NO161374B - ANALOGY PROCEDURE FOR THE PREPARATION OF THERAPEUTICALLY ACTIVE MITOMYCIN ANALOGS. - Google Patents
ANALOGY PROCEDURE FOR THE PREPARATION OF THERAPEUTICALLY ACTIVE MITOMYCIN ANALOGS. Download PDFInfo
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- NO161374B NO161374B NO840433A NO840433A NO161374B NO 161374 B NO161374 B NO 161374B NO 840433 A NO840433 A NO 840433A NO 840433 A NO840433 A NO 840433A NO 161374 B NO161374 B NO 161374B
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- Prior art keywords
- ammonium acetate
- peptides
- tert
- liters
- butyl
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- 238000000034 method Methods 0.000 title claims description 11
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical class C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 title 1
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 29
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 27
- 239000005695 Ammonium acetate Substances 0.000 claims description 27
- 229940043376 ammonium acetate Drugs 0.000 claims description 27
- 235000019257 ammonium acetate Nutrition 0.000 claims description 27
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 24
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 16
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- IDLFZVILOHSSID-OVLDLUHVSA-N corticotropin Chemical group C([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)NC(=O)[C@@H](N)CO)C1=CC=C(O)C=C1 IDLFZVILOHSSID-OVLDLUHVSA-N 0.000 claims description 11
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 10
- 125000006239 protecting group Chemical group 0.000 claims description 10
- 238000000746 purification Methods 0.000 claims description 7
- 150000001413 amino acids Chemical class 0.000 claims description 3
- 125000004432 carbon atom Chemical group C* 0.000 claims description 3
- DNIAPMSPPWPWGF-GSVOUGTGSA-N (R)-(-)-Propylene glycol Chemical compound C[C@@H](O)CO DNIAPMSPPWPWGF-GSVOUGTGSA-N 0.000 claims description 2
- 230000015572 biosynthetic process Effects 0.000 claims description 2
- 230000001376 precipitating effect Effects 0.000 claims description 2
- 238000003786 synthesis reaction Methods 0.000 claims description 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 21
- 239000002244 precipitate Substances 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- 102400000739 Corticotropin Human genes 0.000 description 9
- 101800000414 Corticotropin Proteins 0.000 description 9
- 239000006227 byproduct Substances 0.000 description 9
- 229960000258 corticotropin Drugs 0.000 description 9
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 8
- 239000000872 buffer Substances 0.000 description 8
- 238000001556 precipitation Methods 0.000 description 8
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- 238000004587 chromatography analysis Methods 0.000 description 6
- 239000000055 Corticotropin-Releasing Hormone Substances 0.000 description 5
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- 239000000047 product Substances 0.000 description 5
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- DTQVDTLACAAQTR-UHFFFAOYSA-M Trifluoroacetate Chemical compound [O-]C(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-M 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000000203 mixture Substances 0.000 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 description 4
- CWERGRDVMFNCDR-UHFFFAOYSA-N thioglycolic acid Chemical compound OC(=O)CS CWERGRDVMFNCDR-UHFFFAOYSA-N 0.000 description 4
- 239000011877 solvent mixture Substances 0.000 description 3
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- 230000001476 alcoholic effect Effects 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- ALSPKRWQCLSJLV-UHFFFAOYSA-N azanium;acetic acid;acetate Chemical compound [NH4+].CC(O)=O.CC([O-])=O ALSPKRWQCLSJLV-UHFFFAOYSA-N 0.000 description 2
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- 230000008020 evaporation Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000000859 sublimation Methods 0.000 description 2
- 230000008022 sublimation Effects 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- QWCKQJZIFLGMSD-UHFFFAOYSA-N 2-Aminobutanoic acid Natural products CCC(N)C(O)=O QWCKQJZIFLGMSD-UHFFFAOYSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- QWCKQJZIFLGMSD-GSVOUGTGSA-N D-alpha-aminobutyric acid Chemical compound CC[C@@H](N)C(O)=O QWCKQJZIFLGMSD-GSVOUGTGSA-N 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- QUOGESRFPZDMMT-UHFFFAOYSA-N L-Homoarginine Natural products OC(=O)C(N)CCCCNC(N)=N QUOGESRFPZDMMT-UHFFFAOYSA-N 0.000 description 1
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- QUOGESRFPZDMMT-YFKPBYRVSA-N L-homoarginine Chemical compound OC(=O)[C@@H](N)CCCCNC(N)=N QUOGESRFPZDMMT-YFKPBYRVSA-N 0.000 description 1
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- LRQKBLKVPFOOQJ-YFKPBYRVSA-N L-norleucine Chemical compound CCCC[C@H]([NH3+])C([O-])=O LRQKBLKVPFOOQJ-YFKPBYRVSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 1
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 150000001242 acetic acid derivatives Chemical class 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
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- 235000003704 aspartic acid Nutrition 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
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- 238000011049 filling Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
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- 229960003104 ornithine Drugs 0.000 description 1
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- 239000000126 substance Substances 0.000 description 1
- 125000006253 t-butylcarbonyl group Chemical group [H]C([H])([H])C(C(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
- C07D487/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains three hetero rings
- C07D487/14—Ortho-condensed systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Description
Fremgangsmåte til rensning av syntetisk fremstilte peptider. Process for purification of synthetically produced peptides.
