JPS6112898B2 - - Google Patents
Info
- Publication number
- JPS6112898B2 JPS6112898B2 JP12343883A JP12343883A JPS6112898B2 JP S6112898 B2 JPS6112898 B2 JP S6112898B2 JP 12343883 A JP12343883 A JP 12343883A JP 12343883 A JP12343883 A JP 12343883A JP S6112898 B2 JPS6112898 B2 JP S6112898B2
- Authority
- JP
- Japan
- Prior art keywords
- enzyme
- formula
- acetyltyrosine
- substrate
- measuring
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
- 108090000790 Enzymes Proteins 0.000 claims description 37
- 102000004190 Enzymes Human genes 0.000 claims description 37
- 238000000034 method Methods 0.000 claims description 29
- 230000000694 effects Effects 0.000 claims description 17
- 239000000758 substrate Substances 0.000 claims description 14
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 13
- CAHKINHBCWCHCF-UHFFFAOYSA-N N-acetyltyrosine Chemical compound CC(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 CAHKINHBCWCHCF-UHFFFAOYSA-N 0.000 claims description 7
- CAHKINHBCWCHCF-JTQLQIEISA-N N-acetyl-L-tyrosine Chemical class CC(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 CAHKINHBCWCHCF-JTQLQIEISA-N 0.000 claims description 5
- 238000006482 condensation reaction Methods 0.000 claims description 4
- 230000018044 dehydration Effects 0.000 claims description 4
- 238000006297 dehydration reaction Methods 0.000 claims description 4
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 4
- 230000007062 hydrolysis Effects 0.000 claims description 3
- 238000006460 hydrolysis reaction Methods 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 229940088598 enzyme Drugs 0.000 description 32
- 239000000243 solution Substances 0.000 description 14
- 150000001875 compounds Chemical class 0.000 description 9
- 108010028774 Complement C1 Proteins 0.000 description 8
- 102000016917 Complement C1 Human genes 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 8
- 150000002148 esters Chemical class 0.000 description 8
- 239000000126 substance Substances 0.000 description 8
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 5
- -1 alkyl chlorocarbonate Chemical compound 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- SKAWDTAMLOJQNK-UHFFFAOYSA-N 2-acetamido-3-(4-hydroxyphenyl)propanoic acid ethyl ester Chemical compound CCOC(=O)C(NC(C)=O)CC1=CC=C(O)C=C1 SKAWDTAMLOJQNK-UHFFFAOYSA-N 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 239000012190 activator Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 229960001682 n-acetyltyrosine Drugs 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- 125000005907 alkyl ester group Chemical group 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 125000001584 benzyloxycarbonyl group Chemical group C(=O)(OCC1=CC=CC=C1)* 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- ADFXKUOMJKEIND-UHFFFAOYSA-N 1,3-dicyclohexylurea Chemical compound C1CCCCC1NC(=O)NC1CCCCC1 ADFXKUOMJKEIND-UHFFFAOYSA-N 0.000 description 1
- NGNBDVOYPDDBFK-UHFFFAOYSA-N 2-[2,4-di(pentan-2-yl)phenoxy]acetyl chloride Chemical compound CCCC(C)C1=CC=C(OCC(Cl)=O)C(C(C)CCC)=C1 NGNBDVOYPDDBFK-UHFFFAOYSA-N 0.000 description 1
- NEAQRZUHTPSBBM-UHFFFAOYSA-N 2-hydroxy-3,3-dimethyl-7-nitro-4h-isoquinolin-1-one Chemical compound C1=C([N+]([O-])=O)C=C2C(=O)N(O)C(C)(C)CC2=C1 NEAQRZUHTPSBBM-UHFFFAOYSA-N 0.000 description 1
