JPH03271299A - Production of peptidase synthetic substrate - Google Patents
Production of peptidase synthetic substrateInfo
- Publication number
- JPH03271299A JPH03271299A JP2070338A JP7033890A JPH03271299A JP H03271299 A JPH03271299 A JP H03271299A JP 2070338 A JP2070338 A JP 2070338A JP 7033890 A JP7033890 A JP 7033890A JP H03271299 A JPH03271299 A JP H03271299A
- Authority
- JP
- Japan
- Prior art keywords
- formula
- glycyl
- peptidase
- tin
- dibromo
- 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.)
- Granted
Links
- 108091005804 Peptidases Proteins 0.000 title claims abstract description 12
- 102000035195 Peptidases Human genes 0.000 title claims abstract description 12
- 235000019833 protease Nutrition 0.000 title claims abstract description 10
- 239000000758 substrate Substances 0.000 title claims abstract description 9
- 238000004519 manufacturing process Methods 0.000 title claims description 5
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 16
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 claims abstract description 8
- 125000006239 protecting group Chemical group 0.000 claims abstract description 6
- 150000003752 zinc compounds Chemical class 0.000 claims abstract description 6
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 claims abstract description 3
- 125000005843 halogen group Chemical group 0.000 claims abstract description 3
- 229950003188 isovaleryl diethylamide Drugs 0.000 claims abstract 2
- 239000000126 substance Substances 0.000 claims 2
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 claims 1
- 125000000741 isoleucyl group Chemical group [H]N([H])C(C(C([H])([H])[H])C([H])([H])C([H])([H])[H])C(=O)O* 0.000 claims 1
- 125000001909 leucine group Chemical group [H]N(*)C(C(*)=O)C([H])([H])C(C([H])([H])[H])C([H])([H])[H] 0.000 claims 1
- 125000002987 valine group Chemical group [H]N([H])C([H])(C(*)=O)C([H])(C([H])([H])[H])C([H])([H])[H] 0.000 claims 1
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 abstract description 10
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 abstract description 5
- 235000019253 formic acid Nutrition 0.000 abstract description 5
- 239000002253 acid Substances 0.000 abstract description 3
- 125000001980 alanyl group Chemical group 0.000 abstract description 2
- 230000002255 enzymatic effect Effects 0.000 abstract description 2
- 125000001998 leucyl group Chemical group 0.000 abstract description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 abstract 3
- 238000005259 measurement Methods 0.000 abstract 1
- 125000002114 valyl group Chemical group 0.000 abstract 1
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 15
- SJRJJKPEHAURKC-UHFFFAOYSA-N N-Methylmorpholine Chemical compound CN1CCOCC1 SJRJJKPEHAURKC-UHFFFAOYSA-N 0.000 description 15
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- -1 )curyl Chemical group 0.000 description 8
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 7
- 238000001816 cooling Methods 0.000 description 6
- 239000000706 filtrate Substances 0.000 description 6
- 239000000203 mixture Substances 0.000 description 5
- VFRCXEHNAFUTQC-UHFFFAOYSA-N 2-[[2-(phenylmethoxycarbonylamino)acetyl]amino]acetic acid Chemical compound OC(=O)CNC(=O)CNC(=O)OCC1=CC=CC=C1 VFRCXEHNAFUTQC-UHFFFAOYSA-N 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- 239000000741 silica gel Substances 0.000 description 4
- 229910002027 silica gel Inorganic materials 0.000 description 4
- HPGGPRDJHPYFRM-UHFFFAOYSA-J tin(iv) chloride Chemical compound Cl[Sn](Cl)(Cl)Cl HPGGPRDJHPYFRM-UHFFFAOYSA-J 0.000 description 4
- HFYPXERYZGFDBD-UHFFFAOYSA-N 4-amino-2,6-dibromophenol Chemical compound NC1=CC(Br)=C(O)C(Br)=C1 HFYPXERYZGFDBD-UHFFFAOYSA-N 0.000 description 3
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 229910000037 hydrogen sulfide Inorganic materials 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- UREBWPXBXRYXRJ-UHFFFAOYSA-N ethyl acetate;methanol Chemical compound OC.CCOC(C)=O UREBWPXBXRYXRJ-UHFFFAOYSA-N 0.000 description 2
