400-998-5282
專注多肽 服務(wù)科研
Calcitonin is a peptide hormone involved in calcium and phosphorus homeostasis. Its inhibitory effect on osteoclast activity lowers blood calcium levels and positively influences bone mass density.
編號:156261
CAS號:21215-62-3
單字母:H2N-CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP-CONH2(Disulfide Bridge:C1-C7)
編號: | 156261 |
中文名稱: | 降鈣素Calcitonin, human |
英文名: | Calcitonin, human |
CAS號: | 21215-62-3 |
單字母: | H2N-CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP-CONH2(Disulfide Bridge:C1-C7) |
三字母: | H2N-Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-Tyr-Thr-Gln-Asp-Phe-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro-CONH2(Disulfide Bridge:Cys1-Cys7) |
氨基酸個(gè)數(shù): | 32 |
分子式: | C151H226N40O45S3 |
平均分子量: | 3417.85 |
精確分子量: | 3415.58 |
等電點(diǎn)(PI): | 10.49 |
pH=7.0時(shí)的凈電荷數(shù): | 4.15 |
平均親水性: | -0.67307692307692 |
疏水性值: | 0.02 |
外觀與性狀: | 白色粉末狀固體 |
消光系數(shù): | 1490 |
來源: | 人工化學(xué)合成,僅限科學(xué)研究使用,不得用于人體。 |
純度: | 95%、98% |
鹽體系: | 可選TFA、HAc、HCl或其它 |
生成周期: | 2-3周 |
儲(chǔ)存條件: | 負(fù)80℃至負(fù)20℃ |
標(biāo)簽: | 二硫鍵環(huán)肽 |
Calcitonin is a peptide hormone involved in calcium and phosphorus homeostasis. Its inhibitory effect on osteoclast activity lowers blood calcium levels and positively influences bone mass density.
二硫鍵廣泛存在與蛋白結(jié)構(gòu)中,對穩(wěn)定蛋白結(jié)構(gòu)具有非常重要的意義,二硫鍵一般是通過序列中的2個(gè)Cys的巰基,經(jīng)氧化形成。
形成二硫鍵的方法很多:空氣氧化法,DMSO氧化法,過氧化氫氧化法等。
二硫鍵的合成過程, 可以通過Ellman檢測以及HPLC檢測方法對其反應(yīng)進(jìn)程進(jìn)行監(jiān)測。
如果多肽中只含有1對Cys,那二硫鍵的形成是簡單的。多肽經(jīng)固相或液相合成,然后在pH8-9的溶液中進(jìn)行氧化。
當(dāng)需要形成2對或2對以上的二硫鍵時(shí),合成過程則相對復(fù)雜。盡管二硫鍵的形成通常是在合成方案的最后階段完成,但有時(shí)引入預(yù)先形成的二硫化物是有利于連合或延長肽鏈的。通常采用的巰基保護(hù)基有trt, Acm, Mmt, tBu, Bzl, Mob, Tmob等多種基團(tuán)。我們分別列出兩種以2-Cl樹脂和Rink樹脂為載體合成的多肽上多對二硫鍵形成路線:
二硫鍵反應(yīng)條件選擇
二硫鍵即為蛋白質(zhì)或多肽分子中兩個(gè)不同位點(diǎn)Cys的巰基(-SH)被氧化形成的S-S共價(jià)鍵。 一條肽鏈上不同位置的氨基酸之間形成的二硫鍵,可以將肽鏈折疊成特定的空間結(jié)構(gòu)。多肽分 子通常分子量較大,空間結(jié)構(gòu)復(fù)雜,結(jié)構(gòu)中形成二硫鍵時(shí)要求兩個(gè)半胱氨酸在空間距離上接近。 此外,多肽結(jié)構(gòu)中還原態(tài)的巰基化學(xué)性質(zhì)活潑,容易發(fā)生其他的副反應(yīng),而且肽鏈上其他側(cè)鏈 也可能會(huì)發(fā)生一系列修飾,因此,肽鏈進(jìn)行修飾所選取的氧化劑和氧化條件是反應(yīng)的關(guān)鍵因素, 反應(yīng)機(jī)理也比較復(fù)雜,既可能是自由基反應(yīng),也可能是離子反應(yīng)。
反應(yīng)條件有多種選擇,比如空氣氧化,DMSO氧化等溫和的氧化過程,也可以采用H2O2,I2, 汞鹽等激烈的反應(yīng)條件。
空氣氧化法: 空氣氧化法形成二硫鍵是多肽合成中最經(jīng)典的方法,通常是將巰基處于還原態(tài)的多肽溶于水中,在近中性或弱堿性條件下(PH值6.5-10),反應(yīng)24小時(shí)以上。為了降低分子之間二硫鍵形成的可能,該方法通常需要在低濃度條件下進(jìn)行。
碘氧化法:將多肽溶于25%的甲醇水溶液或30%的醋酸水溶液中,逐滴滴加10-15mol/L的碘進(jìn)行氧化,反應(yīng)15-40min。當(dāng)肽鏈中含有對碘比較敏感的Tyr、Trp、Met和His的殘基時(shí),氧化條件要控制的更精確,氧化完后,立即加入維生素C或硫代硫酸鈉除去過量的碘。 當(dāng)序列中有兩對或多對二硫鍵需要成環(huán)時(shí),通常有兩種情況:
自然隨機(jī)成環(huán): 序列中的Cys之間隨機(jī)成環(huán),與一對二硫鍵成環(huán)條件相似;
定點(diǎn)成環(huán): 定點(diǎn)成環(huán)即序列中的Cys按照設(shè)計(jì)要求形成二硫鍵,反應(yīng)過程相對復(fù)雜。在 固相合成多肽之前,需要提前設(shè)計(jì)幾對二硫鍵形成的順序和方法路線,選擇不同的側(cè)鏈 巰基保護(hù)基,利用其性質(zhì)差異,分步氧化形成兩對或多對二硫鍵。 通常采用的巰基保護(hù) 基有trt, Acm, Mmt, tBu, Bzl, Mob, Tmob等多種基團(tuán)。