- 108090000317 Chymotrypsin Proteins 0.000 description 1
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- FKMJXALNHKIDOD-LBPRGKRZSA-N TAMe Chemical compound NC(=N)NCCC[C@@H](C(=O)OC)NS(=O)(=O)C1=CC=C(C)C=C1 FKMJXALNHKIDOD-LBPRGKRZSA-N 0.000 description 1
- SORGEQQSQGNZFI-UHFFFAOYSA-N [azido(phenoxy)phosphoryl]oxybenzene Chemical compound C=1C=CC=CC=1OP(=O)(N=[N+]=[N-])OC1=CC=CC=C1 SORGEQQSQGNZFI-UHFFFAOYSA-N 0.000 description 1
- MNZMECMQTYGSOI-UHFFFAOYSA-N acetic acid;hydron;bromide Chemical compound Br.CC(O)=O MNZMECMQTYGSOI-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000006149 azo coupling reaction Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000010531 catalytic reduction reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- HLVXFWDLRHCZEI-UHFFFAOYSA-N chromotropic acid Chemical compound OS(=O)(=O)C1=CC(O)=C2C(O)=CC(S(O)(=O)=O)=CC2=C1 HLVXFWDLRHCZEI-UHFFFAOYSA-N 0.000 description 1
- 229960002376 chymotrypsin Drugs 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 239000002274 desiccant Substances 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 229940079919 digestives enzyme preparation Drugs 0.000 description 1
- MKRTXPORKIRPDG-UHFFFAOYSA-N diphenylphosphoryl azide Chemical compound C=1C=CC=CC=1P(=O)(N=[N+]=[N-])C1=CC=CC=C1 MKRTXPORKIRPDG-UHFFFAOYSA-N 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000000921 elemental analysis Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 238000011197 physicochemical method Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 150000003667 tyrosine derivatives Chemical class 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Description
【発明の詳細な説明】
本発明は式（）で示されるチロシン誘導体、
その製造法、及びその化合物を基質として酵素活
性を測定する方法に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention provides a tyrosine derivative represented by the formula (),
The present invention relates to a method for producing the compound and a method for measuring enzyme activity using the compound as a substrate.
式中、Ｒはフエニル基を示す。 In the formula, R represents a phenyl group.
本発明物質（）は新規な化合物であり、酵素
活性を測定する為の試験薬として有用な化合物で
ある。 The substance of the present invention () is a novel compound and is a compound useful as a test drug for measuring enzyme activity.
本発明物質（）はアセチルチロシン（）
とフエノールとの通常の脱水縮合反応によつて製
造することができる。 The substance of the present invention () is acetyltyrosine () It can be produced by a conventional dehydration condensation reaction between phenol and phenol.
本発明を実施するに当つては、上記アセチルチ
ロシン（）とフエノールとの通常の脱水縮合反
応によつて発明物質（）を得ることができる。
本法においてアセチルチロシンの水酸基はカルボ
ベンジルオキシ基等の通常使用される水酸基保護
基で保護してもよく、その場合には脱水縮合反応
後、適当な保護基脱離反応、すなわち、例えば水
酸基保護基がカルボベンジルオキシ基の場合には
パラジウム炭素等による接触還元、または臭化水
素酸酢酸溶液による分解反応等を行うことにより
本発明物質（）を得ることができる。 In carrying out the present invention, the inventive substance (2) can be obtained by a conventional dehydration condensation reaction between the acetyltyrosine (2) and phenol.
In this method, the hydroxyl group of acetyltyrosine may be protected with a commonly used hydroxyl-protecting group such as a carbobenzyloxy group. When the group is a carbobenzyloxy group, the substance of the present invention () can be obtained by catalytic reduction with palladium on carbon or the like, or decomposition reaction with a hydrobromide-acetic acid solution.