Oppfinnelsens gjenstand er en fremgangsmåte til rensning av syntetisk fremstilte peptider med sekvensen 1—18 til 1—25 av corticotropin (ACTH), hvori enkelte aminosyrebyggestener kan avvike fra denne sekvens, idet fremgangsmåten er karakterisert ved at man fraksjonert utfeller de ved syntesen dannede, med tert-butyl- og tert.-butyl-oksykarbonyl-rester beskyttede peptider ved hjelp av vandig ammoniumacetatholdig eddiksyre fra en vandig alifatisk alkohol med 1—3 C-atomer, befrir de anrikede peptider for beskyttelsesgruppene ved behandling med trifluoreddiksyre, oppløser de dannede peptider i fortynnet vandig ammonium-acetatoppløsning og kromatograferer den heri oppløselige del på karboksymetylcellulose. The object of the invention is a method for purifying synthetically produced peptides with the sequence 1-18 to 1-25 of corticotropin (ACTH), in which certain amino acid building blocks may deviate from this sequence, the method being characterized by fractionally precipitating those formed during the synthesis, with tert-butyl- and tert-butyl-oxycarbonyl residues protected peptides using aqueous ammonium acetate-containing acetic acid from an aqueous aliphatic alcohol with 1-3 C atoms, free the enriched peptides of the protecting groups by treatment with trifluoroacetic acid, dissolve the formed peptides in dilute aqueous ammonium acetate solution and chromatograph the portion soluble therein on carboxymethyl cellulose.
Rensningen av corticotropt virk-somme syntetisk fremstilte peptider ved hjelp av kromatografi på karboksymetylcellulose er ofte beskrevet, imidlertid egner denne metode seg ikke for teknisk formål på grunn av de nødvendige store mengder karboksymetylcellulose og eluat. Således krever man ifølge J. Amer. Chem. Soc. 86, side 2715 (1964) for rensning av 164 mg rått a<1>—<19->corticotropin den 100-dobbelte mengde karboksymetylcellulose og 1,2 liter pufferoppløsning, hvorav ca. 200 cm<3> inneholder det ønskede peptid og må lyofiliseres. The purification of corticotropically active synthetically produced peptides by means of chromatography on carboxymethylcellulose is often described, however, this method is not suitable for technical purposes due to the large amounts of carboxymethylcellulose and eluate required. Thus, according to J. Amer, it is required. Chem. Soc. 86, page 2715 (1964) for the purification of 164 mg of crude a<1>—<19->corticotropin the 100-fold amount of carboxymethyl cellulose and 1.2 liters of buffer solution, of which approx. 200 cm<3> contains the desired peptide and must be lyophilized.
Ifølge en annen forskrift (Heiv. Chim. Acta 1^ 6, side 1570 (1963)) renses det ennå According to another regulation (Heiv. Chim. Acta 1^ 6, page 1570 (1963)) it is still purified
med beskyttelsesgrupper utstyrte /31_24-with protective groups equipped /31_24-
corticotropin ved motstrømsfordeling over 400 trinn. Også en slik rensning er bare å gjennomføre med omstendelig arbeide og store oppløsningsmiddelmengder. corticotropin by countercurrent distribution over 400 steps. Such cleaning can also only be carried out with extensive work and large amounts of solvent.