DOI | 名稱 | |
---|---|---|
10.1042/cs0830627 | Colonic absorption of human calcitonin in man | 下載 |
10.1074/jbc.M305719200 | Importance of the amino terminus in secretin family G protein-coupled receptors. Intrinsic photoaffinity labeling establishes initial docking constraints for the calcitonin receptor | 下載 |
10.1373/clinchem.2008.123018 | Method for the selective measurement of amino-terminal variants of procalcitonin | 下載 |
10.1089/adt.2010.0361 | Development of a cyclic adenosine monophosphate assay for Gi-coupled G protein-coupled receptors by utilizing the endogenous calcitonin activity in Chinese hamster ovary cells | 下載 |
10.1021/ac4015116 | High-sensitivity LC-MS/MS quantification of peptides and proteins in complex biological samples: the impact of enzymatic digestion and internal standard selection on method performance | 下載 |
10.1371/journal.pone.0092042 | Prolonged calcitonin receptor signaling by salmon, but not human calcitonin, reveals ligand bias | 下載 |
10.1210/me.2008-0025 | Insights into the structural basis of endogenous agonist activation of family B G protein-coupled receptors | 下載 |
多肽H2N-Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-Tyr-Thr-Gln-Asp-Phe-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro-NH2的合成步驟:
1、合成MBHA樹脂:取若干克的MBHA樹脂(如初始取代度為0.5mmol/g)和1倍樹脂摩爾量的Fmoc-Linker-OH加入到反應(yīng)器中,加入DMF,攪拌使氨基酸完全溶解。再加入樹脂2倍量的DIEPA,攪拌混合均勻。再加入樹脂0.95倍量的HBTU,攪拌混合均勻。反應(yīng)3-4小時(shí)后,用DMF洗滌3次。用2倍樹脂體積的10%乙酸酐/DMF 進(jìn)行封端30分鐘。然后再用DMF洗滌3次,甲醇洗滌2次,DCM洗滌2次,再用甲醇洗滌2次。真空干燥12小時(shí)以上,得到干燥的樹脂{Fmoc-Linker-MHBA Resin},測定取代度。這里測得取代度為 0.3mmol/g。結(jié)構(gòu)如下圖:
2、脫Fmoc:取2.15g的上述樹脂,用DCM或DMF溶脹20分鐘。用DMF洗滌2遍。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-Linker-MBHA Resin 。(此步驟脫除Fmoc基團(tuán),茚三酮檢測為藍(lán)色,Pip為哌啶)。結(jié)構(gòu)圖如下:
3、縮合:取1.93mmol Fmoc-Pro-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3.87mmol DIPEA,1.84mmol HBTU。反應(yīng)30分鐘后,取小樣洗滌,茚三酮檢測為無色。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-Pro-Linker-MBHA 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-Pro-Linker-MBHA Resin
Fmoc-Ala-Pro-Linker-MBHA Resin
H2N-Ala-Pro-Linker-MBHA Resin
Fmoc-Gly-Ala-Pro-Linker-MBHA Resin
H2N-Gly-Ala-Pro-Linker-MBHA Resin
Fmoc-Val-Gly-Ala-Pro-Linker-MBHA Resin
H2N-Val-Gly-Ala-Pro-Linker-MBHA Resin
Fmoc-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
H2N-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
Fmoc-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
H2N-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
Fmoc-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
H2N-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
Fmoc-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
H2N-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
