400-998-5282
专注多肽 服务科研
400-998-5282
专注多肽 服务科研
作为 CCK 的拮抗剂，PP 抑制胰腺的沉淀。PP 还作用于胃肠蠕动、食物摄入和新陈代谢。
编号：158392
CAS号：1816258-36-2
单字母：Biotinyl-APLEPVYPGDNATPEQMAQYAADLRRYINMLTRPRY-CONH2
As antagonist of CCK, PP inhibits pancreatic sedretion. PP also acts on the gastrointestinal motility, food intake, and metabolism.
胰腺多肽(Pancreatic\Pancreastatin)定义
胰多肽（PP）是由十二指肠胰腺内分泌细胞分泌的一种激素。
Pancreatic polypeptide (PP) is a hormone produced by endocrine cells of the duodenal pancreas.
胰抑素（PST）是一种对分泌具有普遍抑制作用的调节肽，来源于嗜铬粒蛋白A，这是一种广泛存在于神经内分泌系统中的糖蛋白。嗜铬粒蛋白A（CgA）也被称为分泌蛋白I，是一种由许多神经内分泌细胞和神经元表达的酸性蛋白。
Pancreastatin (PST) is a regulatory peptide with a general inhibitory effect on secretion, is derived from chromogranin A, a glycoprotein present throughout the neuroendocrine system. Chromogranin A (CgA) is also referred to as secretory protein I, is an acidic protein expressed by many neuroendocrine cells and neurons.
胰腺多肽(Pancreatic\Pancreastatin)的结构特征
PP含有36个氨基酸，分子量为4.2 kDa，等电点为pH 6至7。肽1的氨基酸序列为Gly-Pro-Ser-Gln-Pro-Thr-Tyr-Pro-Gly-Asp-Asp-Ala-Pro-Val-Glu-Asp-Leu-Ile-Arg-Phe-Tyr-Asp-Asn-Leu-Gln-Gln-Tyr-Leu-Asn-Val-Val-Thr-Arg-His-Arg-Tyr-NH2。人CgA是一种由439个残基组成的蛋白质，前面有一个由18个残基组成的信号肽。人CgA与牛CgA的蛋白质序列比较显示，NH2端和COOH端结构域以及潜在的二碱基切割位点高度保守，而中间部分则表现出显著的序列变异（36%）。人CgA中含有的PST序列两侧是蛋白水解加工位点。这表明人CgA可能是人胰抑素分子的前体，也可能是其他尚未鉴定的生物活性肽的前体【2】。
PP contains 36 amino acids, has a molecular weight of 4.2 kDa and the isoelectric point is pH 6 to 7. The amino acid sequence of the peptide 1 is Gly-Pro-Ser-Gln-Pro-Thr-Tyr-Pro-Gly-Asp-Asp-Ala-Pro-Val-Glu-Asp-Leu-Ile-Arg-Phe-Tyr-Asp-Asn-Leu-Gln-Gln-Tyr-Leu-Asn-Val-Val-Thr-Arg-His-Arg-Tyr-NH2. The human CgA is a 439-residue protein preceded by an 18-residue signal peptide. Comparison of the protein sequence of human CgA with that of bovine CgA shows high conservation of the NH2-terminal and COOH terminal domains as well as the potential dibasic cleavage sites, whereas the middle portion shows remarkable sequence variation (36%). The PST sequence contained in human CgA is flanked by sites for proteolytic processing. This suggests that human CgA may be the precursor for a human Pancreastatin molecule and possibly for other, as yet unidentified, biologically active peptides 【2】.
