Solid-phase synthesis njira ya Melanotan I

Mbiri

Melanotan I, yomwe imadziwikanso kuti Afamelanotide, ndi polypeptide yopangidwa ndi 13 amino acid yomwe ili pafupi kuchitapo kanthu pa kafukufuku wamankhwala. Mayina a Chingerezi a Melanotan I ndi Melanotan-1 ndi Afamelanotide. Monga analogi yopangidwa ndi α-melanocyte-stimulating hormone α-MSH), Melanotan I ingagwiritsidwe ntchito pochiza matenda a photosensitivity omwe amayamba chifukwa cha erythropoietic protoporphyria. Pakali pano, mankhwala ambiri a Melanotan I amalembedwa kuti agwiritsidwe ntchito pofufuza okha ndipo sanapangidwe kuti agwiritsidwe ntchito ndi anthu. Mu kaphatikizidwe kachikhalidwe kolimba, kusankha kwa utomoni kumakhudza kwambiri kugwirizana kwake komanso kuyera komaliza kwa chinthucho.

Zambiri Zoyambira

Dzina lachi China: Melanotan 1
Dzina la Chingerezi: Melanotan-1
Nambala ya kampani: GT-A006
Nambala ya CAS: 75921-69-6
Kutsata: SYS{Ne}EH{D-Phe}RWGKPV-NH2
Fomula ya mamolekyu: C78H111N21O19
Kulemera kwa Molecular: 1646.88

 Njira yokonzekera

Ine. Kukonzekera kwa Fmoc-Val-Amino Resin

Gawo 1:Yezerani 11.11g (5mmol) ya utomoni wa Ramage Amide AM ndi digirii yolowa m'malo ya 0.45mmol/g ndikuwonjezera pa choyatsira gawo lolimba. Onjezani DCM kuti mufufuze utomoni kwa mphindi 30, kenako kukhetsa. Sambani katatu ndi DMF. Onjezani yankho la DMF lomwe lili ndi 20% piperidine (v/v) ndikuchitapo kanthu kwa mphindi zisanu. Onjezerani 20% piperidine DMF solution ndikuchitapo kanthu kwa mphindi 10, ndi kusamba kumodzi kwa DMF pakati. Mukamaliza, khetsani ndikusamba katatu ndi DMF.

Gawo 2:Sungunulani 5.09g wa Fmoc-Val-OH, 2.02g wa HOBT (1-hydroxybenzotriazole), ndi 2.32ml wa DIC (N,N'-disopropylcarbodiimide) mu DMF pa 0°C. Pambuyo pa kusungunuka kwathunthu, onjezerani kusakaniza ku gawo la zomwe mukuchita ndikuchita kutentha kwa firiji kwa ola limodzi. Yang'anirani momwe zimayendera pogwiritsa ntchito mayeso a ninhydrin, kuwonetsetsa kuti pali zotsatira zoyipa. Mukamaliza, sambani katatu ndi DMF kuti mupeze Fmoc-Val-amino resin.

II. Kukonzekera kwa Resin Peptide Yotetezedwa Mokwanira ya Melanotan I.

Gawo 1:Onjezani 20% piperidine mu njira ya DMF (chiŵerengero cha voliyumu) ​​ku Fmoc-Val-aminoresin pamwamba. Yankhani kwa mphindi zisanu, kenaka yikani gawo lina la 20% piperidine mu njira ya DMF ndikuchitapo kanthu kwa mphindi khumi. Sambani kamodzi ndi DMF panthawiyi. Zomwezo zikatha, ziume pansi pa vacuum ndikusamba katatu ndi DMF.

