专肽生物合成用于蛋白质-蛋白质相互作用研究的生物素化肽。尽管生物素可以在 N 端或 C 端引入（通过赖氨酸残基），但我们建议使用 N 端修饰，因为它成本低、成功率高、周转时间短且易于操作。因为多肽合成是从 C 端到 N 端合成的，因此，N 端修饰是 SPPS步骤的最后一步，不需要额外的特定缩合步骤。相比之下，C 端修饰需要额外的步骤，并且通常更复杂。当然，原则上生物素可以定位在任何地方。
生物素可以通过多种不同的接头或间隔物与肽分离。尽管如此，还是建议包含一个灵活的间隔物，例如 Ahx（一个 6 碳接头），以使生物素标签更加稳定或灵活。
专肽生物在 N 端或 C 端提供生物素化：生物素-N 端、赖氨酸-生物素-肽中间和赖氨酸-生物素-C 端。
专肽生物还可以使用 Ahx 接头或长碳 (LC) 接头提供生物素化：生物素-Ahx-N 末端、Lys-Ahx-生物素-肽中间、Lys-Ahx-生物素-C-末端。
（生物素结构）
示例：
GRGDS在N端和C端标记生物素的结构展示。
1、GRGDS在N端标记生物素，不增加Ahx 接头
2、GRGDS在N端标记生物素，增加一个Ahx 接头
3、GRGDS在C端标记生物素，不增加Ahx 接头
4、GRGDS在C端标记生物素，增加一个Ahx 接头。
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的合成步骤：
1、合成CTC树脂：称取2.43g CTC Resin（如初始取代度约为1.17mmol/g）和3.41mmol Fmoc-Ser(tBu)-OH于反应器中，加入适量DCM溶解氨基酸（需要注意，此时CTC树脂体积会增大好几倍，避免DCM溶液过少），再加入8.53mmol DIPEA（Mw：129.1,d：0.740g/ml），反应2-3小时后，可不抽滤溶液，直接加入1ml的HPLC级甲醇，封端半小时。依次用DMF洗涤2次，甲醇洗涤1次，DCM洗涤一次，甲醇洗涤一次，DCM洗涤一次，DMF洗涤2次（这里使用甲醇和DCM交替洗涤，是为了更好地去除其他溶质，有利于后续反应）。得到 Fmoc-Ser(tBu)-CTC Resin。结构图如下：
2、脱Fmoc：加3倍树脂体积的20%Pip/DMF溶液，鼓氮气30分钟，然后2倍树脂体积的DMF 洗涤5次。得到 H2N-Ser(tBu)-CTC Resin 。（此步骤脱除Fmoc基团，茚三酮检测为蓝色，Pip为哌啶）。结构图如下：
3、缩合：取8.53mmol Fmoc-Glu(OtBu)-OH 氨基酸，加入到上述树脂里，加适当DMF溶解氨基酸，再依次加入17.06mmol DIPEA，8.1mmol HBTU。反应30分钟后，取小样洗涤，茚三酮检测为无色。用2倍树脂体积的DMF 洗涤3次树脂。（洗涤树脂，去掉残留溶剂，为下一步反应做准备）。得到Fmoc-Glu(OtBu)-Ser(tBu)-CTC Resin。氨基酸：DIPEA：HBTU：树脂=3：6：2.85：1（摩尔比）。结构图如下：
4、依次循环步骤二、步骤三，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
以上中间结构，均可在专肽生物多肽计算器-多肽结构计算器中，一键画出。
最后再经过步骤二得到 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，结构如下：
5、生物素反应连接：在上述树脂中，加入适当DMF后，再加入8.53mmol 生物素到树脂中，再加入17.06mmol DIPEA、8.1mmol HBTU，鼓氮气反应30分钟。用2倍树脂体积的DMF 洗涤3次树脂（洗涤树脂，去掉残留溶剂，为下一步反应做准备）。 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。 结构如下：
6、切割：6倍树脂体积的切割液（或每1g树脂加8ml左右的切割液），摇床摇晃 2小时，过滤掉树脂，用冰无水乙醚沉淀滤液，并用冰无水乙醚洗涤沉淀物3次，最后将沉淀物放真空干燥釜中，常温干燥24小试，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。结构图见产品结构图。
切割液选择：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%
（前两种适合没有容易氧化的氨基酸，例如Trp、Cys、Met。第三种适合几乎所有的序列。）
6、纯化冻干：使用液相色谱纯化，收集目标峰液体，进行冻干，获得蓬松的粉末状固体多肽。不过这时要取小样复测下纯度 是否目标纯度。
7、最后总结：
杭州专肽生物技术有限公司（ALLPEPTIDE https://www.allpeptide.com）主营定制多肽合成业务，提供各类长肽，短肽，环肽，提供各类修饰肽，如：荧光标记修饰（CY3、CY5、CY5.5、CY7、FAM、FITC、Rhodamine B、TAMRA等），功能基团修饰肽（叠氮、炔基、DBCO、DOTA、NOTA等），同位素标记肽（N15、C13），订书肽（Stapled Peptide）,脂肪酸修饰肽（Pal、Myr、Ste），磷酸化修饰肽（P-Ser、P-Thr、P-Tyr），环肽（酰胺键环肽、一对或者多对二硫键环），生物素标记肽，PEG修饰肽，甲基化修饰肽等。
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