胰腺多肽(Pancreatic\Pancreastatin)的作用机制
PP通过其特异性受体分泌并控制分化：在一项研究中，使用小鼠转化成骨细胞系MC3T3-E1细胞研究了PP及其Y受体的调节作用。研究发现，在MC3T3-E1细胞中，细胞接触诱导分化过程中可检测到PP mRNA并增加。此外，在分化过程中，所有类型的NPY家族受体mRNA（Y1、Y2、Y4、Y5和Y6）均增加。还发现，PP在碱性磷酸酶（ALP）mRNA和骨形态发生蛋白-2（BMP-2）mRNA方面刺激了MC3T3-E1细胞的分化。这些发现表明，MC3T3-E1细胞产生并分泌PP，而PP可能通过其特异性受体以自分泌方式刺激MC3T3-E1的分化[3]。PST是一种CgA衍生的肽，已被发现可调节大鼠脂肪细胞中的葡萄糖、脂质和蛋白质代谢。PST通过激活特定的受体-效应子系统（Gaq/11蛋白-PLC-β-PKC经典途径）对胰岛素作用产生总体反调节作用。然而，PST可刺激大鼠脂肪细胞中的基础和胰岛素介导的蛋白质合成。PST剂量依赖性地刺激S6激酶的Thr421/Ser424磷酸化。此外，PST还促进4E-BP1（PHAS-I）中调节位点的磷酸化（Thr37、Thr46）。起始因子eIF4E本身在磷酸化后活性也会增加，在PST刺激下，其Ser209位点被磷酸化。此外，已有研究表明，通过阻止PKC的激活，可以阻断PST对S6激酶和翻译机制的影响。这些结果表明，PST通过激活PKC来刺激蛋白质合成机制，并为所涉及的分子机制提供了一些证据，即S6K的激活和4E-BP1（PHAS-I）以及起始因子eIF4E的磷酸化【4】。
PP is secreted from and controls differentiation through its specific receptors: In a study, the regulatory roles of PP and its Y receptors were studied using MC3T3-E1 cells, a murine transformed osteoblastic cell line. It was found that PP mRNA was detected and increased during cell-contact-induced differentiation in MC3T3-E1 cells. Furthermore, all the types of NPY family receptor mRNAs (Y1, Y2, Y4, Y5, and y6) were found to increase during differentiation. Also it was found that PP stimulated differentiation in MC3T3-E1 cells in terms of alkaline phosphatase (ALP) mRNA and bone morphogenetic protein-2 (BMP-2) mRNA. These findings suggested that MC3T3-E1 cells produce and secrete PP, which may in turn stimulate the differentiation of MC3T3-E1 through its specific receptors in an autocrine manner 3. PST, a CgA-derived peptide, has been found to modulate glucose, lipid, and protein metabolism in rat adipocytes. PST has an overall counter regulatory effect on insulin action by activating a specific receptor–effector system (Gaq/11protein-PLC-ß-PKC classical). However, PST stimulates both basal and insulin-mediated protein synthesis in rat adipocytes. PST dose-dependently stimulates Thr421/Ser424 phosphorylation of S6 kinase. Moreover, PST promotes phosphorylation of regulatory sites in 4E-BP1 (PHAS-I) (Thr37, Thr46). The initiation factor eIF4E itself, whose activity is also increased upon phosphorylation, is phosphorylated in Ser209 by PST stimulation. Also, it has been shown that that these effects of PST on S6 kinase and the translation machinery can be blocked by preventing the activation of PKC. These results indicate that PST stimulates protein synthesis machinery by activating PKC and provides some evidence of the molecular mechanisms involved, i.e., the activation of S6K and the phosphorylation of 4E-BP1 (PHAS-I) and the initiation factor eIF4E 【4】.
胰腺多肽(Pancreatic\Pancreastatin)的功能
小鼠胰多肽调节食物摄入：本研究旨在探讨合成小鼠胰多肽（mPP）对小鼠进食和焦虑行为的影响。研究发现，侧脑室（i.c.v.）注射mPP（0.003-3 nmol）可剂量依赖性地增加食物摄入量。注射后20分钟即可观察到显著增加，并持续4小时。此外，腹腔（i.p.）注射mPP（0.03-30 nmol）可剂量依赖性地减少食物摄入量。注射后20分钟即可观察到显著减少，并持续4小时。在高架十字迷宫测试中，侧脑室注射mPP（0.003-3 nmol）对焦虑行为无影响。这些结果表明，mPP可调节食物摄入量，且脑内的Y4受体可能参与进食行为的调节，但似乎不影响小鼠的焦虑行为【5】。
Mouse pancreatic polypeptide modulates food intake: A study was conducted to investigate the effects of synthetic mouse pancreatic polypeptide (mPP) on feeding and anxiety in mice. It was found that the intracerebroventricular (i.c.v.) injection of mPP (0.003-3 nmol) dose-dependently increased food intake. A significant increase was observed 20 min after i.c.v. injection and continued for 4 h. Further the intraperitoneal (i.p.) injection of mPP (0.03-30 nmol) dose-dependently decreased food intake. A significant decrease was observed 20 min after i.p. injection and continued for 4 h. In the elevated plus maze test, the i.c.v. injection of mPP (0.003-3 nmol) did not affect anxiety behavior. These results suggest that mPP modulates food intake and the Y4 receptor in the brain may contribute to the regulation of feeding, whereas appearing not to influence anxiety in mice 【5】.
