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100016-62-4,Calcitonin, chicken,H2N-Cys-Ala-Ser-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asp-Val-Gly-Ala-Gly-Thr-Pro-NH2,H2N-CASLSTCVLGKLSQELHKLQTYPRTDVGAGTP-NH2,杭州专肽生物的产品

降钙素Calcitonin, chicken

T.Kurihara et al., Peptide Chemistry 1985, Proceedings of the 23rd Symposium, Kyoto, p. 173, Y.Kiso, ed., Protein Res. Foundation, Osaka, (1986)

编号:125878

CAS号:100016-62-4

单字母:H2N-CASLSTCVLGKLSQELHKLQTYPRTDVGAGTP-NH2(Disulfide Bridge:C1-C7)

纠错
  • 编号:125878
    中文名称:降钙素Calcitonin, chicken
    英文名:Calcitonin, chicken
    CAS号:100016-62-4
    单字母:H2N-CASLSTCVLGKLSQELHKLQTYPRTDVGAGTP-NH2(Disulfide Bridge:C1-C7)
    三字母:H2N-Cys-Ala-Ser-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asp-Val-Gly-Ala-Gly-Thr-Pro-NH2(Disulfide Bridge:Cys1-Cys7)
    氨基酸个数:32
    分子式:C145H240N42O46S2
    平均分子量:3371.84
    精确分子量:3369.72
    等电点(PI):10.91
    pH=7.0时的净电荷数:5.15
    平均亲水性:-0.15555555555556
    疏水性值:-0.13
    外观与性状:白色粉末状固体
    消光系数:1490
    来源:人工化学合成,仅限科学研究使用,不得用于人体。
    纯度:95%、98%
    盐体系:可选TFA、HAc、HCl或其它
    生成周期:2-3周
    储存条件:负80℃至负20℃
    标签:二硫键环肽    降钙素(Calcitonins)   

  • T.Kurihara et al., Peptide Chemistry 1985, Proceedings of the 23rd Symposium, Kyoto, p. 173, Y.Kiso, ed., Protein Res. Foundation, Osaka, (1986)

    二硫键广泛存在与蛋白结构中,对稳定蛋白结构具有非常重要的意义,二硫键一般是通过序列中的2个Cys的巯基,经氧化形成。
     

    形成二硫键的方法很多:空气氧化法,DMSO氧化法,过氧化氢氧化法等。
     

    二硫键的合成过程,  可以通过Ellman检测以及HPLC检测方法对其反应进程进行监测。  
       

    如果多肽中只含有1对Cys,那二硫键的形成是简单的。多肽经固相或液相合成,然后在pH8-9的溶液中进行氧化。      
     

    当需要形成2对或2对以上的二硫键时,合成过程则相对复杂。尽管二硫键的形成通常是在合成方案的最后阶段完成,但有时引入预先形成的二硫化物是有利于连合或延长肽链的。通常采用的巯基保护基有trt, Acm, Mmt, tBu, Bzl, Mob, Tmob等多种基团。我们分别列出两种以2-Cl树脂和Rink树脂为载体合成的多肽上多对二硫键形成路线:
     

    二硫键反应条件选择    
     

     二硫键即为蛋白质或多肽分子中两个不同位点Cys的巯基(-SH)被氧化形成的S-S共价键。 一条肽链上不同位置的氨基酸之间形成的二硫键,可以将肽链折叠成特定的空间结构。多肽分 子通常分子量较大,空间结构复杂,结构中形成二硫键时要求两个半胱氨酸在空间距离上接近。 此外,多肽结构中还原态的巯基化学性质活泼,容易发生其他的副反应,而且肽链上其他侧链 也可能会发生一系列修饰,因此,肽链进行修饰所选取的氧化剂和氧化条件是反应的关键因素, 反应机理也比较复杂,既可能是自由基反应,也可能是离子反应。      

    反应条件有多种选择,比如空气氧化,DMSO氧化等温和的氧化过程,也可以采用H2O2,I2, 汞盐等激烈的反应条件。
     

    空气氧化法: 空气氧化法形成二硫键是多肽合成中最经典的方法,通常是将巯基处于还原态的多肽溶于水中,在近中性或弱碱性条件下(PH值6.5-10),反应24小时以上。为了降低分子之间二硫键形成的可能,该方法通常需要在低浓度条件下进行。
     

