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胃泌素释放肽(Gastrin releasing peptide_GRP)的定义

胃泌素释放肽（GRP）是一种27个氨基酸的线性神经肽，在结构和功能上与蛙皮素（BB）相关，介导胃窦胃泌素的神经释放，引起支气管收缩和呼吸道血管舒张，刺激培养细胞的生长和有丝分裂，并可作为肠道中间神经元的兴奋性神经递质。

Gastrin releasing peptide (GRP) is a 27-amino acid linear neuropeptide, structurally and functionally related to bombesin (BB) that mediates neural release of antral gastrin, causes bronchoconstriction and respiratory tract vasodilation, stimulates growth and mitogenesis of cells in culture, and may act as an excitatory neurotransmitter of enteric interneurons.

胃泌素释放肽(Gastrin releasing peptide_GRP)的相关肽

GRP基因编码蛙皮素样胃泌素释放肽家族的成员。在切割信号肽后，其前蛋白被进一步加工以产生27-aa GRP或10-aa neuromedin C【1】。GRP和neuromedin B（NMB）在结构上与BB相关，并存在于哺乳动物小肠内。通过修饰N-乙酰基-GRP-20-27的COOH末端，已经开发出一系列有效的GRP拮抗剂。通过修饰该肽的COOH末端区域获得的该系列中最有效的成员N-乙酰基-GRP-20-26-0CH2CH3阻断GRP刺激的有丝分裂，抑制GRP依赖性胃泌素释放并阻断GRP诱导的升高。体外[Ca2+]i【2】。

The GRP gene encodes a member of the bombesin-like family of gastrin-releasing peptides. Its preproprotein, following cleavage of a signal peptide, is further processed to produce either the 27 aa GRP or the 10 aa neuromedin C【1】.  The GRP and neuromedin B (NMB) are structurally related to BB and exist within the mammalian small intestine. A series of potent GRP antagonists have been developed by modification of the COOH terminus of N-acetyl-GRP-20-27. The most potent member of this series, N-acetyl-GRP-20-26-0CH2CH3, obtained by modification of the COOH-terminal region of this peptide blocks GRP-stimulated mitogenesis, inhibits GRP-dependent release of gastrin and blocks GRP-induced elevation of [Ca2+]i in vitro【2】.

胃泌素释放肽(Gastrin releasing peptide_GRP)的发现

Bombesin是一种最初从欧洲斑点蛙Bombina bombino的皮肤中提取的肽，在哺乳动物中具有深远的生物学效力。这导致了“哺乳动物蛙蛋白”GRP5和NMB的发现【3】。McDonald TJ等人于1978年在猪非胃窦胃组织的提取物中发现了GRP 【4】。

Bombesin is a peptide originally extracted from the skin of the European discoglossid frog Bombina bombino and possessed profound biological potency in mammals. This led to the discovery of the "mammalian bombesins" GRP5 and NMB【3】. McDonald TJ et al., in 1978 discovered GRP in extracts from porcine non-antral gastric tissue【4】.

胃泌素释放肽(Gastrin releasing peptide_GRP)的结构特征

人GRP（hGRP）mRNA编码148个氨基酸的前体，其中包含典型的信号序列，由27或28个氨基酸组成的hGRP和羧基末端延伸肽。hGRP在其羧基末端的两侧是两个碱性氨基酸，其后是用于羧基末端蛋氨酸酰胺化的甘氨酸。hGRP包含两个潜在的内部胰蛋白酶切割位点，可以产生hGRP-（14-27）或hGRP-（18-27）。这两种形式的hGRP可能来源于将pre-proGRP替代蛋白水解加工成GRP-（1-27）和较小的GRP样肽【5】。虽然GRP的残基20-27影响亲本肽与其受体的结合，但COOH末端氨基酸主要负责触发随后的生物反应【5】。

Human GRP (hGRP) mRNA encodes a precursor of 148 amino acids containing a typical signal sequence, hGRP consisting of 27 or 28 amino acids, and a carboxyl-terminal extension peptide. hGRP is flanked at its carboxyl terminus by two basic amino acids, following a glycine used for amidation of the carboxyl-terminal methionine. hGRP contains two potential internal tryptic cleavage sites that could generate hGRP-(14-27) or hGRP-(18-27). The two forms of hGRP probably derive from alternative proteolytic processing of pre-proGRP into both GRP-(1-27) and a smaller GRP-like peptide【5】. While the residues 20-27 of GRP influence binding of the parent peptide to its receptor, the COOH-terminal amino acid is primarily responsible for triggering the subsequent biological response【5】.