Fmoc-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
H2N-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
Fmoc-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
H2N-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
Fmoc-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
H2N-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
Fmoc-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
H2N-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
Fmoc-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
H2N-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
Fmoc-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
H2N-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
Fmoc-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
H2N-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
Fmoc-Asn(Trt)-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
H2N-Asn(Trt)-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
Fmoc-Phe-Asn(Trt)-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
H2N-Phe-Asn(Trt)-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
Fmoc-Asp(OtBu)-Phe-Asn(Trt)-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
H2N-Asp(OtBu)-Phe-Asn(Trt)-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
Fmoc-Gln(Trt)-Asp(OtBu)-Phe-Asn(Trt)-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
H2N-Gln(Trt)-Asp(OtBu)-Phe-Asn(Trt)-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
Fmoc-Thr(tBu)-Gln(Trt)-Asp(OtBu)-Phe-Asn(Trt)-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
H2N-Thr(tBu)-Gln(Trt)-Asp(OtBu)-Phe-Asn(Trt)-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
Fmoc-Tyr(tBu)-Thr(tBu)-Gln(Trt)-Asp(OtBu)-Phe-Asn(Trt)-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
H2N-Tyr(tBu)-Thr(tBu)-Gln(Trt)-Asp(OtBu)-Phe-Asn(Trt)-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
Fmoc-Thr(tBu)-Tyr(tBu)-Thr(tBu)-Gln(Trt)-Asp(OtBu)-Phe-Asn(Trt)-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
H2N-Thr(tBu)-Tyr(tBu)-Thr(tBu)-Gln(Trt)-Asp(OtBu)-Phe-Asn(Trt)-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
Fmoc-Gly-Thr(tBu)-Tyr(tBu)-Thr(tBu)-Gln(Trt)-Asp(OtBu)-Phe-Asn(Trt)-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
H2N-Gly-Thr(tBu)-Tyr(tBu)-Thr(tBu)-Gln(Trt)-Asp(OtBu)-Phe-Asn(Trt)-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
Fmoc-Leu-Gly-Thr(tBu)-Tyr(tBu)-Thr(tBu)-Gln(Trt)-Asp(OtBu)-Phe-Asn(Trt)-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
H2N-Leu-Gly-Thr(tBu)-Tyr(tBu)-Thr(tBu)-Gln(Trt)-Asp(OtBu)-Phe-Asn(Trt)-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
Fmoc-Met-Leu-Gly-Thr(tBu)-Tyr(tBu)-Thr(tBu)-Gln(Trt)-Asp(OtBu)-Phe-Asn(Trt)-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
H2N-Met-Leu-Gly-Thr(tBu)-Tyr(tBu)-Thr(tBu)-Gln(Trt)-Asp(OtBu)-Phe-Asn(Trt)-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
