JPS6384499A - Production of delta sleep inducing peptide by enzyme - Google Patents
Production of delta sleep inducing peptide by enzymeInfo
- Publication number
- JPS6384499A JPS6384499A JP22870186A JP22870186A JPS6384499A JP S6384499 A JPS6384499 A JP S6384499A JP 22870186 A JP22870186 A JP 22870186A JP 22870186 A JP22870186 A JP 22870186A JP S6384499 A JPS6384499 A JP S6384499A
- Authority
- JP
- Japan
- Prior art keywords
- protected
- fragments
- ala
- peptide
- amino acid
- 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.)
- Pending
Links
- 102000004190 Enzymes Human genes 0.000 title claims abstract 3
- 108090000790 Enzymes Proteins 0.000 title claims abstract 3
- 238000004519 manufacturing process Methods 0.000 title claims description 21
- ZRZROXNBKJAOKB-GFVHOAGBSA-N (2s)-2-[[2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[2-[[2-[[(2s)-2-[[(2s)-2-amino-3-(1h-indol-3-yl)propanoyl]amino]propanoyl]amino]acetyl]amino]acetyl]amino]-3-carboxypropanoyl]amino]propanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]pentanedioic acid Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O)=CNC2=C1 ZRZROXNBKJAOKB-GFVHOAGBSA-N 0.000 title claims description 5
- 108010051088 Delta Sleep-Inducing Peptide Proteins 0.000 title claims description 5
- 150000001413 amino acids Chemical class 0.000 claims abstract description 26
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 19
- 239000012634 fragment Substances 0.000 claims abstract description 14
- 238000000034 method Methods 0.000 claims abstract description 14
- 125000006239 protecting group Chemical group 0.000 claims abstract description 6
- 238000006467 substitution reaction Methods 0.000 claims abstract description 6
- 125000001584 benzyloxycarbonyl group Chemical group C(=O)(OCC1=CC=CC=C1)* 0.000 claims abstract description 5
- 239000000470 constituent Substances 0.000 claims abstract description 5
- UYWQUFXKFGHYNT-UHFFFAOYSA-N phenylmethyl ester of formic acid Natural products O=COCC1=CC=CC=C1 UYWQUFXKFGHYNT-UHFFFAOYSA-N 0.000 claims abstract description 5
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims abstract description 4
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims abstract description 4
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 claims abstract description 4
- KQDMENMTYNBWMR-WHFBIAKZSA-N Gly-Asp-Ala Chemical compound [H]NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O KQDMENMTYNBWMR-WHFBIAKZSA-N 0.000 claims abstract 2
- CXUFDWZBHKUGKK-CABZTGNLSA-N Trp-Ala-Gly Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O)=CNC2=C1 CXUFDWZBHKUGKK-CABZTGNLSA-N 0.000 claims abstract 2
- 238000010168 coupling process Methods 0.000 claims abstract 2
- 235000001014 amino acid Nutrition 0.000 claims description 21
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 5
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 4
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 3
- 238000009833 condensation Methods 0.000 claims description 3
- 230000005494 condensation Effects 0.000 claims description 3
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 2
- 235000003704 aspartic acid Nutrition 0.000 claims description 2
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 2
- MHDVGSVTJDSBDK-UHFFFAOYSA-N dibenzyl ether Chemical compound C=1C=CC=CC=1COCC1=CC=CC=C1 MHDVGSVTJDSBDK-UHFFFAOYSA-N 0.000 claims description 2
- 235000013922 glutamic acid Nutrition 0.000 claims description 2
- 239000004220 glutamic acid Substances 0.000 claims description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 2
- 150000002148 esters Chemical class 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 abstract description 11
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 3
- MIJWOJAXARLEHA-WDSKDSINSA-N Ser-Gly-Glu Chemical compound OC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(O)=O MIJWOJAXARLEHA-WDSKDSINSA-N 0.000 abstract 1
- 239000006227 byproduct Substances 0.000 abstract 1
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 84
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 72
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 30
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 24
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 24
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 24
- 239000000047 product Substances 0.000 description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 19
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 18
- 230000008018 melting Effects 0.000 description 16
- 238000002844 melting Methods 0.000 description 16
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 15
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 12
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 12
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 12
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 12
- 108090000526 Papain Proteins 0.000 description 11
- 239000004365 Protease Substances 0.000 description 11
- 238000000921 elemental analysis Methods 0.000 description 11
- 229940055729 papain Drugs 0.000 description 11
- 235000019834 papain Nutrition 0.000 description 11
- 239000002244 precipitate Substances 0.000 description 11
- 239000000243 solution Substances 0.000 description 11
- 238000004458 analytical method Methods 0.000 description 10
- 238000001914 filtration Methods 0.000 description 10
- 239000000203 mixture Substances 0.000 description 10
- 239000003208 petroleum Substances 0.000 description 10
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 7
- 238000003756 stirring Methods 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- 229910000029 sodium carbonate Inorganic materials 0.000 description 6
- 229910052938 sodium sulfate Inorganic materials 0.000 description 6
- 235000011152 sodium sulphate Nutrition 0.000 description 6
- 238000005259 measurement Methods 0.000 description 5
- 239000007974 sodium acetate buffer Substances 0.000 description 5
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 102100022749 Aminopeptidase N Human genes 0.000 description 4
- 108010049990 CD13 Antigens Proteins 0.000 description 4
- -1 benzyl ester Chemical class 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 238000010898 silica gel chromatography Methods 0.000 description 4
- 230000007958 sleep Effects 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000012043 crude product Substances 0.000 description 3
- 229910001873 dinitrogen Inorganic materials 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 238000006911 enzymatic reaction Methods 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 3
- JOVOSQBPPZZESK-UHFFFAOYSA-N phenylhydrazine hydrochloride Chemical compound Cl.NNC1=CC=CC=C1 JOVOSQBPPZZESK-UHFFFAOYSA-N 0.000 description 3
- 229940038531 phenylhydrazine hydrochloride Drugs 0.000 description 3
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000012267 brine Substances 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 2
- 239000010446 mirabilite Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 230000006340 racemization Effects 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- QVHJQCGUWFKTSE-YFKPBYRVSA-N (2s)-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoic acid Chemical compound OC(=O)[C@H](C)NC(=O)OC(C)(C)C QVHJQCGUWFKTSE-YFKPBYRVSA-N 0.000 description 1
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- ZSLUVFAKFWKJRC-IGMARMGPSA-N 232Th Chemical compound [232Th] ZSLUVFAKFWKJRC-IGMARMGPSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 206010062519 Poor quality sleep Diseases 0.000 description 1
- 208000013738 Sleep Initiation and Maintenance disease Diseases 0.000 description 1
- 229910052776 Thorium Inorganic materials 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 108010027597 alpha-chymotrypsin Proteins 0.000 description 1
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 description 1
- 229940040526 anhydrous sodium acetate Drugs 0.000 description 1
- 238000010923 batch production Methods 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 230000005587 bubbling Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- DUYAAUVXQSMXQP-UHFFFAOYSA-N ethanethioic S-acid Chemical compound CC(S)=O DUYAAUVXQSMXQP-UHFFFAOYSA-N 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 150000003949 imides Chemical class 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 206010022437 insomnia Diseases 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 229940098779 methanesulfonic acid Drugs 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000001139 pH measurement Methods 0.000 description 1
- 230000000803 paradoxical effect Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000002953 preparative HPLC Methods 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 210000001747 pupil Anatomy 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 230000037322 slow-wave sleep Effects 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- RSIJVJUOQBWMIM-UHFFFAOYSA-L sodium sulfate decahydrate Chemical compound O.O.O.O.O.O.O.O.O.O.[Na+].[Na+].[O-]S([O-])(=O)=O RSIJVJUOQBWMIM-UHFFFAOYSA-L 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、１９７７年にシェネルベルゲル（Ｓｃｈｏｅ
ｎｅｒｂｔｒｑｅｒ ） 等によりウサギの血液中か
ら単離されたアミノ酸９個からなる徐波睡眠促腫性（デ
ルタ−スリーブ インデューシング）作用を有するペプ
チドを、初めて酵素法により製造することに関する。DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention was first developed by Schönelberger in 1977.
The present invention relates to the production for the first time by an enzymatic method of a peptide having a slow-wave sleep inducing effect (delta-sleeve inducing), which is composed of nine amino acids and is isolated from rabbit blood by E. nerbtrqer et al.
このペプチドは前記した１９７７年の単離に際し、その
構造式が：
Ｈ−Ｔｒｐ −Ａｌａ−Ｇｌ）−Ｇｌ）−Ａｓｐ −ｋ
ｌａ −８ｒｒ−Ｇｌ）−Ｇｌｕ −０：Ｆ（であるこ
とが確認され、爾後その薬理効果の研究と誘導体をも含
めた化学的合成法が相次いで発表されて現在に至ってい
るが、酵素法による製造は現在まで知られていない。When this peptide was isolated in 1977, its structural formula was: H-Trp-Ala-Gl)-Gl)-Asp-k
la -8rr-Gl)-Glu -0:F(, and since then research on its pharmacological effects and chemical synthesis methods including derivatives have been published one after another, and up to the present day, enzymatic methods Its production is unknown to date.
化学的合成法の若干例を示せば次の通りである：Ｈｓｕ
Ｊｅｚｔｎ ｔｔ ａｌ、 、 ： Ｐ ＲＣ−Ｆ
ＲＧ Ｊｏｉｎｔ８ｐｍｐｏｓｉｕｍ ｏｎ Ｎｕｔ
ｌｅｉｔ Ａｃ１ｄｓ ａｎｄ Ｐｒｏｔｅｉｎｓ （
Ａｂｓｔｒａｃｔ） 、 ５ｈａｎ＄ａｉ＋ （１
９７９Ｌ ６８ ６９゜Ｋａｓｔｉｎ、 Ｐ Ｔ ｔ
ｔ ａｌ、 、 ； Ｂｒａｉｎ Ｒｔｓ、 Ｂｕ
ｔム、３゜６９１−６９５ （１９７８）。Some examples of chemical synthesis methods are as follows: Hsu
Jeztn tt al, : P RC-F
RG Joint8pmposium on Nut
leit Ac1ds and Proteins (
Abstract), 5han$ai+ (1
979L 68 69゜Kastin, P T t
Brain Rts, Bu
TM, 3°691-695 (1978).