    碘氧化法:将多肽溶于25%的甲醇水溶液或30%的醋酸水溶液中,逐滴滴加10-15mol/L的碘进行氧化,反应15-40min。当肽链中含有对碘比较敏感的Tyr、Trp、Met和His的残基时,氧化条件要控制的更精确,氧化完后,立即加入维生素C或硫代硫酸钠除去过量的碘。 当序列中有两对或多对二硫键需要成环时,通常有两种情况:
     

    自然随机成环:       序列中的Cys之间随机成环,与一对二硫键成环条件相似;
     

    定点成环:       定点成环即序列中的Cys按照设计要求形成二硫键,反应过程相对复杂。在 固相合成多肽之前,需要提前设计几对二硫键形成的顺序和方法路线,选择不同的侧链 巯基保护基,利用其性质差异,分步氧化形成两对或多对二硫键。       通常采用的巯基保护 基有trt, Acm, Mmt, tBu, Bzl, Mob, Tmob等多种基团。

    Definition
    Calcitonin is a 32-amino acid linear polypeptide hormone produced primarily by the parafollicular cells in humans and ultimobranchial body in many other animals1. It acts to reduce blood calcium (Ca2+), opposing the effects of parathyroid hormone (PTH). Calcitonin is a product of the CALC1 gene and is initially produced as a precursor1.

    Discovery
    Calcitonin was purified in 1962 by Copp and Cheney2. While it was initially considered a secretion of the parathyroid glands, it was later identified as the secretion of the C-cells of the thyroid gland 3 .

    Classification
    CALC1 gene belongs to a superfamily of related protein hormone precursors that includes islet amyloid precursor protein, calcitonin gene-related peptide, and the precursor of adrenomedullin 4 .

    Structural Characteristics
    Human calcitonin is a 32 amino acid peptide and is formed from procalcitonin (Cleavage products: Calcitonin, Katalin and a protein fragment)5. It has an N-terminal disulphide bridge and a C-terminal proline amide residue, shown to potently inhibit bone resorption5. Alternative splicing of the gene coding for calcitonin produces a distantly related peptide of 37 amino acids, called calcitonin gene-related peptide (CGRP) 5.

    Mode of action
    Calcitonin exerts its functions by binding to calcitonin receptor that is a G-protein coupled receptor. Upon binding, the receptor triggers the formation of cAMP, a second messenger which in turn activates various signaling pathways in the target cell (Eg: Osteoblasts) 6 .

    Functions
    Calcitonin is mainly involved in the metabolism of Ca and phosphorous in the cell. Calcitonin secretion is stimulated by rise in Ca levels in the body. It inhibits Ca intake by the intestine and also prevent loss of Ca from the bones during pregnancy and lactation7It also inhibits osteoclast activity in the bones8. This property of calcitonin is utilized for treatment of osteoporosis and osteoarthritis and recently has been tried for bone metastasis1.Procalcitonin is released during severe infection where it is involved in Ca homeostasis. It is also used as a marker for sepsis8.

    References

    1.     Inzerillo AM, Zaidi M, Huang CL (2004). Calcitonin: physiological actions and clinical applications. J Pediatr Endocrinol Metab., 17(7), 931-40.


    2.     Copp DH, Cheney B (1962). Calcitonin-a hormone from the parathyroid which lowers the calcium-level of the blood. Nature, 193, 381–2.


    3.     Stevenson JC, Evans IM (1981). Pharmacology and therapeutic use of calcitonin. Drugs, 21(4), 257-72.


    4.     Zaidi M, Inzerillo AM, Moonga BS, Bevis PJ, Huang CL (2002). Forty years of calcitonin--where are we now? A tribute to the work of Iain Macintyre, FRS, Bone, 30(5), 655-63.


    5.     Andreotti G, Méndez BL, Amodeo P, Morelli MA, Nakamuta H, Motta A (2006). "Structural determinants of salmon calcitonin bioactivity: the role of the Leu-based amphipathic alpha-helix". J. Biol. Chem., 281 (34), 24193–203.