Fmoc-Cys(Trt)-Met-Leu-Gly-Thr(tBu)-Tyr(tBu)-Thr(tBu)-Gln(Trt)-Asp(OtBu)-Phe-Asn(Trt)-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
H2N-Cys(Trt)-Met-Leu-Gly-Thr(tBu)-Tyr(tBu)-Thr(tBu)-Gln(Trt)-Asp(OtBu)-Phe-Asn(Trt)-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
Fmoc-Thr(tBu)-Cys(Trt)-Met-Leu-Gly-Thr(tBu)-Tyr(tBu)-Thr(tBu)-Gln(Trt)-Asp(OtBu)-Phe-Asn(Trt)-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
H2N-Thr(tBu)-Cys(Trt)-Met-Leu-Gly-Thr(tBu)-Tyr(tBu)-Thr(tBu)-Gln(Trt)-Asp(OtBu)-Phe-Asn(Trt)-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
Fmoc-Ser(tBu)-Thr(tBu)-Cys(Trt)-Met-Leu-Gly-Thr(tBu)-Tyr(tBu)-Thr(tBu)-Gln(Trt)-Asp(OtBu)-Phe-Asn(Trt)-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
H2N-Ser(tBu)-Thr(tBu)-Cys(Trt)-Met-Leu-Gly-Thr(tBu)-Tyr(tBu)-Thr(tBu)-Gln(Trt)-Asp(OtBu)-Phe-Asn(Trt)-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
Fmoc-Leu-Ser(tBu)-Thr(tBu)-Cys(Trt)-Met-Leu-Gly-Thr(tBu)-Tyr(tBu)-Thr(tBu)-Gln(Trt)-Asp(OtBu)-Phe-Asn(Trt)-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
H2N-Leu-Ser(tBu)-Thr(tBu)-Cys(Trt)-Met-Leu-Gly-Thr(tBu)-Tyr(tBu)-Thr(tBu)-Gln(Trt)-Asp(OtBu)-Phe-Asn(Trt)-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
Fmoc-Asn(Trt)-Leu-Ser(tBu)-Thr(tBu)-Cys(Trt)-Met-Leu-Gly-Thr(tBu)-Tyr(tBu)-Thr(tBu)-Gln(Trt)-Asp(OtBu)-Phe-Asn(Trt)-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
H2N-Asn(Trt)-Leu-Ser(tBu)-Thr(tBu)-Cys(Trt)-Met-Leu-Gly-Thr(tBu)-Tyr(tBu)-Thr(tBu)-Gln(Trt)-Asp(OtBu)-Phe-Asn(Trt)-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
Fmoc-Gly-Asn(Trt)-Leu-Ser(tBu)-Thr(tBu)-Cys(Trt)-Met-Leu-Gly-Thr(tBu)-Tyr(tBu)-Thr(tBu)-Gln(Trt)-Asp(OtBu)-Phe-Asn(Trt)-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
H2N-Gly-Asn(Trt)-Leu-Ser(tBu)-Thr(tBu)-Cys(Trt)-Met-Leu-Gly-Thr(tBu)-Tyr(tBu)-Thr(tBu)-Gln(Trt)-Asp(OtBu)-Phe-Asn(Trt)-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA Resin
Fmoc-Cys(Trt)-Gly-Asn(Trt)-Leu-Ser(tBu)-Thr(tBu)-Cys(Trt)-Met-Leu-Gly-Thr(tBu)-Tyr(tBu)-Thr(tBu)-Gln(Trt)-Asp(OtBu)-Phe-Asn(Trt)-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA 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-Cys(Trt)-Gly-Asn(Trt)-Leu-Ser(tBu)-Thr(tBu)-Cys(Trt)-Met-Leu-Gly-Thr(tBu)-Tyr(tBu)-Thr(tBu)-Gln(Trt)-Asp(OtBu)-Phe-Asn(Trt)-Lys(Boc)-Phe-His(Trt)-Thr(tBu)-Phe-Pro-Gln(Trt)-Thr(tBu)-Ala-Ile-Gly-Val-Gly-Ala-Pro-Linker-MBHA 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-Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-Tyr-Thr-Gln-Asp-Phe-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro-NH2。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)主營定制多肽合成業(yè)務(wù),提供各類長肽,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、一對或者多對二硫鍵環(huán)),生物素標(biāo)記肽,PEG修飾肽,甲基化修飾肽等。
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