Ｎｏｚａｋｉ、 Ｓ ｅｔ ａｌ、 、 ： Ｂｕｌ
ｌ、 Ｃｈｔｎｔ、、 Ｆ３ｏｔ、 Ｊａｐａｎ。Nozaki, S. et al.: Bul
l, Chtnt, F3ot, Japan.
５５、 ２１６５−２１６８ （１９８２）。55, 2165-2168 (1982).
なお、本ペプチドは一般に徐波型（デルタ）睡眠の誘発
を促進する効果があるとされているが、ネコにおいて逆
説睡眠をも誘発する作用があシ、ヒトにおいては静脈内
注射によって昼間の覚醒レベルが充まシ、夜間の睡眠は
促進される効果が認められ、不眠症治療剤として化学合
成品が市販されている現状である。Although this peptide is generally said to have the effect of promoting the induction of slow-wave (delta) sleep, it also has the effect of inducing paradoxical sleep in cats, and in humans, intravenous injection has been shown to induce daytime wakefulness. It has been found to be effective in improving sleep levels and promoting nighttime sleep, and chemically synthesized products are now on the market as insomnia treatments.
−一般に適用されることではあるが、本デルタ睡眠誘発
ペプチドの製造についても、化学的合成法に比して本発
明の酵素的製造方法は構成アミノ酸のラセミ化がなく、
反応条件が穏やかであるので副反応物の生成をみること
が少く、かつ、反応に際して有害な試薬を使用しなくて
済む、等の長所が数・見られる。-Although generally applicable, the enzymatic production method of the present invention does not cause racemization of the constituent amino acids compared to chemical synthesis methods for the production of the present delta sleep-inducing peptide.
Since the reaction conditions are mild, there are few side-reactants produced, and there is no need to use harmful reagents during the reaction.
より段階的にペプチド鎖化して下記する三つの区分フラ
グメントを製造した：
〔Ｉ〕：保護化したＳｔｒ −Ｇｌ） −Ｇｌｕ 。More stepwise peptide chaining was performed to produce the following three segmented fragments: [I]: Protected Str-Gl)-Glu.
〔■〕：保護化した０１） −Ａ＄ −Ａｌａ 。[■]: Protected 01) -A$ -Ala.
〔■〕：保護化したＴｒｐ−Ａｌａ −Ｇｌ）ここに、
各フラグメントの末端の保護基はフェニルヒドラジドと
した。アミノ酸としてトリプトファンはベンジルオキシ
カルボニル、その他のアミノ酸は第三ブチルオキシカル
ボニルでＮ末端を保護したものを用いた。アスパラギン
酸、グルタミン酸の側鎖カルボキシルはベンジルエステ
ル、セリンの側鎖水酸基はベンジルエーテルとして保護
したものを用いた。ペプチド結合の形成には、カンプリ
ング法及び置換法を併用した。フラグメント結合には置
換法を採用した。[■]: Protected Trp-Ala-Gl) Here,
The terminal protecting group of each fragment was phenylhydrazide. Tryptophan was used as an amino acid with benzyloxycarbonyl, and other amino acids were used with their N-terminals protected with tert-butyloxycarbonyl. The side chain carboxyl of aspartic acid and glutamic acid was protected as benzyl ester, and the side chain hydroxyl group of serine was protected as benzyl ether. A combination of camping method and substitution method was used to form the peptide bond. A substitution method was adopted for fragment conjugation.
なお、説明の便宜上、ペプチド化学の常例に従い、次の
略号符を本明細書でも用いるものとする。For convenience of explanation, the following abbreviations will be used in this specification as well, in accordance with common practice in peptide chemistry.
２：ベンジルオキシカルボニル
Ｂｏｔ：第三ブチルオキシカルボニル
７３ｚｌ ：ペンジル
ＴＦＡ： ）ルフルオロ酢酸
−ＯＭｅ ；メチルエステル
一０ＢＪＩ ：ベンジルエステル
−Ｎ２’ｆ１２Ｐｈ ：フェニルヒドラジド実施例：
Ｈ−Ｔｒｐ−Ａｌａ −Ｇｌｐ −Ｇｌ＆ −Ａｓｐ−
Ａｌａ−Ｂｙ −Ｑ／）二Ｇｌｕ−ＯＲの製造
本実施例の実験は下記する手技によって行なわれた。2: Benzyloxycarbonyl Bot: tert-butyloxycarbonyl 73zl: Penzyl TFA: ) Fluoroacetic acid-OMe; Methyl ester-0BJI: Benzyl ester-N2'f12Ph: Phenylhydrazide Examples:
H-Trp-Ala -Glp -Gl& -Asp-
Preparation of Ala-By-Q/)2Glu-OR The experiment of this example was conducted by the following procedure.
融点ニヤマド社製ＭＰ−２１型融点測定装置、未補正
旋光度：堀場５ＥＰＡ−２００
薄層クロマトグラフィー（ＴＬＣ）：メルク社製キーゼ
ルゲル６０
展開溶媒Ｒｆ１；クロロホルム：メタノール＝１０ ：
１
Ｒ／２ ； ｎ−ブタノール：酢酸：水＝４：１：１
アミノ酸分析：日立８３５型アミノ酸分析計試料は封管
中、６Ｎ塩酸で１１ＣＩ’Ｃ１１８時間加水分解した。Melting point: MP-21 type melting point measuring device manufactured by Niyamad Co., Ltd., uncorrected optical rotation: Horiba 5EPA-200 Thin layer chromatography (TLC): Kieselgel 60 manufactured by Merck & Co., Ltd. Developing solvent Rf1: Chloroform: methanol = 10:
1 R/2; n-butanol:acetic acid:water=4:1:1 Amino acid analysis: Hitachi Model 835 amino acid analyzer The sample was hydrolyzed with 6N hydrochloric acid for 118 hours in a sealed tube.
トリプトファンを含むペプタイドは封管中、４Ｍノタン
スルホン酸で１１０°０１１８時間加水分解した。Peptides containing tryptophan were hydrolyzed with 4M notane sulfonic acid in a sealed tube for 110 hours.
アミノペプチダーゼＭによる本発明の目的ペプチドの加
水分解ニアミノペプチダーゼＭの０，０５Ｍトリス塩酸
緩衝液（ｐＨ７，７３）の溶液（２Ｕ量）で３７°Ｃ１
２４時間ペプチドを消化し、アミノ酸分析を行なった。Hydrolysis of the target peptide of the present invention by aminopeptidase M. A solution (2U volume) of niaminopeptidase M in 0.05M Tris-HCl buffer (pH 7.73) at 37°C.
Peptides were digested for 24 hours and amino acid analysis was performed.
ＰＨ測定ニガラス電極により測定
ＨＰＬＣ（高速液体クロマトグラフィー）：装置；高滓
ＬＣ−４Ａ、検出器；高滓ＳＰＤ２ Ａｓ
カラム； Ｎｕｔｌｅｏｓｉ１５Ｃ１Ｂ （０，４Ｘ
２０ ｃＭ）流速９１肩ｌ／分
検出器２２０ ｎｍ
溶出；１４チメタノール／Ｑ、１チＴＦＡ試薬：保護ア
ミノ酸はＬ−型を用い、ペプチド研究所（大阪）よシ購
入。パパインは和光紬薬（１：３５０）、α−キモトリ
プシンはシグマ（…型）そしてアミノペプチダーゼＭは
ペーリンガーマンハイムよりそれぞれ購入した。その他
の試薬は特級品を使用。PH measurement Measured using a Niglass electrode HPLC (High Performance Liquid Chromatography): Apparatus; Takashi LC-4A, Detector; Takashi SPD2 As column; Nutleosi15C1B (0,4X
20 cM) Flow rate: 91 l/min Detector: 220 nm Elution: 14 timeethanol/Q, 1 time TFA Reagent: L-type protected amino acid was used, purchased from Peptide Research Institute (Osaka). Papain was purchased from Wako Tsumugi (1:350), α-chymotrypsin was purchased from Sigma (... type), and aminopeptidase M was purchased from Peringer Mannheim. Other reagents are of special grade.
分取ＨＰＬＣ：
装置；高滓Ｌ Ｃ−４Ａ 、検出器：高滓ＳＰＤＡＳ
カラム； Ｎｕｃｌｔｏｓｉｌ ５ＣＩＢ （１，ＯＸ
２５ ｆｆ１）流速Ｈ５ｒｘｌ／分
検出ｉ ２８０ ｎｍ
溶出；Ａ メタノール
Ｂ Ｏ２８チ酢酸
０−→２分 １５チＡ
２→１７分 １５ ２１％Ａ
保持時間１６分付近の主ピークを分取した。Preparative HPLC: Apparatus: Takashi L C-4A, Detector: Takashi SPDAS Column; Nucltosil 5CIB (1,OX
25 ff1) Flow rate H5rxl/min Detection i 280 nm Elution; A Methanol B O28 thiacetic acid 0-→2 minutes 15% A 2→17 minutes 15 21% A The main peak around the retention time of 16 minutes was fractionated.
