Tag nrho cov o ntawm cov resin
Nyhav 2.0 g dawb paug Wang resin nyob rau hauv ib tug huv si thiab qhuav cov tshuaj tiv thaiv raj, ntxiv 15 mL DMF, chav tsev kub ua kom 30 min txog.
Noj thawj cov amino acid
Nyob rau hauv chav tsev kub, lim tawm cov kuab tshuaj los ntawm cov kauj ruam dhau los ntawm cov xuab zeb core thiab ntxiv 1 mmol 5 molar tshaj C Thawj amino acid thaum kawg, 5-fold molar tshaj DMAP, 5 molar tshaj DIC, DMF Ua hnyav chav tsev kub tshuaj tiv thaiv 3 h. Siv tom qab cov tshuaj tiv thaiv tiav DMF Ntxuav 4 ~ 6 zaug, 5-6 zaug txhua zaus mL. Tom qab ntawd ntxiv qhov tsim nyog ntawm pyridine thiab acetic anhydride nrog lub ntim piv ntawm 1: 1, thiab ua rau 30 feeb. Siv tom qab cov tshuaj tiv thaiv tiav DMF Ntxuav 4 ~ 6 zaug, 5-6 zaug txhua zaus mL. (Qhov cuam tshuam: Thaiv tawm cov microdots nquag ntawm cov khoom tsis muaj kev cuam tshuam.)
Fmoc Tawm ntawm pawg tiv thaiv
Tshem tawm cov kuab tshuaj los ntawm cov kauj ruam dhau los los ntawm kev nqus pom, thiab ntxiv 10 mL 20% ntawm piperidine DMF Cov tshuaj ntxiv rau cov resin, N2 Do 10 min Thaum kawg, lim cov tshuaj thiab ntxiv 10 mL 20% ntawm piperidine DMF tov, N2 Tshuab thiab do 5 min Ces lim tawm cov tshuaj thiab rov ua haujlwm no ob zaug. DMF Ntxuav 4 zaug, ntxuav 2 zaug nrog methanol, 5-6 zaug ib zaug mL.
Kev ntsuam xyuas cov nyhuv tshem tawm ntawm ninhydrin
Tshem tawm ib qho me me ntawm cov resin, ntxuav nws peb zaug nrog methanol, ntxiv ninhydrin, KCN, Ib tee ntawm cov tshuaj phenol, 105℃-110℃Cua sov 5 min, tig xiav tsaus yog cov tshuaj tiv thaiv zoo, qhia tias kev tshem tawm tiav thiab cov kauj ruam tom ntej ntawm cov tshuaj tiv thaiv tuaj yeem ua tiav.; Yog tias nws tsis muaj xim, nws txhais tau hais tias pawg tiv thaiv tsis tau raug tshem tawm tag nrho, thiab kev ua haujlwm deprotection saum toj no yuav tsum tau rov ua dua.
Tom qab ntawd qhov thib ob amino acid thiab Fmoc Protection pawg tshem tawm
Qhov hnyav 3 zaug molar tshaj C Qhov thib ob amino acid thaum kawg, 3-fold molar tshaj HBTU thiab 3-fold molar tshaj ntawm HOBT Hauv cov tshuaj tiv thaiv raj, ntxiv cov nyiaj tsim nyog DMF Tom qab cov tshuaj tau yaj tag, ntxiv 10-fold molar tshaj (ntshiab) DIEA, Chav ntsuas kub 40 min, siv DMF Ntxuav 4-6 zaug txhua zaus, Siv ib qho me me ntawm cov resin siv Test nrog ninhydrin nrhiav reagent thiab nws yuav tshwm sim tsis muaj xim, tom qab ntawd ntxiv 10 mL 20% ntawm piperidine DMF kev tshem tawm Fmoc, Ua li no ob zaug rau 10 min, 5min, tom qab siv DMF Ntxuav 4 zaug, ntxuav 2 zaug nrog methanol, 5-6 zaug txhua zaus mL. Tshem tawm ib qho me me ntawm cov resin siv Siv cov ninhydrin detection reagent los xyuas nws. Yog tias qhov kuaj pom yog xiav, koj tuaj yeem mus rau kauj ruam tom ntej ntawm cov tshuaj tiv thaiv.
Los ntawm kev sib piv, rov ua cov kauj ruam 2.6 kom txog thaum lub synthesis N Tshem tawm cov amino acid kawg ntawm qhov kawg Fmoc Tiv thaiv puag, ces ntws.
Resin shedding thiab ntshiab khoom sib cais thiab nrhiav kom tau
Thaum kawg, siv cov tshuaj trifluoroacetic acid txiav (95% TFA: 2% TIS: 2% EDT: 1% H2O) Txiav 2 h, Lim cov tshuaj tiv thaiv nrog nqus thiab tau txais cov tshuaj trifluoroacetic acid ntawm polypeptide. Qhuav lub lysate nrog nitrogen kom ntau li ntau tau, precipitate nrog ether, centrifuge, thiab ces ntxuav nrog ether rau 3 vib nas this. ~ 5 zaug, cov khoom dawb tau txais. Tom qab dissolve nws nyob rau hauv dej ntshiab, HPLC Desalt thiab purify, khov - qhuav thiab precipitate crystals, siv ib tug me me MS tshuaj ntsuam.