Rensningsfremgangsmåten ifølge oppfinnelsen beror nå på kombinasjonen av to enkle trinn som er gjennomførbare med små tekniske komplikasjoner, og som fore-går på ideell måte. Ved den fraksjonerte utfelling kan man fjerne nettopp de biprodukter og de ved siste reaksjon ikke omsatte utgangsprodukter som forstyrrer ved den etterfølgende kromatografi på karboksymetylcellulose og ville betinge en betraktelig større mengde elueringsmiddel. Kromatograferingen er derfor uvanlig en-kel, den krever bare den fire- til fem-dobbelte vektmengde karboksymetylcellulose, beregnet på anvendt peptid, og følge-lig et i forhold til de kjente fremgangs-måter meget mindre volum eluat. Fremgangsmåten er også fordelaktig fordi den kan anvendes når det ikke anvendes noe høyrenset utgangsprodukt ved de siste reaksjonstrinn til fremstilling av de beskyttede peptidsekvenser. The cleaning method according to the invention is now based on the combination of two simple steps which are feasible with little technical complications, and which take place in an ideal way. With the fractional precipitation, it is possible to remove precisely the by-products and the starting products not converted in the last reaction which interfere with the subsequent chromatography on carboxymethyl cellulose and would require a considerably larger amount of eluent. Chromatography is therefore unusually simple, it only requires four to five times the weight amount of carboxymethyl cellulose, calculated for the peptide used, and consequently a much smaller volume of eluate compared to the known methods. The method is also advantageous because it can be used when no highly purified starting product is used in the last reaction steps to produce the protected peptide sequences.
For rensningen egner seg peptider av aminosyrekvens 1—18 til 1—25 av corticotropin, f. eks. de som fremstilles ifølge Chem. Ber 97 1207 (1964), spesielt ifølge den i Z. Naturforschg. 19 b, 858 (1964) nevnte variant, eller ifølge Heiv. Chim. Acta 46 1550 (1963). Disse peptider adskiller seg lite fra hverandre med hensyn til basisitet og oppløselighet i de ifølge oppfinnelsen anvendte oppløsningsblandinger, således at de alle kan renses etter samme skjema. Også analoger lar seg rense etter denne fremgangsmåte. I slike analoge er enkelte aminosyrer utvekslet mot andre tilsvarende byggede, såvidt ACTH-aktiviteten bibeholdes. For eksempel lar serin seg ut-veksle mot glycin eller alanin, tyrosin mot fenylalanin, metionin mot a-aminosmør-syre, leucin eller norleucin, tryptofan mot fenylalanin eller tyrosin, glutaminsyre mot asparaginsyre, lysin mot ornitin, arginin eller homoarginin. Peptides of amino acid residues 1-18 to 1-25 of corticotropin are suitable for the purification, e.g. those prepared according to Chem. Ber 97 1207 (1964), especially according to that in Z. Naturforschg. 19 b, 858 (1964) mentioned variant, or according to Heiv. Chim. Acta 46 1550 (1963). These peptides differ little from each other with regard to basicity and solubility in the solution mixtures used according to the invention, so that they can all be purified according to the same scheme. Analogues can also be purified using this procedure. In such analogues, certain amino acids are exchanged for others of similar structure, as long as the ACTH activity is retained. For example, serine can be exchanged for glycine or alanine, tyrosine for phenylalanine, methionine for α-aminobutyric acid, leucine or norleucine, tryptophan for phenylalanine or tyrosine, glutamic acid for aspartic acid, lysine for ornithine, arginine or homoarginine.
Ved rensningen går man eksempelvis frem på følgende måte: Man oppløser 1 del av den ennå med tert.-butyl- og tert.butyl-oksykarbonyl-beskyttelsesgrupper utstyrte rå peptidblan-ding ved oppvarmning i 15—20 deler av en vandig alkohol med 1—3 C-atomer, fortrinnsvis metanol, som fortrinnsvis inneholder 10—20 % vann og ca. 1 % ammoniumacetat. For example, the purification process is as follows: 1 part of the crude peptide mixture still equipped with tert-butyl and tert-butyl oxycarbonyl protective groups is dissolved by heating in 15-20 parts of an aqueous alcohol with 1- 3 C atoms, preferably methanol, which preferably contains 10-20% water and approx. 1% ammonium acetate.