［１−） ： Ｂｏｃ−Ｓｙ （”ｆｉＱＢｚｌ ）
−Ｇｌｐ −Ｇｌｕ （ＯＢｚ／ ） −Ｎ２．Ｈ２Ｐ
ｈの合成
（イ）Ｂｏｔ −Ｇｌｕ （ＯＢｇ／ ）−Ｎ２Ｈ２Ｒ
＆の製造フェニルヒドラジン塩酸塩（８，６８，６０ミ
リ−Ｅ： ル） 、Ｂｏｃ −Ｇｌｕ （細ｘ／ ）
−ＯＨ（６，７５ｔ、２０ミリモル）に２Ｍ酢酸ナトリ
ウム緩衝液（ｐＨ５，０）４００ｍｌ及びメタノール１
５０ｓｔ／を加え、加温溶解した。２Ｎ水酸化ナトリウ
ム（約２０ｆｆｌ）で所５．０に調整し、窒素ガスを数
分間バブリングした。２−メルカプトエタノール（３，
１０？）、パパイン（５，００？）を加え、室温で１８
時間攪拌した。生成した沈澱をｐ取、水洗浄後、酢酸エ
チル（約２００ｍ１）に溶解、分液ロート上、酢酸エチ
ル層を冷０．５Ｎ塩酸、５チ炭酸水素ナトリウム、飽和
食塩水で順次洗浄後、芒硝上乾燥、濾過後、酢酸エチル
を減圧留去し、残渣を酢酸エチルと石油エーテルで再結
晶した。[1-): Boc-Sy (”fiQBzl)
-Glp -Glu (OBz/ ) -N2. H2P
Synthesis of h (a) Bot -Glu (OBg/ )-N2H2R
Production of & phenylhydrazine hydrochloride (8,68,60m-E:L), Boc-Glu (fine x/)
-OH (6,75 t, 20 mmol) in 400 ml of 2M sodium acetate buffer (pH 5,0) and methanol 1
50st/ was added and dissolved by heating. The temperature was adjusted to 5.0 with 2N sodium hydroxide (approximately 20 ffl), and nitrogen gas was bubbled in for several minutes. 2-mercaptoethanol (3,
10? ), add papain (5,00?) and heat at room temperature for 18
Stir for hours. The generated precipitate was collected, washed with water, dissolved in ethyl acetate (approximately 200 ml), placed on a separating funnel, and the ethyl acetate layer was washed successively with cold 0.5N hydrochloric acid, 5% sodium bicarbonate, and saturated saline, and then added with Glauber's salt. After drying and filtration, ethyl acetate was distilled off under reduced pressure, and the residue was recrystallized from ethyl acetate and petroleum ether.
収量８．１０１ｉ′（収率９５％）、融点１１３．５−
１１５°Ｃ１Ｒ７１０，６５、〔６３１％５−１６．イ
（ＣＣ１５、メタノール）。Yield 8.101i' (95% yield), melting point 113.5-
115°C1R710,65, [631%5-16. (CC15, methanol).
元素分析（イ）： Ｃ２３Ｈ２９Ｎ３０５として理論値
： Ｃ１６４，６２、Ｈ，６，８４、ＮＳ９．８３測定
値：Ｃ１６４，３１；Ｈ，６，９Ｃ１；Ｎ、 ９．７４
（ｏ） Ｂｏｔ −Ｇｌ＆ −Ｇｌｕ （０Ｂｚｌ ）
−Ｎ２ Ｈ２Ｐｈ の製造前工程の生成物Ｂｏｃ
−Ｃ４１ｕ （ＯＢＺ／ ）−Ｎ２Ｉ（２ＰＡ （６，
４１５’、１５ミリモル）をＴＦＡ（１Ｂ＋！Ｉｌ）で
室温３０分間処理し、脱Ｂｏｔ化した。過剰の’Ｉ’Ｆ
Ａを減圧留去し、水酸化カリウム上３時間減圧乾燥した
。これに２Ｍ酢酸ナトリウム緩衝液（ｐＴ（５，０）５
５ｔｘｔ、メタノール２５ｍ１を加え、次いでＢｘ −
Ｇｌ？ −ＯＨ（２，６３°１．１５ミリモル）を加え
た。Elemental analysis (a): Theoretical value as C23H29N305: C164,62, H, 6,84, NS9.83 Measured value: C164,31; H, 6,9C1; N, 9.74
(o) Bot -Gl& -Glu (0Bzl)
- Product Boc of the pre-production process of N2 H2Ph
-C41u (OBZ/ )-N2I(2PA (6,
415′, 15 mmol) was treated with TFA (1B+!Il) for 30 minutes at room temperature to debotify it. Excessive 'I'F
A was distilled off under reduced pressure, and the residue was dried under reduced pressure over potassium hydroxide for 3 hours. This was added with 2M sodium acetate buffer (pT(5,0)5
Add 5txt and 25ml of methanol, then Bx-
Gl? -OH (2.63° 1.15 mmol) was added.
２Ｎ水酸化ナトリウム（約５ｇ／）でｐＴ（５，０に調
整し、２−メルカプトエタノール（０，８ＯＳ’）及び
パパイン（２，２！Ｍ’）を加え、室温で２４時間攪ト
リウム、飽和食塩水で順次洗浄後、芒硝上乾燥した。濾
過後、酢酸エチルを減圧下留去、残渣をシリカゲルカラ
ムクロマトグラフィー（３Ｘ２５Ｃ＊）に付し、クロロ
ホルム−メタノール（５０：１）で溶出した。目的物を
含むフラクションを濃縮し、メタノールと石油エーテル
で再結晶した。Adjust pT (5,0) with 2N sodium hydroxide (approximately 5 g/), add 2-mercaptoethanol (0,8OS') and papain (2,2!M'), stir at room temperature for 24 hours, and saturated with thorium. After washing successively with brine, it was dried over sodium sulfate. After filtration, ethyl acetate was distilled off under reduced pressure, and the residue was subjected to silica gel column chromatography (3X25C*) and eluted with chloroform-methanol (50:1). Purpose The fraction containing the product was concentrated and recrystallized from methanol and petroleum ether.
収量３，６３５’（収率５０ｔ１６）、融点５８−５９
．５°Ｃ、Ｉｔ／１０．５３、〔α〕Ｄ−１５，２（Ｃ
Ｃ１５、メタノ−・ル）。Yield 3,635' (yield 50t16), melting point 58-59
．． 5°C, It/10.53, [α]D-15,2(C
C15, methanol).
元素分析＠）：Ｃ２５Ｈ３２Ｎ４０６として理論値：
Ｃ，６１，９７ＨＨ１６，６６１Ｎ１１１．５６測定値
： Ｃ１６１，８８、Ｈ，６，７＋ １Ｎ１１１．３９
（ハ）Ｂｘ−８ｒｒ（Ｂｚｌ ）−（４−Ｇｌｕ （Ｏ
Ｂｚ／ ）−Ｎ２Ｈ２Ｐｈの製造
前工程の生成物Ｂｏｔ： −Ｇｌｐ −Ｇｌｕ （０Ｂ
ｔｒｒｌ ） −Ｎ２Ｈ２Ｐｈ（２，９１Ｆ、６ミリモ
ル）をＴＦＡ（８笥ｔ）で室温６０分間処理し、脱Ｂｏ
Ｃ化した。過剰のＴＦＡを減圧留去し、水酸化カリウム
上３時間減圧乾燥した。これに０．２ Ｍ炭酸ナトリウ
ム１０ｍ／ｓ メタノール１２ｍ１を加えた後、２Ｎ水
酸化ナトリウム（約０．５笥ｔ）でｐＨ８に調整した。Elemental analysis @): Theoretical value as C25H32N406:
C,61,97HH16,661N111.56 Measured value: C161,88, H,6,7+ 1N111.39
(c) Bx-8rr(Bzl)-(4-Glu(O
Product Bot of the pre-production process of Bz/ )-N2H2Ph: -Glp -Glu (0B
trrl) -N2H2Ph (2,91F, 6 mmol) was treated with TFA (8 tons) at room temperature for 60 minutes to remove Bo
It became C. Excess TFA was distilled off under reduced pressure, and the residue was dried under reduced pressure over potassium hydroxide for 3 hours. After adding 0.2 M sodium carbonate at 10 m/s and 12 ml of methanol to this, the pH was adjusted to 8 with 2N sodium hydroxide (about 0.5 tons).
Ｂｏｃ −Ｓｙ （＆／ ） −〇Ｎし （３，７１Ｓ
’、１２ミリモル）、２−メルカプトエタノール（０，
２５ｒ）及びパパイン（０，９０２）を加え、３７°Ｃ
で９０分間攪拌した。反応液に酢酸エチル（約１５０ｍ
／）を加え、分液ロート上、冷０．５Ｎ塩酸、５％炭酸
水素ナトリウム、飽和食塩水で順次洗浄後、芒硝上乾燥
した。濾過後、酢酸エチルを減圧下留去、残渣にエーテ
ルを加え沈澱させ、残存ＢｏＣ−Ｓｒｒ （Ｂｚｌ ）
−ＯＭｚを除去した。固体の残渣をメタノール−エー
テル−石油エーテルで再沈澱した。Boc -Sy (&/ ) -〇Nshi (3,71S
', 12 mmol), 2-mercaptoethanol (0,
25r) and papain (0,902) and heated to 37°C.
The mixture was stirred for 90 minutes. Ethyl acetate (approximately 150 m
/) was added, and the mixture was washed on a separatory funnel with cold 0.5N hydrochloric acid, 5% sodium bicarbonate, and saturated saline in this order, and then dried over Glauber's sulfate. After filtration, ethyl acetate was distilled off under reduced pressure, ether was added to the residue to precipitate it, and the remaining BoC-Srr (Bzl)
- OMz was removed. The solid residue was reprecipitated with methanol-ether-petroleum ether.
収量２．７８１（収率７０％）、融点１３９−１４０°
Ｃ，Ｒ／ｉ ０．９５、 Ｃ（ＩＥｐ）”
−７１” （００，５、メタノール）。Yield 2.781 (yield 70%), melting point 139-140°
C, R/i 0.95, C(IEp)”
-71” (00,5, methanol).