- YMAWOPBAYDPSLA-UHFFFAOYSA-N glycylglycine Chemical compound [NH3+]CC(=O)NCC([O-])=O YMAWOPBAYDPSLA-UHFFFAOYSA-N 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- HJRODLUQNAKSNI-VIFPVBQESA-N (2S)-5-(diaminomethylideneamino)-N-(3,5-dibromo-4-hydroxyphenyl)-2-nitramidopentanamide Chemical compound [N+](=O)([O-])N[C@@H](CCCNC(N)=N)C(=O)NC1=CC(=C(C(=C1)Br)O)Br HJRODLUQNAKSNI-VIFPVBQESA-N 0.000 description 1
- KPYXMALABCDPGN-HYOZMBHHSA-N (4s)-5-[[(2s)-6-amino-1-[[(2s,3s)-1-[[(2s)-1-[[(2s)-1-[[(2s)-1-[[(2s)-1-[[(2r)-1-[[2-[[2-[[(1s)-3-amino-1-carboxy-3-oxopropyl]amino]-2-oxoethyl]amino]-2-oxoethyl]amino]-1-oxo-3-sulfanylpropan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-1-oxopropan-2-yl]a Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)CNC(=O)CNC(=O)[C@H](CS)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN)CC1=CC=C(O)C=C1 KPYXMALABCDPGN-HYOZMBHHSA-N 0.000 description 1
- HDCHHZLVYAEBBF-UHFFFAOYSA-N 2-[[2-(methoxycarbonylamino)acetyl]amino]acetic acid Chemical compound COC(=O)NCC(=O)NCC(O)=O HDCHHZLVYAEBBF-UHFFFAOYSA-N 0.000 description 1
- YOETUEMZNOLGDB-UHFFFAOYSA-N 2-methylpropyl carbonochloridate Chemical compound CC(C)COC(Cl)=O YOETUEMZNOLGDB-UHFFFAOYSA-N 0.000 description 1
- 229910000497 Amalgam Inorganic materials 0.000 description 1
- OBMZMSLWNNWEJA-XNCRXQDQSA-N C1=CC=2C(C[C@@H]3NC(=O)[C@@H](NC(=O)[C@H](NC(=O)N(CC#CCN(CCCC[C@H](NC(=O)[C@@H](CC4=CC=CC=C4)NC3=O)C(=O)N)CC=C)NC(=O)[C@@H](N)C)CC3=CNC4=C3C=CC=C4)C)=CNC=2C=C1 Chemical compound C1=CC=2C(C[C@@H]3NC(=O)[C@@H](NC(=O)[C@H](NC(=O)N(CC#CCN(CCCC[C@H](NC(=O)[C@@H](CC4=CC=CC=C4)NC3=O)C(=O)N)CC=C)NC(=O)[C@@H](N)C)CC3=CNC4=C3C=CC=C4)C)=CNC=2C=C1 OBMZMSLWNNWEJA-XNCRXQDQSA-N 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 108090000270 Ficain Proteins 0.000 description 1
- 108010008488 Glycylglycine Proteins 0.000 description 1
- CKRZKMFTZCFYGB-UHFFFAOYSA-N N-phenylhydroxylamine Chemical compound ONC1=CC=CC=C1 CKRZKMFTZCFYGB-UHFFFAOYSA-N 0.000 description 1
- 101710176384 Peptide 1 Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 125000004429 atom Chemical group 0.000 description 1
- 125000001584 benzyloxycarbonyl group Chemical group C(=O)(OCC1=CC=CC=C1)* 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- FZFAMSAMCHXGEF-UHFFFAOYSA-N chloro formate Chemical compound ClOC=O FZFAMSAMCHXGEF-UHFFFAOYSA-N 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229940039227 diagnostic agent Drugs 0.000 description 1
- 239000000032 diagnostic agent Substances 0.000 description 1
- 239000000428 dust Substances 0.000 description 1
- 238000000921 elemental analysis Methods 0.000 description 1
- POTUGHMKJGOKRI-UHFFFAOYSA-N ficin Chemical compound FI=CI=N POTUGHMKJGOKRI-UHFFFAOYSA-N 0.000 description 1
- 235000019836 ficin Nutrition 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 229940043257 glycylglycine Drugs 0.000 description 1
- 239000001046 green dye Substances 0.000 description 1
- 210000003127 knee Anatomy 0.000 description 1
- 239000012280 lithium aluminium hydride Substances 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- UKVIEHSSVKSQBA-UHFFFAOYSA-N methane;palladium Chemical compound C.[Pd] UKVIEHSSVKSQBA-UHFFFAOYSA-N 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 229940012957 plasmin Drugs 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 238000001226 reprecipitation Methods 0.000 description 1
- 150000003839 salts Chemical group 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 239000012279 sodium borohydride Substances 0.000 description 1
- 229910000033 sodium borohydride Inorganic materials 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- YJGJRYWNNHUESM-UHFFFAOYSA-J triacetyloxystannyl acetate Chemical compound [Sn+4].CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O YJGJRYWNNHUESM-UHFFFAOYSA-J 0.000 description 1
- ITMCEJHCFYSIIV-UHFFFAOYSA-N triflic acid Chemical compound OS(=O)(=O)C(F)(F)F ITMCEJHCFYSIIV-UHFFFAOYSA-N 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Low-Molecular Organic Synthesis Reactions Using Catalysts (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
【発明の詳細な説明】
吏果圭史剋里公藪
本発明は臨床検査の分野で広く用いられているペプチダ
ーゼの酵素活性測定に好適なペプチダーゼ合成基質の効
率のよい改良製造法に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an improved and efficient method for producing a peptidase synthetic substrate suitable for measuring the enzymatic activity of peptidase, which is widely used in the field of clinical testing.
