400-998-5282
專注多肽 服務(wù)科研
編號(hào):145846
CAS號(hào):
單字母:H2N-SGRHRFDPFC-C(Acm)-EVICDDGTSVKLCT-OH
多肽H2N-Ser-Gly-Arg-His-Arg-Phe-Asp-Pro-Phe-Cys-Cys(Acm)-Glu-Val-Ile-Cys-Asp-Asp-Gly-Thr-Ser-Val-Lys-Leu-Cys-Thr-COOH的合成步驟:
1、合成CTC樹脂:稱取2.69g CTC Resin(如初始取代度約為1.06mmol/g)和3.42mmol Fmoc-Thr(tBu)-OH于反應(yīng)器中,加入適量DCM溶解氨基酸(需要注意,此時(shí)CTC樹脂體積會(huì)增大好幾倍,避免DCM溶液過少),再加入8.55mmol DIPEA(Mw:129.1,d:0.740g/ml),反應(yīng)2-3小時(shí)后,可不抽濾溶液,直接加入1ml的HPLC級(jí)甲醇,封端半小時(shí)。依次用DMF洗滌2次,甲醇洗滌1次,DCM洗滌一次,甲醇洗滌一次,DCM洗滌一次,DMF洗滌2次(這里使用甲醇和DCM交替洗滌,是為了更好地去除其他溶質(zhì),有利于后續(xù)反應(yīng))。得到 Fmoc-Thr(tBu)-CTC Resin。結(jié)構(gòu)圖如下:
2、脫Fmoc:加3倍樹脂體積的20%Pip/DMF溶液,鼓氮?dú)?0分鐘,然后2倍樹脂體積的DMF 洗滌5次。得到 H2N-Thr(tBu)-CTC Resin 。(此步驟脫除Fmoc基團(tuán),茚三酮檢測(cè)為藍(lán)色,Pip為哌啶)。結(jié)構(gòu)圖如下:
3、縮合:取8.55mmol Fmoc-Cys(Trt)-OH 氨基酸,加入到上述樹脂里,加適當(dāng)DMF溶解氨基酸,再依次加入17.11mmol DIPEA,8.13mmol HBTU。反應(yīng)30分鐘后,取小樣洗滌,茚三酮檢測(cè)為無色。用2倍樹脂體積的DMF 洗滌3次樹脂。(洗滌樹脂,去掉殘留溶劑,為下一步反應(yīng)做準(zhǔn)備)。得到Fmoc-Cys(Trt)-Thr(tBu)-CTC Resin。氨基酸:DIPEA:HBTU:樹脂=3:6:2.85:1(摩爾比)。結(jié)構(gòu)圖如下:
4、依次循環(huán)步驟二、步驟三,依次得到
H2N-Cys(Trt)-Thr(tBu)-CTC Resin
Fmoc-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
H2N-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
Fmoc-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
H2N-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
Fmoc-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
H2N-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
Fmoc-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
H2N-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
Fmoc-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
H2N-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
Fmoc-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
H2N-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
Fmoc-Asp(OtBu)-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
H2N-Asp(OtBu)-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
Fmoc-Asp(OtBu)-Asp(OtBu)-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
H2N-Asp(OtBu)-Asp(OtBu)-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
Fmoc-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
H2N-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
Fmoc-Ile-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
H2N-Ile-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
Fmoc-Val-Ile-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
H2N-Val-Ile-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
Fmoc-Glu(OtBu)-Val-Ile-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
H2N-Glu(OtBu)-Val-Ile-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
Fmoc-Cys(Acm)-Glu(OtBu)-Val-Ile-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
H2N-Cys(Acm)-Glu(OtBu)-Val-Ile-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
Fmoc-Cys(Trt)-Cys(Acm)-Glu(OtBu)-Val-Ile-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
H2N-Cys(Trt)-Cys(Acm)-Glu(OtBu)-Val-Ile-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
Fmoc-Phe-Cys(Trt)-Cys(Acm)-Glu(OtBu)-Val-Ile-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
H2N-Phe-Cys(Trt)-Cys(Acm)-Glu(OtBu)-Val-Ile-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