胃泌素释放肽(Gastrin releasing peptide_GRP)的作用机理

GRP的作用是通过GRP受体介导的。该受体是一种糖基化的7-跨膜G蛋白偶联受体，可激活磷脂酶C信号通路。该受体在许多癌症中异常表达，例如肺癌，结肠癌和前列腺癌【6】。

The effects of GRP are mediated through the GRP receptor. This receptor is a glycosylated, 7-transmembrane G-protein coupled receptor that activates the phospholipase C signaling pathway. The receptor is aberrantly expressed in numerous cancers such as those of the lung, colon, and prostate【6】.

胃泌素释放肽(Gastrin releasing peptide_GRP)的功能

GRP由迷走神经的神经节后纤维释放，神经支配胃的G细胞并刺激它们释放胃泌素。GRP可以通过动员细胞内钙的特定GRP受体直接刺激主要细胞释放胃蛋白酶原。GRP作为肿瘤标志物在小细胞肺癌的诊断中具有突出的作用。它调节胃肠道和中枢神经系统的许多功能，包括平滑肌细胞收缩和上皮细胞增殖，并且是肿瘤组织的有效促分裂原6。

GRP is released by the post-ganglionic fibres of the vagus nerve, which innervate the G cells of the stomach and stimulate them to release gastrin. GRP can directly stimulate pepsinogen release from chief cells by a specific GRP receptor that mobilizes intracellular calcium. GRP has a prominent role as a tumor marker in the diagnosis of small-cell lung carcinoma. It regulates numerous functions of the gastrointestinal and central nervous systems, including smooth muscle cell contraction, and epithelial cell proliferation and is a potent mitogen for neoplastic tissues6.

胃泌素释放肽(Gastrin releasing peptide_GRP)的相关文献

1. Chandan R, Newell SM, Brown DR (1988).Actions of gastrin-releasing peptide and related mammalian and amphibian peptides on ion transport in the porcine proximal jejunum. Regul Pept, 23(1):1-14.

2. Heimbrook DC, Saari WS, Balishin NL, Friedman A, Moore KS, Reimen MW, Kiefer DM, Rotberg NS, Wallen JW, Oliff A(1989). Carboxyl-terminal Modification of a Gastrin Releasing Peptide Derivative Generates Potent Antagonists. J Biol Chem, 264(19):11258-11262.

3. Preston SR, Woodhouse LF, Jones-Blackett S, Wyatt JI, Primrose JN(1993). Shaun R. Preston, Linda F. Woodhouse, Steven Jones-Blackett, Judy I. Wyatt, and John N. Primrose.1993. High Affinity Binding Sites for Gastrin Releasing Peptide on Human Gastric Cancer and Menetrier's Mucosa. Cancer Res, 53(21):5090-5092.

4. McDonald TJ, Nilsson G, Vagne M, Ghatei M, Bloom SR, Mutt V.1978. A gastrin releasing peptide from the porcine nonantral gastric tissue. Gut, 19(9):767-774.

5. Spindel ER, Chin WW, Price J, Rees LH, Besser GM, Habener JF(1984). Cloning and characterization of cDNAs encoding human gastrin-releasing peptide. Proc. Nati. Acad. Sci. USA 81(18):5699-5703.

6. Martínez A, Zudaire E, Julián M, Moody TW, Cuttitta F (2005). Gastrin-releasing peptide (GRP) induces angiogenesis and the specific GRP blocker 77427 inhibits tumor growth in vitro and in vivo. Oncogene, 24(25):4106-4113.

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。

多肽Biotin-Val-Pro-Leu-Pro-Ala-Gly-Gly-Gly-Thr-Val-Leu-Thr-Lys-Met-Tyr-Pro-Arg-Gly-Asn-His-Trp-Ala-Val-Gly-His-Leu-Met-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2.89g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2.6mmol Fmoc-Met-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5.2mmol DIPEA，2.47mmol 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-Met-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-Met-Linker-MBHA Resin

Fmoc-Leu-Met-Linker-MBHA Resin

H2N-Leu-Met-Linker-MBHA Resin

Fmoc-His(Trt)-Leu-Met-Linker-MBHA Resin

H2N-His(Trt)-Leu-Met-Linker-MBHA Resin

Fmoc-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

H2N-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

Fmoc-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

H2N-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

Fmoc-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

H2N-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

Fmoc-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

H2N-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

Fmoc-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

H2N-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

Fmoc-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

H2N-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

Fmoc-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

H2N-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

Fmoc-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

H2N-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

Fmoc-Pro-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

H2N-Pro-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

Fmoc-Tyr(tBu)-Pro-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

H2N-Tyr(tBu)-Pro-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

Fmoc-Met-Tyr(tBu)-Pro-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

H2N-Met-Tyr(tBu)-Pro-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

Fmoc-Lys(Boc)-Met-Tyr(tBu)-Pro-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

H2N-Lys(Boc)-Met-Tyr(tBu)-Pro-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