逆坐芝垣旦主プ塁じ
ペプチダーゼ合成基質は、ペプチダーゼによりＣ−末端
が切断され、発色基を遊離し、発色に関与するので、ペ
プチダーゼ酵素の活性に基づく臨床検査の分野では広く
用いられている。The peptidase synthetic substrate is cleaved at the C-terminus by peptidase, liberating a chromogenic group and participating in color development, so it is widely used in the field of clinical tests based on the activity of peptidase enzymes. There is.
かかるペプチダーゼの合成基質の代表的なものとして、
−数式
（Ｒ，はα−アミノ保護基、
Ｒ，およびＲ１はグリシル基、アラニル基、）くリル基
、ロイシル基またはインロイシル基、ＸおよびＹはハロ
ゲン原子を意味する）で示されるペプチドが挙げられる
。Representative synthetic substrates for such peptidases include:
- Peptides represented by the formula (R is an α-amino protecting group, R and R1 are glycyl, alanyl, )curyl, leucyl or inleucyl groups, and X and Y are halogen atoms. It will be done.
−数式（■）で表わされる化合物、例えば、Ｎ−ベンジ
ルオキシカルボニル−グリシル−グリシル−Ｌ−アルギ
ニル−３，５−ジブロモ−４−ヒドロキシアニリン［式
（Ｉ）中、Ｒ３は（□ＣＨ，０ＣＯ−
Ｒ１およびＲ，： −ＮＨＣＨ，Ｃ０−ＸおよびＹはＢ
ｒを意味する］は、ペプチダーゼ酵素の作用により発色
基である３、５−ジブロモ−４−ヒドロキシアニリン（
Ｄ Ｂ ＨＡ）を遊離し、Ｎ−エチル−Ｎ−（３−メチ
ルフェニル）−Ｎ”−スクシニルエチレンジアミン（Ｅ
ＭＳＥ）とカップリングし、緑色色素を生じる。かくし
て、その吸光度を測定することにより酵素活性を知るこ
とができる。- A compound represented by formula (■), for example, N-benzyloxycarbonyl-glycyl-glycyl-L-arginyl-3,5-dibromo-4-hydroxyaniline [in formula (I), R3 is (□CH,0CO - R1 and R,: -NHCH, C0-X and Y are B
3,5-dibromo-4-hydroxyaniline, which is a chromogenic group, is produced by the action of a peptidase enzyme.
D B HA) and N-ethyl-N-(3-methylphenyl)-N”-succinylethylenediamine (E
MSE) to produce a green dye. Thus, enzyme activity can be determined by measuring its absorbance.
このように−数式（１）で表わされる合成基質は非常に
重要であるが収率よく高純度な合成基質を得ることは困
難である。As described above, the synthetic substrate represented by the formula (1) is very important, but it is difficult to obtain a highly purified synthetic substrate with good yield.
従来、−数式（Ｉ）のペプチドは、−数式１１ Ｃ＝Ｎ（Ｎｏｔ） ＮＨ。Conventionally, the peptide of formula (I) has the formula (11) C=N (Not) N.H.
（式中、Ｒ１，Ｒ１、Ｒ１、ＸおよびＹは前記と同じで
ある）
で示される保護ペプチドを一般に、
パラジウム−炭素やラネーニ・ノケル触媒で還元して製
造されている［（ストライドライザー著、監訳、湯用泰
秀、有機化学解説■、９７６頁、広１書店（昭和５４年
）コ
しかしながら、パラジウム−炭素やラネーニ・７ケルを
触媒として還元したとき、発色基中の７％ロゲン原子が
変化し、酵素の作用が不十分となる。(In the formula, R1, R1, R1, Supervised translation, Yasuhide Yuyo, Organic Chemistry Commentary ■, 976 pages, Koichi Shoten (1976) However, when palladium-carbon or Raneni 7-kel is used as a catalyst to reduce the 7% rogen atoms in the color-forming group change. However, the action of the enzyme becomes insufficient.