Ved avkjøling kan det opptre en utfelling som består av ikke-omsatte utgangsprodukter og biprodukter. I dette til-felle frafiltrerer man utfellingen og be-handler den med omtrent halvparten av det foran nevnte oppløsningsmiddel på samme måte. Upon cooling, a precipitate may occur consisting of unconverted starting products and by-products. In this case, the precipitate is filtered off and treated with approximately half of the aforementioned solvent in the same way.
Den dannede oppløsning resp. de forenede filtrater av adskilt utfelling blandes kaldt med flere ganger, fortrinnsvis 2—4 ganger, mengden vann som omtrent inneholder 2 % eddiksyre og ammoniumacetat. Derved utfelles et fnokket bunnfall som praktisk talt inneholder det samlede bio-logisk aktive peptid i beskyttet form. Ytterligere biprodukter forblir i oppløsning. For å unngå tap av denne utfelling som har en liten, men merkbar oppløselighet i den etter utfellingen tilstedeværende opp-løsningsmiddelblanding, er det fordelaktig å inndampe de forenede filtrater før blan-dingen med den vandige eddiksyre-ammo-niumacetatoppløsning til halvparten til -2/3 av det opprinnelige volum. Det trenges da mindre av den til utfelling anvendte vandige eddiksyre-ammoniumacetat-oppløs-ning, fortrinnsvis 2—3 ganger mengden, referert til den inndampede alkoholiske oppløsning. The formed solution resp. the combined filtrates of separated precipitation are mixed cold with several times, preferably 2-4 times, the amount of water containing approximately 2% acetic acid and ammonium acetate. Thereby, a fine precipitate is precipitated which practically contains the total biologically active peptide in a protected form. Additional by-products remain in solution. In order to avoid loss of this precipitate, which has a small but noticeable solubility in the solvent mixture present after the precipitation, it is advantageous to evaporate the combined filtrates before mixing with the aqueous acetic acid-ammonium acetate solution to half to -2/ 3 of the original volume. Less is then needed of the aqueous acetic acid-ammonium acetate solution used for precipitation, preferably 2-3 times the amount, referred to the evaporated alcoholic solution.
Utfellingen samles, vaskes med den til utfelling anvendte oppløsningsmiddel-blanding og tørkes i vakuum ved 40°C. Innholdet av eddiksyre og ammoniumacetat i den til utfellingen anvendte opp-løsningsmiddelblanding kan varieres innen vide grenser. Den vandige oppløsning kan inneholde inntil 10 % ammoniumacetat, fortrinnsvis 1—3 %. Hertil setter man så meget eddiksyre at pH ligger innen gren-sene 3—7, fortrinnsvis 4—6,5. The precipitate is collected, washed with the solvent mixture used for precipitation and dried in a vacuum at 40°C. The content of acetic acid and ammonium acetate in the solvent mixture used for the precipitation can be varied within wide limits. The aqueous solution can contain up to 10% ammonium acetate, preferably 1-3%. Add enough acetic acid to this that the pH is within the range of 3-7, preferably 4-6.5.
Den tørkede utfelling, som inneholder det ønskede peptid, blir for avspaltning av beskyttelsesgruppen behandlet med eksempelvis den 10-dobbelte mengde trifluoreddiksyre som inneholder 0,1—0,3 % tioglykolsyre, i 1 time ved værelsestempera-tur. Deretter utfeller man med det 10-dobbelte volum eter (beregnet på trifluoreddiksyre) det for beskyttelsesgrupper be-fridde peptid som trifluoracetat. The dried precipitate, which contains the desired peptide, is treated with, for example, a 10-fold amount of trifluoroacetic acid containing 0.1-0.3% thioglycolic acid for 1 hour at room temperature to remove the protective group. The deprotected peptide is then precipitated with the 10-fold volume of ether (calculated on trifluoroacetic acid) as trifluoroacetate.
