TW402606B - New fluorogenic substrates for assay of angiotensin converting enzyme - Google Patents

Abstract

Disclosed are new fluorogenic substrates for assay of angiotensin converting enzyme, a process for preparing them and methods for using them to assay angiotensin converting enzyme and to screen antihypertensive agents which inhibit angiotensin converting enzyme.

Description

No. 8 3 1 Ο 8 Ο 8 Patent No. 8 please file the Chinese manual to revise page E of December U5 December 1 丨 3606 5. Description of the invention (

, Renin [22] and other endopeptidases EDANS, and the recipient is very suitable for endogenous enzymes (e X 〇pept ί dase) The purpose of the present invention is to raise the fluorescent substrate, K can be divided into K Another object is a simple and sensitive method for analyzing a further blood pressure drug of the present invention, which is an antihypertensive drug. . The providers in these matrices are D i \ B C Y L connected to the C-terminus. Although these matrix analyses are not suitable for applications other than A C R, etc. The invention provides a novel analysis of A (: E activity with good enzyme kinetic properties. Pi 丨 侫 provides a method for using the novel fluorescent-based ACE activity of the present invention. The system provides a method for detecting ACE inhibition. Quickly and surely screen the schematic diagram of ACE inhibitory activity. Figure 1 Series M Fluorescence Spectrometer Detecting Wine UCE Hydrolyzed ES-FWF (5 (2-Please read the note on the back ¥ Write this page to order the center of the Ministry of Economic Affairs Standards Bureau employee consumer cooperatives print 睽 ethyl :) Amine] naphthalene-1-sulfonyl succinyl- phenylalanine-tryptophan-clavulanic acid matrix) Figure 2 1¾ W. SS- FWF matrix screening results of AC E inhibitors. Detailed description of the invention _Explanation The present invention has found that EDANS and · tryptophan are good fluorescent extractor-recipient pairs, and W can be used to design an intramolecular quenching fluorescent matrix. It is K, and the present invention provides a fluorescent substrate having the formula: fc — L k — A Α ~ AA a — AA 3 — AA λ where E is ί5-[2 -aminoethyl) amino] naphthalene-1 Written as EDANS group), sulfonic group (condensed

vU 8-This paper size applies Chinese National Standard (CNS) A4 specification (210 > < 297 mm) A7 B7 a〇S6Q6 V. Description of the invention (1% 聃 section 蟪 The present invention is used to analyze vasoconstriction Novel fluorescent matrix with Angiotensin Converting Enzyme activity and preparation method thereof * Use this novel fluorescent matrix to analyze angiotensin converting enzyme activity and screen for heparin and glucagon converting enzyme inhibitor antihypertensive drugs Method °-发明 背 # Free tube shrinkin converting enzyme (ijigiotensin CLonvgrting ELuzyme 'is abbreviated as ACE, and its classification number is EC 3.4.15.1, which is a diipeptidyl carboxypeptidase. It is also found in ~ * netalloprotease containing fresh metalloproteinases, which can catalyze the release of a peptide at the C-terminus of the hydrolyzed matrix [1]. Its most well-known physiological function involves renin-angiotensin- The physiological response of the system (Lenin-Lngiotensin-.S_ysteni, referred to as RAS) converts the non-physiological decapeptide Angiotensin I into Angiotensin E. This catalysis The hydrolysis reaction is as follows:

Asp-A, g-Val-Tyr-Ile-His-Pro-Phe-His_Leu (ifil tube contraction

Prime I) ^ ACE

Asp-A 「s, -Val-Tyr-Ile-His-Pr.o-Phe (angiotensin Π) + His ~ Leu [2] Angiotensin] I is the most important function transmitter in RAS, which can trigger A variety of physiological reactions, including vasoconstriction and stimulation of the adrenal cortex to release sodium aldosterone, which increases blood pressure; therefore, 'it is closely related to the cause of hypertension [3,4] ° Please Read the note below and fill in% This page r Printed by the shellfish consumer cooperation department of the Central Bureau of Standards of the Ministry of Economic Affairs 83.3. 10,000 Β7—

Patent No. 8 3 1 0 S Ο 8 No. 8 is filed for 49 you 6¾ 馔 Books Rev. E page (IFeb 85) A7 V. Description of the invention (gi-based, such as unary, binary or polyunsaturated and unsaturated Esters of aliphatic carboxylic acids; aldehyde groups; carboxyl groups, such as those derived from mono-, di- or poly-saturated and unsaturated aliphatic carboxylic acids; and other similar functional groups. For example * Won.g, SS (19 9 1) Chetnistry of Protein Conjugation and (: ross-L 丨 nking · CRCP ress, [nc · [2:;]] 卽 revealed a variety of functional groups that can be condensed with amine groups, the full text of this document and the Taiwen, as References herein. At least two functional groups possessed by the compound may be the same or different as long as they can be inducted with the amine groups of EDANS and amino acids, respectively. Therefore, the bridging system according to the present invention is derived from having at least two functional groups. Each compound is the same or different functional group selected from the group consisting of halo, alkoxy, ester, aldehyde, carboxyl and similar functional groups. Please read the precautions on this page and fill in this page. Dioxane, 4-penta 6-chlorochlorocarboxanase 3 -Propane, 2, bromine 1, such as ii. Rattan; guanxi 1 'chloroane 1' di, di-diary, same as tributyl, 5- phytic acid acid alkane 3-chloroane 1. ^ 合 二Dichloromethane is used for the conversion of ethylene 2, dibutyl-ι / ίpentene halide and more than 1 4 chloroalkane-V halogen, butane or two, 1.tripentane, chloro chloride and other two groups, 4 chloride .6. Tossa, M amine 1, propane 2 'di 1 or dichloride and proton, 1.5-¾ dicarboxylic acid vinegar and cananane diammonium, 1, ^., Perdichlorine with ethyl 3-triane '㈣ 物 M' acid 簏 two-phase Chun; 1..3-butane-1 combination, two or three less by two, 2, 鲟] one, fenenoic acid as 2-alkane 1, two exchange , 6 Alcohol-like. Limited 1, propane, 4-tril halo-di-cis: Tart. With molybdenum, 1.4-finic acid, melamine, 2-propane, 2-tw, dichlorogas, ethyl 3-bromoane l.Ia. Propionoids? !! Too much chlorine 1. · tributane, ja, dioxin, dioxane-1, alkane chloride_: tired acid for use 'combined 2-alkane 2. dibutene Pentamate is suitable for 1, propane 1..4-bromo.1 dichloromethane, 1.triamenedioic acid-, this paper size applies Chinese National Standard (CNS) A4 specification (210X297 (Mm) ^ 02606 A7 B7 V. Description of the invention (2) It is currently recommended to treat hypertension with M Agents can be divided into four major categories: ⑴ calcium ion antagonists, ⑵ACE inhibitors, ⑶ diuretics, and ⑷; s-sympathetic nerve inhibitors. Among them, ACE inhibitors have the ability to inhibit ACE from converting angiotensin I to heparin The function of ϋ, and prevent the rise of blood pressure, is a kind of good anti-hypertensive 槩 [5]. A simple and sensitive ACE activity analysis method is an indispensable tool for the development of ACE inhibitor antihypertensive drugs. At present, the most commonly used base is glycosaminoglycan, which releases the equine matrix and separates with glycosyl glycine. acid. Quantitative determination of glycine 醯 released by Μ [8] c All of the above methods Screening for hypertension drugs Analysis of ACE activity • Leucine as the reaction matrix, the rate of uric acid. This measuring method measures the absorption of ACE at a wavelength of 228 nm and measures the similarity of ACE activity. This matrix is one of the three bases of glycine M hydrate when analyzing ACE activity. Make the photogenicity of horse uric acid with water [6,7]. The substance is horse urinary chromeglycinamine, which undergoes ACE catalyzed hydrolysis and the ketone (ninhydrin) reaction is quite time-consuming, and is not suitable for use as a large amount of ACE inhibitors. If a compound can absorb light at one wavelength, please read the note on the back ^^! ^ And fill in ^ -this page)-Pack. Order 0. The Ministry of Economic Affairs Central Standard Bureau Staff Consumer Cooperative printed compound has fluorescent Optical properties, can be used for the detection and analysis of fluorenyl-diaminoamine [methyl-leucine as the reaction matrix, or the hydrolysis of the substrate by the sister amine dibenzyl (o-phthalaldehyde, OPA) [9, (f 1 uorescamine) [11] and other fluorescent properties to determine the activity of ACE by fluorescent reaction [12]. A wavelength of light, then this. For example, the aforementioned method for measuring ACE activity in K equine urine is based on the reaction of leucine dipeptide with ortho-10] or dance amine compounds, and then the private paper size applies the Chinese National Standard (CNS) A4 specification (2 丨 0X297). ) 83. 3.10,000 No. 8 3 1 〇 8 〇 8 No. 8 Patent Examination Case Chinese Manual Amendment Page (December 1985) A7 _2Na --- 5. Description of Invention (Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs

ΗΜΡ DC Μ Η B t HBTIJ ί) IEADMS 0 ACE ί] 1 y > GA 1 a > Price V ai 'V Leu * L ί U, Ϊ Asp > 0 fi iu, KA rg > RL ys, K His, HP he 1 FT yr · Y Try * y P r ο > P KS

1-hydroxypyridotriazole 2-(1 fluorene-cagetriazole-1 -yl) -1,1,3,3-tetramethylurea iron hexafluorophosphate diisopropylethylamine dimethylphosphine Contractin-Converting Enzyme Glycine Alanine _ Valine Acid _ Leucine Isobutyric Acid Aspartic Acid Branexic Acid Spermic Acid Sadonic Acid Lysine Histamine Cage Propionate Tryptophan Acid Tryptophan Amino acid 5.-[(2-Fluoroethyl) fluorenyl-Laptonylnaphthalene 6 yellow 1 9-(Please read the precautions on the back before filling this page)

This paper size is in accordance with Chinese National Standard (CNS) A4 (210X297 mm) Central Government Standards Bureau of the Ministry of Economic Affairs w: Industrial and Consumer Cooperative Cooperatives 4_〇β ___ V. Description of Invention (3) Another type of fluorescence analysis method The activity of hydrolyzing enzymes is determined by the change in fluorescence intensity of intramolecularly quenched fluorogenic substrate [13]. It is different from the aforementioned mechanism of wild light analysis. It is related to resonance energy transfer (resonance eng.ergy transfer). 0. Although resonance energy transfer is also called non-radiative energy transfer, it means that an excited fluorescent substance will absorb it. Although the energy can be transferred to another within a proper distance of the same system-the phenomenon of fluorescent quality * the former is called the donor * the latter is called the acceptor [13,14]. In general, the emission spectrum of the provider often overlaps with the excitation spectrum of the receiver. The most transfer of resonance energy is not caused by direct impact of molecules. When energy transfer occurs in a system, the system is excited by the wavelength of the absorption peak of the provider, and when the emission wavelength of M is detected, its fluorescence intensity is significantly reduced. This phenomenon is called quenching [14] ]. However, in this system, fluorescence can be measured when it is irradiated at another wavelength, such as the receiver's emission wavelength. The efficiency of resonance energy transfer can be expressed by the following formula [15]:, p _ 1 F _ Γο e_i '~ --- i ~~ 6F. r + r〇 where F and the fluorescence intensity of the provider in the presence and absence of the recipient, respectively, "is the distance between the center of the provider and the recipient, and between the provider and the recipient when the transfer efficiency is 50 It can be known from the above formula that the energy transfer efficiency is subject to the emission spectrum of the provider and the recipient, please read the note on the back before filling in ^ 'this page) Order © Standard Chinese paper (CNS) A4 size (210X29) * 7 mm) 83. 3.10,000 i030〇6 A7 B7 V. Description of the invention (4) The overlapping part of the absorption spectrum and the s separation between the two fluorescent substances are affected. The hydroxyl experiment confirmed that The use of the donor-acceptor pair can show the distance between the two fluorescent substances. "Intramolecular quenching fluorescent matrix J system has a pair of energy providers and recipients at the same time. The fluorescent property of the donor is quenched in the molecule, and there is a bond between the provider and the recipient that can be cleaved by the enzyme. The reason is that when the knot is cleaved by the enzyme * because of the quenching between the provider and the recipient. The system terminates and emits fluorescence [13]. Such molecules have been used in water Analysis of enzyme activity. Intramolecular quenching fluorescent matrix peptides known to be useful for ACE active tillers include p-nitrobenzylaminocarbonylglycinamido-fluorene-tryptamine-glycolyl-glycolic acid [16]. O-Aminobenzyl [Gly-Glyamine-P-nitro-L-amphetamine-L-Proline [17], 1-Dimethylaminonaphthalene-5-sulfonyl-glycine (Please read first Note on the back ^^ 3, then fill it like Benwa). Azatoki-p-nitro-L-phenylalanine [18] and 1,7-di-ming- 2,3,5,6-tetramethyl-1H, 7H-pyrazolo [1,21] pyrazole (referred to as bimane) [19,20]. Among them, the bieane matrix has difficulty in dissolving due to the hydrophobicity of the molecule The problem is that it must be dissolved in the ammonium solvent when used. The most commonly used organic solvent for dissolving bimane is dimethylimide (DMS0) [19,20] ° Although the DHS0 added to the analysis mixture is not high, its It can significantly reduce the activity of ACE, which affects the accuracy of the analysis. In addition, 5-[(2-aminoethyl) amino] naphthalene-1 -sulfonic acid (abbreviated as EDANS) and 4-(4-dimethylamine Amine, phenylphenylazo) benzoic acid (abbreviated as DABCYL) is also widely used in endopeptides Quenching fluorescent substrate of endopeptidase, .M analysis, such as HIV-1 protease [21], HAV-3C protease 〇 Private paper scale is applicable to China National Standard (CNS) A4 (210X297 mm) 83 . 3.10,000 Patent No. 8 3 1 Ο 8 Ο 8 Patent No. 8 please apply for Chinese manual to revise page E U5 December 1 丨 3606 5. Description of the invention (

, Renin [22] and other endopeptidases EDANS, and the recipient is very suitable for endogenous enzymes (e X 〇pept ί dase) The purpose of the present invention is to raise the fluorescent substrate, K can be divided into K Another object is a simple and sensitive method for analyzing a further blood pressure drug of the present invention, which is an antihypertensive drug. . The providers in these matrices are D i \ B C Y L connected to the C-terminus. Although these matrix analyses are not suitable for applications other than A C R, etc. The invention provides a novel analysis of A (: E activity with good enzyme kinetic properties. Pi 丨 侫 provides a method for using the novel fluorescent-based ACE activity of the present invention. The system provides a method for detecting ACE inhibition. Quickly and surely screen the schematic diagram of ACE inhibitory activity. Figure 1 Series M Fluorescence Spectrometer Detecting Wine UCE Hydrolyzed ES-FWF (5 (2-Please read the note on the back ¥ Write this page to order the center of the Ministry of Economic Affairs Standards Bureau employee consumer cooperatives print 睽 ethyl :) Amine] naphthalene-1-sulfonyl succinyl- phenylalanine-tryptophan-clavulanic acid matrix) Figure 2 1¾ W. SS- FWF matrix screening results of AC E inhibitors. Detailed description of the invention _Explanation The present invention has found that EDANS and · tryptophan are good fluorescent extractor-recipient pairs, and W can be used to design an intramolecular quenching fluorescent matrix. It is K, and the present invention provides a fluorescent substrate having the formula: fc — L k — A Α ~ AA a — AA 3 — AA λ where E is ί5-[2 -aminoethyl) amino] naphthalene-1 Written as EDANS group), sulfonic group (condensed

v.U 8-This paper size applies Chinese National Standard (CNS) A4 specification (210 > < 297 mm) 403606 A7 B7 V. Description of the invention (6)