また、水素化ホウ素ナトリウムや水素化リチウムアルミ
ニウムを用いて還元することも知られているが、式（Ｉ
Ｉ）のペプチドに含まれるニトロ基が十分に脱離しない
。It is also known to reduce using sodium borohydride or lithium aluminum hydride, but the formula (I
The nitro group contained in the peptide I) is not removed sufficiently.
本発明者らは、このような問題点を解決し、効率のよい
製造法を見出すため種々の還元剤を検討した結果、式（
ｎ）のペプチドを酸性下、錫あるいは亜鉛あるいはこれ
らの塩の存在下で還元することにより、目的とする式（
１）のペプチドを容易ビ高収率、高純度でかつ経済的に
得ることができることを見出した。In order to solve these problems and find an efficient production method, the present inventors investigated various reducing agents and found that the formula (
The desired formula (
It has been found that the peptide 1) can be easily obtained in high yield, high purity, and economically.
課題を解決するための手段
本発明は、−数式（ｎ）で示される保護ペプチドを錫あ
るいは亜鉛化合物の存在下で還元することにより、Ｎ−
末端のα−アミノ保護基、ＸおよびＹを脱離することな
く一般式（１）で示されるペプチドを得るペプチダーゼ
合成基質の製造法を提供するものである。Means for Solving the Problems The present invention provides - by reducing the protected peptide represented by formula (n) in the presence of a tin or zinc compound, N-
The present invention provides a method for producing a peptidase synthetic substrate that yields a peptide represented by general formula (1) without removing the terminal α-amino protecting group, X and Y.
さらに詳しくは、例えば式（ＩＩ）のペプチドをギ酸、
塩酸等の酸に溶解し、過剰の錫あるいは亜鉛化合物を加
え、３０〜６０’Ｃで３０分〜５時間反応させることに
より、６０％以上の収率で式（Ｉ）のペプチドを得るこ
とができる。用途によりトリフルオロメタンスルホン酸
等の酸で処理することにより種々の塩の形の式（１）の
ペプチドを得ることも可能である。More specifically, for example, the peptide of formula (II) is treated with formic acid,
The peptide of formula (I) can be obtained in a yield of 60% or more by dissolving it in an acid such as hydrochloric acid, adding an excess of tin or zinc compound, and reacting at 30 to 60'C for 30 minutes to 5 hours. can. Depending on the purpose, it is also possible to obtain the peptide of formula (1) in various salt forms by treatment with an acid such as trifluoromethanesulfonic acid.
出発物質として用いる式（Ｉ［）のペプチドは通常のペ
プチド合成の手法で製造できる。例えば、グリシル−グ
リシンのＮ−末端αアミ７基をベンジルオキシカルボニ
ル基で保護し、Ｎ−ベンジルオキシカルボニル−グリシ
ル−グリシンを得る。The peptide of formula (I[) used as a starting material can be produced by conventional peptide synthesis techniques. For example, the N-terminal α-ami7 group of glycyl-glycine is protected with a benzyloxycarbonyl group to obtain N-benzyloxycarbonyl-glycyl-glycine.
一方、ｔ−ブトキシカルボニル−Ｌ−ニトロアルギニン
と３，５−ジブロモ−４−ヒドロキシアニリンをＮ−メ
チルモルホリン、インブチルクロロホルメート存在下、
縮合し、ｔ−ブトキシカルボニル−Ｌ−ニトロアルギニ
ル−３，５−ジブロモ−４−ビトロキシアニリンを得る
。ついで、トリフルオロ酢酸で処理し、トリフルオロ酢
酸り一ニト亡アルギニルー３．５−ジブロモ−４−ヒド
ロキシアニリンを得る。これを前記のＮ−ベンジルオキ
シカルボニル−グリシル−グリシンとＮ−メチルモルホ
リン、インブチルクロロホルメート、トリエチルアミン
の存在下で縮合させ、目的とす６Ｘ（１）のＮ−ベンジ
ルオキシカルボニル−グリシル−グリシル−し−ニトロ
アルギニル−３゜５−ジブロモ−４−ヒドロキシアニリ
ンを得る。On the other hand, t-butoxycarbonyl-L-nitroarginine and 3,5-dibromo-4-hydroxyaniline in the presence of N-methylmorpholine and inbutylchloroformate,
Condensation is performed to obtain t-butoxycarbonyl-L-nitroarginyl-3,5-dibromo-4-bitroxyaniline. It is then treated with trifluoroacetic acid to obtain trifluoroacetic acid-reduced arginyl-3,5-dibromo-4-hydroxyaniline. This is condensed with the above-mentioned N-benzyloxycarbonyl-glycyl-glycine in the presence of N-methylmorpholine, inbutylchloroformate, and triethylamine, and the desired N-benzyloxycarbonyl-glycyl-glycine of 6X (1) is obtained. -Nitroarginyl-3°5-dibromo-4-hydroxyaniline is obtained.