    6.     Purdue BW, Tilakaratne N, Sexton PM (2002). Molecular pharmacology of the calcitonin receptor. Recept. Channels, 8 (3-4), 243–55.


    7.     Woodrow JP, Sharpe CJ, Fudge NJ, Hoff AO, Gagel RF, Kovacs CS (2006). Calcitonin plays a critical role in regulating skeletal mineral metabolism during lactation. Endocrinology, 147(9), 4010-21.


    8.     BalcI C, Sungurtekin H, Gürses E, Sungurtekin U, Kaptanoglu B (2003). Usefulness of procalcitonin for diagnosis of sepsis in the intensive care unit. Crit Care, 7 (1), 85–90


    Definition

    Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide with potent receptor mediated vasodilatory and cardioexcitatory properties1.

    Discovery

    It was discovered when alternative processing of RNA transcripts from the calcitonin gene were shown to result in the production of distinct mRNAs encoding CGRP2. A human form of CGRP was isolated from thyroid tissue of patients with medullary thyroid carcinoma3.

    Classification

    CGRP belongs to the regulatory-peptide family that also includes adrenomedullin and amylin4.

    Structural Characteristics

    CGRP consists of an amino-terminal disulphide bridge linked loop between amino acids 2 and 7 followed by alpha helix between amino acids 8 and 18 and a poorly defined turn between residues 19 and 215.  The carboxy and amino terminals of CGRP can interact independently with its receptors5.

    Mode of action

    CGRP exerts its function by binding to two G-protein coupled receptors, CGRP1 and CGRP2.  One of the major functions of CGRP is vasodilation of cardiac muscles5.  In order to achieve this, CGRP first binds to CGRP1 receptor which results in the production of cAMP which in turn activates Protein Kinase A (PKA)6.  PKA phosphorylates and opens potassium channels that cause relaxation of muscles6.

    Functions

    CGRP is widely distributed in the central and peripheral nervous systems5.  It produces vascular relaxation via binding to CGRP1 receptor5.  Studies in mice have shown that CGRP may play a role in controlling blood pressure5.  CGRP also protects tissue injury through its vasodilatory functions.  Through its activity as a vasodilator, CGRP influence the activity of inflammatory cells by recruiting more cells at the site of inflammation7.  CGRP plays a role in migraine as it is found that its levels raise during painful phases of the disease8.  CGRP plays a protective role in cardiac tissue.  The infusion of CGRP is beneficial in increasing cardiac output and lowering blood pressure in patients with congestive heart failure5.

    References

    1.     Tortorella C, Macchi C, Forneris M and Nussdorfer GG (2001). Calcitonin gene-related peptide (CGRP), acting via CGRP type 1 receptors, inhibits potassium-stimulated aldosterone secretion and enhances basal catecholamine secretion from rat adrenal gland. Int. J Mol. Med., 8(3), 261-4.

    2.     Amara SG, Jonas V, Rosenfeld MG, Ong ES and Evans RM (1982). Alternative RNA processing in calcitonin gene expression generates mRNAs encoding different polypeptide products. Nature, 298, 240–244.

    3.     Aiyar N, Rand K, Elshourbagy NA, Zeng Z, Adamou JE, Bergsma DJ, and Li Y (1996). A cDNA encoding the Calcitonin Generelated peptide type 1 receptor. J Biol Chem., 271, 11325–11329.

    4.     Bell D and McDermott BJ (1996). Calcitonin gene-related peptide in the cardiovascular system: characterization of receptor populations and their (patho)physiological significance. Pharmacol Rev., 48, 253–288.

    5.     Susan DB and Andrew DG (2004). Vascular Actions of Calcitonin Gene-Related Peptide and Adrenomedullin. Physiol Rev., 84, 903-934.

    6.     Hirata Y, Takagi Y, Takata S, Fukuda Y, Yoshimi H, and Fujita T (1988). Calcitonin gene-related peptide receptor in cultured vascular smooth muscle and endothelial cells. Biochem Biophys Res Commun., 151, 1113–1121.

