專肽生物_定制多肽合成服務(wù)_多肽合成定制服務(wù)_多肽合成廠家_多肽合成公司

專肽生物合成用于蛋白質(zhì)-蛋白質(zhì)相互作用研究的生物素化肽。盡管生物素可以在 N 端或 C 端引入（通過賴氨酸殘基），但我們建議使用 N 端修飾，因為它成本低、成功率高、周轉(zhuǎn)時間短且易于操作。因為多肽合成是從 C 端到 N 端合成的，因此，N 端修飾是 SPPS步驟的最后一步，不需要額外的特定縮合步驟。相比之下，C 端修飾需要額外的步驟，并且通常更復(fù)雜。當然，原則上生物素可以定位在任何地方。

生物素可以通過多種不同的接頭或間隔物與肽分離。盡管如此，還是建議包含一個靈活的間隔物，例如 Ahx（一個 6 碳接頭），以使生物素標簽更加穩(wěn)定或靈活。

專肽生物在 N 端或 C 端提供生物素化：生物素-N 端、賴氨酸-生物素-肽中間和賴氨酸-生物素-C 端。
專肽生物還可以使用 Ahx 接頭或長碳 (LC) 接頭提供生物素化：生物素-Ahx-N 末端、Lys-Ahx-生物素-肽中間、Lys-Ahx-生物素-C-末端。

（生物素結(jié)構(gòu)）

示例：
GRGDS在N端和C端標記生物素的結(jié)構(gòu)展示。

1、GRGDS在N端標記生物素，不增加Ahx 接頭

2、GRGDS在N端標記生物素，增加一個Ahx 接頭

3、GRGDS在C端標記生物素，不增加Ahx 接頭

4、GRGDS在C端標記生物素，增加一個Ahx 接頭。

多肽Biotin-Leu-Cys-Leu-Leu-Gly-Asp-Phe-Phe-Arg-Lys-Ser-Lys-Glu-Lys-Ile-Gly-Lys-Glu-Phe-Lys-Arg-Ile-Val-Gln-Arg-Ile-Lys-Asp-Phe-Leu-Arg-Asn-Leu-Val-Pro-Arg-Thr-Glu-Ser-COOH的合成步驟：

1、合成CTC樹脂：稱取2.43g CTC Resin（如初始取代度約為1.17mmol/g）和3.41mmol Fmoc-Ser(tBu)-OH于反應(yīng)器中，加入適量DCM溶解氨基酸（需要注意，此時CTC樹脂體積會增大好幾倍，避免DCM溶液過少），再加入8.53mmol DIPEA（Mw：129.1,d：0.740g/ml），反應(yīng)2-3小時后，可不抽濾溶液，直接加入1ml的HPLC級甲醇，封端半小時。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-Ser(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，鼓氮氣30分鐘，然后2倍樹脂體積的DMF 洗滌5次。得到 H2N-Ser(tBu)-CTC 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8.53mmol Fmoc-Glu(OtBu)-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17.06mmol DIPEA，8.1mmol 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)做準備）。得到Fmoc-Glu(OtBu)-Ser(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-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Gly-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Gly-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Ile-Gly-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Ile-Gly-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Lys(Boc)-Ile-Gly-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Lys(Boc)-Ile-Gly-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Glu(OtBu)-Lys(Boc)-Ile-Gly-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Glu(OtBu)-Lys(Boc)-Ile-Gly-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Ile-Gly-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Ile-Gly-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Ile-Gly-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Ile-Gly-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Lys(Boc)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Ile-Gly-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Lys(Boc)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Ile-Gly-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Arg(Pbf)-Lys(Boc)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Ile-Gly-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Arg(Pbf)-Lys(Boc)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Ile-Gly-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Phe-Arg(Pbf)-Lys(Boc)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Ile-Gly-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Phe-Arg(Pbf)-Lys(Boc)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Ile-Gly-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Phe-Phe-Arg(Pbf)-Lys(Boc)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Ile-Gly-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Phe-Phe-Arg(Pbf)-Lys(Boc)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Ile-Gly-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Asp(OtBu)-Phe-Phe-Arg(Pbf)-Lys(Boc)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Ile-Gly-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Asp(OtBu)-Phe-Phe-Arg(Pbf)-Lys(Boc)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Ile-Gly-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Gly-Asp(OtBu)-Phe-Phe-Arg(Pbf)-Lys(Boc)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Ile-Gly-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Gly-Asp(OtBu)-Phe-Phe-Arg(Pbf)-Lys(Boc)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Ile-Gly-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Leu-Gly-Asp(OtBu)-Phe-Phe-Arg(Pbf)-Lys(Boc)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Ile-Gly-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Leu-Gly-Asp(OtBu)-Phe-Phe-Arg(Pbf)-Lys(Boc)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Ile-Gly-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Leu-Leu-Gly-Asp(OtBu)-Phe-Phe-Arg(Pbf)-Lys(Boc)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Ile-Gly-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Leu-Leu-Gly-Asp(OtBu)-Phe-Phe-Arg(Pbf)-Lys(Boc)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Ile-Gly-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Cys(Trt)-Leu-Leu-Gly-Asp(OtBu)-Phe-Phe-Arg(Pbf)-Lys(Boc)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Ile-Gly-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

H2N-Cys(Trt)-Leu-Leu-Gly-Asp(OtBu)-Phe-Phe-Arg(Pbf)-Lys(Boc)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Ile-Gly-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin

Fmoc-Leu-Cys(Trt)-Leu-Leu-Gly-Asp(OtBu)-Phe-Phe-Arg(Pbf)-Lys(Boc)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Ile-Gly-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC 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-Leu-Cys(Trt)-Leu-Leu-Gly-Asp(OtBu)-Phe-Phe-Arg(Pbf)-Lys(Boc)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Ile-Gly-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTC Resin，結(jié)構(gòu)如下：

5、生物素反應(yīng)連接：在上述樹脂中，加入適當DMF后，再加入8.53mmol 生物素到樹脂中，再加入17.06mmol DIPEA、8.1mmol HBTU，鼓氮氣反應(yīng)30分鐘。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)做準備）。 得到Biotin-Leu-Cys(Trt)-Leu-Leu-Gly-Asp(OtBu)-Phe-Phe-Arg(Pbf)-Lys(Boc)-Ser(tBu)-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Ile-Gly-Lys(Boc)-Glu(OtBu)-Phe-Lys(Boc)-Arg(Pbf)-Ile-Val-Gln(Trt)-Arg(Pbf)-Ile-Lys(Boc)-Asp(OtBu)-Phe-Leu-Arg(Pbf)-Asn(Trt)-Leu-Val-Pro-Arg(Pbf)-Thr(tBu)-Glu(OtBu)-Ser(tBu)-CTCResin。 結(jié)構(gòu)如下：

6、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Biotin-Leu-Cys-Leu-Leu-Gly-Asp-Phe-Phe-Arg-Lys-Ser-Lys-Glu-Lys-Ile-Gly-Lys-Glu-Phe-Lys-Arg-Ile-Val-Gln-Arg-Ile-Lys-Asp-Phe-Leu-Arg-Asn-Leu-Val-Pro-Arg-Thr-Glu-Ser-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 是否目標純度。

7、最后總結(jié)：

杭州專肽生物技術(shù)有限公司（ALLPEPTIDE https://www.allpeptide.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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