Lk is a linking arm * pseudo-derived from a compound containing at least two functional groups that can be condensed with an amine group, Ah is a direct bond, or an amino acid or amine group formed by any protein other than tryptophan Residues of acid derivatives, AA2 is the residue of any amino acid or amino acid derivative of any protein other than tryptophan, " * AA3 and AA4 are the amino acids of any protein Or amino acid derivative residues, but at least one of AA3 and AA 4 is: tryptophan residue. 5-[(2-Aminoethyl) amino] naphthalene-1 -sulfonic acid (EDANS) knot (please read the note on the back to fill this page) ~-wear. The structure is as follows: h2 tr HN ^ φόs〇3h , Ch2

Line C. Printed by the Shellfish Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs. It is used as the energy provider fluorescent base in the novel fluorescent matrix of the present invention. In the novel fluorescent group of the present invention, Lk represents an earning substance, which is derived from a compound having at least two functional groups which can be condensed with amine groups of EDANS and amino acid residues of the matrix peptide molecule, respectively. Functional groups that can be condensed with -NH2 radicals are well known in the art and include, but are not limited to, halo radicals * such as gas radicals, aryl radicals, bromo and iodine radicals; fluorenyl radicals, such as those derived from mono- or di- or Polyethylenic group of polyvalent saturated and unsaturated aliphatic carboxylic acid; 9-Applicable for private paper size-Chinese Standard (CNS) A4 (210X297) 8.3.10,000 Β7—

Patent No. 8 3 1 0 S Ο 8 No. 8 is filed for 49 you 6¾ 馔 Books Rev. E page (IFeb 85) A7 V. Description of the invention (gi-based, such as unary, binary or polyunsaturated and unsaturated Esters of aliphatic carboxylic acids; aldehyde groups; carboxyl groups, such as those derived from mono-, di- or poly-saturated and unsaturated aliphatic carboxylic acids; and other similar functional groups. For example * Won.g, SS (19 9 1) Chetnistry of Protein Conjugation and (: ross-L 丨 nking · CRCP ress, [nc · [2:;]] 卽 revealed a variety of functional groups that can be condensed with amine groups, the full text of this document and the Taiwen, as References herein. At least two functional groups possessed by the compound may be the same or different as long as they can be inducted with the amine groups of EDANS and amino acids, respectively. Therefore, the bridging system according to the present invention is derived from having at least two functional groups. Each compound is the same or different functional group selected from the group consisting of halo, alkoxy, ester, aldehyde, carboxyl and similar functional groups. Please read the precautions on this page and fill in this page. Dioxane, 4-penta 6-chlorochlorocarboxanase 3 -Propane, 2, bromine 1, such as ii. Rattan; guanxi 1 'chloroane 1' di, di-diary, same as tributyl, 5- phytic acid acid alkane 3-chloroane 1. ^ 合 二Dichloromethane is used for the conversion of ethylene 2, dibutyl-ι / ίpentene halide and more than 1 4 chloroalkane-V halogen, butane or two, 1.tripentane, chloro chloride and other two groups, 4 chloride .6. Tossa, M amine 1, propane 2 'di 1 or dichloride and proton, 1.5-¾ dicarboxylic acid vinegar and cananane diammonium, 1, ^., Perdichlorine with ethyl 3-triane '㈣ 物 M' acid 簏 two-phase Chun; 1..3-butane-1 combination, two or three less by two, 2, 鲟] one, fenenoic acid as 2-alkane 1, two exchange , 6 Alcohol-like. Limited 1, propane, 4-tril halo-di-cis: Tart. With molybdenum, 1.4-finic acid, melamine, 2-propane, 2-tw, dichlorogas, ethyl 3-bromoane l.Ia. Propionoids? !! Too much chlorine 1. · tributane, ja, dioxin, dioxane-1, alkane chloride_: tired acid for use 'combined 2-alkane 2. dibutene Pentamate is suitable for 1, propane 1..4-bromo.1 dichloromethane, 1.triamenedioic acid-, this paper size applies Chinese National Standard (CNS) A4 specification (210X297 (Mm) 403606 V. Description of the invention (8) A7 B7 Shellfish Consumption Printed acid di- or polyesters such as oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, succinic acid, fumaric acid, tartaric acid, or dialkyl esters of citric acid, such as Dimethyl, diethyl, dipropyl, dibutyl and similar esters, or trialkyl citrate, such as trimethyl acetate, triethyl, tripropyl, tributyl and similar depleted esters , Or bis-H-hy'droxysucciniaidyl esters, 1-> 13-nitfrophenyl esters or bis-i ^ idoesters, And similar esters; acid anhydrides, such as oxalic anhydride * malonic anhydride, succinic anhydride, glutaric anhydride, adipic anhydride, and similar anhydrides; dialdehydes, such as oxalic acid * malonic acid aldehyde, succinaldehyde, glutaraldehyde , Adipaldehyde and the like; di- or polycarboxylic acids, such as oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, succinic acid, fumaric acid, tartaric acid or. Clavulanic acid and similar carboxylic acids; and similar compounds. Suitable. Compounds having at least two different functional groups which can be condensed with an amine group for use in the present invention include, but are not limited to, di- or poly-substituted alkanes with different halogen atoms, such as dibromo-2-chloroethane * 1- Bromo-3-chloropropane and the like; uronic acid • such as malonic acid, butyranoic acid, valeric acid, hexanal acid, etc .; monoesters of di- or polycarboxylic acids and di- or polyesters of polycarboxylic acids, Such as oxalic acid, malonic acid, panic acid, glutaric acid, adipic acid, succinic acid, fumaric acid, _lithic acid or citric acid monoester and citric acid diester; and two or more Carboxylic acid monohalide or polycarboxylic acid dihalide compounds such as oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, maleic acid, fumaric acid, tartaric acid or citric acid Chlorine and dichloride citrate; and similar compounds. Preferably, the U-link arm of the novel fluorescent substrate of the present invention is derived from the two (please read the note on the back before filling this page) 柒. Order

Line C. This paper size is in accordance with Chinese National Standard (CNS) A4 (210X297 cm) 83. 3.1〇, 〇〇 403606 A7 B7 V. Description of the invention (9) 倨 Compounds with the same functional group are selected from dihalides Alkanes, halogenated dicarboxylic acid compounds, alkyl dicarboxylic acid esters, or bis-N-hydroxysuccinimide imide esters * bis-nitrophenyl esters or bis-imide esters • Dicarboxylic acids or anhydrides. More preferably, they are derived from acid anhydrides, especially M succinic anhydride. In contrast, a specific embodiment of the present invention is that the hydroxyl group is linked to Έ-DANS and oligopeptide molecules by succinic dipyridone. The novel fluorescent matrix of the present invention is a hydrocarbon-modified oligopeptide matrix * which may be a modified tripeptide molecule having three amino acid residues or a modified tetrapeptide molecule having tetra® amino acid residues. When it is a fluorene-modified tripeptide molecule, AAα is a direct bond. When it is a modified tetrapeptide molecule, AAI, AA2 can be the same or different, and can be any amine_acid residue without fluorescence, which can also be any protein amine other than tryptophan M Residues of amino acids or amino acid derivatives, please read the note on the back and then fill in 5?-This page), ya printed by the Consumers' Cooperative of the Central Government Bureau of the Ministry of Economic Affairs. At least one of them is tryptophan amino acid derived from the amino acid amino acid of the present invention, hydroxy * alanine glutamine