脱水縮合反応を行うにあたつては原料物質を適
当な溶媒に溶解し、DCC（ジシクロヘキシルカ
ーボジイミド）、DPPA（ジフエニルフオスフオ
リルアジド）、クロル炭酸アルキル等のエステル
活性化剤を加え、これにフエノールを加え、必要
に応じトリエチルアミン等の塩基を加え、撹拌す
ることにより製造することができる。又、良く知
られた酸クロライド法やフエノールのスルフイツ
ト体としてよく知られたフエニル化剤（Ber.49、
2339、1916）を用いることによつても製造するこ
とができるが、上記エステル活性化剤による方法
が好ましい。使用し得る溶媒は、クロロホルム、
ジクロルメタン、ジメチルホルムアミド、テトラ
ヒドロフラン等通常使用されるもので、原料物質
を溶解するものであれば良い。反応温度は０゜〜
40℃で良い。反応終了後、通常行われる処理方法
により、反応液より目的化合物を得ることができ
る。すなわち、例えばDCCをエステル活性化剤
として製造した場合、析出するジシクロヘキシル
尿素を取して除き、塩酸水溶液、NaOH水溶
液、及び飽和食塩水で洗浄し、無水硫酸マグネシ
ウム等の乾燥剤で乾燥後、溶媒を留去することに
より得ることができる。目的化合物は、所望によ
り、再結晶、クロマトグラフイー等により精製す
ることができる。 To carry out the dehydration condensation reaction, the raw material is dissolved in a suitable solvent, an ester activator such as DCC (dicyclohexylcarbodiimide), DPPA (diphenylphosphoryl azide), or alkyl chlorocarbonate is added. It can be produced by adding phenol to the solution, adding a base such as triethylamine if necessary, and stirring. In addition, the well-known acid chloride method and the well-known phenylating agent (Ber. 49 ,
2339, 1916), but the method using the above-mentioned ester activator is preferred. Solvents that can be used include chloroform,
Any commonly used dichloromethane, dimethylformamide, tetrahydrofuran, etc. may be used as long as it dissolves the raw material. Reaction temperature is 0°~
40℃ is good. After the reaction is completed, the target compound can be obtained from the reaction solution by a commonly used treatment method. That is, for example, when DCC is produced as an ester activator, precipitated dicyclohexyl urea is removed, washed with an aqueous hydrochloric acid solution, an aqueous NaOH solution, and saturated saline, dried with a drying agent such as anhydrous magnesium sulfate, and then washed with a solvent. It can be obtained by distilling off. The target compound can be purified by recrystallization, chromatography, etc., if desired.
本発明物質（）は、酵素と接触させることに
より基質として働き、一定時間後酵素により水解
されて遊離したフエノールを測定することによ
り、酵素の活性を測定することが出来る。酵素の
活性を測定するという事は酵素製剤の規格、血中
酵素パターンの測定による診断、血中酵素濃度の
測定等の為に非常に重要な事である。従来、酵素
活性を測定するためには種々の方法が知られてい
る。その一つの方法として、アミノ酸のアルキル
エステルを基質として、酵素と接触させ、そのエ
ステルの水解の程度により活性を測定するという
方法がある。例えば、ヘステリン法として良く知
られている方法がその一つである。これは、酵素
とアミノ酸のアルキルエステルを接触させ、一定
時間後に残存するエステル部分をヒドロキシルア
ミンによりヒドロキサム酸とし、過クロル鉄と反
応させ発色させ、その発色を吸光度として測定
し、その結果より、酵素のエステル水解能、すな
わち酵素の活性を測定するという方法である。そ
の他、基質を用いエステルの水解能を指標とする
方法には、クロモトロプ酸法などがあるが、これ
らの方法では、ある程度の酵素量を必要とし、酵
素が低濃度である場合、又は低活性の酵素の測定
には難があつた。そこで、発明者は、酵素に対し
てアフイニテイを持ち、更に定量法が簡便であり
かつ検出感度の良い、という三つの条件を兼ね備
えた基質としての化合物を検索することにより、
従来よりも、非常に優れた化合物を見出すことが
できた。 The substance of the present invention () acts as a substrate when brought into contact with an enzyme, and after a certain period of time, it is hydrolyzed by the enzyme and the liberated phenol is measured, thereby making it possible to measure the activity of the enzyme. Measuring enzyme activity is very important for purposes such as standardization of enzyme preparations, diagnosis by measuring blood enzyme patterns, and measurement of blood enzyme concentration. Conventionally, various methods are known for measuring enzyme activity. One method is to use an alkyl ester of an amino acid as a substrate, contact it with an enzyme, and measure the activity based on the degree of hydrolysis of the ester. For example, a method well known as the Hesterin method is one of them. This is done by bringing an enzyme into contact with an alkyl ester of an amino acid, converting the remaining ester portion after a certain period of time into hydroxamic acid using hydroxylamine, reacting with iron perchloride to develop a color, and measuring the color development as absorbance. This method measures the ester water-degrading ability of the enzyme, that is, the activity of the enzyme. Other methods that use a substrate and use the water-degrading ability of esters as an indicator include the chromotropic acid method, but these methods require a certain amount of enzyme and are difficult to use when the enzyme is at a low concentration or has low activity. Measuring enzymes was difficult. Therefore, the inventor searched for a compound as a substrate that had affinity for the enzyme, was easy to quantify, and had high detection sensitivity.
We were able to discover a compound that is far superior to conventional ones.
