolide substrates for vertebrate collagenase
    US5097014A|1992-03-17|Chromogenic tripeptides
    US4629695A|1986-12-16|Peptide derivatives and use thereof as substrates for quantitatively assaying enzymes
    JP2929555B2|1999-08-03|Chromogenic substrate
    同族专利:
    公开号 | 公开日
    EP0004256B1|1982-07-21|
    DK147495C|1985-07-15|
    PL213217A1|1980-02-11|
    US4279810A|1981-07-21|
    US4335204A|1982-06-15|
    ES477501A1|1979-11-16|
    DE2963358D1|1982-09-09|
    PL116682B1|1981-06-30|
    CA1136124A|1982-11-23|
    EP0004256A1|1979-09-19|
    JPS55500076A|1980-02-14|
    ATA78279A|1982-03-15|
    AT368773B|1982-11-10|
    AU4394579A|1979-08-16|
    NO790375L|1979-08-08|
    WO1979000602A1|1979-08-23|
    SE7801373L|1979-08-08|
    JPS6345397B2|1988-09-09|
    DK420879A|1979-10-05|
    IL56520D0|1979-03-12|
    JPS63173600A|1988-07-18|
    FI790376A|1979-08-08|
    DD142550A5|1980-07-02|
    DK147495B|1984-09-03|
    ZA79424B|1980-01-30|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    SE380257B|1972-05-02|1975-11-03|Bofors Ab|NEW DIAGNOSTIC OPERATING SUBSTRATES WITH HIGH SPECIFICITY FOR THROMBIN AND OTHER PROTEOLYTIC ENZYMES OF THE PEPTIDYL-PEPTIDE HYDROLASES|
    IL42124A|1972-05-02|1977-02-28|Kabi Ab|Substrate for the determination of proteolytic enzymes|
    DE2527932C2|1974-07-02|1983-04-21|Pentapharm AG, 4052 Basel|Acid addition salts of tripeptide derivatives and their use as substrates for the determination of plasma kallikrein|
    SE407058B|1974-12-05|1979-03-12|Kabi Ab|NEW CHROMOGENA ENZYME SUBSTRATE FOR SERINE PROTEASES|
    CH622286A5|1975-06-23|1981-03-31|Pentapharm Ag|
    SE407571B|1975-07-11|1979-04-02|Kabi Ab|NEW CHROMOGENA ENZYME SUBSTRATE FOR SERINE PROTEASES|
    SE437153B|1976-12-01|1985-02-11|Kabi Ab|SPECIFIC CHROMOGENA ENZYM SUBSTATE FOR SERINE PROTEASES|
    US4215047A|1977-06-06|1980-07-29|Ajinomoto Company Incorporated|7--4-methylcoumarins|US4275153A|1978-08-03|1981-06-23|American Hospital Supply Corporation|Analytical fluorogenic substrates for proteolytic enzymes|
    US4336186A|1978-08-03|1982-06-22|Gargiulo Robert J|Analytical fluorogenic substrates for proteolytic enzymes|
    US4409140A|1979-04-23|1983-10-11|Smith Robert E|Substrates and method for determining enzymes|
    DE3061860D1|1979-04-24|1983-03-17|Marcel Jozefonvicz|Process for the determination of proteases and antiproteases|
    DE2936543A1|1979-09-10|1981-04-09|Behringwerke Ag, 3550 Marburg|CHROMOGENIC COMPOUNDS|
    CA1161432A|1980-02-12|1984-01-31|Lars G. Svendsen|Tripeptide derivatives and their application inassaying enzymes|
    DE3164437D1|1980-08-25|1984-08-02|Kabivitrum Ab|Peptide substrates for determination of protease activity|
    FR2497798B1|1981-01-09|1984-03-30|Pharmindustrie|
    US4388233A|1981-05-15|1983-06-14|The Regents Of The University Of California|Synthetic substrates for enzyme analysis|
    US4510241A|1981-09-03|1985-04-09|Mallinckrodt, Inc.|Peptide-type substrates useful in the quantitative determination of endotoxin|