Novel fluorescein, ACE light matrix can catalyze the invention of novel oligomers-those called tryptophan acid residues, the so-called eggs, including lysine * aspartic acid, glycine-another residue. White matter sister Arginine, Methionine, Amino acid, M tryptophan is the fluorescent substance of energy receiver. The oligopeptide is hydrolyzed to release a C-terminal dipeptide. A diamino acid residue at the C-terminus of the fluorescent matrix. That is, at least one of αα3 and ααλ is any amino acid or amino acid of any protein, including naturally occurring proteins, including amino acids, leucine and isoleucine, lysine, and stupid. Alanine, tyrosine * valine, asparagine, cysteine, glutamate, proline, 4-hydroxyproline, serine

Line C-12-83. 3.10,000 sheet size is applicable to China National Standards (CMS) Α4 specifications (210X297 gong) A7 B7 V. Fenming instructions (10) Printed by the consumer consumer cooperatives of the Central Government Bureau of the Ministry of Economic Affairs Amino acids and tryptophan, etc .; and amino acid derivatives such as (H «-tB〇c) -lysine * D-alanine, D-glutamic acid, n-valine, 4- (E) -Butenyl-4 (R) _methylmethyl-L-glycine, N-methyl alimic acid (MeLeu) »limbyl isobutyric acid (ct -methylalanine, A ib) or 3 -(S) -hydroxy-4-(S) -amino-6-methylheptanoic acid (Sta). Preferably, if Ααα is not directly amine, it is an amine acid residue which is the same as or different from AA2, and the two are independently selected from phenylalanine, leucine, alanine, glycine, lysine and ( Ne-t-Boc) -amidate residue. One of AA3 and A / U is a tryptophan residue, and the other is selected from tryptophan, alanine, phenylalanine, leucine, alanine, glycine, glutamic acid, spermine Acid * proline, lysine and (He lysine residue. Preferably, it is a direct 鐽 | / A2 is selected from phenylalanine, leucine, alanine, glycine • lysine And (N «-tB〇C) _ lysine residues. When AA3 is a tryptophan residue, AA4 is selected from chromic acid, phenylalanine, leucine, glycine · lysine Amino acid, alanine, glutamic acid, arginine, and proline, and when AA4 tryptophan residue, AA3 is selected from tryptophan · epiamine, phenylalanine * leucine, alanine, Glycine, heteroamine, (Ne-t-Boc) -lysine residues. More preferably, Ah is a direct amidine, A is a phenylalanine residue, and one of AA3 and A / U is tryptamine. Acid residues, the other is selected from tryptophan, phenylalanine, glycine *, lysine, and leucine residues. Especially when AA3 is a tryptophan residue, AA4 It is selected from tryptophan, phenylalanine, glycine, amino acid, lysine and leucine * And when AA4 tryptophan residues, AA3 is selected from tryptophan and ammonium. -13-This paper size is applicable to China National Standard (CNS) M specifications (210X297 mm) 83. 3. 10,000 ( Please read the note on the back before filling in this page. I-Binding and Binding ο Line 402606 A7 B7 V. Description of the invention (11) The best substrate of the present invention has AAAϊ as a direct bond, A A2 is a phenylalanine residue * AA3 is Modified tripeptide molecules with tryptophan residues and AA4 being phenylalanine residues. The matrix of the present invention can be prepared by any conventional method, for example, a tripeptide or tetrapeptide molecule can be prepared by a solid phase method, and then A functional group of at least two linking arm compounds capable of condensing with an amine · functional group is conjugated with the OC-amine group of the three- or four-fe molecule, and then a peptide bond synthesis method is used to connect EDAHS to Satoshi For example, you can use the following procedure to prepare the preferred elegant embodiment of the present invention "EDAHS-succinyl-peptide": (Please read the note on the back-install-fill in this page ) '-5

Printed by the Consumer Standards Cooperative of the Central Bureau of Standards of the Ministry of Economics of the People's Republic of China 14 813. 10,000 Private paper standards are applicable to the Chinese National Standard (CNS) A4 specifications (210X297 mm) Na_ A7 B7 V. Fenming Instructions (I2) 〇 10 millimeters Molten peptide (^^) + 5 ml DO (Μ

+200 μl Et3N \ l added 0.2-0.3 millimolar succinic anhydride Μ

+1 ml of DCM 反应 react at room temperature for 1 hour \ 1 overdose V rinse with DCM 2-3 times dry 酸 acidification

V Add 5ml 3N HC1 and stir for 30 minutes (read the note on the back and then fill in this page). Packing. Ordering / drying and drying succinyl-peptide one (§ ||) + 1ml 2M DLEA / NMP ν +2 ml 0.4 5M HBTU / HOBt / DMF, // reaction for 20 minutes ^ add

〇4 mM EDANS Μ 亳 liter NMP +30 micro liter Et3N line printing reaction of the Central Standards Bureau Shellfish Consumer Cooperative of the Ministry of Economic Affairs 3 hours' 1 over 遽 NIT rinse and dry ^ TFA cleavage EDANS-axle brewing base-peptide -15-This paper is applicable to China National Standard (CNS) A4 (210X297mm) 83. 3. 10,000 402606 Β7 V. Description of the invention (13) Printed by the Staff Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs. Definitions of the abbreviations For details, please refer to the description below. In the novel fluorescent matrix of the present invention, the EDAHS group located at the H-terminus of the matrix molecule can transfer the absorbed energy to the acceptor fluorescein tryptophan residue at the C-terminus of the matrix molecule. Therefore, its fluorescent properties are quenched in the complete fluorescent matrix of the present invention. When the matrix of the present invention is cleaved by the ACE present in the test system, it will release the receptor containing tryptophan residues at the C-terminus. The dipeptide • At this time the quenching system is terminated • Fluorescence can be released. Therefore, the novel fluorescent substrate of the present invention can be used to analyze ACE activity. It has been verified by the invention * The novel fluorescent substrate of the present invention has good enzyme kinetic properties * Kcat / Km values and ACE analysis The matrix has a kcat / Km value that is equal to or higher • Can (more) quickly-and sensitively detect ACE activity. In addition, the novel fluorescent matrix of the present invention has good solubility, ranging from 7-40 g / liter water-soluble buffer, It can directly test the kinetic parameters of enzymes without using any amidine solvents to overcome the difficulty of using organic solvents that would affect the activity of ACE in the prior art containing Binane matrix. It is M. Another aspect of the present invention provides a method of using the present invention. Invented a novel fluorescent matrix method for analyzing ACE activity. When the novel fluorescent matrix of the present invention is used to analyze ACE activity, the matrix can be formulated into an appropriate concentration matrix solution with a suitable buffer solution. For example, it can be used as 8.2 and contains 0.2 MNaCl and 0.6MNa2SO < 1 in 50 mM Hepes buffer, the matrix was prepared into a 15 W Μ matrix solution. Then take an appropriate amount of matrix solution (such as 0.5 liters or 1.0 ml) and place the test sample (such as 20 microliters) In a quartz colorimetric tube for measuring fluorescence, use a fluorometer to measure the fluorescence change at 5 or 6 time intervals. -16-This paper size applies to China National Standard (CNS) Α4 specifications (210 × 297 mm) 83. 3. 10,000 (Please read the note on the back ^ 3¾Fill this page-0, β 403606 V. Description of the invention (l4) A7 B7 The Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs prints any fluorescent light Photometers can be used, the better one is a fluorescent photometer with an on / off function. For example, you can set the light source to turn on once every minute and irradiate for 1 second and 5 minutes each time to complete the activity test. You can also shorten the exposure The interval * shortens the test time. It has been proven that the light source can be set to turn on every 15 seconds. The activity test of a sample can be completed accurately within 1 minute. -The obtained fluorescence change data can be converted into a product concentration library as follows: E = 132.8C-2.7 where E is the measured fluorescence value and C is the product concentration (μM). Then based on the data of 5 or 6 points, use the regression equation to find the slope, and you can know the cost of the substrate per minute. Furthermore * As mentioned above, ACE inhibitors are a good class of antihypertensive drugs. A simple and sensitive method for analyzing ACE activity is an indispensable tool for the development of antihypertensive drugs such as ACE inhibitors. From the foregoing description, when analyzing the ACE activity using the novel fluorescent matrix of the present invention, the activity test of a sample can be completed within 5 minutes, and the shortest can be completed within 1 minute. It is Kr. The present invention further provides a method for quickly and accurately screening A CE inhibitors for antihypertensive drugs. The method specifically uses the novel fluorescent matrix of the present invention to detect and administer ACE inhibitors for antihypertensive drugs. Residual ACE activity after ACE enzyme solution. The method for detecting the substance with ACE inhibitory activity using the novel fluorescent matrix sieve of the present invention is similar to the method for analyzing the ACE activity described above. The matrix can be formulated with a suitable concentration (such as the 50mM Hepes element solution described above) to a suitable concentration (such as -17-The size of this paper is applicable to the National Standard of China (CNS) A4 (210X297 mm) 83.3.10,000 (Please read the note on the back ^^ before filling out this page) 40 ^ 606 5. Description of the invention (l5) Matrix solution of A7 B7 15 «Μ · Mix appropriate amount of matrix solution (such as 0.5 ml), enzyme solution (such as 20 microliters containing 2.6x10_4 units) and dilute the test sample to an appropriate concentration (such as 100 micrometers) Liters) or an equal volume of distilled water or tritium solution (blank control sister) · K fluorescence meter to determine the change in fluorescence. When the amount of fluorescence change of the test sample is lower than that of the blank control *, it can be inferred that the sample has A C € -inhibitory activity. It is also possible to directly dilute the sample to be tested or further dilute the sample that has been preliminarily determined as ACE inhibitor to 5 different concentrations. • Add them to the reaction solution containing two substrates with different matrix degrees. The reciprocal number is plotted against the measured degree of the sample, and the Ki value is obtained from the intersection of the two regression lines to confirm the inhibition ability of the sample to be tested. -The various objects and effects of the present invention will be further explained in the following non-limiting examples. The meanings of the abbreviations used herein and in the examples below are as follows: ΗMP-resin-(4-hydroxymethylphenoxymethyl) resin Fmoc-9-fluorenylmethoxycarbonyl TFA-trifluoroacetic acid 〇c --Butyl-Third butyl Please read the note on the back to fill in this page% Order a Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economy