元素分析鍾） ： Ｃ３５Ｈ４３Ｎ５０８・６Ｈ２０と
して理論値： Ｃ１５Ｂ、７３ ｉＨ，６，９０、Ｎ、
９．７８測定値： Ｃ１５８，９８、Ｈ，６，８５、
Ｎ、９７５アミノ酸分析： Ｓ”（０，９８） 、Ｇｌ
ｕ（Ｑ、９９）、Ｇｌｐ（ｉ、ｏｏ）
（１） ： Ｂｏｔ−Ｇｌ＆−ＡＱ （ＯＢｚ／ ）
−Ａｌａ−ＯＭｚの合成（イ）Ｂｔｘ −Ａｌａ−Ｎ２
Ｈ２ＰｈＯ製造フエニルヒドラジン塩酸塩（２１，６９
ｆ、１５０ミリモル）及びＢｏｔニーＡｌａ −ＯＨ（
９，４６？、５０ミリモル）を２Ｍ酢酸ナトリウム緩衝
液（ｐＨ５゜０）１７５ｍｌに溶解、２Ｎ水酸化ナトリ
ウム（約７５ｍ１＞でｐＩ（５に調整し、窒素ガスを数
分間バブリングした。２−メルカプトメタノール（２，
００！ｉ’）及びパパイン（ｔｏｏＦ）を加え、室温で
１８時間攪拌した。生成した沈澱を炉取し、水で洗浄後
、酢酸エチル（約２００ｔｌ）に溶解した。分液ロート
上、酢酸エチル層を冷０．５Ｎ塩酸、５％炭酸水素ナト
リウム、飽和食塩水で順次洗浄後、芒硝上乾燥した。濾
過後、酢酸エチルを減圧留去し、残渣を酢酸エチルと石
油エーテルで再結晶した。Elemental analysis): Theoretical value as C35H43N508/6H20: C15B, 73 iH, 6,90, N,
9.78 measurement value: C158,98, H,6,85,
N, 975 amino acid analysis: S”(0,98), Gl
u(Q, 99), Glp(i, oo) (1): Bot-Gl&-AQ (OBz/ )
-Synthesis of Ala-OMz (a) Btx -Ala-N2
H2PhO production phenylhydrazine hydrochloride (21,69
f, 150 mmol) and Botney Ala-OH (
9,46? , 50 mmol) was dissolved in 175 ml of 2M sodium acetate buffer (pH 5°0), the pI was adjusted to 5 with 2N sodium hydroxide (approx.
00! i') and papain (tooF) were added and stirred at room temperature for 18 hours. The generated precipitate was collected in an oven, washed with water, and then dissolved in ethyl acetate (approximately 200 tl). The ethyl acetate layer on the separating funnel was washed successively with cold 0.5N hydrochloric acid, 5% sodium bicarbonate, and saturated brine, and then dried over sodium sulfate. After filtration, ethyl acetate was distilled off under reduced pressure, and the residue was recrystallized from ethyl acetate and petroleum ether.
収量１３．３８Ｓ’（収率９６チ）、融点１５３−１５
４．５°Ｃ，Ｒ７１０，５１、〔α）ｊ５−４９．６’
（ＣＣ１５、メタノール）。Yield 13.38S' (yield 96T), melting point 153-15
4.5°C, R710,51, [α)j5-49.6'
(CC15, methanol).
元素分析（イ）： Ｃ１４Ｈ２１Ｎ３０３として理論値
： Ｃ，６０，２０ｉＨ，７，５８ｉＮ、 １５．０４
測定値： Ｃ，５８，９７、Ｒ１７，５３１Ｎ１１５．
２２（ｏ）ＢＯｃ−Ａｙ／（ＯＢＺ／） −Ａ／（２−
Ｎ２Ｈ２ＰＡ ｔ７）製造前工程の生成物Ｂａｔ −
Ａｌａ −Ｎ２Ｈ２Ｐｈ （４，１９ｆ、１５ミリモル
）をＴＦＡ（１５笥ｔ）で室温３０分間処理し、脱Ｂｏ
ｔ化した。過剰のＴＦＡを減圧留去し、水酸化カリウム
上３時間減圧乾燥した。これを０．２Ｍ炭酸ナトリウム
６０ゴに溶解した後、２Ｎ水酸化ナトリウム（約３Ｂｙ
ｌ）でｐＨ９に調整した。ここに、酢酸エチル６０肩ｔ
％ＢＫ−Ａｓｐ（ＯＢ、”／）−〇ル （６，７５グ、
２０ミリモル）、次いで２−メルカプトエタノール（ｔ
２０ｓ’）及びパパイン（３，０Ｏｆ）を加え、室温で
１８時間攪拌した。Elemental analysis (a): Theoretical value as C14H21N303: C, 60, 20iH, 7, 58iN, 15.04
Measured value: C, 58,97, R17,531N115.
22(o)BOc-Ay/(OBZ/) -A/(2-
N2H2PA t7) Product of pre-production process Bat −
Ala-N2H2Ph (4.19f, 15 mmol) was treated with TFA (15 tons) for 30 minutes at room temperature to remove Bo
It became t. Excess TFA was distilled off under reduced pressure, and the residue was dried under reduced pressure over potassium hydroxide for 3 hours. After dissolving this in 60 grams of 0.2M sodium carbonate, 2N sodium hydroxide (approximately 3By
1) to adjust the pH to 9. Here, 60 t of ethyl acetate
%BK-Asp(OB,”/)-〇ru (6,75g,
20 mmol), then 2-mercaptoethanol (t
20s') and papain (3,0Of) were added and stirred at room temperature for 18 hours.
反応液に酢酸エチル（約１００１１Ｉｌ）を加え、分液
ロート上、冷０．５Ｎ塩酸、５％炭酸水素ナトリウム、
飽和食塩水で順次洗浄後、芒硝上乾燥した。Ethyl acetate (approximately 10011 Il) was added to the reaction solution, and placed on a separating funnel with cold 0.5N hydrochloric acid, 5% sodium hydrogen carbonate,
After washing successively with saturated brine, it was dried over sodium sulfate.
濾過後、酢酸エチルを減圧留去、残渣にエーテル−石油
エーテル（ｉ：５ｖ／ｖ） 混液を加えて沈澱させ、
残存ＢθＣ−Ｍ戸（ＯＢｚ／ ） −０Ｐｙしを除去し
た。After filtration, ethyl acetate was distilled off under reduced pressure, and a mixture of ether-petroleum ether (i:5v/v) was added to the residue for precipitation.
The remaining BθC-M (OBz/)-0Py was removed.
固体の残渣を酢酸エチルと石油エーテルで再結晶した。The solid residue was recrystallized from ethyl acetate and petroleum ether.
収量ｔ７２ｒ（収率６５％）、融点１１４．５−１１５
°Ｃ，Ｒ７１０，５６、〔α）ｊ５−３３．７” （Ｃ
Ｃ１５、メタノール）
元素分析帳） ： Ｃ２５Ｈ３２Ｎ４０６・Ｒ２０とし
て理論値： ０．５９．７５ ｉｎ、 ６．８２ ｉＮ
、 １１．１５測定値： Ｃ，５９，９５、ｎ、 ６．
８８ 、Ｎ、 １１．１１（ハ）Ｂｏｅ−Ｇ／） −１
’ｕｌ） （ＯＢＪ／ ）−Ａ／（Ｚ −Ｎ２Ｈ２Ｐｈ
の製造前工程の生成物ＢＯｔ −Ａｓｐ （０Ｂｚ
ｌ ） −Ａｌａ −Ｎ２Ｈ２Ｐｈ（４，８４１ｉ′、
１０ミリモル）をＴＦＡ （１５笥ｔ）で室温３０分間
処理し、脱ｆ３ｏｔ化した。過剰のＴＥＡを減圧留去し
、水酸化カリウム上３時間減圧乾燥した。これに０．２
Ｍ炭酸ナトリウム１３ｍ／、メタノール６ ｍｌを加
えた後、２Ｎ水酸化ナトリウム（約１耐）でｐＨＢに調
整し、ｆ３ｏｔ −Ｇｌｐ−０Δしく３．７８ＳＦ、２
０ミリモル）、２−メルカプトエタノール（１，０ＣＮ
ｉＩ）及びパパイン（１，５０１）を加え、３７°Ｃで
３０分間攪拌した。反応液に酢酸エチル（約１５０ｍ／
）を加え、分液ロート上、冷０．５Ｎ塩酸、５チ炭酸水
素ナトリウム、飽和食、 塩水で順次洗浄後、芒硝上
乾燥した。濾過後、酢酸エチルを減圧下留去、残渣をシ
リカゲルカラムクロマトグラフィー（３Ｘ２５０）に付
し、クロロホルム−メタノール（５０：１）で溶出した
。Yield t72r (yield 65%), melting point 114.5-115
°C, R710,56, [α)j5-33.7" (C
C15, methanol) Elemental analysis book): Theoretical value as C25H32N406/R20: 0.59.75 in, 6.82 iN
, 11.15 measurement value: C, 59,95, n, 6.
88, N, 11.11 (c) Boe-G/) -1
'ul) (OBJ/ )-A/(Z -N2H2Ph
The product BOt -Asp (0Bz
l ) -Ala -N2H2Ph (4,841i',
10 mmol) was treated with TFA (15 tons) for 30 minutes at room temperature to remove f3ot. Excess TEA was distilled off under reduced pressure, and the residue was dried under reduced pressure over potassium hydroxide for 3 hours. 0.2 to this
After adding 13 m/m of M sodium carbonate and 6 ml of methanol, the pH was adjusted to pHB with 2N sodium hydroxide (approx.