用いる錫あるいは亜鉛化合物としては、例えば、錫粉末
、塩化錫、酢酸錫、亜鉛粉末、亜鉛アマルガムなどが挙
げられる。これらは一般に、式（ＩＩ）にペプチドに対
して５倍モル以上、好ましくは、８〜１０倍モル程度で
用いられる。Examples of the tin or zinc compound used include tin powder, tin chloride, tin acetate, zinc powder, and zinc amalgam. These are generally used in formula (II) in an amount of at least 5 times the molar amount, preferably about 8 to 10 times the molar amount, relative to the peptide.
得られた式（【）のペプチドは、例えば、常法に従って
クロマトグラフィー、再結晶、再沈澱などにより精製さ
れる。The obtained peptide of formula ([) is purified, for example, by chromatography, recrystallization, reprecipitation, etc. according to conventional methods.
式（Ｉ）のペプチドはトリプシン、プラスミン、ブｃナ
ーゼ、フィシン、コラゲナーゼ等のペプチダーゼにより
Ｃ末端が切断され、発色基を遊離する。この現象は、臨
床診断薬に広く応用できる。The peptide of formula (I) is cleaved at the C-terminus by a peptidase such as trypsin, plasmin, bucnase, ficin, collagenase, etc. to release a chromogenic group. This phenomenon can be widely applied to clinical diagnostic agents.
哀塵悉
以下、参考例、実施例を挙げて具体的に説明するが、こ
れには本発明を限定するものではない。The present invention will be specifically described below with reference to reference examples and examples, but the present invention is not limited thereto.
参考例１
１ｊＫ法で得たＮ−ベンジルオキシカルボニル−グリシ
ル−グリシン５．１８９（１９，５ミリモル）を無水ジ
メチルホルムアミド（ＤＭＦ）ｌｏ４ｘ１２に溶解し、
冷却下（−１５°Ｃ）、これにＮ−メチルモルホリン２
．１４１０とインブチルクロロホルメート２．６５ｘ（
ｌを加えた。ついで、トリフルオロＤＭＬ−ニトロアル
ギニル３．５−ジブロモ−４−ヒドロキシアニリン１１
．９９の無水ＤＭＦ８３ｚ（ｌ溶液にトリエチルアミン
２．８５ｘ（ｌを加えた溶液を、冷却下（−１５℃）、
滴下した。滴下終了後、室温で２時間反応させた。反応
終了後、減圧濃縮し、残渣をシリカゲルクロマトグラフ
ィー（クロロホルム：メタノール＝５ ： １）で精製
し、Ｎ−ベンジルオキシカルボニル−グリシル−グリシ
ル−し−ニトロアルギニル−３，５−ジブロモ−４−ヒ
ドロキシアニリン１１．６９（収率８３％）を得た。Reference Example 1 5.189 (19.5 mmol) of N-benzyloxycarbonyl-glycyl-glycine obtained by the 1jK method was dissolved in anhydrous dimethylformamide (DMF) lo4x12,
Under cooling (-15°C), this was treated with N-methylmorpholine 2
．． 1410 and inbutyl chloroformate 2.65x (
Added l. Then, trifluoroDML-nitroarginyl 3,5-dibromo-4-hydroxyaniline 11
．． A solution of 99 anhydrous DMF 83z (l) and triethylamine 2.85x (l) was added under cooling (-15°C).
dripped. After the dropwise addition was completed, the mixture was allowed to react at room temperature for 2 hours. After the reaction was completed, it was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (chloroform:methanol = 5:1) to give N-benzyloxycarbonyl-glycyl-glycyl-nitroarginyl-3,5-dibromo-4-hydroxyaniline. 11.69 (yield 83%) was obtained.