t-B 〇c EDAMS DCC DAMP DMF 5- [(2-limylethyl) amino] naphthalene-i-dicyclohexylcarbonyldiimidedimethylaminopyridine hydrazone, N-dimethylformamidine Amine 18 This paper size applies to China National Standards (CNS) A4 (210X297 public variable) 83. 3.'10,000 No. 8 3 1 Ο 8 Ο 8 No. 8 Patent Examination Case Chinese Manual Amendment Page (December 85 ） Α7 _2na-— 5. Description of the Invention (Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs

ΗΜΡ DC Μ Η B t HBTIJ ί) IEADMS 0 ACE ί] 1 y > GA 1 a > Price V ai 'V Leu * L ί U, Ϊ Asp > 0 fi iu, KA rg > RL ys, K His, HP he 1 FT yr · Y Try * y P r ο > P KS

1-hydroxypyridotriazole 2-(1 fluorene-cagetriazole-1 -yl) -1,1,3,3-tetramethylurea iron hexafluorophosphate diisopropylethylamine dimethylphosphine Contractin-Converting Enzyme Glycine Alanine _ Valine Acid _ Leucine Isobutyric Acid Aspartic Acid Branexic Acid Spermic Acid Sadonic Acid Lysine Histamine Cage Propionate Tryptophan Acid Tryptophan Amino acid 5.-[(2-Fluoroethyl) fluorenyl-Laptonylnaphthalene 6 yellow 1 9-(Please read the precautions on the back before filling this page)

This paper size applies to Chinese National Standard (CNS) A4 specification (210X297 mm) A7 B7 V. Description of invention (l7)

Bin-1, 7 -bis Bf-2,3,5,6 -tetramethyl-1H, 7H -pyrazolo [1,2-a] pyrazolyl Hip Equine urine.

Abz-benzamidine

Dns'-1-Dimethylaminonaphthalene-5-sulfofluorenyl • FWf--Phenylalanine-Phenylalanine-Phenylalanine FWCT-Phenylalanine-Tryptophan-Glycine FWH-Phenylalanine- Tryptophan-Family FWK-Phenylalanine-tryptophan-lysine FWL-Phenylalanine-Tryptophan-Leucine FHW-Phenylalanine-Fermentine-Tryptophan -FUF, FS-FUn. KS-FUH RS-FUK »F. ^-FW). And RS-FHtf Xidan 楢 simultaneously use automatic and manual methods to prepare peptide matrix. Among the reagents used in the preparation of peptides, HMP (4-hydroxymethylphenoxymethyl) resin (0.92¾ Moraxon / g), Faoc-L_Gly-〇H »Faoc-L-Leu_0H > Fraoc-L- Phe-〇H · 1? 1 »〇 <:-1 ^ -1 >" -011 and? 111〇 (;-1 ^-} 1 丨 5 (trityl) -0} 1 were purchased from the United States Applied Biosystems (A pp 1 ied Bisystems); F boc-T rp-p-Isooxybenzyl ferment resin (0.76 dang / g), Fmoc-H is (trityl)-p-alkane Oxybenzyl alcohol resin (0.61 equivalent / g) and FraOC-LyS (Boc) -p-alkoxybenzyl ferment resin (0.42 equivalent / g) were purchased from the American Peninsula Corporation; trifluoroacetic acid (TFA) and Ethyl dithiol was purchased from the American company Aldrich; stupid _________- 20 -_ This paper size applies to Chinese national standards (CNS> A4 specification (21〇χ297 公 楚) 83. 3.10 000 (Please read the note on the back first * — 装--t (refill this page) Printed by the Shellfish Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs, 3606 A7 B7 Printed by the Shellfish Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (w) Methyl ether and succinic anhydride Purchased from Merck, Germany; EDANS From Sigma Company in the United States. First, P he-T rp-G was prepared in a 430A type automatic belly-belt synthesizer provided by Applied Biosystems in the United States. Y 'P he-T "p-L e U, Phe-Trp ~ Phe »Phe-Trp-His, Phe-Trp-Lys and. Ph-H is-Trp tripeptide. Use standard coupling method [24], although it is 0.25 mol. Use recommended reaction conditions. Briefly In other words, the synthesis of the winner is the use of Fboc-L-amino acid-p-alkoxybenzyl resin or HHP resin which can produce the C-terminus. * This resin can successfully couple the first Fmoc-amino acid to Resin. If you want to load the automatic synthesizer, add 0.25 奄 Moore HHP resin to the reaction tank; M di 嗄 hexylcarbonyldiimide (DCC) activates 1 millimolar Fnoc-amino acid into a symmetric anhydride Then, it is coupled with the resin for about an hour. Because the HMP resin has a hydroxyl group at the active position, 0.1 M dimethyl.aminopyridine (DMAP) in Ν, Ν -dimethylformamide solution (DMF) is added. ), Catalyzing the reaction. At the beginning of each cycle, a K weak base (for example, 20% hexahydropyridine) removes the Fmoc- group that protects the amino acid α-amino group. The standard reaction volume is 1.0 millimolar of dried amino acid contained in a reaction cassette dissolved in 1.2 ml of N-methylpyramine (NMP), 0.4-0.8 ml of dichloromethane (DCM). And 1 ml of a 1 M solution of hydroxybenzotriazole (HOBt) in NMP. Transfer the solution into the activation tank. Then add 1 liter of 1M DCC in NMP solution. After about 50 minutes of activation, the HOB t active ester was transferred into a reaction tank. The activated Fmoc-amino acid can react with the N-terminus of the amidine in the extension. Peptide bonds are formed. 430A automatic peptide synthesizer is extended by -21 per equivalent-This paper size is applicable to China National Standard (CNS) A4 (210X297 gigabytes) 83. 3.10,000 (Please read the note on the back * --- Then fill in ^ 'this page)