0 mmol), 2-mercaptoethanol (1,0CN
iI) and papain (1,501) were added and stirred at 37°C for 30 minutes. Add ethyl acetate (approx. 150ml/
) was added, and the mixture was washed on a separatory funnel with cold 0.5N hydrochloric acid, 5% sodium bicarbonate, saturated food, and brine, and then dried over Glauber's sulfate. After filtration, ethyl acetate was distilled off under reduced pressure, and the residue was subjected to silica gel column chromatography (3×250) and eluted with chloroform-methanol (50:1).
目的物を含むフラクションを濃縮し、酢酸エチルと石油
エーテルで再沈澱した。The fraction containing the target product was concentrated and reprecipitated with ethyl acetate and petroleum ether.
収量２．３８５Ｆ（収率４４チ）、融点１５０．５−１
５２°Ｃ，Ｒ７１０，４１、〔α）２５−２６．４７
（ＯＣ１５、メタノール）
元素分析（１）：Ｃ２７Ｈ３５Ｎ５０７ として理論
値： Ｃ，５９，８８ｉＨ，６，５１士Ｎ、１２．９３
測定値： Ｃ，５９，７３、Ｈ，６，３７、Ｎ、 １２
．９６アミノ酸分析： Ｉ’−Ｋｌ’（，１，０６）
、Ｇｌ）（１，００）、Ａｌａ（１，０１）
に）Ｂｏｔ −Ｇｌ）−ｋ戸（’０Ｂｚｌ ） −Ａｌ
ａ −ＯＭｔ の製造前工程の生成物Ｂｔｙｔ −Ｇ
ｌ）−Ａｓ／） （０Ｂｚｌ ） −Ｎａ −Ｎ２Ｈ２
Ｐ／ｒ （２，１７ｙ、４ミリモル）をジクロロメタ
ン（２００肩／）に溶解し、ピリジン（４１６ｆｉ）を
加え、以下遮光してＮ−ブロモコノ・り酸イミドＣ７６
Ｂｌ１ｆ、４．３２ミリモル）を加え、２０分間激しく
攪拌した。反応液を分液ロートに移し、冷０．５Ｎ塩酸
、５ｑ６炭酸水素ナトリウム、水で順次洗浄後、芒硝上
乾燥、濾過後、ジクロロメタンを減圧留去、残渣にジク
ロロメタン（１２０ｍ？）、無水メタノール（１２，４
Ｂｚｌ）を加え、−夜撹拌した（これまで遮光下）。反
応液を分液ロートに移し、冷０．５ Ｎ塩酸、５条炭酸
水素ナトリウム、水で順次洗浄後、芒硝上乾燥した。濾
過後ジクロロメタンを減圧留去、残渣をシリカゲルカラ
ムクロマトグラフィー（２，５×２０ＣＩＲ）に付し、
クロロホルム−メタノール（１５０：１）で溶出した。Yield 2.385F (yield 44T), melting point 150.5-1
52°C, R710,41, [α)25-26.47
(OC15, methanol) Elemental analysis (1): Theoretical value as C27H35N507: C, 59,88iH, 6,51N, 12.93
Measured values: C, 59, 73, H, 6, 37, N, 12
．． 96 amino acid analysis: I'-Kl' (,1,06)
, Gl) (1,00), Ala(1,01) ni)Bot -Gl) -k('0Bzl) -Al
The product Btyt -G of the pre-production step of a -OMt
l)-As/) (0Bzl)-Na-N2H2
P/r (2,17y, 4 mmol) was dissolved in dichloromethane (200 mmol), pyridine (416fi) was added, and N-bromoconophosphoric acid imide C76 was protected from light.
Bl1f, 4.32 mmol) was added and stirred vigorously for 20 minutes. The reaction solution was transferred to a separatory funnel, washed successively with cold 0.5N hydrochloric acid, 5q6 sodium bicarbonate, and water, dried over sodium sulfate, filtered, and dichloromethane was distilled off under reduced pressure. 12,4
Bzl) was added and stirred overnight (until now protected from light). The reaction solution was transferred to a separatory funnel, washed successively with cold 0.5 N hydrochloric acid, 5-line sodium bicarbonate, and water, and then dried over Glauber's sulfate. After filtration, dichloromethane was distilled off under reduced pressure, and the residue was subjected to silica gel column chromatography (2.5 x 20 CIR).
Elution was with chloroform-methanol (150:1).
目的物を含むフラクションを濃縮し、酢酸エチルと石油
エーテルで再結晶した。The fraction containing the target product was concentrated and recrystallized from ethyl acetate and petroleum ether.
収量１．０３ｆ（収率５５％）、融点８８．５−８９．
５℃、Ｒ／１ ０．５３、〔αＩＤ５２２．８’ （Ｃ
Ｏ３５、メタノール）
元素分析＠）二Ｃ２２Ｈ３１Ｎ３０８として理論値：Ｃ
，５６，７６、Ｎ１６．７１１Ｎ１９．０．３測定値：
Ｃ１５６，６５、Ｈ，６，６９ｉ”Ｌ ９．１５アミ
ノ酸分析： Ａ５戸（１，０６） 、’ Ｇ４（１，０
０）、Ａｌａ（１，００）
［： ｌ［） ： Ｚ−Ｔｒｐ−ｋｌａ −０４−ＯＭ
ｅ （Ｄ合成（イ）Ｂｏｔ −Ｇｌ）−Ｎ２ Ｎ２
Ｐｈの製造フェニルヒドラジン塩酸塩（２１，６９１，
１５０ミリモル） 、ＢＯｔ−Ｇｌ＆−ＯＨ（８，７６
ｔ、５０ミリモル）及び無水酢酸ナトリウム（２８Ｐ）
を２Ｍ酢酸ナトリウム緩衝液（ｐＩ（５，０）１７０ｍ
ｌ？に溶解（ｐＨは５であった）、窒素ガスを数分間バ
ブリングした後、２−メルカプトエタノール（７５０Ｐ
）及びパパイン（ａ、ｏｏｙ）を加え、室温で１８時間
清押した。生成した沈澱を戸数し、水で洗浄後、酢酸エ
チル（約２００ｍ１）に溶解し、分液ロート上、酢酸エ
チル層を冷０．５Ｎ塩酸、５チ炭酸水素ナトリウム、飽
和食塩水で順次洗浄後、芒硝上乾燥した。濾過後、酢酸
エチルを減圧留去し、残渣を酢酸エチルと石油エーテル
で再結晶した。Yield 1.03f (yield 55%), melting point 88.5-89.
5℃, R/1 0.53, [αID522.8' (C
O35, methanol) Elemental analysis @) Theoretical value as 2C22H31N308: C
,56,76,N16.711N19.0.3 Measured value:
C156,65, H,6,69i"L 9.15 amino acid analysis: A5 (1,06), 'G4 (1,0
0), Ala(1,00) [: l[): Z-Trp-kla-04-OM
e (D synthesis (a) Bot -Gl) -N2 N2
Production of Ph phenylhydrazine hydrochloride (21,691,
150 mmol), BOt-Gl&-OH (8,76
t, 50 mmol) and anhydrous sodium acetate (28P)
in 2M sodium acetate buffer (pI(5,0) 170m
l? (pH was 5), and after bubbling nitrogen gas for several minutes, 2-mercaptoethanol (750P
) and papain (a, ooy) were added and pressed at room temperature for 18 hours. Separate the generated precipitate, wash it with water, dissolve it in ethyl acetate (about 200 ml), place it on a separating funnel, and wash the ethyl acetate layer with cold 0.5N hydrochloric acid, 5% sodium bicarbonate, and saturated saline in sequence. , dried over mirabilite. After filtration, ethyl acetate was distilled off under reduced pressure, and the residue was recrystallized from ethyl acetate and petroleum ether.
収量１１．４１ｆ°（収率８６係）、融点１２３−１２
４°Ｃ５Ｒ７１０，４５
元素分析（働：Ｃ１３Ｈ１９Ｎ３０３として理論値：
０．５８．８５ 、Ｈ，７，２２、Ｎ１１５．８４測定
値＝０１５８．３７；Ｈ１７２０；Ｎ１１５．９２（ｏ
） Ｂ＆ −Ａｌａ −Ｇｌ）−Ｎ２ Ｎ２ Ｐｈ
の製造前工程の生成物Ｂθｃ −Ｇｌｐ−Ｎ２Ｈ２Ｐｈ
（１０，６１？、４０ミリモル）をＴＦＡ（１５肩ｌ）
で室温３０分間処理し、脱Ｂｏｃ化した。過剰のＴＦＡ
を減圧留去し、水酸化カリウム上３時間減圧乾燥した。Yield 11.41f° (yield 86%), melting point 123-12
4°C5R710,45 Elemental analysis (Working: C13H19N303 Theoretical value:
0.58.85, H, 7, 22, N115.84 measurement value = 0158.37; H1720; N115.92 (o
) B& -Ala -Gl)-N2 N2 Ph
The product Bθc-Glp-N2H2Ph of the pre-production step
(10,61?, 40 mmol) in TFA (15 shoulder l)
was treated at room temperature for 30 minutes to remove Boc. Excess TFA
was distilled off under reduced pressure and dried over potassium hydroxide under reduced pressure for 3 hours.
これを２Ｍ酢酸ナトリウム緩衝液（ｐＩ（５，０）３０
ゴに溶解し、Ｂｏｃ−Ａｌａ −ＯＨ（３，７８Ｓ’、
２０ミリモル）を加え溶かした後、２Ｎ水酸化ナトリウ
ム（約５ｍ／）でｐＩ（５に調整し、窒素ガスを数分間
バブリングした。２−メルカプトエタノール（２，００
２）及びパパイン（２，００２）を加え、室温で２４時
間攪拌、生成した沈澱を戸数した後、ｐｔＬに２−メル
カプトエタノール（０，５０ｆ）及びパパイン（ｏ、５
ｏｆ）を追加し、更に室温で２４時間攪拌した。生成し
た沈澱を炉取し、先の沈澱と合わせ水で洗浄、５チクエ
ン酸、５チ炭酸水素ナトリウム、水で順次バッチ法で洗
浄した。次いでメタノールとエーテルで再結晶した。This was mixed with 2M sodium acetate buffer (pI (5,0) 30
Boc-Ala-OH (3,78S',
After adding and dissolving 2-mercaptoethanol (2,00 mmol), the pI was adjusted to 5 with 2N sodium hydroxide (approximately 5 m/m), and nitrogen gas was bubbled for several minutes.
