Secretin (28-54), human 是由 27 個(gè)氨基酸組成的多肽，作用于人分泌素受體 (secretin receptor)。
Secretin (28-54), human is a 27-amino acid residue C-terminally amidated peptide, which acts on human secretin receptors[1].

多肽H2N-His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu-Leu-Ser-Arg-Leu-Arg-Glu-Gly-Ala-Arg-Leu-Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-NH2的合成步驟：

1、合成MBHA樹(shù)脂：取若干克的MBHA樹(shù)脂（如初始取代度為0.5mmol/g）和1倍樹(shù)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(shù)脂2倍量的DIEPA，攪拌混合均勻。再加入樹(shù)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(shù)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(shù)脂{Fmoc-Linker-MHBA Resin}，測(cè)定取代度。這里測(cè)得取代度為 0.3mmol/g。結(jié)構(gòu)如下圖：

2、脫Fmoc：取2.93g的上述樹(shù)脂，用DCM或DMF溶脹20分鐘。用DMF洗滌2遍。加3倍樹(shù)脂體積的20%Pip/DMF溶液，鼓氮?dú)?0分鐘，然后2倍樹(shù)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(cè)為藍(lán)色，Pip為哌啶）。結(jié)構(gòu)圖如下：

3、縮合：取2.64mmol Fmoc-Val-OH 氨基酸，加入到上述樹(shù)脂里，加適當(dāng)DMF溶解氨基酸，再依次加入5.27mmol DIPEA，2.51mmol HBTU。反應(yīng)30分鐘后，取小樣洗滌，茚三酮檢測(cè)為無(wú)色。用2倍樹(shù)脂體積的DMF 洗滌3次樹(shù)脂。（洗滌樹(shù)脂，去掉殘留溶劑，為下一步反應(yīng)做準(zhǔn)備）。得到Fmoc-Val-Linker-MBHA Resin。氨基酸：DIPEA：HBTU：樹(shù)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-Val-Linker-MBHA Resin

Fmoc-Leu-Val-Linker-MBHA Resin

H2N-Leu-Val-Linker-MBHA Resin

Fmoc-Gly-Leu-Val-Linker-MBHA Resin

H2N-Gly-Leu-Val-Linker-MBHA Resin

Fmoc-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

H2N-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

Fmoc-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

H2N-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

Fmoc-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

H2N-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

Fmoc-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

H2N-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

Fmoc-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

H2N-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

Fmoc-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

H2N-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

Fmoc-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

H2N-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

Fmoc-Ala-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

H2N-Ala-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

Fmoc-Gly-Ala-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

H2N-Gly-Ala-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

Fmoc-Glu(OtBu)-Gly-Ala-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

H2N-Glu(OtBu)-Gly-Ala-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

Fmoc-Arg(Pbf)-Glu(OtBu)-Gly-Ala-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

H2N-Arg(Pbf)-Glu(OtBu)-Gly-Ala-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

Fmoc-Leu-Arg(Pbf)-Glu(OtBu)-Gly-Ala-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

H2N-Leu-Arg(Pbf)-Glu(OtBu)-Gly-Ala-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

Fmoc-Arg(Pbf)-Leu-Arg(Pbf)-Glu(OtBu)-Gly-Ala-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

H2N-Arg(Pbf)-Leu-Arg(Pbf)-Glu(OtBu)-Gly-Ala-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

Fmoc-Ser(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Glu(OtBu)-Gly-Ala-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

H2N-Ser(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Glu(OtBu)-Gly-Ala-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

Fmoc-Leu-Ser(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Glu(OtBu)-Gly-Ala-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

H2N-Leu-Ser(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Glu(OtBu)-Gly-Ala-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

Fmoc-Glu(OtBu)-Leu-Ser(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Glu(OtBu)-Gly-Ala-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

H2N-Glu(OtBu)-Leu-Ser(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Glu(OtBu)-Gly-Ala-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

Fmoc-Ser(tBu)-Glu(OtBu)-Leu-Ser(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Glu(OtBu)-Gly-Ala-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

H2N-Ser(tBu)-Glu(OtBu)-Leu-Ser(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Glu(OtBu)-Gly-Ala-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

Fmoc-Thr(tBu)-Ser(tBu)-Glu(OtBu)-Leu-Ser(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Glu(OtBu)-Gly-Ala-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

H2N-Thr(tBu)-Ser(tBu)-Glu(OtBu)-Leu-Ser(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Glu(OtBu)-Gly-Ala-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

Fmoc-Phe-Thr(tBu)-Ser(tBu)-Glu(OtBu)-Leu-Ser(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Glu(OtBu)-Gly-Ala-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

H2N-Phe-Thr(tBu)-Ser(tBu)-Glu(OtBu)-Leu-Ser(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Glu(OtBu)-Gly-Ala-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Glu(OtBu)-Leu-Ser(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Glu(OtBu)-Gly-Ala-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

H2N-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Glu(OtBu)-Leu-Ser(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Glu(OtBu)-Gly-Ala-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

Fmoc-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Glu(OtBu)-Leu-Ser(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Glu(OtBu)-Gly-Ala-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

H2N-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Glu(OtBu)-Leu-Ser(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Glu(OtBu)-Gly-Ala-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

Fmoc-Asp(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Glu(OtBu)-Leu-Ser(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Glu(OtBu)-Gly-Ala-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

H2N-Asp(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Glu(OtBu)-Leu-Ser(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Glu(OtBu)-Gly-Ala-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

Fmoc-Ser(tBu)-Asp(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Glu(OtBu)-Leu-Ser(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Glu(OtBu)-Gly-Ala-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

H2N-Ser(tBu)-Asp(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Glu(OtBu)-Leu-Ser(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Glu(OtBu)-Gly-Ala-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin

Fmoc-His(Trt)-Ser(tBu)-Asp(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Glu(OtBu)-Leu-Ser(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Glu(OtBu)-Gly-Ala-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-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(huà)出。

最后再經(jīng)過(guò)步驟二得到 H2N-His(Trt)-Ser(tBu)-Asp(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Glu(OtBu)-Leu-Ser(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Glu(OtBu)-Gly-Ala-Arg(Pbf)-Leu-Gln(Trt)-Arg(Pbf)-Leu-Leu-Gln(Trt)-Gly-Leu-Val-Linker-MBHA Resin，結(jié)構(gòu)如下：

5、切割：6倍樹(shù)脂體積的切割液（或每1g樹(shù)脂加8ml左右的切割液），搖床搖晃 2小時(shí)，過(guò)濾掉樹(shù)脂，用冰無(wú)水乙醚沉淀濾液，并用冰無(wú)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-His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu-Leu-Ser-Arg-Leu-Arg-Glu-Gly-Ala-Arg-Leu-Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-NH2。結(jié)構(gòu)圖見(jiàn)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(méi)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(guò)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://tsjxdd.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(shū)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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