This potent CRF antagonist is used as a reference substance in CRF studies.

這種強效的CRF拮抗劑被用作CRF研究的參比物質。

很多蛋白在細胞中非常容易被降解，或被標記，進而被選擇性地破壞。但含有部分D型氨基酸的多肽則顯示了很強的抵抗蛋白酶降解能力。

多肽H2N-DPhe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Nle-Glu-Ile-Ile-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。結構如下圖：

2、脫Fmoc：取1.2g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）。結構圖如下：

3、縮合：取1.08mmol Fmoc-Ile-OH 氨基酸，加入到上述樹脂里，加適當DMF溶解氨基酸，再依次加入2.16mmol DIPEA，1.03mmol HBTU。反應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，為下一步反應做準備）。得到Fmoc-Ile-Linker-MBHA Resin。氨基酸：DIPEA：HBTU：樹脂=3：6：2.85：1（摩爾比）。結構圖如下：

4、依次循環(huán)步驟二、步驟三，依次得到

H2N-Ile-Linker-MBHA Resin

Fmoc-Ile-Ile-Linker-MBHA Resin

H2N-Ile-Ile-Linker-MBHA Resin

Fmoc-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Nle-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Nle-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Glu(OtBu)-Nle-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Glu(OtBu)-Nle-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Leu-Glu(OtBu)-Nle-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Leu-Glu(OtBu)-Nle-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Val-Leu-Glu(OtBu)-Nle-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Val-Leu-Glu(OtBu)-Nle-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Glu(OtBu)-Val-Leu-Glu(OtBu)-Nle-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Glu(OtBu)-Val-Leu-Glu(OtBu)-Nle-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Nle-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Nle-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Nle-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Nle-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Nle-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Nle-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-His(Trt)-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Nle-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-His(Trt)-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Nle-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-DPhe-His(Trt)-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Nle-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

以上中間結構，均可在專肽生物多肽計算器-多肽結構計算器中，一鍵畫出。

最后再經過步驟二得到 H2N-DPhe-His(Trt)-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Nle-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin，結構如下：

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-DPhe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Nle-Glu-Ile-Ile-NH2。結構圖見產品結構圖。

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測下純度 是否目標純度。

7、最后總結：

杭州專肽生物技術有限公司（ALLPEPTIDE http://tsjxdd.com）主營定制多肽合成業(yè)務，提供各類長肽，短肽，環(huán)肽，提供各類修飾肽，如：熒光標記修飾（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)），生物素標記肽，PEG修飾肽，甲基化修飾肽等。

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