2) and papain (2,002) were added, stirred at room temperature for 24 hours, and the resulting precipitate was removed.
of) was added, and the mixture was further stirred at room temperature for 24 hours. The generated precipitate was collected in a furnace, combined with the previous precipitate, washed with water, and sequentially washed with 5-thicitric acid, 5-thionic sodium bicarbonate, and water in a batch method. It was then recrystallized from methanol and ether.
収量４．５１Ｓ’（収率６７％）、融点２１８．５−２
１９°Ｃ％Ｒ７ｉ ０．４０、〔α〕’１５−５．６
’（Ｃ０５、メタノール）
元素分析（％） ： Ｃ１６Ｈ２４Ｎ４０４として理論
値：ｃ、５７．１３ｉＨ，７，１９，Ｎ、１６．６６測
定値：ｃ、 ５７．５１ 、Ｈ，７，１１、Ｎ、 １６
．７４（ハ）Ｚ −Ｔｒｐ −ｋｌａ −Ｇｌ）−Ｎ２
Ｈ２Ｐｈの製造前工程の生成物Ｂｏｔ −Ａｌａ −
Ｇｌｐ−Ｎ２Ｈ２Ｐｈ（４，０４７，１２ミリモル）を
ＴＦＡ （１０肩ｌ）で室温３０分間処理し、脱Ｂｗ化
した。過剰のＴＦＡを減圧留去し、水酸化カリウム上３
時間減圧乾燥した。Yield 4.51S' (yield 67%), melting point 218.5-2
19°C%R7i 0.40, [α]'15-5.6
'(C05, methanol) Elemental analysis (%): Theoretical value as C16H24N404: c, 57.13iH, 7, 19, N, 16.66 Measured value: c, 57.51, H, 7, 11, N, 16
．． 74(c)Z -Trp -kla -Gl)-N2
Product Bot -Ala- of the pre-production process of H2Ph
Glp-N2H2Ph (4,047,12 mmol) was treated with TFA (10 liters) at room temperature for 30 minutes to remove Bw. Excess TFA was distilled off under reduced pressure and dissolved on potassium hydroxide.
Dry under reduced pressure for an hour.
これに２Ｍ酢酸ナトリウム（１００＊ｌ）、メタノール
（１２ｍｉ？） 及ＵＺ−Ｔｒｐ−ＯＨ（４，０６ｔ、
１２ミリモル）を加え、加温溶解した（ｐＨは７であっ
た）。α−キモトリプ７ン（１，２０ｆ）を加え３７°
Ｃで１８時間攪拌した。反応液に水（１００ｍ／）を加
え、得られる粉末を５チクエン酸、５係炭酸水素ナトリ
ウム、水で順次バッチ法で洗浄した。得られた粗生成物
をシリカゲルカラムクロマトグラフィー（３Ｘ２５ｃ１
ｎ）に付し、クロロホルム−メタノール（４０：１）で
溶出した。目的物を含むフラクションを濃縮し、メタノ
ールとエーテルで再結晶した。To this, 2M sodium acetate (100*l), methanol (12mi?) and UZ-Trp-OH (4,06t,
12 mmol) was added and dissolved by heating (pH was 7). Add α-chymotrypone (1,20f) and 37°
The mixture was stirred at C for 18 hours. Water (100 m/m) was added to the reaction solution, and the resulting powder was washed sequentially with 5% citric acid, 5% sodium bicarbonate, and water in a batch method. The obtained crude product was subjected to silica gel column chromatography (3X25c1
n) and eluted with chloroform-methanol (40:1). The fraction containing the target product was concentrated and recrystallized from methanol and ether.
収量２．４７１（収率３７％）、融点２１２．５−２１
３８°Ｃ，Ｒ／１ ０．３３、〔α〕２５ ａ、 ｏ
ｏ（ＣＯ３５、ＤＭＦ’）
元素分析＠）：Ｃ６０Ｈ３２Ｎ６０５・２Ｈ２０として
理論値： Ｃ，６０，８０ｉＨ，６，１２、Ｎ、 １４
．１８測定値： ０．６１．９４ 、Ｈ，６，３８、Ｎ
、 １４．４０アミノ酸分析： Ｃ７）（１，００）
、Ａｌａ（［］、９６）、Ｔ’／’（０，８８）
に）Ｚ −Ｔｒｆｉ −Ａｌａ −Ｇｌｐ −ＯＭｔ
ノ製造前工程の生成物Ｚ−ＴＱ−Ａｌａ −（Ｊｌｐ−
Ｎ２Ｈ２Ｐｈ（２，２３ｆ、４ミリモル）をジオキサン
（３６ｔ／）ニ溶解、塩化第二鉄（Ｆｒ（ｕ３−６Ｈ２
０） １２．８ ｔを水２９ｔｘｔに溶かした液を上の
液に６５°Ｃで攪拌しつつ徐々に滴下した。１５分間攪
拌後、２Ｎ水酸化ナトリウムを加えてｐＨ８とし、生じ
た沈澱を２、００ Ｏｒ、 ｌ）、ｍ、、５分間遠心分
離し、上澄液と沈澱とに分離した。沈澱に０．１Ｎ炭酸
すｌ−ＩＪウム（３０πｔ）を加え、同様に遠心分離し
、上澄液を得た。雨上澄液を合し、１０％クエン酸を加
えて酸性とし、生じた粉末を５％クエン酸、水で順次バ
ッチ法で洗浄、粗生成物をメタノールとエーテルで再沈
澱した。１．２６ｆのＺ−Ｔｒｐ−Ａｌａ −Ｇｌ＆
−０Ｈ（収率６７チ）を得だ。Yield 2.471 (yield 37%), melting point 212.5-21
38°C, R/1 0.33, [α]25 a, o
o (CO35, DMF') Elemental analysis @): Theoretical value as C60H32N605/2H20: C, 60, 80iH, 6, 12, N, 14
．． 18 Measured value: 0.61.94, H, 6,38, N
, 14.40 amino acid analysis: C7) (1,00)
,Ala([],96),T'/'(0,88))Z -Trfi -Ala -Glp -OMt
The product Z-TQ-Ala-(Jlp-
N2H2Ph (2.23f, 4 mmol) was dissolved in dioxane (36t/) and ferric chloride (Fr(u3-6H2
0) A solution prepared by dissolving 12.8 t in 29 txt of water was gradually added dropwise to the above solution while stirring at 65°C. After stirring for 15 minutes, 2N sodium hydroxide was added to adjust the pH to 8, and the resulting precipitate was centrifuged for 5 minutes at 2,00 Or, m) to separate the supernatant and the precipitate. 0.1N sodium carbonate (30πt) was added to the precipitate and centrifuged in the same manner to obtain a supernatant. The rain supernatants were combined and made acidic by adding 10% citric acid, and the resulting powder was washed sequentially with 5% citric acid and water in a batch process, and the crude product was reprecipitated with methanol and ether. 1.26f Z-Trp-Ala-Gl&
-0H (yield: 67 cm) was obtained.
このもの１．１７ ｆ （２，５ミリモル）をジクロロ
メタン（６０Ｍ／）中、４−ジメチルアミノピリジン（
２８〜）、無水メタノール（５００縛）、１−エチル−
５−（３−ジメチルアミンプロピル）−カルボジイミド
塩酸塩（５２５〜）でＯ’０．２時間、次いで室温で一
夜攪拌し、メチル化を行なった。ジクロロメタンを減圧
留去し、残渣に酢酸エチル（１００ｍ／）を加え、分液
ロート上、５％クエン酸、５チ炭酸水素ナトリウム、水
で順次洗浄後、芒硝上乾燥した。濾過後、酢酸エチルを
減圧留去し、酢酸エチルと石油エーテルで再結晶した。1.17 f (2.5 mmol) of this product was dissolved in 4-dimethylaminopyridine (
28~), anhydrous methanol (500 bound), 1-ethyl-
Methylation was carried out by stirring with 5-(3-dimethylaminepropyl)-carbodiimide hydrochloride (525~) for O'0.2 hour and then overnight at room temperature. Dichloromethane was distilled off under reduced pressure, and ethyl acetate (100 m/s) was added to the residue, which was washed on a separating funnel with 5% citric acid, 5% sodium bicarbonate, and water in order, and then dried over sodium sulfate. After filtration, ethyl acetate was distilled off under reduced pressure, and the residue was recrystallized from ethyl acetate and petroleum ether.