PST和CgA对不同促胰岛素剂刺激的胰岛素释放的影响：使用离体灌流大鼠胰腺，比较了猪胰高血糖素样肽（PST）对促胰岛素剂、胰高血糖素、八肽胆囊收缩素（CCK-8）、胃抑肽素（GIP）和L-精氨酸刺激的胰岛素释放的影响，以及牛嗜铬粒蛋白A（CGA）的影响。PST显著增强了胰高血糖素刺激的胰岛素释放（对照组第一阶段：12.5±0.9 ng/8分钟；第二阶段：34.5±1.6 ng/25分钟；胰抑素组第一阶段：16.5±1.1 ng/8分钟；第二阶段：44.0±2.2 ng/25分钟），而CGA则无效。同样，CGA也不影响CCK-8或GIP刺激的胰岛素释放。这些发现表明，PST在胰高血糖素存在时刺激胰岛素释放。由于PST对胰岛素释放有多种影响，且这些影响取决于胰岛素效应剂的局部浓度，因此PST可能参与B细胞胰岛素释放的精细调节【6】。
Effects of PST and CgA on insulin release stimulated by various insulinotropic agents: The effects of porcine PST on insulin release stimulated by insulinotropic agents, glucagon, cholecystokinin-octapeptide (CCK-8), gastric inhibitory polypeptide (GIP) and L-arginine, were compared to those of bovine chromogranin A (CGA) using the isolated perfused rat pancreas. PST significantly potentiated glucagon-stimulated insulin release (first phase: 12.5 ± 0.9 ng/8 min; second phase: 34.5 ± 1.6 ng/25 min in controls; 16.5 ± 1.1 ng/8 min and 44.0 ± 2.2 ng/25 min in pancreastatin group), whereas CgA was ineffective. Similarly, CGA did not affect insulin release stimulated by CCK-8 or GIP. These findings suggest that PST stimulates insulin release in the presence of glucagon. Because PST can have multiple effects on insulin release, which are dependent upon the local concentration of insulin effectors, PST may participate in the fine tuning of insulin release from B cells 【6】
胰腺多肽(Pancreatic\Pancreastatin)的相关文献
1. Kimmel JR, Hayden LJ, Pollock HG (1975). Isolation and characterization of a new pancreatic polypeptide hormone. J. Biol. Chem., 250(24):9369-9376.
2. Konecki DS, Benedum UM, Gerdes HH, Huttner WB (1987). The Primary Structure of Human Chromogranin A and Pancreastatin. J. Biol. Chem., 262(35):17026-17030.
3. Hosaka H, Nagata A, Yoshida T, Shibata T, Nagao T, Tanaka T, Saito Y, Tatsuno I (2008). Pancreatic polypeptide is secreted from and controls differentiation through its specific receptors in osteoblastic MC3T3-E1 cells. Peptides, 29(8):1390-1395.
4. González-Yanes C, Sánchez-Margalet V (2002). Pancreastatin, a chromogranin A-derived peptide, activates protein synthesis signaling cascade in rat adipocytes. Biochemical and Biophysical Research Communications, 299(4):525-531.
5. Asakawa A (1999). Mouse pancreatic polypeptide modulates food intake, while not influencing anxiety in mice. Peptides, 20(12):1445-1448.
6. Ishizuka J, Tatemoto K, Cohn DV, Thompson JC, Greeley GH Jr (1991). Effects of pancreastatin and chromogranin A on insulin release stimulated by various insulinotropic agents. Regulatory Peptides, 34(1):25-32.