Line C ^ 02606 A7 B7 V. Description of the invention (19) The peptide chain reacts with 4 equivalents of activated amino acid. The standard time for a complete cycle is 92 minutes. After synthesis, ‘K hexahydropyridine deprotected the peptide-resin * and then washed with NMP and DCM. The post-synthesis procedure was performed using 0.10 mol. After carefully washing 3 times with 5 ml-L NMP and 3 times with 5 ml of DCM, the resin was floated in 5 ml of DCH and 200 µl of tri-Z amine. Add succinic anhydride (0.2-0.3 mol) dissolved in DCM. After one hour of reaction, the resin was filtered, washed with DCM, and dried under vacuum. K5 liters of 3H HCI acidified the peptide-resin with extended ligation for 30 minutes. Then • 2 (1Η-benzotriazol-1-yl) -1,1,3,3-tetramethyl can be used (please read note 111 ^ 3 on the back and fill in 3? -This page) The Ministry of Central Standards Bureau's Consumer Cooperatives printed the printed glandular gland extensions in the chemical industry, which should be quickly connected to the two. Adding the original coupling washing peptide-hexafluorophosphate (HBTU) as an activator, the EDANS group is connected to the peptide to form a peptide bond. HBTU is a new coupling reagent of amino acid and Fmoc / NMP. K HBTU activation is more complete than K carbonyldiimide as a catalyst, and the cycle time is shorter and the coupling efficiency is greater. The millimolar BTT was pre-dissolved in 2 ml of 0.5 M HOBt and DMF solution. Isopropylethylamine (DIEA) was added to the vacuum-dried acidified extension peptide-resin to initiate activation. The activation reaction was carried out for 20 minutes and a coupling of 0.4 mol EDAHS in triethylamine was performed for three hours. After synthesis, M SMP and DCM were pre-dissolved in. The resin was then dried overnight under vacuum at room temperature. K 10 liters of TFA: ethylenedithiol; anisole (95: 1.25 · · 3.75) was finally cracked for 1.5 hours. The solid support was filtered and rinsed with a little TF A lysis solution. The filtrate was collected and placed in 200 ml of cold B @@ * to form a precipitate. Then collect the Shen-5 Π line -22-83 with a sintered glassware of 10-15wM. 3. 10,000 This paper size uses the Chinese National Standard (CNS) A4 ^ Luo (210X297 Gongao). 5. Description of the invention (20) A7 B7 Printed by Yokohama Consumers Cooperative of the Central Bureau of Standards, Ministry of Economic Affairs * Wash thoroughly with ether. The dried product was redissolved in 20S! Acetic acid, filtered, and freeze-dried. Κ Reverse phase HPLC (Inertsil 5 0DS-2, 1 0.7 X 250 am) Passivated the crude matrix. MO.1% TFA in aqueous solution is mobile phase A. 80% acetonitrile 戾 0.1% TFA is used as mobile phase B. Use a gradient of 20% B to 90¾ B for 15 minutes, a gradient of 90% B to 90¾ B for 15 to 17 minutes, and a gradient of 9 (UB to 205ί B for 17 to 19 minutes. Flow rate is 3 liters / tamping shovel. Collect the desired substrate The eluate at the position of the absorption peak appeared. Then the collected sample was freeze-dried and the molecular weight was determined by a fast atomic impact mass spectrometer (JEOL JMS-HX 110 FAB-MS). The prepared ES-FWF, ES-FWG, ES_ -FWH, ES-FWK. ES-FVIL and ES-FHW, with a solubility of 7-40 g / l in water-soluble buffer solution. For band analysis, use Hitachi (Tokyo, Japan) F2000 fluorescence photometer to record fluorescence data, wavelength Set to 286 na (excitation light) and 360 nm (emission light). Detect the rate of enzyme hydrolysis of ES-FWF, ES-FWG, ES-FWH. ES-FWK, ES-FWL and ES-FHW. Preparation ACE enzyme solution, its activity is ί.3 xlO-2 units / ml. The concentration of Xuesu protein is estimated using Bio-Rad protein analysis kits, and K-bovine albumin is used as the standard. ACE (purchased from Sigma Chemicals, The source is rabbit lung) with a specific activity of 0.2 units per g of protein. Amino acid is used as the matrix to analyze the activity. One unit of enzyme activity refers to the enzyme required to catalyze the formation of 1.0 micromolar uric acid per minute (at 37 Ό in Hepes at 50ra M pH 8.3 and 0.3M HaCl)- 23-The size of the private paper is in accordance with the Chinese National Standard (〇 Bi) 8-4 specifications (210 > < 297 mm) 83. 3.10,000 (please read Note 1 on the back first-installation--I #Fill this page) Order ^ ίν · line 03606 A7 B7 V. Description of the invention (21) A 20 microliter enzyme solution was added to K0.05 M Hepes buffer (pH 8.2 · 0.2M NaCl and 1 M Na2S〇4). 0.5 liter of matrix solution (2 X 10-β_6.5 X 0_SM) was placed in a quartz colorimetric tube for fluorescence measurement, and K was used to measure fluorescence. When no enzyme was present, the fluorescence intensity was not Increasing A in the presence of enzymes * continuously records fluorescence with increasing time (36 0 nb emission ... 286 ηη excitation). Because the product (probably the matrix itself) performs photolysis, the beam of the laser monophotometer should not be too large Or, you can use pulsed lighting to automatically detect backlash, with a time interval of 150 seconds, as shown in Figure 1. (Within 10% of the initial hydrolysis) Establish the relationship between the hydrolysis rate and the substrate. Concentration. Measure the fluorescence of the solution containing the true dipeptide under the same conditions, and use it as the calibration curve to express the released dipeptide as the slope of the curve. Of the concentration. The Km value was calculated using the Lineweaver-Burk mapping method. From the average of two independent tests, five points of approximately equal distance on the 1 / [S] horizontal axis were obtained. Measure similar kinetic parameters of other matrices. Continuous photofluorescence analysis was used to measure the dynamic parameters of ES-FWF, ES-FWG, ES-FWH, ES-FWK, ES-FWL and ES-FHW matrix analysis ACE. The results are shown in Table I. In order to compare the fluorescent matrix used in analyzing and analyzing ACE activity, the parameter values reported in the literature were used as controls. (Please read the note on the back-install ----then fill out this page) ΤΓ Γ Printed by the Consumer Cooperatives of the Central Bureau of Standards, Ministry of Economic Affairs-24-This paper size applies to China National Standard (CNS) Α4 size (210x297 Public plague] '83. 3.10,000

