多肽H2N-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2的合成步骤：
1、合成MBHA树脂：取若干克的MBHA树脂（如初始取代度为0.5mmol/g）和1倍树脂摩尔量的Fmoc-Linker-OH加入到反应器中，加入DMF，搅拌使氨基酸完全溶解。再加入树脂2倍量的DIEPA，搅拌混合均匀。再加入树脂0.95倍量的HBTU，搅拌混合均匀。反应3-4小时后，用DMF洗涤3次。用2倍树脂体积的10%乙酸酐/DMF 进行封端30分钟。然后再用DMF洗涤3次，甲醇洗涤2次，DCM洗涤2次，再用甲醇洗涤2次。真空干燥12小时以上，得到干燥的树脂{Fmoc-Linker-MHBA Resin}，测定取代度。这里测得取代度为 0.3mmol/g。结构如下图：
2、脱Fmoc：取2.46g的上述树脂，用DCM或DMF溶胀20分钟。用DMF洗涤2遍。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-Linker-MBHA 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2.21mmol Fmoc-Asn(Trt)-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4.43mmol DIPEA，2.1mmol 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-Asn(Trt)-Linker-MBHA 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-Asn(Trt)-Linker-MBHA Resin
Fmoc-Leu-Asn(Trt)-Linker-MBHA Resin
H2N-Leu-Asn(Trt)-Linker-MBHA Resin
Fmoc-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
H2N-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
Fmoc-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
H2N-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
Fmoc-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
H2N-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
Fmoc-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
H2N-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
Fmoc-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
H2N-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
Fmoc-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
H2N-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
Fmoc-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
H2N-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
Fmoc-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
H2N-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
Fmoc-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
H2N-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
Fmoc-Met-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
H2N-Met-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
Fmoc-Gln(Trt)-Met-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
H2N-Gln(Trt)-Met-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
Fmoc-Lys(Boc)-Gln(Trt)-Met-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
H2N-Lys(Boc)-Gln(Trt)-Met-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
Fmoc-Arg(Pbf)-Lys(Boc)-Gln(Trt)-Met-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
H2N-Arg(Pbf)-Lys(Boc)-Gln(Trt)-Met-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
Fmoc-Leu-Arg(Pbf)-Lys(Boc)-Gln(Trt)-Met-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
H2N-Leu-Arg(Pbf)-Lys(Boc)-Gln(Trt)-Met-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
Fmoc-Arg(Pbf)-Leu-Arg(Pbf)-Lys(Boc)-Gln(Trt)-Met-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
H2N-Arg(Pbf)-Leu-Arg(Pbf)-Lys(Boc)-Gln(Trt)-Met-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
Fmoc-Thr(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Lys(Boc)-Gln(Trt)-Met-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
H2N-Thr(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Lys(Boc)-Gln(Trt)-Met-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
Fmoc-Tyr(tBu)-Thr(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Lys(Boc)-Gln(Trt)-Met-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
H2N-Tyr(tBu)-Thr(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Lys(Boc)-Gln(Trt)-Met-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
Fmoc-Asn(Trt)-Tyr(tBu)-Thr(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Lys(Boc)-Gln(Trt)-Met-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
H2N-Asn(Trt)-Tyr(tBu)-Thr(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Lys(Boc)-Gln(Trt)-Met-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
Fmoc-Asp(OtBu)-Asn(Trt)-Tyr(tBu)-Thr(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Lys(Boc)-Gln(Trt)-Met-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
H2N-Asp(OtBu)-Asn(Trt)-Tyr(tBu)-Thr(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Lys(Boc)-Gln(Trt)-Met-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
Fmoc-Thr(tBu)-Asp(OtBu)-Asn(Trt)-Tyr(tBu)-Thr(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Lys(Boc)-Gln(Trt)-Met-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
H2N-Thr(tBu)-Asp(OtBu)-Asn(Trt)-Tyr(tBu)-Thr(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Lys(Boc)-Gln(Trt)-Met-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
Fmoc-Phe-Thr(tBu)-Asp(OtBu)-Asn(Trt)-Tyr(tBu)-Thr(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Lys(Boc)-Gln(Trt)-Met-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
H2N-Phe-Thr(tBu)-Asp(OtBu)-Asn(Trt)-Tyr(tBu)-Thr(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Lys(Boc)-Gln(Trt)-Met-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
Fmoc-Val-Phe-Thr(tBu)-Asp(OtBu)-Asn(Trt)-Tyr(tBu)-Thr(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Lys(Boc)-Gln(Trt)-Met-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
H2N-Val-Phe-Thr(tBu)-Asp(OtBu)-Asn(Trt)-Tyr(tBu)-Thr(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Lys(Boc)-Gln(Trt)-Met-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
Fmoc-Ala-Val-Phe-Thr(tBu)-Asp(OtBu)-Asn(Trt)-Tyr(tBu)-Thr(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Lys(Boc)-Gln(Trt)-Met-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
H2N-Ala-Val-Phe-Thr(tBu)-Asp(OtBu)-Asn(Trt)-Tyr(tBu)-Thr(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Lys(Boc)-Gln(Trt)-Met-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA Resin
Fmoc-Asp(OtBu)-Ala-Val-Phe-Thr(tBu)-Asp(OtBu)-Asn(Trt)-Tyr(tBu)-Thr(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Lys(Boc)-Gln(Trt)-Met-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA 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-Asp(OtBu)-Ala-Val-Phe-Thr(tBu)-Asp(OtBu)-Asn(Trt)-Tyr(tBu)-Thr(tBu)-Arg(Pbf)-Leu-Arg(Pbf)-Lys(Boc)-Gln(Trt)-Met-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-Ile-Leu-Asn(Trt)-Linker-MBHA 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-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2。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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