実施例１
参考例１で得たＮ−ベンジルオキシカルボニル−クリシ
ル−グリシル−し−ニトロアルギニル−３，５−ジブロ
モ−４−ヒドロキシアニリン１１゜２９（１５，７ミリ
モル）を６０％ギ酸２２５ＲＱに溶解し、塩化錫２３．
９９を加え５０℃で１時間撹拌し、反応させた。Example 1 N-benzyloxycarbonyl-crysyl-glycyl-nitroarginyl-3,5-dibromo-4-hydroxyaniline 11°29 (15.7 mmol) obtained in Reference Example 1 was dissolved in 60% formic acid 225RQ. , tin chloride23.
99 was added thereto, and the mixture was stirred at 50°C for 1 hour to react.
反応終了後、濾過し、濾液を水２２５ｍ１２で希釈し、
硫化水素ガスを吹き込み沈殿を生成させ、濾去した。濾
液を減圧濃縮した後、メタノール−水、さらにメタノー
ル−酢酸エチルより再結晶させ、Ｎ−ベンジルオキシカ
ルボニル−グリシル−グリシル−し−アルギニル−３，
５−ジブロモ−４−ヒドロキシアニリンの白色結晶７．
１９（収率６４％）を得た。After the reaction was completed, it was filtered and the filtrate was diluted with 225 ml of water.
Hydrogen sulfide gas was blown into the solution to form a precipitate, which was filtered off. After concentrating the filtrate under reduced pressure, it was recrystallized from methanol-water and then methanol-ethyl acetate to give N-benzyloxycarbonyl-glycyl-glycyl-arginyl-3,
White crystals of 5-dibromo-4-hydroxyaniline7.
19 (yield 64%) was obtained.
融点：１３０−１３４℃
［αコ Ｄ”ニー１４．３°（ｃ＝１．０． メタノ
ール）
元素分析：
実測値（％） Ｃ：４０．７３． Ｈ：４．２７．
Ｎ：１３．８５計算値（％） Ｃ：４０．７１． Ｈ
：４．２６． Ｎ：Ｌ３．２５ＮＭＲ（ＣＤ、ＯＤ）：
δ １．５３〜２．０７（ｍ、４Ｈ）、３．２１（ｔ。Melting point: 130-134°C [α Co D” knee 14.3° (c = 1.0. Methanol) Elemental analysis: Actual value (%) C: 40.73. H: 4.27.
N: 13.85 Calculated value (%) C: 40.71. H
:4.26. N:L 3.25 NMR (CD, OD): δ 1.53-2.07 (m, 4H), 3.21 (t.
２Ｈ，Ｊ＝６．４Ｈｚ）、３．８２ （ｓ、２Ｈ）、３
゜９２ （ＡＢｑ、２Ｈ，Ｊ＝１７．６Ｈｚ、 Δ、Ａ
Ｂ＝２６．０Ｈｚ）、４．４８ （ｄｄ、ＬＨ，Ｊ＝４
．６゜８．４Ｈｚ）、５．０７ （ｓ、２Ｈ）、７．３
２（ｓ。2H, J=6.4Hz), 3.82 (s, 2H), 3
゜92 (ABq, 2H, J=17.6Hz, Δ, A
B=26.0Hz), 4.48 (dd, LH, J=4
．． 6°8.4Hz), 5.07 (s, 2H), 7.3
2 (s.
５Ｈ）、７．８１ （ｓ、２Ｈ）
参考例２
常法で得たメトキシカルボニル−グリシル−グリシン１
．７９を２４ｘ（ｌの無水ＤＭＦに溶解し、これに、冷
却下（−１５℃）でＮ−メチルモルホリン２．２＊（！
とイソブチルクロロホルメート２．６ｘＱを加えた。5H), 7.81 (s, 2H) Reference Example 2 Methoxycarbonyl-glycyl-glycine 1 obtained by a conventional method
．． 79 was dissolved in 24x(l) of anhydrous DMF and added with 2.2*(!) of N-methylmorpholine under cooling (-15°C).
and 2.6xQ of isobutyl chloroformate were added.
ついで、さらに、トリフルオロ酢酸Ｌ−ニドＣアルギニ
ル−３，５−ジブロモ−４−ヒドロキシアニリン６．０
９を無水ＤＭＦ６０ｆｆｉ！２に溶解し、トリエチルア
ミン４．２ｚ（ｌを加えた溶液を冷却下（−１５℃）滴
下した。滴下終了後、室温で１時間反応させた。Then, further, trifluoroacetic acid L-nide C arginyl-3,5-dibromo-4-hydroxyaniline 6.0
9 with anhydrous DMF60ffi! A solution containing 4.2z (l) of triethylamine was added dropwise under cooling (-15°C). After the dropwise addition was completed, the reaction was allowed to proceed at room temperature for 1 hour.