本発明を実施するに当つては、酵素と一定量の
化合物（）を、適当な緩衝液中で接触させ、一
定温度で、一定時間後に遊離したフエノールを測
定することにより、酵素の活性を測定することが
できる。緩衝液はその酵素の至適PHを有する適当
な緩衝液でよい。又、反応温度、反応時間ともに
適当な一定条件でよいが、25〜37℃で30分後に測
定するのが望ましい。フエノールを測定する方法
は従来、良く知られたガスクロマトグラフイーま
たは薄層クロマトグラフイー等の物理化学的方
法、過クロル鉄反応、ジアゾカツプリング反応、
FVB（フアーストバイオレツトＢソルト）法ま
たは４−アミノアンチピリン法等の化学的方法の
いずれの方法を使用してもよいが、フエノールの
測定の場合、反応液に４−アミノアンチピリンを
加え発色させ分光光度計により吸光度として測定
する方法がその簡便さおよび検出感度においてよ
り好ましい方法である。本法は単一酵素系におけ
る測定のみならず、種々の酵素が含まれた場合の
測定にも使用できる。すなわち、例えば血清中に
含まれる酵素活性を測定する場合、血清を適当な
プレパラートに添加し、これを電気泳動等により
酵素を分離し、これを本発明物質の溶液に浸し適
当な時間後、更に上記発色試薬を加えることによ
り、従来見ることができなかつた血中酵素パター
ンを見ることができる。この方法によれば、種々
の病態に起因する酵素パターンの変動を見ること
ができる。 In carrying out the present invention, the activity of the enzyme is measured by bringing the enzyme and a certain amount of the compound () into contact with each other in an appropriate buffer solution, and measuring the released phenol after a certain period of time at a certain temperature. can do. The buffer may be a suitable buffer having the optimum pH of the enzyme. Further, the reaction temperature and reaction time may be set under appropriate constant conditions, but it is preferable to measure after 30 minutes at 25 to 37°C. Conventional methods for measuring phenol include well-known physicochemical methods such as gas chromatography or thin layer chromatography, perchloriron reaction, diazo coupling reaction,
Either the FVB (first violet B salt) method or a chemical method such as the 4-aminoantipyrine method may be used, but in the case of measuring phenol, 4-aminoantipyrine is added to the reaction solution to develop color. A method of measuring absorbance using a spectrophotometer is a more preferable method due to its simplicity and detection sensitivity. This method can be used not only for measurements in a single enzyme system, but also for measurements involving various enzymes. That is, for example, when measuring the enzyme activity contained in serum, the serum is added to a suitable preparation, the enzymes are separated from this by electrophoresis, etc., this is immersed in a solution of the substance of the present invention, and after an appropriate time, further By adding the above-mentioned coloring reagent, it is possible to see enzyme patterns in the blood that were previously impossible to see. According to this method, changes in enzyme patterns caused by various pathological conditions can be observed.
本発明物質は、種々の酵素のうち、特に、炎症
系の酵素として良く知られた、C1エステラー
ゼ、又はキモトリプシンの優れた基質として作用
し、アセチルチロシンフエニルエステルを基質と
して、C1エステラーゼを測定した場合、従来、
酵素基質として知られたアセチルチロシンエチル
エステル、又はトシルアルギニンメチルエステル
を用い、ヘステリン法により測定した場合に比
べ、高い検出感度を有していた。 The substance of the present invention acts as an excellent substrate for C1 esterase or chymotrypsin, which is well known as an inflammatory enzyme among various enzymes, and C1 esterase was measured using acetyl tyrosine phenyl ester as a substrate. In the case, conventionally,
The detection sensitivity was higher than that measured by the hesterin method using acetyl tyrosine ethyl ester or tosyl arginine methyl ester, which are known enzyme substrates.
次に本発明の実施例をあげ、更に詳細に説明す
る。 Next, examples of the present invention will be given and explained in more detail.
実施例 １
Ｎ−アセチルチロシンフエニルエステルの合成
Ｎ−アセチルチロシン22.3ｇをＮ・N′−ジメチ
ルホルムアミド125mlに溶かしフエノール10.0
ｇ、トリエチルアミン16mlを加え氷冷撹拌下Ｎ・
N′−ジシクロヘキシルカーボジイミド26.7ｇを加
え、１時間撹拌した後室温にもどしさらに一昼夜
撹拌する。反応後析出するＮ・N′−ジシクロヘ
キシル尿素をろ過して除き、母液に酢酸エチルを
加え、10％クエン酸、飽和重そう水、飽和食塩水
で洗浄した後、無水硫酸マグネシウムで乾燥した
後、溶媒を留去、残渣をシリカゲルに吸着させ３
％メタノール含有クロロホルムにて溶出してくる
フラクシヨンを集め、エーテルを加えて析出する
結晶を集める。Example 1 Synthesis of N-acetyltyrosine phenyl ester Dissolve 22.3g of N-acetyltyrosine in 125ml of N・N'-dimethylformamide and add 10.0ml of phenol.
g, add 16 ml of triethylamine and stir under ice cooling with N.
