Bombinin-Like Peptide (BLP-1) 是來自鈴蟾屬的抗微生物 (antimicrobial) 肽。
Bombinin-Like Peptide (BLP-1) is an antimicrobial peptide from Bombina species.

Gibson BW, et al. Bombinin-like peptides with antimicrobial activity from skin secretions of the Asian toad, Bombina orientalis. J Biol Chem. 1991 Dec 5;266(34):23103-11. : https://www.ncbi.nlm.nih.gov/pubmed/1744108

多肽H2N-Gly-Ile-Gly-Ala-Ser-Ile-Leu-Ser-Ala-Gly-Lys-Ser-Ala-Leu-Lys-Gly-Leu-Ala-Lys-Gly-Leu-Ala-Glu-His-Phe-Ala-Asn-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小時后，用DMF洗滌3次。用2倍樹脂體積的10%乙酸酐/DMF 進行封端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2.74g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，鼓氮氣30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，茚三酮檢測為藍色，Pip為哌啶）。結(jié)構(gòu)圖如下：

3、縮合：取2.47mmol Fmoc-Asn(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4.93mmol DIPEA，2.34mmol 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-Asn(Trt)-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-Asn(Trt)-Linker-MBHA Resin

Fmoc-Ala-Asn(Trt)-Linker-MBHA Resin

H2N-Ala-Asn(Trt)-Linker-MBHA Resin

Fmoc-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

H2N-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

Fmoc-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

H2N-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

Fmoc-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

H2N-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

Fmoc-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

H2N-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

Fmoc-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

H2N-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

Fmoc-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

H2N-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

Fmoc-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

H2N-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

Fmoc-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

H2N-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

Fmoc-Leu-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

H2N-Leu-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

Fmoc-Gly-Leu-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

H2N-Gly-Leu-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

Fmoc-Lys(Boc)-Gly-Leu-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

H2N-Lys(Boc)-Gly-Leu-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

Fmoc-Leu-Lys(Boc)-Gly-Leu-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

H2N-Leu-Lys(Boc)-Gly-Leu-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

Fmoc-Ala-Leu-Lys(Boc)-Gly-Leu-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

H2N-Ala-Leu-Lys(Boc)-Gly-Leu-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

Fmoc-Ser(tBu)-Ala-Leu-Lys(Boc)-Gly-Leu-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

H2N-Ser(tBu)-Ala-Leu-Lys(Boc)-Gly-Leu-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

Fmoc-Lys(Boc)-Ser(tBu)-Ala-Leu-Lys(Boc)-Gly-Leu-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

H2N-Lys(Boc)-Ser(tBu)-Ala-Leu-Lys(Boc)-Gly-Leu-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

Fmoc-Gly-Lys(Boc)-Ser(tBu)-Ala-Leu-Lys(Boc)-Gly-Leu-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

H2N-Gly-Lys(Boc)-Ser(tBu)-Ala-Leu-Lys(Boc)-Gly-Leu-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

Fmoc-Ala-Gly-Lys(Boc)-Ser(tBu)-Ala-Leu-Lys(Boc)-Gly-Leu-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

H2N-Ala-Gly-Lys(Boc)-Ser(tBu)-Ala-Leu-Lys(Boc)-Gly-Leu-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

Fmoc-Ser(tBu)-Ala-Gly-Lys(Boc)-Ser(tBu)-Ala-Leu-Lys(Boc)-Gly-Leu-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

H2N-Ser(tBu)-Ala-Gly-Lys(Boc)-Ser(tBu)-Ala-Leu-Lys(Boc)-Gly-Leu-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

Fmoc-Leu-Ser(tBu)-Ala-Gly-Lys(Boc)-Ser(tBu)-Ala-Leu-Lys(Boc)-Gly-Leu-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

H2N-Leu-Ser(tBu)-Ala-Gly-Lys(Boc)-Ser(tBu)-Ala-Leu-Lys(Boc)-Gly-Leu-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

Fmoc-Ile-Leu-Ser(tBu)-Ala-Gly-Lys(Boc)-Ser(tBu)-Ala-Leu-Lys(Boc)-Gly-Leu-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

H2N-Ile-Leu-Ser(tBu)-Ala-Gly-Lys(Boc)-Ser(tBu)-Ala-Leu-Lys(Boc)-Gly-Leu-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

Fmoc-Ser(tBu)-Ile-Leu-Ser(tBu)-Ala-Gly-Lys(Boc)-Ser(tBu)-Ala-Leu-Lys(Boc)-Gly-Leu-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

H2N-Ser(tBu)-Ile-Leu-Ser(tBu)-Ala-Gly-Lys(Boc)-Ser(tBu)-Ala-Leu-Lys(Boc)-Gly-Leu-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

Fmoc-Ala-Ser(tBu)-Ile-Leu-Ser(tBu)-Ala-Gly-Lys(Boc)-Ser(tBu)-Ala-Leu-Lys(Boc)-Gly-Leu-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

H2N-Ala-Ser(tBu)-Ile-Leu-Ser(tBu)-Ala-Gly-Lys(Boc)-Ser(tBu)-Ala-Leu-Lys(Boc)-Gly-Leu-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

Fmoc-Gly-Ala-Ser(tBu)-Ile-Leu-Ser(tBu)-Ala-Gly-Lys(Boc)-Ser(tBu)-Ala-Leu-Lys(Boc)-Gly-Leu-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

H2N-Gly-Ala-Ser(tBu)-Ile-Leu-Ser(tBu)-Ala-Gly-Lys(Boc)-Ser(tBu)-Ala-Leu-Lys(Boc)-Gly-Leu-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

Fmoc-Ile-Gly-Ala-Ser(tBu)-Ile-Leu-Ser(tBu)-Ala-Gly-Lys(Boc)-Ser(tBu)-Ala-Leu-Lys(Boc)-Gly-Leu-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

H2N-Ile-Gly-Ala-Ser(tBu)-Ile-Leu-Ser(tBu)-Ala-Gly-Lys(Boc)-Ser(tBu)-Ala-Leu-Lys(Boc)-Gly-Leu-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

Fmoc-Gly-Ile-Gly-Ala-Ser(tBu)-Ile-Leu-Ser(tBu)-Ala-Gly-Lys(Boc)-Ser(tBu)-Ala-Leu-Lys(Boc)-Gly-Leu-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-Linker-MBHA Resin

以上中間結(jié)構(gòu)，均可在專肽生物多肽計算器-多肽結(jié)構(gòu)計算器中，一鍵畫出。

最后再經(jīng)過步驟二得到 H2N-Gly-Ile-Gly-Ala-Ser(tBu)-Ile-Leu-Ser(tBu)-Ala-Gly-Lys(Boc)-Ser(tBu)-Ala-Leu-Lys(Boc)-Gly-Leu-Ala-Lys(Boc)-Gly-Leu-Ala-Glu(OtBu)-His(Trt)-Phe-Ala-Asn(Trt)-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，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-Gly-Ile-Gly-Ala-Ser-Ile-Leu-Ser-Ala-Gly-Lys-Ser-Ala-Leu-Lys-Gly-Leu-Ala-Lys-Gly-Leu-Ala-Glu-His-Phe-Ala-Asn-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，進行凍干，獲得蓬松的粉末狀固體多肽。不過這時要取小樣復(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://tsjxdd.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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