    7.     Lambrecht BN (2001). Immunologists getting nervous: neuropeptides, dendritic cells and T cell activation.  Respir Res., 2, 133–138.

    8.     Durham, P (2006). Calcitonin gene-related peptide (CGRP) and migraine. Headache, 48: S3–8.

  • Coast, G. et al. J. Exp. Biol. 204, 1795 (2001);

    Kurihara, T. et al. Peptide Chem. Proceedings of the 23rd Symposium, Kyoto, p.173, Y. Kiso, ed., Protein Res. Foundation, Osaka (1986)

  • 多肽H2N-Cys-Ala-Ser-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asp-Val-Gly-Ala-Gly-Thr-Pro-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2.19g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1.97mmol Fmoc-Pro-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3.94mmol DIPEA,1.87mmol 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-Pro-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-Pro-Linker-MBHA Resin

    Fmoc-Thr(tBu)-Pro-Linker-MBHA Resin

    H2N-Thr(tBu)-Pro-Linker-MBHA Resin

    Fmoc-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    H2N-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    Fmoc-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    H2N-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    Fmoc-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    H2N-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    Fmoc-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    H2N-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    Fmoc-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    H2N-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    Fmoc-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    H2N-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    Fmoc-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    H2N-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    Fmoc-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    H2N-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    Fmoc-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    H2N-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    Fmoc-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    H2N-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    Fmoc-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    H2N-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    Fmoc-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    H2N-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    Fmoc-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    H2N-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    Fmoc-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    H2N-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    Fmoc-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    H2N-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    Fmoc-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    H2N-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    Fmoc-Gln(Trt)-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    H2N-Gln(Trt)-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    Fmoc-Ser(tBu)-Gln(Trt)-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    H2N-Ser(tBu)-Gln(Trt)-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    Fmoc-Leu-Ser(tBu)-Gln(Trt)-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    H2N-Leu-Ser(tBu)-Gln(Trt)-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    Fmoc-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    H2N-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    Fmoc-Gly-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    H2N-Gly-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    Fmoc-Leu-Gly-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    H2N-Leu-Gly-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    Fmoc-Val-Leu-Gly-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    H2N-Val-Leu-Gly-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    Fmoc-Cys(Trt)-Val-Leu-Gly-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    H2N-Cys(Trt)-Val-Leu-Gly-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    Fmoc-Thr(tBu)-Cys(Trt)-Val-Leu-Gly-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    H2N-Thr(tBu)-Cys(Trt)-Val-Leu-Gly-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    Fmoc-Ser(tBu)-Thr(tBu)-Cys(Trt)-Val-Leu-Gly-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    H2N-Ser(tBu)-Thr(tBu)-Cys(Trt)-Val-Leu-Gly-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    Fmoc-Leu-Ser(tBu)-Thr(tBu)-Cys(Trt)-Val-Leu-Gly-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    H2N-Leu-Ser(tBu)-Thr(tBu)-Cys(Trt)-Val-Leu-Gly-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    Fmoc-Ser(tBu)-Leu-Ser(tBu)-Thr(tBu)-Cys(Trt)-Val-Leu-Gly-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    H2N-Ser(tBu)-Leu-Ser(tBu)-Thr(tBu)-Cys(Trt)-Val-Leu-Gly-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    Fmoc-Ala-Ser(tBu)-Leu-Ser(tBu)-Thr(tBu)-Cys(Trt)-Val-Leu-Gly-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    H2N-Ala-Ser(tBu)-Leu-Ser(tBu)-Thr(tBu)-Cys(Trt)-Val-Leu-Gly-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-Linker-MBHA Resin

    Fmoc-Cys(Trt)-Ala-Ser(tBu)-Leu-Ser(tBu)-Thr(tBu)-Cys(Trt)-Val-Leu-Gly-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-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-Cys(Trt)-Ala-Ser(tBu)-Leu-Ser(tBu)-Thr(tBu)-Cys(Trt)-Val-Leu-Gly-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr(tBu)-Pro-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-Cys-Ala-Ser-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asp-Val-Gly-Ala-Gly-Thr-Pro-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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