After adding 26.7 g of N'-dicyclohexylcarbodiimide and stirring for 1 hour, the mixture was returned to room temperature and further stirred overnight. After the reaction, N-N'-dicyclohexylurea precipitated was removed by filtration, ethyl acetate was added to the mother liquor, and the mixture was washed with 10% citric acid, saturated aqueous sodium chloride, and saturated brine, and dried over anhydrous magnesium sulfate. The solvent was distilled off and the residue was adsorbed on silica gel.
Collect the fractions eluted with chloroform containing % methanol, add ether and collect the precipitated crystals.
収量9.0ｇ、収率30％、融点116〜118℃
IR（KBr）cm-1；3250、1750、1640
MS；ｍ／e299M＋
元素分析 C17H17NO4（299.31）として
理論値 Ｃ、68.21 Ｈ、5.73 Ｎ、4.68
実測値 Ｃ、68.33 Ｈ、5.88 Ｎ、4.66
実施例 ２
Ｎ−アセチルチロシンフエニルエステルを基質
としたC1エステラーゼの活性測定法
C1エステラーゼの希釈液0.5mlにＮ−アセチル
チロシンフエニルエステル溶液（14.9mg／10ml
H2O）0.5ml、及び４−アミノアンチピリン溶液
（134mg／200mlリン酸緩衝液）２mlを加え37℃で
30分反応させる。更にフエリシアン化カリウム溶
液（500mg／200mlクエン酸緩衝液）２mlを加え37
℃で30分インキユベーシヨンし、発色を分光光度
計により吸光度（500nｍ）として測定し酵素に
より水解されて遊離したフエノールを定量する。
この遊離したフエノール量が酵素の活性度を示
す。Yield 9.0g, yield 30%, melting point 116-118℃ IR (KBr) cm -1 ; 3250, 1750, 1640 MS; m/e299M+ Elemental analysis C 17 H 17 NO 4 (299.31) Theoretical value C, 68.21 H , 5.73 N, 4.68 Actual value C, 68.33 H, 5.88 N, 4.66 Example 2 Method for measuring the activity of C1 esterase using N-acetyl tyrosine phenyl ester as a substrate Add N-acetyl tyrosine phenyl to 0.5 ml of a diluted solution of C1 esterase. Ester solution (14.9mg/10ml
Add 0.5 ml of H 2 O) and 2 ml of 4-aminoantipyrine solution (134 mg/200 ml phosphate buffer) and heat at 37℃.
Incubate for 30 minutes. Furthermore, add 2 ml of potassium ferricyanide solution (500 mg/200 ml citrate buffer).
After incubation at ℃ for 30 minutes, color development is measured as absorbance (500 nm) using a spectrophotometer to quantify the amount of phenol released by hydrolysis by the enzyme.
The amount of released phenol indicates the activity of the enzyme.
参考例 １
Ｎ−アセチルチロシンエチルエステルを基質と
したC1エステラーゼの活性測定法。（ヘステリ
ン法）
C1エステラーゼの希釈液0.5mlにＮ−アセチル
チロシンエチルエステル溶液（10μmoles／0.4ml
５％DMSO）0.4ml、及びリン酸緩衝液（PH7.4）
0.1mlを加える。37℃で30分間インキユベーシヨ
ンした後、ヒドロキシルアミン溶液（2M−
NH2OH・HClおよび3.5MNaOHの等量混合物）
1.5ml加え室温で15分間放置する。これに18％ト
リクロル酢酸１ml、4N塩酸１ml、及び10％塩化
第二鉄１mlを加え充分にかくはんした後3000r.p.
m.で10分間遠心分離する。上澄液の発色を分光
光度計により吸光度（530nｍ）として測定す
る。この値はC1エステラーゼによつて水解され
ずに残つた基質の量に相関するもので酵素の活性
は基質のみの値（対照）から酵素反応を行なつた
後に得られる値を引いた値に相当する。実施例２
及び参考例１で測定したC1エステラーゼの各濃
度段階における結果を図面に示すが前者の方法は
後者の方法に比べ約10倍の感度を有していること
が分る。図面中〇印は実施例２の方法による標準
曲線を、×印は参考例１の方法による標準曲線を
示す。Reference Example 1 A method for measuring the activity of C1 esterase using N-acetyltyrosine ethyl ester as a substrate. (Hesterin method) Add N-acetyl tyrosine ethyl ester solution (10 μmoles/0.4 ml) to 0.5 ml of C1 esterase diluted solution.