収量０．９ＣＮＦ（収率７５チ）、融点１５１−１５２
．５°Ｃ１〔α）Ｄ−１２，５（ＣＤ、５、メタノール
）、几７ｉ０．４２
元素分析（（６）： Ｃ２５Ｈ２ＢＮ４０１６ とし
て理論値：Ｃ，６２，４９逼Ｈ，５，８７ｉＮ、 １１
．６６測定値： Ｃ，６２，５４、ｕ、 ５．５９ ｉ
Ｎ、 １１．８６アミノ酸分析： Ｇ４（１，００）
、Ａｌａ（１，０１）、Ｔヶ（０，９２）
［■ 〕 〕Ｚ−Ｔｒｐ−Ａ７２−０４−Ｇｌ）−１ｕ
ｐ（ＯＢｚｌ）、−Ａｌａ−８ｔｒ（Ｂｄ ） −Ｇｌ
）−Ｇｌｕ（ＯＢｚｌ ）−Ｎ２Ｈ２Ｐｈの合成（〔Ｉ
〕、〔■〕及び〔■〕フラクションの縮合）
（イ）Ｂｏｔ： −Ｇｌｐ −ｋｓｐ （０Ｂｚｌ ）
−Ａｌａ −５ｔｒ（Ｂｚｔ ） −Ｇｌｐ−Ｇｌｕ
（０＆／ ）−Ｎ２Ｈ２Ｐｈの製造（〔１〕と（ｎ）
フラクションの縮合）先に製造したＣＩ）フラクション
すなわちＢｏｔ −８ｔｒ（Ｂｚｌ ） −Ｇｌ＆−Ｇ
ｌｕ （ＯＢｘｌ ） −Ｎ２Ｈ２ＰＡ （４６３Ｗ
／、０．７ミリモル）をＴＦＡ （３ｍｌ）で室温３０
分間処理し、脱ｆＪｏｔ化した。過剰のＴＦＡを減圧留
去し、水酸化カリウム上３時間減圧乾燥した。ここに０
．２ Ｍ炭酸ナトリウム（１０，５ｒｔ）、エタノ−シ
ル（３，５ｙｔ／）を加えた後、２Ｎ塩酸（約２００１
／）でｐＨＦ３に調整した。Ｂｘ −Ｇｌｐ−Ａｓｐ（
ＯＢｚｌ ） −Ａｌａ −ＯＭｅ（９７７ｑ、２．１
ミリモル）、２−メルカプトエタノール（０，３５２
）、パパイン（０，３５７）を加え、３７°Ｃで１時間
攪拌した。反応液に酢酸エチル（約１５０ｍ１）を加え
、分液ロート上、冷０．５Ｎ塩酸、５チ炭酸水素ナトリ
ウム、飽和食塩水で順次洗浄後、芒硝上乾燥し、沖過後
、酢酸エチル減圧留去し、残渣をメタノールとエーテル
で再沈澱した。Yield 0.9CNF (yield 75cm), melting point 151-152
．． 5°C1 [α) D-12,5 (CD, 5, methanol), 7i0.42 Elemental analysis ((6): Theoretical value as C25H2BN4016: C,62,49〼H,5,87iN, 11
．． 66 measurement value: C, 62, 54, u, 5.59 i
N, 11.86 amino acid analysis: G4 (1,00)
, Ala (1,01), T (0,92) [■ ] ] Z-Trp-A72-04-Gl)-1u
p(OBzl), -Ala-8tr(Bd) -Gl
)-Glu(OBzl)-N2H2Ph synthesis ([I
], [■] and [■] Condensation of fractions) (a) Bot: -Glp -ksp (0Bzl)
-Ala -5tr(Bzt) -Glp-Glu
Production of (0&/)-N2H2Ph ([1] and (n)
Condensation of fractions) previously prepared CI) fractions i.e. Bot-8tr(Bzl)-Gl&-G
lu (OBxl) -N2H2PA (463W
/, 0.7 mmol) in TFA (3 ml) at room temperature.
It was processed for 1 minute to remove fJot. Excess TFA was distilled off under reduced pressure, and the residue was dried under reduced pressure over potassium hydroxide for 3 hours. 0 here
．． After adding 2M sodium carbonate (10.5rt) and ethanolyl (3.5yt/), 2N hydrochloric acid (approx.
/) to adjust the pH to 3. Bx-Glp-Asp(
OBzl ) -Ala -OMe (977q, 2.1
mmol), 2-mercaptoethanol (0,352
) and papain (0,357) were added, and the mixture was stirred at 37°C for 1 hour. Ethyl acetate (approximately 150 ml) was added to the reaction solution, washed sequentially with cold 0.5N hydrochloric acid, 5% sodium bicarbonate, and saturated brine on a separating funnel, dried over sodium sulfate, filtered, and ethyl acetate was distilled off under reduced pressure. The residue was reprecipitated with methanol and ether.
収量４１０〜（収率５９チ）、融点２０４−２０５°Ｃ
，（αゾ５−１０．８°（Ｃ０，５、ＤＭＦ）、Ｒ／’
ｉｏ、４５
元素分析（４）： Ｃ５１Ｈ６２Ｎ８０１３・５Ｈ２０
として理論値： Ｃ，５６，４５、Ｈ１６，６９、Ｎ、
１０．３３測定値： Ｃ，５７，０３１Ｈ１６，９２
；Ｎ、 １０．０２アミノ酸分析： Ａｓ、ｏ（１，ｏ
１ ）、５ｅｒ（０，９５）−Ｇｌｐ −Ｇｌｕ （
ＯＢＪ／ ）−Ｎ２Ｈ２ＰｈＯ製造（〔■〕フラクショ
ンの製造）
前工程の生成物Ｂｅｘ −Ｇｌｐ −Ａｓ１ｒ （０Ｂ
ｚｌ ） −、ｋｌａ −８ｅｒ（Ｂｚｌ ） −Ｇｌ
ｐ −Ｇｌｕ （ＯＢｘｌ ）−Ｎ２Ｈ２ＰＡ （２
９９ｔｎダ、０．６ミリモル）をＴＦＡ（２ｍｌ）で室
温３０分間処理し、脱ＢＫ化した。過剰のＴＦＡを減圧
留去し、水酸化カリウム上３時間減圧乾燥した。ここに
０．２Ｍ炭酸ナトリウム（１，５ｍ／）、エタノール（
４，５肩りを加えた後、２Ｎ塩酸（約５０ＩＣで１））
Ｉ Ｂに調整した。フラクション（Ｉｔ）すなわチｚ−
Ｔｒｐ−Ａｌｌｌ −０４−ＯＭｅ （４３２’ｆｌ、
０．９ミリモル）、２−メルカプトエタノール（５０Ｍ
ダ）、パパイン（５０１１１ｆ）を加え、６７°Ｃで２
時間攪拌した。Yield: 410 ~ (yield: 59 cm), melting point: 204-205°C
, (αzo5-10.8° (C0,5, DMF), R/'
io, 45 Elemental analysis (4): C51H62N8013・5H20
Theoretical values: C, 56,45, H16,69, N,
10.33 Measured value: C, 57,031H16,92
;N, 10.02 Amino acid analysis: As, o(1, o
1), 5er(0,95)-Glp-Glu (
OBJ/ )-N2H2PhO production ([■] fraction production) Previous step product Bex -Glp -As1r (0B
zl ) −, kla −8er(Bzl ) −Gl
p-Glu (OBxl)-N2H2PA (2
99tnda, 0.6 mmol) was treated with TFA (2 ml) at room temperature for 30 minutes to remove BK. Excess TFA was distilled off under reduced pressure, and the residue was dried under reduced pressure over potassium hydroxide for 3 hours. Here, 0.2M sodium carbonate (1.5m/), ethanol (
After adding 4,5 drops, 2N hydrochloric acid (1 at about 50 IC))
Adjusted to IB. Fraction (It) i.e.
Trp-All-04-OMe (432'fl,
0.9 mmol), 2-mercaptoethanol (50M
), add papain (50111f) and heat at 67°C for 2 hours.
Stir for hours.
反応液に水（２０ｍ／）を加えた後、５％クエン酸、５
係炭酸水素ナトリウム、水、メタノールで順次バッチ法
で洗浄した。得られた粗生成物をジメチルホルムアミド
（Ｄ Ｍ Ｆ ）とメタノールで再沈澱した。After adding water (20m/) to the reaction solution, 5% citric acid, 5%
The mixture was washed sequentially with sodium bicarbonate, water, and methanol in a batch manner. The obtained crude product was reprecipitated with dimethylformamide (DMF) and methanol.
収量１８１ダ（収率４５チ）、融点２３６．５−２３
Ｚ ５°Ｃ１几ｆ１ ０、 〔α）２５− １ ３．
６” （ＣＣ１５、ＤΔ（Ｆ）
元素分析（ｅｌＡ： ｃ７０Ｈ７８Ｎ１２ｏ１６・６Ｈ
２ｏ として理論値： Ｃ，５７，９２１Ｈ１６，２５
、Ｎ、 １１．５８測定値：ｃ、５７４３；Ｈ，６，１
２ｉＮ、１１．４７アミノ酸分析： Ａｊ７（１，０９
） 、＆ｙ（０，９６）、Ｇｌｕｅｌ、 ０３） 、Ｇ
４（３，００） 、Ａｌａ（２，０１）、Ｔｒｌ’Ｃ０
，９１）
〔■〕目的化合物Ｈ−Ｔｒｐ −Ａｌａ −Ｇｌｐ −
Ｇｌｐ −Ａｓｐ −Ａｌａ −Ｓｅｒ −Ｇｌｐ −
０＆ −ＯＨの取得（保護基の離脱及び精製）
前工程の生成物Ｚ −Ｔｒｐ−Ａｌａ −Ｇｌｐ −Ｇ
ｌ＆−ＡＳ、ａ（ＯＢｘｌ）−Ａｌａ−８ｅｒ（Ｂｚｌ
）−Ｇｉｇ−Ｇｌｕ（ＯＢｚｌ）−Ｎ２Ｈ２Ｐｈ（１０
９，８’＃、００８２ミリモル）をＤ Ｍ Ｆ ６ ｍ
ｌに溶解、塩化第二鉄溶液（塩化第二鉄・ろ水和物３２
７を水７２ｍ１に溶解して調製）１ｍｌを加え、６５°
Ｃ１３０分間反応させた。反応後、水５０ｙｎｔを加え
、遠心分離（３０００ｒ ｐｍ、１０分間）した。上清
液を除去し、沈澱に水３０ａ＋／を加え攪拌した後、再
度遠心分離（３，０００ｒ、 ｌ’、ｍ、、１０分間）
しだ。上清°液を除去し、沈澱を減圧下シリカゲル上で
乾燥し、脱ヒドラジド体１０４．７ｑを得た。これにギ
酸８ｍｌを加え溶解し、ギ酸アンモニウム８４ｑ１パラ
ジウム黒１ｏ５ｍｙを加え、室温で２時間還元した。次
いで水１０ｍ／を加え、更に３０分間還元した。ｐ過に
よりパラジウム黒を除去し、ろ液を減圧下約１／３容ま
で濃縮した。Yield 181 da (yield 45 chi), melting point 236.5-23
Z 5°C1㇠f1 0, [α)25-1 3.