反応終了後、減圧濃縮後、残渣をシリカゲルカラムで精
製し、メトキシカルボニルーグリシルーグワシルーし一
ニトロアルギニルー３．５−ジブロモ−４−ヒドロキシ
アニリン１．２８ｙ（収率２０％）を得た。After the reaction was completed, the residue was concentrated under reduced pressure and purified with a silica gel column, and methoxycarbonyl-glycyl-glycyl was mixed to obtain 1.28y of mononitroarginyl-3,5-dibromo-4-hydroxyaniline (yield 20%). .
Ｘ塵廻１
参考例２で得たメトキシカルボニル−グリシル−グリシ
ル−Ｌ−ニトロアルギニル−３，５−ジブａモー４−ヒ
ドロキシアニリン１．０９を６０％ギ酸２０ｍ１！に溶
解し、塩化錫（ｆｆ）２．３７９を加え、５０℃で３時
間撹拌して反応させた。反応衿了後、濾過し、濾液に硫
化水素を吹き込み、濾液を減圧濃縮後、メタノール−酢
酸エチルで再沈澱させた。シリカゲルカラムで精製し、
メトキシカルボニル−グリシル−グリシル−Ｌ−アルギ
ニル−３，５−ジブロモ−４−ヒドロキシアニリン０゜
６１９（収率６２％）を得た。X-dust 1 1.09 methoxycarbonyl-glycyl-glycyl-L-nitroarginyl-3,5-dibu-am-4-hydroxyaniline obtained in Reference Example 2 was added to 20 ml of 60% formic acid! 2.379 of tin chloride (ff) was added thereto, and the mixture was stirred at 50° C. for 3 hours to react. After the reaction was completed, it was filtered, hydrogen sulfide was blown into the filtrate, the filtrate was concentrated under reduced pressure, and then reprecipitated with methanol-ethyl acetate. Purified with silica gel column,
Methoxycarbonyl-glycyl-glycyl-L-arginyl-3,5-dibromo-4-hydroxyaniline 0°619 (yield 62%) was obtained.
ＮＭＲ（ＣＤ、≠ＯＤ）：
δ １．３５〜２．２９ （ＩＩ＋、４Ｈ） 、 ３
．２３ （２Ｈ）、３．６８ （ｓ、３Ｈ）、３．８４
（ｓ、２Ｈ）。NMR (CD, ≠OD): δ 1.35-2.29 (II+, 4H), 3
．． 23 (2H), 3.68 (s, 3H), 3.84
(s, 2H).
３．９５ （ｓ、２Ｈ）、４．５１ （ｎ、ＩＨ）、７
．８１ （Ｓ、２Ｈ）
参考例３
常法で得たＮ−ペンジルオ亭ジカルボニルーβ−アラニ
ル−グリシン２．８９を５６１４の無水ＤＭＦに溶解し
、これに冷却下（−１５℃）でＮ−メチルモルホリン１
１１１２とインブチルクロロホルメ−）１３．８１１１
２を加えた。ついで、トリフルオロ酢ＭＬ−ニトロアル
ギニル−３，５−ジブロモ−４−ヒドロキシアニリン６
、Ｏｆを無水ＤＭＦ６０１１１２に溶解し、トリエチル
アミン４．２Ｍσを加えた溶液を冷却下（−１５℃）で
滴下した。滴下終了後、室温で１時間反応させた。反応
終了後、減圧濃縮し、残渣をシリカゲルカラムで精製し
、Ｎ−ベンジルオキシカルボニル−β−アラニル−グリ
シル−Ｌ−ニトロアルギニル−３，５−ジブロモ−４−
ヒドロキシアニリン４．０９（収１５５％）を得た。3.95 (s, 2H), 4.51 (n, IH), 7
．． 81 (S, 2H) Reference Example 3 2.89% of N-penzylotei dicarbonyl-β-alanyl-glycine obtained by a conventional method was dissolved in 5614 anhydrous DMF, and N-methylmorpholine was added to this under cooling (-15°C). 1
1112 and inbutylchloroforme) 13.8111
Added 2. Then, trifluoroacetic acid ML-nitroarginyl-3,5-dibromo-4-hydroxyaniline 6
, Of was dissolved in anhydrous DMF601112, and a solution containing 4.2 Mσ of triethylamine was added dropwise under cooling (-15°C). After the dropwise addition was completed, the mixture was allowed to react at room temperature for 1 hour. After the reaction was completed, it was concentrated under reduced pressure, and the residue was purified with a silica gel column to obtain N-benzyloxycarbonyl-β-alanyl-glycyl-L-nitroarginyl-3,5-dibromo-4-
4.09 hydroxyaniline (yield: 155%) was obtained.