5% DMSO) 0.4ml, and phosphate buffer (PH7.4)
Add 0.1ml. After incubation at 37°C for 30 minutes, hydroxylamine solution (2M−
Equivalent mixture of NH 2 OH HCl and 3.5M NaOH)
Add 1.5ml and leave at room temperature for 15 minutes. To this, 1 ml of 18% trichloroacetic acid, 1 ml of 4N hydrochloric acid, and 1 ml of 10% ferric chloride were added, stirred thoroughly, and then heated at 3000 r.p.
Centrifuge for 10 min at m. The color development of the supernatant liquid is measured as absorbance (530 nm) using a spectrophotometer. This value correlates to the amount of substrate remaining without being hydrolyzed by C1 esterase, and the enzyme activity is equivalent to the value obtained by subtracting the value obtained after carrying out the enzyme reaction from the value of substrate only (control). do. Example 2
The results at each concentration level of C1 esterase measured in Reference Example 1 are shown in the figure, and it can be seen that the former method has about 10 times the sensitivity than the latter method. In the drawings, ○ marks indicate the standard curve obtained by the method of Example 2, and × marks indicate the standard curve obtained by the method of Reference Example 1.
図面はC1エスラーゼ活性の標準曲線を示す。 The figure shows a standard curve for C1 esrase activity.
Claims (1)
の脱水縮合反応させることを特徴とする式（）
する式（） （式中Ｒはフエニル基を示す）で示されるアセチ
ルチロシン誘導体の製造方法。 ３ 式（） （式中Ｒはフエニル基を示す）で示されるアセチ
ルチロシン誘導体を基質として酵素と接触させる
ことを特徴とする酵素活性の測定方法。 ４ 式（）で示されるアセチルチロシン誘導体
を基質として酵素と接触させ、一定時間後、酵素
により水解されて遊離したフエノールを測定する
ことを特徴とする特許請求の範囲第３項に記載の
酵素活性の測定方法。[Claims] 1 Formula () (In the formula, R represents a phenyl group.) An acetyltyrosine derivative represented by the following. 2 formula () Formula () characterized by a normal dehydration condensation reaction of acetyltyrosine and phenol shown by
expression () A method for producing an acetyltyrosine derivative represented by the formula (wherein R represents a phenyl group). 3 formula () A method for measuring enzyme activity, which comprises contacting an acetyltyrosine derivative represented by the formula (wherein R represents a phenyl group) with an enzyme as a substrate. 4. Enzyme activity according to claim 3, characterized in that the acetyltyrosine derivative represented by formula () is brought into contact with an enzyme as a substrate, and after a certain period of time, the phenol released by hydrolysis by the enzyme is measured. How to measure.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP12343883A JPS5925363A (en) | 1983-07-08 | 1983-07-08 | Tyrosine derivative |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP12343883A JPS5925363A (en) | 1983-07-08 | 1983-07-08 | Tyrosine derivative |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP12887277A Division JPS593989B2 (en) | 1977-10-27 | 1977-10-27 | Tyrosine derivative |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS5925363A JPS5925363A (en) | 1984-02-09 |
| JPS6112898B2 true JPS6112898B2 (en) | 1986-04-10 |
Family
ID=14860585
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP12343883A Granted JPS5925363A (en) | 1983-07-08 | 1983-07-08 | Tyrosine derivative |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS5925363A (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102219706B (en) * | 2011-04-21 | 2013-08-21 | 宁波市镇海海德生化科技有限公司 | Method for preparing acetyl tyrosine ethyl ester monohydrate and product of acetyl tyrosine ethyl ester monohydrate |
-
1983
- 1983-07-08 JP JP12343883A patent/JPS5925363A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5925363A (en) | 1984-02-09 |
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