Fmoc-Pro-Phe-Cys(Trt)-Cys(Acm)-Glu(OtBu)-Val-Ile-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
H2N-Pro-Phe-Cys(Trt)-Cys(Acm)-Glu(OtBu)-Val-Ile-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
Fmoc-Asp(OtBu)-Pro-Phe-Cys(Trt)-Cys(Acm)-Glu(OtBu)-Val-Ile-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
H2N-Asp(OtBu)-Pro-Phe-Cys(Trt)-Cys(Acm)-Glu(OtBu)-Val-Ile-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
Fmoc-Phe-Asp(OtBu)-Pro-Phe-Cys(Trt)-Cys(Acm)-Glu(OtBu)-Val-Ile-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
H2N-Phe-Asp(OtBu)-Pro-Phe-Cys(Trt)-Cys(Acm)-Glu(OtBu)-Val-Ile-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
Fmoc-Arg(Pbf)-Phe-Asp(OtBu)-Pro-Phe-Cys(Trt)-Cys(Acm)-Glu(OtBu)-Val-Ile-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
H2N-Arg(Pbf)-Phe-Asp(OtBu)-Pro-Phe-Cys(Trt)-Cys(Acm)-Glu(OtBu)-Val-Ile-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
Fmoc-His(Trt)-Arg(Pbf)-Phe-Asp(OtBu)-Pro-Phe-Cys(Trt)-Cys(Acm)-Glu(OtBu)-Val-Ile-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
H2N-His(Trt)-Arg(Pbf)-Phe-Asp(OtBu)-Pro-Phe-Cys(Trt)-Cys(Acm)-Glu(OtBu)-Val-Ile-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
Fmoc-Arg(Pbf)-His(Trt)-Arg(Pbf)-Phe-Asp(OtBu)-Pro-Phe-Cys(Trt)-Cys(Acm)-Glu(OtBu)-Val-Ile-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
H2N-Arg(Pbf)-His(Trt)-Arg(Pbf)-Phe-Asp(OtBu)-Pro-Phe-Cys(Trt)-Cys(Acm)-Glu(OtBu)-Val-Ile-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
Fmoc-Gly-Arg(Pbf)-His(Trt)-Arg(Pbf)-Phe-Asp(OtBu)-Pro-Phe-Cys(Trt)-Cys(Acm)-Glu(OtBu)-Val-Ile-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
H2N-Gly-Arg(Pbf)-His(Trt)-Arg(Pbf)-Phe-Asp(OtBu)-Pro-Phe-Cys(Trt)-Cys(Acm)-Glu(OtBu)-Val-Ile-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
Fmoc-Ser(tBu)-Gly-Arg(Pbf)-His(Trt)-Arg(Pbf)-Phe-Asp(OtBu)-Pro-Phe-Cys(Trt)-Cys(Acm)-Glu(OtBu)-Val-Ile-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin
以上中間結(jié)構(gòu),均可在專肽生物多肽計(jì)算器-多肽結(jié)構(gòu)計(jì)算器中,一鍵畫出。
最后再經(jīng)過步驟二得到 H2N-Ser(tBu)-Gly-Arg(Pbf)-His(Trt)-Arg(Pbf)-Phe-Asp(OtBu)-Pro-Phe-Cys(Trt)-Cys(Acm)-Glu(OtBu)-Val-Ile-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Gly-Thr(tBu)-Ser(tBu)-Val-Lys(Boc)-Leu-Cys(Trt)-Thr(tBu)-CTC Resin,結(jié)構(gòu)如下:
5、切割:6倍樹脂體積的切割液(或每1g樹脂加8ml左右的切割液),搖床搖晃 2小時(shí),過濾掉樹脂,用冰無水乙醚沉淀濾液,并用冰無水乙醚洗滌沉淀物3次,最后將沉淀物放真空干燥釜中,常溫干燥24小試,得到粗品H2N-Ser-Gly-Arg-His-Arg-Phe-Asp-Pro-Phe-Cys-Cys(Acm)-Glu-Val-Ile-Cys-Asp-Asp-Gly-Thr-Ser-Val-Lys-Leu-Cys-Thr-COOH。結(jié)構(gòu)圖見產(chǎn)品結(jié)構(gòu)圖。
切割液選擇:1)TFA:H2O=95%:5%
2)TFA:H2O:TIS=95%:2.5%:2.5%
3)三氟乙酸:茴香硫醚:1,2-乙二硫醇:苯酚:水=87.5%:5%:2.5%:2.5%:2.5%
(前兩種適合沒有容易氧化的氨基酸,例如Trp、Cys、Met。第三種適合幾乎所有的序列。)
6、純化凍干:使用液相色譜純化,收集目標(biāo)峰液體,進(jìn)行凍干,獲得蓬松的粉末狀固體多肽。不過這時(shí)要取小樣復(fù)測(cè)下純度 是否目標(biāo)純度。
7、最后總結(jié):
杭州專肽生物技術(shù)有限公司(ALLPEPTIDE http://amynixphotography.com)主營(yíng)定制多肽合成業(yè)務(wù),提供各類長(zhǎng)肽,短肽,環(huán)肽,提供各類修飾肽,如:熒光標(biāo)記修飾(CY3、CY5、CY5.5、CY7、FAM、FITC、Rhodamine B、TAMRA等),功能基團(tuán)修飾肽(疊氮、炔基、DBCO、DOTA、NOTA等),同位素標(biāo)記肽(N15、C13),訂書肽(Stapled Peptide),脂肪酸修飾肽(Pal、Myr、Ste),磷酸化修飾肽(P-Ser、P-Thr、P-Tyr),環(huán)肽(酰胺鍵環(huán)肽、一對(duì)或者多對(duì)二硫鍵環(huán)),生物素標(biāo)記肽,PEG修飾肽,甲基化修飾肽等。
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