実施ｆ１１３
参考例２で得たＮ−ベンジルオキシカルボニル−β−ア
ラニル−グリシル−し−ニトロアルギニル−３，５−ジ
ブロモ−４−ヒドロキシアニリン２．０９を６０％ギ酸
４０ＩＱに溶解し、塩化錫（ｎ）４．１６ｇを加え、５
０℃で２時間攪拌し、反応させた。反応終了後、濾液に
硫化水素を吹き込み沈殿を生成させ濾去した。濾液を減
圧濃縮後、メタノール酢酸エチルで再沈澱させ、シリカ
ゲルカラムで精製し、Ｎ−ベンジルオキシカルボニル−
β−アラニル−グリシル−Ｌ−ニトロアルギニル−３，
５−ジブロモ−４−ヒドロキシアニリン１．１９９（収
率６０％）を得た。Implementation f113 2.09% of N-benzyloxycarbonyl-β-alanyl-glycyl-thi-nitroarginyl-3,5-dibromo-4-hydroxyaniline obtained in Reference Example 2 was dissolved in 60% formic acid 40IQ, and tin chloride (n ) Add 4.16g, 5
The mixture was stirred at 0° C. for 2 hours to react. After the reaction was completed, hydrogen sulfide was blown into the filtrate to form a precipitate, which was filtered off. After concentrating the filtrate under reduced pressure, it was reprecipitated with methanol and ethyl acetate, and purified with a silica gel column to obtain N-benzyloxycarbonyl-
β-alanyl-glycyl-L-nitroarginyl-3,
1.199 of 5-dibromo-4-hydroxyaniline (yield 60%) was obtained.
ＮＭＲ（ＣＤ、ＯＤ）： δ ｌ、４７〜２．２５（園、４Ｈ）、２．５１（ｔ。NMR (CD, OD): δ l, 47-2.25 (Sono, 4H), 2.51 (t.
２Ｈ，Ｊ＝６．１Ｈｚ）、３．２２（２Ｈ）、３．４５
（ｔ、２Ｈ，Ｊ＝６．１Ｈｚ）、３．８９ （ｓ、２
Ｈ）、４．５２ （ｍ、ＬＨ）、５．０９（ｓ、２Ｈ）
。2H, J=6.1Hz), 3.22 (2H), 3.45
(t, 2H, J=6.1Hz), 3.89 (s, 2
H), 4.52 (m, LH), 5.09 (s, 2H)
.
Claims (1)
基、ロイシル基またはイソロイシル基、 ＸおよびＹはハロゲン原子を意味する） で示される保護ペプチドを錫あるいは亜鉛化合物の存在
下で還元して、Ｎ−末端のα−アミノ保護基、Ｘおよび
Ｙを脱離することなしに、一般式で示される ▲数式、化学式、表等があります▼ （Ｒ＿１：式中、Ｒ＿１、Ｒ＿２、Ｒ＿３、ＸおよびＹ
は前記と同じである） で示されるペプチドを得ることを特徴とするペプチダー
ゼ合成基質の製造法。(1) General formula ▲ Numerical formula, chemical formula, table, etc. ▼ (In the formula, R_1 is an α-amino protecting group, R_2 and R_3 are glycyl, alanyl, valyl, leucyl, or isoleucyl groups, and X and Y are (meaning a halogen atom) is reduced in the presence of a tin or zinc compound to reduce the protected peptide represented by the general formula without removing the N-terminal α-amino protecting group, X and Y. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (R_1: In the formula, R_1, R_2, R_3, X and Y
is the same as above) A method for producing a peptidase synthetic substrate, characterized by obtaining a peptide represented by:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2070338A JPH0742312B2 (en) | 1990-03-20 | 1990-03-20 | Method for producing peptidase synthetic substrate |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2070338A JPH0742312B2 (en) | 1990-03-20 | 1990-03-20 | Method for producing peptidase synthetic substrate |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH03271299A true JPH03271299A (en) | 1991-12-03 |
| JPH0742312B2 JPH0742312B2 (en) | 1995-05-10 |
Family
ID=13428532
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2070338A Expired - Fee Related JPH0742312B2 (en) | 1990-03-20 | 1990-03-20 | Method for producing peptidase synthetic substrate |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0742312B2 (en) |
-
1990
- 1990-03-20 JP JP2070338A patent/JPH0742312B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0742312B2 (en) | 1995-05-10 |
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