Dette stoff oppløses eksempelvis i 50 —100 ganger mengden 0,1 molar ammoniumacetat. Man filtrerer fra et lite resi-duum og bringer oppløsningen på den omtrent 4-dobbelte vektmengde karboksymetylcellulose (beregnet på trifluoracetatet) som befinner seg i en kromatograferings-søyle. Deretter eluerer man peptidene med ammoniumacetatpuffer av økende puffer-konsentrasjon mellom 0,1 og 0,3 m og en fra 8 til 9 stigende pH. Konsentrasjonen av det peptid som trer ut av søylen be-stemmes ved måling av dets UV-absorp-sjon ved 254 eller 275. m^. Etter noen biprodukter fremkommer til slutt peptidet med ACTH-aktivitet. Ved lyofilisering får man de ønskede peptider i ren form som acetater. This substance is dissolved, for example, in 50-100 times the amount of 0.1 molar ammonium acetate. One filters from a small residue and brings the solution to the approximately 4-fold weight amount of carboxymethylcellulose (calculated on the trifluoroacetate) which is in a chromatography column. The peptides are then eluted with ammonium acetate buffer of increasing buffer concentration between 0.1 and 0.3 m and an increasing pH from 8 to 9. The concentration of the peptide that emerges from the column is determined by measuring its UV absorption at 254 or 275 m^. After some by-products, the peptide with ACTH activity finally emerges. By lyophilization, the desired peptides are obtained in pure form as acetates.
I en ytterligere forenklet variant av fremgangsmåten oppløser man det rå ennå med beskyttelsesgrupper utstyrte peptid, som beskrevet, i 80—90 %-ig alkohol, blander imidlertid nå med en gang med 2—3 ganger mengden vann med det angitte innhold av eddiksyre og ammoniumacetat. Man får således det søkte peptid forurenset med de peptider som ville ha falt ut ved avkjøling av den alkoholiske oppløsning. Etter avspaltning av beskyttelsesgruppene og opp-tagelse i 0,1 molar ammoniumacetat forblir disse utgangs- og biprodukter uopp-løst og frafiltreres. Den videre rensning ved hjelp av kromatografi på karboksymetylcellulose foretas på den beskrevne måte. In a further simplified variant of the method, the crude peptide, still equipped with protective groups, is dissolved, as described, in 80-90% alcohol, but now mixed with 2-3 times the amount of water with the indicated content of acetic acid and ammonium acetate . The desired peptide is thus obtained contaminated with the peptides that would have precipitated upon cooling the alcoholic solution. After removal of the protective groups and absorption in 0.1 molar ammonium acetate, these starting and by-products remain undissolved and are filtered off. The further purification by means of chromatography on carboxymethyl cellulose is carried out in the manner described.
Eksempel 1. Example 1.
30 g rått, ennå med tert.-butyl og tert.-butyloksykarbonyl-beskyttelsesgrupper utstyrte, /?'—<23->corticotropin-23-amid, som ble fremstilt ifølge Chem. Ber. 97 1207 30 g of crude, still with tert-butyl and tert-butyloxycarbonyl protecting groups equipped, /?'-<23->corticotropin-23-amide, which was prepared according to Chem. Pray. 97 1207
(1964) med den i Z. Naturforsch., 19 b, (1964) with that in Z. Naturforsch., 19 b,
858 (1964) beskrevne variant, oppløses varmt i 4,5 liter 90 %-ig metanol som inneholder 35 g ammoniumacetat. Ved avkjø-ling utfelles geleaktig bunnfall som suges fra og vaskes med 250 cm<3> 90 %-ig metanol. Utfellingen behandles igjen på samme måte med 2 liter varm 90%-ig metanol og suges fra etter avkjøling. Man får, alt 858 (1964) described variant, is dissolved hot in 4.5 liters of 90% methanol containing 35 g of ammonium acetate. On cooling, a gel-like precipitate precipitates which is sucked off and washed with 250 cm<3> of 90% methanol. The precipitate is treated again in the same way with 2 liters of hot 90% methanol and sucked off after cooling. You get everything
etter råproduktets renhetsgrad, inntil 50 g utgangs- og biprodukter. De forenede filtrater inndampes i vakuum til 4 liter og blandes med 8 liter vann, som pr. liter inneholder 20 cm3 eddiksyre og 20 cm» ammoniumacetat. Herved utfelles et fnokket bunnfall, som etter noen tids henstand kaldt suges fra og vaskes med en blanding av 30 % metanol og 70 % av den til utfelling anvendte ammoniumacetatpuffer. Utbytte: 180 g — 230 g beskyttet forrenset /3<1-23.>corticotropin-23-amid. Fra filtratet kan det etter inndampning og avsublimering av ammoniumacetatet fremstilles ytterligere 65 g utgangs- og biprodukter. depending on the purity of the raw product, up to 50 g of starting and by-products. The combined filtrates are evaporated in vacuum to 4 liters and mixed with 8 liters of water, which per liter contains 20 cm3 of acetic acid and 20 cm» of ammonium acetate. This precipitates a friable precipitate, which after a period of time is coldly sucked off and washed with a mixture of 30% methanol and 70% of the ammonium acetate buffer used for precipitation. Yield: 180 g — 230 g protected pre-purified /3<1-23.>corticotropin-23-amide. After evaporation and sublimation of the ammonium acetate, a further 65 g of starting and by-products can be produced from the filtrate.