6” (CC15, DΔ(F) Elemental analysis (elA: c70H78N12o16・6H
Theoretical value as 2o: C, 57,921H16,25
, N, 11.58 Measured value: c, 5743; H, 6,1
2iN, 11.47 amino acid analysis: Aj7 (1,09
) , &y(0,96), Gluel, 03) , G
4(3,00),Ala(2,01),Trl'C0
,91) [■] Target compound H-Trp -Ala -Glp -
Glp -Asp -Ala -Ser -Glp -
Obtaining 0 & -OH (removal of protecting group and purification) Product of previous step Z -Trp-Ala -Glp -G
l&-AS, a(OBxl)-Ala-8er(Bzl
)-Gig-Glu(OBzl)-N2H2Ph(10
9,8'#, 0082 mmol) in D M F 6 m
Ferric chloride solution (ferric chloride filtrate 32
7 (prepared by dissolving 72 ml of water), add 1 ml of
The reaction was carried out for 130 minutes. After the reaction, 50 ynt of water was added and centrifuged (3000 rpm, 10 minutes). Remove the supernatant, add 30a+/ of water to the precipitate, stir, and centrifuge again (3,000r, l', m, 10 minutes).
Shida. The supernatant liquid was removed, and the precipitate was dried on silica gel under reduced pressure to obtain 104.7q of dehydrazide. To this, 8 ml of formic acid was added and dissolved, 84q1 ammonium formate and 105 my of palladium black were added, and the mixture was reduced at room temperature for 2 hours. Then, 10 m/ml of water was added and the mixture was further reduced for 30 minutes. Palladium black was removed by p-filtration, and the filtrate was concentrated to about 1/3 volume under reduced pressure.
この溶液を数回に分けて分取ＨＬＰＣに注入して、目的
化合物を含む分画を集めた。減圧下約１／２容まで濃縮
後、凍結乾燥し、題記の目的化合物３０．２Ｗ（収率４
４チ）を得た。This solution was divided into several parts and injected into a preparative HLPC to collect fractions containing the target compound. After concentrating to about 1/2 volume under reduced pressure and freeze-drying, 30.2W of the title target compound (yield 4
4) was obtained.
本目的化合物の物性値及び純度は下記する通りである：
融点２１３−２１５°Ｃ（分解）、（／Ｚ 〕２５−１
７．３−（Ｃ０，２、Ｈ２Ｏ）、Ｒ／’２０．１４にン
ヒドリン試薬）
元素分析（％） ： Ｃ３５Ｈ４８Ｎ１ｏＯｉ５．ＣＨ
３ＣＯ０Ｈ，５Ｈ２０理論値： Ｃ，４４，４９ｉ”Ｌ
６．２６ 、Ｎ、 １４．０２測定瞳： Ｃ，４４，
２６ｉＨ，６，４１ＢＮ、 １４．１５アミノ酸分析：
メタンスルホン酸分解 ん戸（１，１２）、５ｅｒ（１
，０６）、Ｇｌｕ（１，０６）、Ｇｌ）（３，００）
、Ａｌａ（１，９９） 、Ｔｒ）（０，８４） 、Ｇ４
の回収率は８０％。The physical properties and purity of the target compound are as follows: Melting point: 213-215°C (decomposed), (/Z) 25-1
7.3-(C0,2, H2O), R/'20.14 to nhydrin reagent) Elemental analysis (%): C35H48N1oOi5. CH
3CO0H, 5H20 theoretical value: C, 44, 49i”L
6.26, N, 14.02 measurement pupil: C,44,
26iH, 6,41BN, 14.15 amino acid analysis:
Methanesulfonic acid decomposition Ndo (1, 12), 5er (1
,06), Glu(1,06), Gl)(3,00)
,Ala(1,99),Tr)(0,84),G4
The recovery rate was 80%.
アミノペプチターゼＭ消化二ＡＳ戸（１，１１）、５ｙ
（１，０５） 、Ｇｌｕ（１，０４） 、Ｇ４（３，０
０）、Ａｌ’（２，０３） 、Ｔｒ／（０，９９） 、
Ｇｌｐ ＬＤ回収率は７３％。Aminopeptidase M digestion two AS doors (1,11), 5y
(1,05), Glu(1,04), G4(3,0
0), Al'(2,03), Tr/(0,99),
Glp LD recovery rate was 73%.
本品の純度はＨＰＬＣ法により測定した結果はほぼ１０
０％であり、まだ化学的に合成された本ペプタイド（ペ
プチド研究所、大阪より購入）品とＨＬＰＣ上で同一位
置に溶出された。The purity of this product is approximately 10% as measured by HPLC method.
0% and was still eluted at the same position on HLPC as the chemically synthesized peptide (purchased from Peptide Institute, Osaka).
さらに、本品はアミノペプチターゼＭ消化の結果より、
すべてがＬ体からなり、ラセミ化は生じておらず、かつ
、アスパラギン酸残基での転位は認められなかった。Furthermore, this product is based on the results of aminopeptidase M digestion.
All of them were L-configured, no racemization occurred, and no rearrangement at aspartic acid residues was observed.
かくして、本発明の酵素法により製造されたデルタ睡眠
誘発ペプチド（ＤＳＩＰ）は化学的合成品のものとは異
なり、光学活性体として特異な生理効果を発揮するもの
として期待される。Thus, the delta sleep-inducing peptide (DSIP) produced by the enzymatic method of the present invention is different from chemically synthesized products and is expected to exhibit unique physiological effects as an optically active substance.
（特許出願人 東宝薬品工業株式会社）（代理人 弁理
士 糟谷 安）(Patent applicant: Toho Pharmaceutical Industries, Ltd.) (Representative: Patent attorney Yasu Kasuya)
Claims (1)
て行く方法により、［１］保護されたＳｅｒ−Ｇｌｙ−
Ｇｌｕ、［２］保護されたＧｌｙ−Ａｓｐ−Ａｌａ及び
［３］保護されたＴｒｐ−Ａｌａ−Ｇｌｙなる三つのフ
ラグメントを製造する第一段階と、この三つのフラグメ
ントを縮合させる第二段階と、次いで保護基を順次離脱
させて行く第三段階とよりなることを特徴とする公知有
用ペプチドの製造法。 ２、各構成アミノ酸より段階的にペプチド結合させて三
つのフラグメントを製造するに当ってはカップリング法
と置換法とを併用し、これら三つのフラグメント間の縮
合は置換法により行なうことを特徴とする特許請求の範
囲第一項記載の公知有用ペプチドの製造法。 ３、三つのフラグメントのカルボキシル末端の保護基は
フェニルヒドラジドとし、アミノ酸としてトリプトファ
ンはベンジルオキシカルボニル、その他のアミノ酸は第
三ブチルオキシカルボニルでＮ末端を保護し、アスパラ
ギン酸及びグルタミン酸の側鎖カルボキシルはベンジル
エステル、そしてセリンの側鎖水酸基はベンジルエーテ
ルとして保護したものを用いることを特徴とする特許請
求の範囲第一項及び同第二項記載の公知有用ペプチドの
製造法。[Claims] 1. In the production of the known delta sleep-inducing peptide: [There is an amino acid sequence], each constituent amino acid is first linked stepwise using an enzyme to elongate the peptide chain. [1] Protected Ser-Gly-
A first step of producing three fragments: Glu, [2] protected Gly-Asp-Ala and [3] protected Trp-Ala-Gly, a second step of condensing these three fragments, and then A method for producing a known useful peptide, comprising a third step of sequentially removing protecting groups. 2. A coupling method and a substitution method are used in combination to produce three fragments by stepwise peptide bonding from each constituent amino acid, and the condensation between these three fragments is carried out by a substitution method. A method for producing a known useful peptide according to claim 1. 3. The carboxyl-terminal protecting group of the three fragments is phenylhydrazide, the amino acid tryptophan is protected with benzyloxycarbonyl, the other amino acids are protected with tert-butyloxycarbonyl, and the side chain carboxyls of aspartic acid and glutamic acid are benzyloxycarbonyl. The method for producing a known useful peptide according to claims 1 and 2, characterized in that the ester and the side chain hydroxyl group of serine are protected as benzyl ether.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP22870186A JPS6384499A (en) | 1986-09-26 | 1986-09-26 | Production of delta sleep inducing peptide by enzyme |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP22870186A JPS6384499A (en) | 1986-09-26 | 1986-09-26 | Production of delta sleep inducing peptide by enzyme |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS6384499A true JPS6384499A (en) | 1988-04-15 |
Family
ID=16880446
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP22870186A Pending JPS6384499A (en) | 1986-09-26 | 1986-09-26 | Production of delta sleep inducing peptide by enzyme |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS6384499A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5143854A (en) * | 1989-06-07 | 1992-09-01 | Affymax Technologies N.V. | Large scale photolithographic solid phase synthesis of polypeptides and receptor binding screening thereof |
| US7056666B2 (en) | 1990-12-06 | 2006-06-06 | Affymetrix, Inc. | Analysis of surface immobilized polymers utilizing microfluorescence detection |
-
1986
- 1986-09-26 JP JP22870186A patent/JPS6384499A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5143854A (en) * | 1989-06-07 | 1992-09-01 | Affymax Technologies N.V. | Large scale photolithographic solid phase synthesis of polypeptides and receptor binding screening thereof |
| US7056666B2 (en) | 1990-12-06 | 2006-06-06 | Affymetrix, Inc. | Analysis of surface immobilized polymers utilizing microfluorescence detection |
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