生物素可以与亲和素或者链霉亲和素有力结合，结合强度甚至接近共价键。生物素标记的肽通常用于免疫测定，组织细胞化学和基于荧光的流式细胞术。标记的抗生物素抗体也可以用来结合生物素化多肽。生物素标记常连接在赖氨酸侧链或者N末端。通常在多肽和生物素之间使用6-氨基己酸作为纽带，纽带能够灵活结合底物，并且在有空间位阻的情况下能结合地更好。专肽生物根据需求，向客户提供具有不同位点生物素标记多肽的定制合成。
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。
| DOI | 名称 | |
|---|---|---|
| 10.1038/sj.bjp.0704604 | Neuropeptide Y, Y1, Y2 and Y4 receptors mediate Y agonist responses in isolated human colon mucosa | 下载 |
| 10.1210/jc.2003-030630 | Pancreatic polypeptide reduces appetite and food intake in humans | 下载 |
| 10.1002/ijc.20948 | Neuropeptide Y receptors in renal cell carcinomas and nephroblastomas | 下载 |
| 10.1038/oby.2006.180 | Physiological evidence for the involvement of peptide YY in the regulation of energy homeostasis in humans | 下载 |
| 10.1016/j.peptides.2012.06.008 | Surface masking shapes the traffic of the neuropeptide Y Y2 receptor | 下载 |
| 10.1002/oby.20534 | PYY3-36 and pancreatic polypeptide reduce food intake in an additive manner via distinct hypothalamic dependent pathways in mice | 下载 |
| 10.3390/ijms15034856 | Dimers of G-protein coupled receptors as versatile storage and response units | 下载 |
| 10.1021/acs.jmedchem.6b00310 | Optically Pure, Structural, and Fluorescent Analogues of a Dimeric Y4 Receptor Agonist Derived by an Olefin Metathesis Approach | 下载 |
| 10.1021/acs.bioconjchem.6b00376 | Identification of a Cyanine-Dye Labeled Peptidic Ligand for Y1R and Y4R, Based upon the Neuropeptide Y C-Terminal Analogue, BVD-15 | 下载 |
| 10.1016/0167-0115(93)90435-b | Proteolytic processing of neuropeptide Y and peptide YY by dipeptidyl peptidase IV | 下载 |
| 10.1371/journal.pone.0120346 | Cytotoxic helix-rich oligomer formation by melittin and pancreatic polypeptide | 下载 |
多肽Biotin-Ala-Pro-Leu-Glu-Pro-Val-Tyr-Pro-Gly-Asp-Asn-Ala-Thr-Pro-Glu-Gln-Met-Ala-Gln-Tyr-Ala-Ala-Asp-Leu-Arg-Arg-Tyr-Ile-Asn-Met-Leu-Thr-Arg-Pro-Arg-Tyr-NH2的合成步骤：
1、合成MBHA树脂：取若干克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2.1g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1.89mmol Fmoc-Tyr(tBu)-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3.78mmol DIPEA，1.8mmol 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-Tyr(tBu)-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-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
H2N-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
H2N-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
H2N-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
H2N-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
H2N-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
H2N-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
H2N-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
H2N-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
H2N-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
H2N-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
H2N-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
H2N-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
H2N-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
H2N-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
H2N-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
H2N-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
H2N-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
H2N-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
H2N-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Gln(Trt)-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
H2N-Gln(Trt)-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Glu(OtBu)-Gln(Trt)-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
H2N-Glu(OtBu)-Gln(Trt)-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Pro-Glu(OtBu)-Gln(Trt)-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
H2N-Pro-Glu(OtBu)-Gln(Trt)-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Thr(tBu)-Pro-Glu(OtBu)-Gln(Trt)-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