Til avspaltning av beskyttelsesgrupper oppløser man 180 g av det beskyttede, forrensede /^-^-corticotropin-23-amid i 1,8 liter trifluoreddiksyre som inneholder 3 cm<3> tioglykolsyre, og lar det henstå i 1 time ved værelsetemperatur. Deretter utfelles peptid-trifluoracetatet med 18 liter peroksydfri eter, vaskes med eter og tørkes i vakuum. Utbytte 174 g. To remove protective groups, 180 g of the protected, prepurified /^-^-corticotropin-23-amide are dissolved in 1.8 liters of trifluoroacetic acid containing 3 cm<3> of thioglycolic acid, and allowed to stand for 1 hour at room temperature. The peptide trifluoroacetate is then precipitated with 18 liters of peroxide-free ether, washed with ether and dried in a vacuum. Yield 174 g.
100 g av dette forrensede peptid-trifluoracetat omrøres med 10 liter 0,1 molar ammoniumacetatpuffer i 10 min. ved værelsetemperatur, og den fra uoppløst frafiltrerte oppløsning filtreres ved en passeringshastighet på 5 liter/time over en med samme puffer ekvilibrert søyle av 400 g karboksymetylcellulose (søylevolum 2,5—3 liter, tilsvarende en søylediameter på ca. 8 cm og en fyllhøyde på 50—60 cm). Ved samme passeringshastighet eluerer man deretter under mellomkopling av et blandekar med 10 liter 0,1 molar ammoniumacetatpuffer med følgende oppløs-ninger (pH innstilt med ammoniakk): 100 g of this prepurified peptide-trifluoroacetate is stirred with 10 liters of 0.1 molar ammonium acetate buffer for 10 minutes. at room temperature, and the solution filtered from undissolved is filtered at a passage rate of 5 litres/hour over a column of 400 g of carboxymethyl cellulose equilibrated with the same buffer (column volume 2.5-3 litres, corresponding to a column diameter of approx. 8 cm and a filling height of 50-60 cm). At the same passage speed, one then elutes with intermediate connection of a mixing vessel with 10 liters of 0.1 molar ammonium acetate buffer with the following solutions (pH adjusted with ammonia):
a) 10 liter 0,2 molar ammoniumacetat, . pH 8,0; b) 10 liter 0,25 molar ammoniumacetat, pH .8,5 og ... c) ca. 20 liter 0,3 molar ammoniumacetat, pH 9,0. UV-absorpsjonskurven viser først fire svakere topper som tilsvarer biproduktene. Like etter tilsetningen av 0,3 molar ammoniumacetat øker absorpsjonen igjen. Øk-ningen tilsvarer de rene ACTH-aktive peptider. Den til denne langstrakte topp tilsvarende del av eluatet (ca. 15 liter) lyofiliseres deretter to ganger. Det volumi-nøse produkt utdriver man med 500 cm» vannfritt aceton, suger fra med en gang, vasker med aceton og peroksydfri eter og tørker i vakuum. Utbytte 62 g, kromato grafisk rent fi<1>—<23->corticotropin-23-amid, som acetat med ca. 100 I.E./mg ACTH-aktivitet. i Eksempel 2. 300 g rått, ennå med tert.-butyl-og tert.-butyloksykarbonyl-beskyttelsesgrupper utstyrte, i81-23-corticotropin-23-amid, som ble fremstilt ifølge Chem. Ber. 97 1207 (1964) med den i Z. Naturforsch. 19 b, 858 (1964) beskrevne variant, opp-løses varmt i 4 liter 90 %-ig metanol som inneholder 30 g ammoniumacetat. Deretter blander man under kraftig omrøring med 8 liter vann, som pr. liter inneholder 20 cm<3> eddiksyre og 20 g ammoniumacetat. Derved utfelles et fnokket bunnfall, som etter noen tids henstand kaldt suges fra og vaskes med en blanding av 30 % metanol og 70 % av den til utfelling anvendte ammoniumacetatpuffer. Utbytte: 228 g beskyttet forrenset /^-^-corticotropin-23- amid. Fra filtratet kan det etter inndampning og avsublimering av ammoniumacetat utvinnes ytterligere 67 g utgangs- og biprodukter. a) 10 liters of 0.2 molar ammonium acetate, . pH 8.0; b) 10 liters of 0.25 molar ammonium acetate, pH .8.5 and ... c) approx. 