多肽H2N-His-Ser-Gly-Ile-Asn-Ser-Ser-Asn-Ala-Glu-Val-Leu-Ala-Leu-Phe-Asn-Val-Thr-Glu-Met-Asp-Ala-Gly-Glu-Tyr-COOH的合成步骤：
1、合成CTC树脂：称取2.49g CTC Resin（如初始取代度约为0.68mmol/g）和2.03mmol Fmoc-Tyr(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5.08mmol 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-Tyr(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-Tyr(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5.08mmol 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10.16mmol DIPEA，4.83mmol 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)-Tyr(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)-Tyr(tBu)-CTC Resin
Fmoc-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
H2N-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
Fmoc-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
H2N-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
Fmoc-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
H2N-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
Fmoc-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
H2N-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
Fmoc-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
H2N-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
Fmoc-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
H2N-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
Fmoc-Val-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
H2N-Val-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
Fmoc-Asn(Trt)-Val-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
H2N-Asn(Trt)-Val-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
Fmoc-Phe-Asn(Trt)-Val-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
H2N-Phe-Asn(Trt)-Val-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
Fmoc-Leu-Phe-Asn(Trt)-Val-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
H2N-Leu-Phe-Asn(Trt)-Val-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
Fmoc-Ala-Leu-Phe-Asn(Trt)-Val-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
H2N-Ala-Leu-Phe-Asn(Trt)-Val-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
Fmoc-Leu-Ala-Leu-Phe-Asn(Trt)-Val-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
H2N-Leu-Ala-Leu-Phe-Asn(Trt)-Val-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
Fmoc-Val-Leu-Ala-Leu-Phe-Asn(Trt)-Val-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
H2N-Val-Leu-Ala-Leu-Phe-Asn(Trt)-Val-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
Fmoc-Glu(OtBu)-Val-Leu-Ala-Leu-Phe-Asn(Trt)-Val-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
H2N-Glu(OtBu)-Val-Leu-Ala-Leu-Phe-Asn(Trt)-Val-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
Fmoc-Ala-Glu(OtBu)-Val-Leu-Ala-Leu-Phe-Asn(Trt)-Val-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
H2N-Ala-Glu(OtBu)-Val-Leu-Ala-Leu-Phe-Asn(Trt)-Val-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
Fmoc-Asn(Trt)-Ala-Glu(OtBu)-Val-Leu-Ala-Leu-Phe-Asn(Trt)-Val-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
H2N-Asn(Trt)-Ala-Glu(OtBu)-Val-Leu-Ala-Leu-Phe-Asn(Trt)-Val-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
Fmoc-Ser(tBu)-Asn(Trt)-Ala-Glu(OtBu)-Val-Leu-Ala-Leu-Phe-Asn(Trt)-Val-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
H2N-Ser(tBu)-Asn(Trt)-Ala-Glu(OtBu)-Val-Leu-Ala-Leu-Phe-Asn(Trt)-Val-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
Fmoc-Ser(tBu)-Ser(tBu)-Asn(Trt)-Ala-Glu(OtBu)-Val-Leu-Ala-Leu-Phe-Asn(Trt)-Val-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
H2N-Ser(tBu)-Ser(tBu)-Asn(Trt)-Ala-Glu(OtBu)-Val-Leu-Ala-Leu-Phe-Asn(Trt)-Val-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
Fmoc-Asn(Trt)-Ser(tBu)-Ser(tBu)-Asn(Trt)-Ala-Glu(OtBu)-Val-Leu-Ala-Leu-Phe-Asn(Trt)-Val-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
H2N-Asn(Trt)-Ser(tBu)-Ser(tBu)-Asn(Trt)-Ala-Glu(OtBu)-Val-Leu-Ala-Leu-Phe-Asn(Trt)-Val-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
Fmoc-Ile-Asn(Trt)-Ser(tBu)-Ser(tBu)-Asn(Trt)-Ala-Glu(OtBu)-Val-Leu-Ala-Leu-Phe-Asn(Trt)-Val-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
H2N-Ile-Asn(Trt)-Ser(tBu)-Ser(tBu)-Asn(Trt)-Ala-Glu(OtBu)-Val-Leu-Ala-Leu-Phe-Asn(Trt)-Val-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
Fmoc-Gly-Ile-Asn(Trt)-Ser(tBu)-Ser(tBu)-Asn(Trt)-Ala-Glu(OtBu)-Val-Leu-Ala-Leu-Phe-Asn(Trt)-Val-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
H2N-Gly-Ile-Asn(Trt)-Ser(tBu)-Ser(tBu)-Asn(Trt)-Ala-Glu(OtBu)-Val-Leu-Ala-Leu-Phe-Asn(Trt)-Val-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
Fmoc-Ser(tBu)-Gly-Ile-Asn(Trt)-Ser(tBu)-Ser(tBu)-Asn(Trt)-Ala-Glu(OtBu)-Val-Leu-Ala-Leu-Phe-Asn(Trt)-Val-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
H2N-Ser(tBu)-Gly-Ile-Asn(Trt)-Ser(tBu)-Ser(tBu)-Asn(Trt)-Ala-Glu(OtBu)-Val-Leu-Ala-Leu-Phe-Asn(Trt)-Val-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin
Fmoc-His(Trt)-Ser(tBu)-Gly-Ile-Asn(Trt)-Ser(tBu)-Ser(tBu)-Asn(Trt)-Ala-Glu(OtBu)-Val-Leu-Ala-Leu-Phe-Asn(Trt)-Val-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(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-His(Trt)-Ser(tBu)-Gly-Ile-Asn(Trt)-Ser(tBu)-Ser(tBu)-Asn(Trt)-Ala-Glu(OtBu)-Val-Leu-Ala-Leu-Phe-Asn(Trt)-Val-Thr(tBu)-Glu(OtBu)-Met-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-CTC Resin，结构如下：
5、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H2N-His-Ser-Gly-Ile-Asn-Ser-Ser-Asn-Ala-Glu-Val-Leu-Ala-Leu-Phe-Asn-Val-Thr-Glu-Met-Asp-Ala-Gly-Glu-Tyr-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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