H2N-Thr(tBu)-Pro-Glu(OtBu)-Gln(Trt)-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Ala-Thr(tBu)-Pro-Glu(OtBu)-Gln(Trt)-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
H2N-Ala-Thr(tBu)-Pro-Glu(OtBu)-Gln(Trt)-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Asn(Trt)-Ala-Thr(tBu)-Pro-Glu(OtBu)-Gln(Trt)-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
H2N-Asn(Trt)-Ala-Thr(tBu)-Pro-Glu(OtBu)-Gln(Trt)-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Asp(OtBu)-Asn(Trt)-Ala-Thr(tBu)-Pro-Glu(OtBu)-Gln(Trt)-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
H2N-Asp(OtBu)-Asn(Trt)-Ala-Thr(tBu)-Pro-Glu(OtBu)-Gln(Trt)-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Gly-Asp(OtBu)-Asn(Trt)-Ala-Thr(tBu)-Pro-Glu(OtBu)-Gln(Trt)-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
H2N-Gly-Asp(OtBu)-Asn(Trt)-Ala-Thr(tBu)-Pro-Glu(OtBu)-Gln(Trt)-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Pro-Gly-Asp(OtBu)-Asn(Trt)-Ala-Thr(tBu)-Pro-Glu(OtBu)-Gln(Trt)-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
H2N-Pro-Gly-Asp(OtBu)-Asn(Trt)-Ala-Thr(tBu)-Pro-Glu(OtBu)-Gln(Trt)-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Tyr(tBu)-Pro-Gly-Asp(OtBu)-Asn(Trt)-Ala-Thr(tBu)-Pro-Glu(OtBu)-Gln(Trt)-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
H2N-Tyr(tBu)-Pro-Gly-Asp(OtBu)-Asn(Trt)-Ala-Thr(tBu)-Pro-Glu(OtBu)-Gln(Trt)-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Val-Tyr(tBu)-Pro-Gly-Asp(OtBu)-Asn(Trt)-Ala-Thr(tBu)-Pro-Glu(OtBu)-Gln(Trt)-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
H2N-Val-Tyr(tBu)-Pro-Gly-Asp(OtBu)-Asn(Trt)-Ala-Thr(tBu)-Pro-Glu(OtBu)-Gln(Trt)-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Pro-Val-Tyr(tBu)-Pro-Gly-Asp(OtBu)-Asn(Trt)-Ala-Thr(tBu)-Pro-Glu(OtBu)-Gln(Trt)-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
H2N-Pro-Val-Tyr(tBu)-Pro-Gly-Asp(OtBu)-Asn(Trt)-Ala-Thr(tBu)-Pro-Glu(OtBu)-Gln(Trt)-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Glu(OtBu)-Pro-Val-Tyr(tBu)-Pro-Gly-Asp(OtBu)-Asn(Trt)-Ala-Thr(tBu)-Pro-Glu(OtBu)-Gln(Trt)-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
H2N-Glu(OtBu)-Pro-Val-Tyr(tBu)-Pro-Gly-Asp(OtBu)-Asn(Trt)-Ala-Thr(tBu)-Pro-Glu(OtBu)-Gln(Trt)-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Leu-Glu(OtBu)-Pro-Val-Tyr(tBu)-Pro-Gly-Asp(OtBu)-Asn(Trt)-Ala-Thr(tBu)-Pro-Glu(OtBu)-Gln(Trt)-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
H2N-Leu-Glu(OtBu)-Pro-Val-Tyr(tBu)-Pro-Gly-Asp(OtBu)-Asn(Trt)-Ala-Thr(tBu)-Pro-Glu(OtBu)-Gln(Trt)-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Pro-Leu-Glu(OtBu)-Pro-Val-Tyr(tBu)-Pro-Gly-Asp(OtBu)-Asn(Trt)-Ala-Thr(tBu)-Pro-Glu(OtBu)-Gln(Trt)-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
H2N-Pro-Leu-Glu(OtBu)-Pro-Val-Tyr(tBu)-Pro-Gly-Asp(OtBu)-Asn(Trt)-Ala-Thr(tBu)-Pro-Glu(OtBu)-Gln(Trt)-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA Resin
Fmoc-Ala-Pro-Leu-Glu(OtBu)-Pro-Val-Tyr(tBu)-Pro-Gly-Asp(OtBu)-Asn(Trt)-Ala-Thr(tBu)-Pro-Glu(OtBu)-Gln(Trt)-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-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-Ala-Pro-Leu-Glu(OtBu)-Pro-Val-Tyr(tBu)-Pro-Gly-Asp(OtBu)-Asn(Trt)-Ala-Thr(tBu)-Pro-Glu(OtBu)-Gln(Trt)-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHA 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1.89mmol 生物素到树脂中，再加入3.78mmol DIPEA、1.8mmol 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-Ala-Pro-Leu-Glu(OtBu)-Pro-Val-Tyr(tBu)-Pro-Gly-Asp(OtBu)-Asn(Trt)-Ala-Thr(tBu)-Pro-Glu(OtBu)-Gln(Trt)-Met-Ala-Gln(Trt)-Tyr(tBu)-Ala-Ala-Asp(OtBu)-Leu-Arg(Pbf)-Arg(Pbf)-Tyr(tBu)-Ile-Asn(Trt)-Met-Leu-Thr(tBu)-Arg(Pbf)-Pro-Arg(Pbf)-Tyr(tBu)-Linker-MBHAResin。 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Biotin-Ala-Pro-Leu-Glu-Pro-Val-Tyr-Pro-Gly-Asp-Asn-Ala-Thr-Pro-Glu-Gln-Met-Ala-Gln-Tyr-Ala-Ala-Asp-Leu-Arg-Arg-Tyr-Ile-Asn-Met-Leu-Thr-Arg-Pro-Arg-Tyr-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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