Gawo 2:Sungunulani 5.06g Fmoc-Pro-OH, 2.02g HOBT (1-hydroxybenzotriazole), ndi 2.32ml DIC (N,N'-disopropylcarbodiimide) mu DMF pa 0°C. Pambuyo pa kusungunuka kwathunthu, onjezerani kusakaniza ku gawo la zomwe mukuchita ndikuchita kutentha kwa firiji kwa ola limodzi. Yang'anirani momwe momwe zimayendera pogwiritsa ntchito mayeso a ninhydrin (Kaiser test) mpaka zotsatira zoyipa zitapezeka. Mukamaliza, sambani katatu ndi DMF kuti mupeze Fmoc-Pro-Val-aminoresin.

Gawo 3:Potsatira njira yomwe ikufotokozedwa mu Gawo 2, sequentially phatikizani amino acid iliyonse malinga ndi ndondomeko ya Melanotan I. Mukamaliza kugwirizanitsa kwa Ac-Ser (tBu) -OH, sambani katatu ndi DMF, katatu ndi DCM (dichloromethane), ndi katatu ndi methanol. Yanikani pansi pa vacuum kuti mupeze 23.89g ya peptide yotetezedwa kwathunthu ya Melanotan I.

III. Kukonzekera kwa Melanotan I Crude Peptide
23.89 g ya peptide yotetezedwa mokwanira ndi Melanotan I idayikidwa mu botolo lozungulira pansi. Pa 0 ° C, 250 ml ya cocktail yokonzedweratu yokonzekera (yopangidwa ndi TFA: thioanisole: phenol: triisopropylsilane: madzi = 82.5: 7.5: 5: 3: 2 ndi voliyumu) ​​pang'onopang'ono anawonjezera pansi pa kugwedeza. The osakaniza anachita pa otsika kutentha kwa 0,5 hours, kenako anachita firiji kwa 2 hours. Njira ya cleavage idasefedwa pansi pa vacuum. Njirayi idawonjezedwa pang'onopang'ono ku 2 L ya ayezi-wozizira wa anhydrous diethyl ether akugwedezeka. Peptide yosakanizidwayo inasiyanitsidwa ndi kusefera, kutsukidwa katatu ndi madzi oundana a diethyl ether, ndipo 8.55 g ya Melanotan I crude peptide inapezedwa.

IV. Kukonzekera kwa Melanotan I Yoyeretsedwa Peptide
Gawo 1:Sungunulani peptide ya Melanotan I yaiwisi mu 100 ml ya madzi oyeretsedwa ndikusefa kudzera mu nembanemba ya 0.45 μm kuti mupeze yankho lopanda peptide.

Gawo 2:Yeretsani peptide yopanda mafuta ya Melanotan I pogwiritsa ntchito chromatography yamadzimadzi (HPLC). Gwiritsani ntchito gawo la DAC-HB50 dynamic axial compression column ndi mobile phase A (0.05% trifluoroacetic acid aqueous solution) ndi mobile phase B (0.05% trifluoroacetic acid mu acetonitrile). Chitani gradient elution kuti apatukane ndi kuyeretsedwa. Dziwani zachitsanzocho pogwiritsa ntchito chowunikira cha UV ndikusonkhanitsa yankho la peptide lomwe likugwirizana ndi zomwe mukufuna kuzigawo.

Gawo 3:Pambuyo pa kuyeretsedwa kwa HPLC, pezani 180 ml ya Melanotan I trifluoroacetate solution ndi chiyero choposa 99%. Yang'anani yankho la 50 ml kudzera mu evaporation yozungulira.

Gawo 4:Gwirizanitsani ndime ya chromatography ndi madzi opangidwa ndi deionized ndikunyamula 50 ml ya Melanotan I trifluoroacetate solution yoyeretsedwa (kuyera> 99%). Elute kwa mphindi 50 mu 2% acetic acid aqueous solution system. Yang'anani zomwe mwasonkhanitsa mpaka 80 ml kudzera mu mpweya wozungulira. Yambani kuumitsa musanawume ndi kuumitsa kuti muthe kupeza 5.33 g ya Melanotan I yoyeretsedwa peptide, ndi zokolola za 32.33%.