20 liters of 0.3 molar ammonium acetate, pH 9.0. The UV absorption curve first shows four weaker peaks corresponding to the by-products. Shortly after the addition of 0.3 molar ammonium acetate, the absorption increases again. The increase corresponds to the pure ACTH-active peptides. The portion of the eluate corresponding to this elongated peak (approx. 15 litres) is then lyophilized twice. The voluminous product is extracted with 500 cc of anhydrous acetone, suctioned off at once, washed with acetone and peroxide-free ether and dried in a vacuum. Yield 62 g, chromato graphically pure fi<1>—<23->corticotropin-23-amide, as acetate with approx. 100 I.U./mg ACTH activity. in Example 2. 300 g of crude, still with tert-butyl and tert-butyloxycarbonyl protecting groups equipped, i81-23-corticotropin-23-amide, which was prepared according to Chem. Pray. 97 1207 (1964) with that in Z. Naturforsch. 19 b, 858 (1964) described variant, is dissolved hot in 4 liters of 90% methanol containing 30 g of ammonium acetate. It is then mixed with vigorous stirring with 8 liters of water, which per liter contains 20 cm<3> acetic acid and 20 g ammonium acetate. Thereby, a crumbly precipitate is precipitated, which after a period of time is coldly sucked off and washed with a mixture of 30% methanol and 70% of the ammonium acetate buffer used for precipitation. Yield: 228 g of protected prepurified /^-^-corticotropin-23-amide. After evaporation and sublimation of ammonium acetate, a further 67 g of starting and by-products can be recovered from the filtrate.
Beskyttelsesgruppenes avspaltning The separation of the protection groups
gjennomføres som beskrevet i eksempel 1. carried out as described in example 1.
Til kromatografering på karboksymetylcellulose blir 130 g av peptid-trifluoracetatet omrørt med 10 liter 0,1 molar ammoniumacetatpuffer i 10 minutter ved værelsetemperatur, og den fra uoppløst (inntil 35 g) frafiltrerte oppløsning videre-behandlet som i eksempel 1. Man får 60 g kromatografisk rent /?'-<23->corticotropin-23-amid som acetat med ca. 100 I.E./mg ACTH-aktivitet. For chromatography on carboxymethyl cellulose, 130 g of the peptide trifluoroacetate is stirred with 10 liters of 0.1 molar ammonium acetate buffer for 10 minutes at room temperature, and the solution filtered from undissolved (up to 35 g) is further processed as in example 1. 60 g is obtained chromatographically pure /?'-<23->corticotropin-23-amide as acetate with approx. 100 I.U./mg ACTH activity.
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| JPS63150282A (en) * | 1986-12-13 | 1988-06-22 | Kyowa Hakko Kogyo Co Ltd | Mitomycin derivative |
| JPS63246379A (en) * | 1987-03-31 | 1988-10-13 | Kyowa Hakko Kogyo Co Ltd | 7-n,8-n-ethylenemitomycin 8-imines |
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| JPS5439098A (en) * | 1977-08-31 | 1979-03-24 | Kyowa Hakko Kogyo Co Ltd | Mitomycin c derivatives |
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| NZ199617A (en) * | 1981-05-15 | 1985-08-30 | University Patents Inc | Azirino(2',3',:3,4)pyrrolo(1,2-a)indole-4,7-dione derivatives and pharmaceutical compositions |
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