    JPS619840B2|1981-10-14|1986-03-26|Nitto Boseki Co Ltd|
    US4448715A|1981-11-02|1984-05-15|University Of Miami|Tagged pyroglu-L-Phe-L-Arg derivatives, substrates and assays for kallikrein|
    DE3211254A1|1982-03-26|1983-09-29|Boehringer Mannheim Gmbh, 6800 Mannheim|METHOD FOR DETECTING THE PRESENCE OF AN ALLERGY AND FOR SPECIFIC DETECTING THE ALLERGY RESPONSIBLE FOR THE ALLERGY|
    US4491541A|1982-11-10|1985-01-01|Farmitalia Carlo Erba|Peptides|
    DE3244030A1|1982-11-27|1984-05-30|Behringwerke Ag, 3550 Marburg|CHROMOGENIC COMPOUNDS, METHOD FOR THEIR PRODUCTION AND THEIR USE|
    DE3484912D1|1983-06-03|1991-09-19|Pentapharm Ag|PEPTIDE DERIVATIVES AND THE USE THEREOF AS SUBSTRATES FOR THE QUANTITATIVE DETERMINATION OF ENZYMES.|
    JPH059068B2|1984-07-04|1993-02-03|Nitto Boseki Co Ltd|
    CA1293591C|1985-01-11|1991-12-24|Charles A. Kettner|Peptide substrates for detecting virus-specified protease activity|
    US4801534A|1985-05-28|1989-01-31|Coulter Electronics, Inc.|Water soluble zanthylium derivative substrates|
    US4694070A|1985-05-28|1987-09-15|Coulter Electronics, Inc.|Water soluble xanthylium derivatives substrates|
    US5231006A|1986-10-16|1993-07-27|Behringwerke Aktiengesellschaft|Method for the determination of plasminogen|
    DE3635191A1|1986-10-16|1988-04-21|Behringwerke Ag|METHOD FOR DETERMINING PLASMINOGENS|
    SE8701801L|1987-04-30|1988-10-31|Kabivitrum Ab|PROCEDURE FOR IDENTIFYING MICRO-ORGANISMS|
    US5115099A|1988-06-14|1992-05-19|Nitto Boseki Co., Ltd.|Substrates for determination of enzyme activity and intermediates for synthesis of the substrates as well as process for producing the intermediates|
    JPH06104079B2|1988-07-14|1994-12-21|日東紡績株式会社|New enzyme activity measurement substrate|
    DE3839379A1|1988-11-22|1990-05-23|Hoechst Ag|METHOD FOR PRODUCING TRIPEPTIDES|
    US5191065A|1988-11-22|1993-03-02|Hoechst Aktiengesellschaft|Process for the preparation of tripeptides|
    SE8904188D0|1989-12-12|1989-12-12|Kabivitrum Ab|CHROMOGENIC SUBSTRATE|
    JP2769243B2|1990-02-19|1998-06-25|ノボノルディスクアクティーゼルスカブ|Method for recovering purified EPI protein from solution, especially fermentation solution|
    US5871946A|1995-05-18|1999-02-16|Coulter Corporation|Method for determining activity of enzymes in metabolically active whole cells|
    US5776720A|1995-05-18|1998-07-07|Coulter Corporation|Assay reagent|
    US5698411A|1995-05-18|1997-12-16|Coulter Corporation|Method for determining activity of enzymes in metabolically active whole cells|
    US5733719A|1995-05-18|1998-03-31|Coulter Corporation|Method of making an assay compound|
    WO2001094332A1|2000-06-02|2001-12-13|Regents Of The University Of California|Profiling of protease specificity using combinatorial fluorogenic substrate libraries|
    GB0208989D0|2002-04-19|2002-05-29|Amersham Biosciences Uk Ltd|Methods for measuring enzyme activity|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    SE7801373A|SE7801373L|1978-02-07|1978-02-07|EASY SPLABLE SUBSTRATE FOR QUANTIFIATION OF PROTEASES|
    [返回顶部]