V. Description of the invention (22)

Table I Matrix Km (M) kcat (seconds-1) kcat / Km (M-l seconds-1) References 1. ES * -FWF 4.5 x I. 6 2.1 4.6 x 105. The present invention 2. ES-FWG 2.3 X 10-5 2.0 8.9 x 104 The present invention 3. ES-FWH 4.9 x 10-δ 0.8 1.7 x 105. Existing invention 4. ES = FWK 2.4 x 10 -5 3.8 1.6 xl05 The present invention 5. ES-FWL 2.7 X 10-6 0.9 3.2 xl05 The present invention 6. ES-FHW 1.5 X 10-5 2.0 1.3 xl05. The present invention 7. Bim-GWL 1.7 x 10-4 1.2 7.0 x 103 [19] 8. Bim-FWL 5.9 x 10-5 3.1 5.2 x 10 ^ [19] 9. Bim-FWP 2.9 x 10-5 2.5 8.7 x 104 [19] 10. Bim -FF (N02) P 1.7 x 10-5 5.Γ 3.0 x I〇5 [20] 11. Bim-FW (N02) P 1.9 x! 0-5 5.4 2.9 XI〇5 [20] 12. Bim-FWP 2.6 xi〇-5 6.9 2.6 xl05 [20] 13. Z (N02) -GWG 9.0 xl0-4 [16] 14. Abz-GF (N2) P 1.9 x 10-4 1.5 7.7 xl03 [17] 15. Dns-GF (N〇2) P 4.2 xl0-4 4.1 9.6 xl03 [18] (Please read Note 1 on the back first 丨 Install!-F \ Fill this page again) Order Θ Line Economy The Kb value shown in Table I printed by the Ministry of Standards and Staff's Consumer Cooperatives represents the affinity of the enzyme to the substrate. The lower the KB value, the greater the affinity of the enzyme to the substrate. The kcat value is the maximum number of matrix molecules that convert a matrix to a product per activation site per unit time. When the enzyme is saturated with the substrate * Iccat value can represent the catalytic rate of the enzyme. However, when most enzymes are operating in vivo, they are not in a saturated matrix concentration, and the ratio of matrix maize / Kb is usually in the range of 0.1 to 10. If the matrix concentration is lower than the saturated state, the enzyme The true response rate is lower than the measured value -25-This paper size applies to the Chinese National Standard (CNS) A4 specification (210 > < 297 gong) 83.3. 10,000 ^ 02606 A7 B7 V. Description of the invention (23 Central Standard of the Ministry of Economic Affairs) Produced by local co-workers and consumers. Therefore, the general pseudo K kcat / Kb value indicates the enzyme catalytic efficiency lower than the degree of saturated substrate. As shown in Table I, the fluorescent-based gastric device of the present invention is equivalent to conventional substrates or A higher kcat / Km value indicates that it is a good matrix for ACE activity analysis. Although some conventional matrices (matrix 10-12) have a -kcat / Km value comparable to that of the matrix of the present invention, its _ contains Bimane hydrophobicity Molecules * have poor solubility. Prepared by ACR_Xizhan 抡 K 50bH Hepes buffer solution to prepare ES-FWF matrix solutions with a concentration of 20 β Μ and 10 u Μ, respectively, and add inhibitors to make 20wM ES-FWF based hydrazone solution Contains 0, 15, 30, 45 and 60wM respectively 10 μM ES-FWF matrix solution contains inhibitors of 0, 15, 30, 36. 45 wM, respectively. Take 0.5 ml of ES-FWF matrix reaction solution containing different inhibitors of Mae, and place them in a fluorescent assay. In the colorimetric tube used for light, the setting value of the fluorescence spectrophotometer is ex286 > era360 · Add 20w 1 ACE, and start revolting. When the reaction is terminated, collect the fluorescence change data every second. Bell fluorescence change value is converted into fluorescence change value per minute, and converted into product change per minute through the equation £ = 132.8 C-2. 7 Although the reciprocal 1 / V of the concentration of the sample to be measured is plotted, we can get 'Figure 2 According to the figure, the inhibition constant is Ki = 53.59WM. Ju Lao Xuan Xian 1. Skeggs, LT, Harsh, WH, Kahn, J. R., & Shumway, NP (1954) J. Exp. Med. 99 , 275-282 2. Johnston, (1990) Drugs 39 (Suppl. 1), 26 This paper ruler is applicable to China National Standard (CNS) A4 (210X297 mm) 83. 3.10,000 (Please read the (Note one-install--fill out this page) tr

Line C 606 A7 B7 Printed by the Shellfish Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (24) 21-31 〇3 · Ehlers, H, RV · & Riordan, JF (1989) Biochemistry 28, 5311-5317 〇4 · Beneteau-Burnat, B. & B audi π, B. (19 91) Critical Reviews in Clinical Laboratory Sciences 28, 337-356 ° • 5 .-- Cheng Hongyou (1 989) Chemical Industry Information Monthly, 9, 1-16. 6 ·-Cushman, D. W · & Cheung, S ··· (1971) Biochea., Pharmacol, 20, 1637-1648 ° 7. Ryan, JW, Chung, A., Ammons t C. & Carlton, ML (1977) Biochera. J. 167, 501-5504. 8. Dorer, FEf Kahn, JR, Lentz, KE, Levine, M. & Skeggs, LT (1976) Biochiia. B ί ο P hy s. Acta 429, 220-228 ° 9. Piquilloud Y, Reinharz A. & amp Roth H. (1970) Biochim. Biophys. Acta 206, 136-142 0 10. Friehand J. & Silverstein E. (1976) Am. J. Clin. Pathol. 66, 41 6-424 〇11. Conroy JM , Lai CY (1978) Anal. Biochen # 87, '5 56-561 ^ 12 K warts, E., Beukenvekd, G., & Gazendam, J. (1982) Ann · Clin · Biochera. 19, 227-232 . 13. Yaron, A., Carmel, A., & Katchalski-Katzir, E. (1979) Anal. Biochem. 95, 228-235. 14. David Freifelder, Physical Biochemistry 2nd -27-(Please read Note 1 on the back-Equipment-? Fill out this K) '

TT

C. Line The size of this paper is applicable to Chinese National Standard (CNS) A4 specification (210X297 mm) 83.3.10,000 A7 B7 to 02606 today 5. Description of invention (25)

Ed. Pp537-556 0 15. Forster, T. (1948) Ann. Physik. 2, 55-75 ° 16. Persson, A. & Wilson, I. Β (1977) Anal, Biochem. 83, 296 -303 0 17. Carnel, A. & Yaron, A. (1978) Eur. J. Biochea. • 87, 265-273 〇 1 & 7 F 1 em i nger, G., Go 1 denbe rg, D., & Ya rοn, A. * (1981) FEBS Lett. 135 / 131-134 ^ 19. Sato, E, (Nishikawa, S., & Kanaoka, Y. (1989) C he π *. P hara. Bull. 37, 145-147 ° 2 0. Sato, E., H a 11 ori, H., Nishikawa, Sf &

Kanaoka, Y. (19 91) C he m. P harm. Bull. 39, 2146-2148 ° 21. Hatayoshi, ED, Wang, GT, Krafft, GA & Erickson, J. (1 990) Science 247, 954 -958 ° 22. Ma ggiora »LL, Smith * CW, & Zhang, Z. A. A general Method for the synthesis of fluorogenic protease substrates using sol id-phase peptide methods ° 23. Wong, SS (1991) Chemistry of Protein Conjugation and Cross-Linking. CRC Press, Inc.

O 24. Model 4 3 0 A peptide synthesizer manual, 6-109-6-188 〇 This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) (Please read the note on the back first: fill out this page) Printed by Shellfish Consumer Cooperatives, Central Bureau of Standards, Ministry of Economic Affairs 83. 3. 10,000

Claims (1)