Fmoc-Thr(tBu)-Lys(Boc)-Met-Tyr(tBu)-Pro-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

H2N-Thr(tBu)-Lys(Boc)-Met-Tyr(tBu)-Pro-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

Fmoc-Leu-Thr(tBu)-Lys(Boc)-Met-Tyr(tBu)-Pro-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

H2N-Leu-Thr(tBu)-Lys(Boc)-Met-Tyr(tBu)-Pro-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

Fmoc-Val-Leu-Thr(tBu)-Lys(Boc)-Met-Tyr(tBu)-Pro-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

H2N-Val-Leu-Thr(tBu)-Lys(Boc)-Met-Tyr(tBu)-Pro-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

Fmoc-Thr(tBu)-Val-Leu-Thr(tBu)-Lys(Boc)-Met-Tyr(tBu)-Pro-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

H2N-Thr(tBu)-Val-Leu-Thr(tBu)-Lys(Boc)-Met-Tyr(tBu)-Pro-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

Fmoc-Gly-Thr(tBu)-Val-Leu-Thr(tBu)-Lys(Boc)-Met-Tyr(tBu)-Pro-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

H2N-Gly-Thr(tBu)-Val-Leu-Thr(tBu)-Lys(Boc)-Met-Tyr(tBu)-Pro-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

Fmoc-Gly-Gly-Thr(tBu)-Val-Leu-Thr(tBu)-Lys(Boc)-Met-Tyr(tBu)-Pro-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

H2N-Gly-Gly-Thr(tBu)-Val-Leu-Thr(tBu)-Lys(Boc)-Met-Tyr(tBu)-Pro-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

Fmoc-Gly-Gly-Gly-Thr(tBu)-Val-Leu-Thr(tBu)-Lys(Boc)-Met-Tyr(tBu)-Pro-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

H2N-Gly-Gly-Gly-Thr(tBu)-Val-Leu-Thr(tBu)-Lys(Boc)-Met-Tyr(tBu)-Pro-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

Fmoc-Ala-Gly-Gly-Gly-Thr(tBu)-Val-Leu-Thr(tBu)-Lys(Boc)-Met-Tyr(tBu)-Pro-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

H2N-Ala-Gly-Gly-Gly-Thr(tBu)-Val-Leu-Thr(tBu)-Lys(Boc)-Met-Tyr(tBu)-Pro-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

Fmoc-Pro-Ala-Gly-Gly-Gly-Thr(tBu)-Val-Leu-Thr(tBu)-Lys(Boc)-Met-Tyr(tBu)-Pro-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

H2N-Pro-Ala-Gly-Gly-Gly-Thr(tBu)-Val-Leu-Thr(tBu)-Lys(Boc)-Met-Tyr(tBu)-Pro-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

Fmoc-Leu-Pro-Ala-Gly-Gly-Gly-Thr(tBu)-Val-Leu-Thr(tBu)-Lys(Boc)-Met-Tyr(tBu)-Pro-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

H2N-Leu-Pro-Ala-Gly-Gly-Gly-Thr(tBu)-Val-Leu-Thr(tBu)-Lys(Boc)-Met-Tyr(tBu)-Pro-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

Fmoc-Pro-Leu-Pro-Ala-Gly-Gly-Gly-Thr(tBu)-Val-Leu-Thr(tBu)-Lys(Boc)-Met-Tyr(tBu)-Pro-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

H2N-Pro-Leu-Pro-Ala-Gly-Gly-Gly-Thr(tBu)-Val-Leu-Thr(tBu)-Lys(Boc)-Met-Tyr(tBu)-Pro-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-Linker-MBHA Resin

Fmoc-Val-Pro-Leu-Pro-Ala-Gly-Gly-Gly-Thr(tBu)-Val-Leu-Thr(tBu)-Lys(Boc)-Met-Tyr(tBu)-Pro-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-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-Val-Pro-Leu-Pro-Ala-Gly-Gly-Gly-Thr(tBu)-Val-Leu-Thr(tBu)-Lys(Boc)-Met-Tyr(tBu)-Pro-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-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2.6mmol 生物素到树脂中，再加入5.2mmol DIPEA、2.47mmol 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-Val-Pro-Leu-Pro-Ala-Gly-Gly-Gly-Thr(tBu)-Val-Leu-Thr(tBu)-Lys(Boc)-Met-Tyr(tBu)-Pro-Arg(Pbf)-Gly-Asn(Trt)-His(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-Leu-Met-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-Val-Pro-Leu-Pro-Ala-Gly-Gly-Gly-Thr-Val-Leu-Thr-Lys-Met-Tyr-Pro-Arg-Gly-Asn-His-Trp-Ala-Val-Gly-His-Leu-Met-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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