Patent No. 83108088 asks Zhaibianyuan to revise too (December 1985) Α ·· Β8 CS VI. Supplement of patent application scope * A fluorescent matrix with the following formula E-L k-AAAA: AA 3 ~ kk 4, where E is 5-L (L k is a carboxydiazyl group * A / W is a direct mirror, or a residue of an anthracene or an amino acid derivative of any protein other than tryptophan , A A2 is the residue of amino acid or amino acid derivative of any protein except tryptophan, A As and A A4 are the amino 2-aminoethyl) amino groups of any protein ] Naphthalene-1-sulfonic acid group (please read the notes on the back before filling this page) z 3. Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs Ervine is tryptophan, and the residues of white sticks are based on the amino acid residues, but at least the second side of the patent scope is required. Please apply for the scope of patents, ΑΑι is a straight acid or a histamine residue, glycine for the scope of patents, and AiU for the scope of patents, and for patents, and AA 4 for the group acids Or the fluorenyl acid derivative is the fluorescent matrix of the tryptophan residue 1 item, in which the carboxylic acid group 1 or 2 of the fluorescent matrix is connected to the microscope * AA2 is propylpropylamine, and AiU 侫 is selected from the group consisting of color, ionamine Acid or histamine residues of the three fluorescent substrates, their alanine residues. Fluorescent matrix of 3 items. Amino acid residues. Fluorescent matrix of 3 items. Amino acid residues. , Where Lk is a succinic acid residue * AA3 amine acid, propylamine. Chinese AA3 is tryptophan, where AA 3 is tryptophan, where AA 3 is tryptophan. The size of the paper is applicable to Chinese National Standard (CNS) A4. Standard (210X297 mm) Patent No. 83108088. Please revise it too (85 years) December) Α ·· Β8 CS VI. Supplement of patent application scope * A fluorescent matrix E-L k-AAAA with the following formula: AA 3 ~ kk 4, where E is 5-L (L k is carboxydi Twisted group * A / W is a direct mirror, or the residue of an anthracene or amino acid derivative of any protein other than tryptophan, A A2 is any protein composition other than tryptophan Residues of amino acids or amino acid derivatives, A As and A A4 are amine 2-aminoethyl) amino] naphthalene-1 -sulfonic acid groups (please read the Note: Please fill in this page again. Z 3. Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs. According to the application of the acid residues, the residues of the residues are applied, but at least the second surface of the patent scope is required. Please apply for the scope of patents, ΑΑι is a straight acid or a histamine residue, glycine for the scope of patents, and AiU for the scope of patents, and for patents, and AA 4 for the group acids Or the fluorenyl acid derivative is the fluorescent matrix of the tryptophan residue 1 item, in which the carboxylic acid group 1 or 2 of the fluorescent matrix is connected to the microscope * AA2 is propylpropylamine, and AiU 侫 is selected from the group consisting of color, ionamine Acid or histamine residues of the three fluorescent substrates, their alanine residues. Fluorescent matrix of 3 items. Amino acid residues. Fluorescent matrix of 3 items. Amino acid residues. , Where Lk is a succinic acid residue * AA3 amine acid, propylamine. Chinese AA3 is tryptophan, of which AA 3 is tryptophan, of which AA 3 is tryptophan. The size of the paper is applicable to Chinese National Standard (CNS) A4 Regulation. (210X297 mm) No. 83100422. June) A8 B8 C8 D8 89,6 ... month-τΤ. —.1 dagger supplement. Xi W- Λ · |-Patent application scope A variety of compounds with the following structures and their pharmaceutically acceptable 'acceptable salts OX' '-A. ^ 1-. ^ 2-A_ ^ J-CFn-C-. ^ A4-AA5-OY where: (a) -AA1- ^ is selected from the group consisting of fluorenyl, valinyl, Isoleumine and white (b)-AA 2-selected from the group consisting of propylamine groups; (c)-AA 3-selected from the group consisting of Benzylamine and p-fat (d) -AA4- are selected from the group consisting of: tert-butylglycineamine group; group: benzylamine and naphthalene group: spermine Groups: propylamine, amphetamine, and phenylamine; groups: propylamine with phenylamine, leukoamine and phenylpropylamine (please read the precautions on the back before filling this page), 1T printed by the Employees' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 2. 3. 4. Fujiki, Isoba Amine, Glyceryl, Val, Naphthylamine, and Cyclohexylpropylamine; (e) -AA5-selected from the group consisting of spermine, high-carbon spermine And amine groups; (f)-X 'is selected from the group consisting of: gold_alkoxycarbonyl, endo-bornyloxycarbonyl, naphthyloxycarbonyl; (g)-Y is hydrogen or methyl. The compound according to item 1 of the patent application is attached to the base. The compound according to item 2 of the patent application. The compound according to item 3 of the scope of application, wherein -X 'is a gold-alkoxy truncation group. Among them-AA5- is spermine, where -AA 3-is amine. Paper size is applicable to Chinese National Standard (CNS) A4 specification (210 X 297 mm) Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs ^ 02606. Sf C8 6. Scope of patent application 7. According to the patent application, the fluorescent substrate of item 3, wherein A Α 3 is a chromic acid residue and AA 4 is a leucine residue. 8- The fluorescent substrate according to item 3 of the scope of patent application, wherein A A 3 is a tryptophan residue and AAd is a lysine residue. a Fluorescent substrate based on item 3 of the patent application, where AAA3 is a histamine residue and A is a tryptophan residue. itt A method for preparing a fluorescent substrate according to item 1 of the scope of the patent application, which includes the following steps: (1) making a peptide of the following formula AAi-AAa_AA3-Art4 where i \ Α τ-AA 4 is defined as in the patent application Scope item 1; and (2) the peptide and 5-II2-aminoethyl) amino] naphthalene-sulfonic acid are then linked with a compound having two carboxylate groups. 11. The method according to item 10 of the scope of patent application, wherein the drawing of A A 1-A A 2 _ A A _ A A 4 is made and transmitted by solid-phase synthesis. 12- The method according to item 10 of the application, wherein the compound is a carboxylic anhydride. 13. The method according to item 12 of the scope of patent application, wherein the carboxylic acid ii is succinic anhydride. W—A method for analyzing the activity of angiotensin conversion. 1¾ Mix the sample to be tested with the fluorescent matrix in the first patent application scope, and use a fluorescence spectrometer to analyze the change in fluorescence. 15. —A method for screening anti-hypertensive drugs of angiotensin-converting enzyme inhibitors, which is to test the drug and angiotensin-converting enzyme, and according to the patent issued by the patent '-2-This paper applies Chinese national standards ( CNS) A4 gauge (210X297 mm) (Please read the precautions on the back before filling this page) 402606 is 6. Application for patent scope Photoluminescence spectrophotometer for photoluminescence nu and mixed base light · Fluorescent ΙΙί οι Quantitative perimeter change Sr (Please read the precautions on the back before filling this page) Economy The standard printed by the Consumers' Cooperative of the Ministry of Standards of the People's Republic of China is applicable to the Chinese National Standard (CNS) A4 regulations, and the standard is (210X297 mm) No. 83100422. Chinese patent application (8 June 2009) A8 B8 C8 D8 89,6 .. month-τΤ. —.1 dagger supplement. Xi W- Λ · |-Application scope Patent compounds with the following structures and their pharmaceutically acceptable 'acceptable salts OX'-A. ^ 1- . ^ 2-A_ ^ J-CFn-C-. ^ A4-AA5-OY where: (a) -AA1- ^ is selected from the group consisting of fluorenyl, valinyl, isoleucine and white (b)-AA 2-selected from the group consisting of the following groups: (c)-AA 3-selected from the group consisting of the group consisting of the following groups, p-foxylbenzylamine and p- Fat (d) -AA4- is selected from the group consisting of: tert-butylglycineamine group; group: benzylamine group and naphthalene group: spermine group, lysylamine group Base Alanine base, leukoamine base, benzylamine (please read the precautions on the back before filling out this page), 1T printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 2.3.4. Amine, Glyceryl, Val, Naphthylamine, and Cyclohexylpropylamine; (e) -AA5-selected from the group consisting of spermine, high-carbon spermine And amine groups; (f)-X 'is selected from the group consisting of: gold_alkoxycarbonyl, endo-bornyloxycarbonyl, naphthyloxycarbonyl; (g)-Y is hydrogen or methyl. The compound according to item 1 of the patent application is attached to the base. The compound according to item 2 of the patent application. The compound according to item 3 of the scope of application, wherein -X 'is a gold-alkoxy truncation group. Among them-AA5- is spermine, where -AA 3-is amine. The paper size is applicable to Chinese National Standard (CNS) A4 specification (210 X 297 mm) 402606 as B8 C8 D8 6. Application for patent scope 5. According to the scope of patent application The compound of item 3, wherein -AA 2-is amphetamine and M-AA 4-is leucine. 6. The compound according to item 5 of the scope of the patent application, wherein -A A 1-is a 3-butylglycinamidinyl group, -X 'is adamantyloxycarbonyl group, and -Y is a methyl group. 7. The compound according to item 3 of the patent application, wherein -Y is methyl. (Please read the precautions on the back before filling out this page), 1T Printed by Shelley Consumer Cooperative, Central Bureau of Standards, Ministry of Economic Affairs This paper size applies to China National Standard (CNS) A4 (210 X 297 mm)