胎盘组织提取物中主要的阿片肽。Dynorphin A (1-8) 是 kappa 受体最可能的天然配体。
编号：117068
CAS号：75790-53-3
单字母：H2N-YGGFLRRI-OH
Dynorphin A (1-8) 是胎盘组织提取物中主要的阿片肽。Dynorphin A (1-8) 是 kappa 受体最可能的天然配体。Dynorphin A (1-8) 抑制 3H-Bremazocine 与纯化的 kappa 受体的结合 (IC50=303 nM)。
Dynorphin A (1-8) is the predominant opioid peptide identified in placental tissue extracts. Dynorphin A (1-8) is the most likely natural ligand of the kappa receptor. The binding of 3H-Bremazocine to the purified kappa receptor is inhibited by Dynorphin A (1-8) (IC50=303 nM).
Dynorphins是一类阿片肽，由前体蛋白proynorphin产生。在被前蛋白转化酶2（PC2）切割后，释放出多种活性肽：强啡肽A、强啡肽B和α/β-新内啡肽。Dynorphins主要通过阿片受体（KOR）发挥作用，KOR是一种G蛋白偶联受体。强啡肽已被证明是疼痛反应的调节剂，参与药物成瘾和食欲控制。
Dynorphins are a class of opioid peptides that arise from the precursor protein prodynorphin. Upon cleavage by proprotein convertase 2 (PC2), multiple active peptides are released: dynorphin A, dynorphin B, and α/β-neo-endorphin. Dynorphins exert their effects primarily through the Κ-opioid receptor (KOR), a G-protein-coupled receptor. Dynorphin has been shown to be a modulator of pain response, involved in drug addiction and appetite control.
Definition
Dynorphins are a class of endogenous opioid peptides produced in many different parts of the brain, including the hypothalamus, the hippocampus and the spinal cord, and have many different physiological actions, depending upon the site of production.
Related peptides
Dynorphins arise from the precursor protein prodynorphin. When prodynorphin is cleaved during processing by proprotein convertase 2 (PC2), multiple active peptides are released: dynorphin A, dynorphin B, “big dynorphin” and a/ß-neo-endorphin1.
Discovery
Dynophin was discovered in the mid 1970's in the laboratory of Avram Goldstein, one of the most important researchers in the field of opioid receptors and endogenous opioid peptides. The molecular identification was achieved by Goldstein in collaboration with the Japanese biochemist, Shinro Tachibana for purification, and M. Hunkapiller and L. Hood, who performed the microsequencing.
Structural characteristics
A 4,000-dalton dynorphin (also called the “Big dynorphin”) was isolated from porcine pituitary. It has 32 amino acids, with a heptadecapeptide (17 amino acid sequence), called dynorphin A, at its amino terminus and a related tridecapeptide (13 amino acid sequence), dynorphin B, at its carboxyl terminus. The two peptides are separated by the "processing signal" Lys-Arg2.
Mechanism of action
Dynorphins primarily exert their effects through a G-protein coupled receptor called the ?-opioid receptor (KOR) 3. Although KOR is the primary receptor for all dynorphins, the peptides do have some affinity for the µ-opioid receptor (MOR), d-opioid receptor (DOR), N-methyl-D-aspartic acid (NMDA)-type glutamate receptor, and bradykinin receptor. Different dynorphins show different receptor selectivities and potencies at receptors. Both big dynorphin and dynorphin A are more potent and more selective than dynorphin B. Dynorphin decreases dopamine release by binding to KORs on dopamine nerve terminals, which leads to drug tolerance and withdrawal symptoms.
Functions
Dynorphins modulate pain response. They can significantly inhibit morphine- or beta-endorphin-induced analgesia4. Dynorphins inhibit dopamine release that would counter the pleasurable effects of cocaine5. They are important in maintaining homeostasis through appetite control and circadian rhythms6. In addition to their role in weight control, dynorphins have also been found to regulate body temperature7.
References
1. Day, R., Lazure, C., Basak, A., Boudreault, A., Limperis, P., Dong, W., et al. (1998). Prodynorphin processing by proprotein convertase 2. Cleavage at single basic residues and enhanced processing in the presence of carboxypeptidase activity. J Biol. Chem., 273(2), 829-836.
2. W Fischli, A Goldstein, M W Hunkapiller, and L E Hood (1982). Isolation and amino acid sequence analysis of a 4,000-dalton dynorphin from porcine pituitary. PNAS, 79 (17), 5435-5437.
3. Nyberg, F. & Hallburg, M. (2007). Neuropeptides in hyperthermia. Progress in brain research, 162:277-93.
4. FC Tulunay, MF Jen, JK Chang, HH Loh and NM Lee, (1981). Possible regulatory role of dynorphin on morphine- and beta-endorphin- induced analgesia. American Society for Pharmacology and Experimental Therapeutics, 219 (2), 296-298.
5. Clavin, W. (2005). Dynorphin: Nature’s Own Antidote to Cocaine (and Pleasure?).
6. Przewlocki, R., Lason, W., Konecka, A. M., Gramsch, C., Herz, A., & Reid, L. D. (1983). The opioid peptide dynorphin, circadian rhythms, and starvation. Science, 219(4580), 71-73.
7. Xin, L., Geller, E. B., & Adler, M. W. (1997). Body temperature and analgesic effects of selective mu and kappa opioid receptor agonists microdialyzed into rat brain. Journal of Pharmacology and Experimental Therapeutics, 281(1), 499-507.
Agbas A, Ahmed MS, Millington W, et al. Dynorphin A(1-8) in human placenta: amino acid sequence determined by tandem mass spectrometry. Peptides. 1995;16(4):623-627. : https://pubmed.ncbi.nlm.nih.gov/7479294/
Ahmed MS, Zhou DH, Cavinato AG, Maulik D. Opioid binding properties of the purified kappa receptor from human placenta. Life Sci. 1989;44(13):861-871. : https://pubmed.ncbi.nlm.nih.gov/2538693/
多肽H2N-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-COOH的合成步骤：
1、合成CTC树脂：称取0.52g CTC Resin（如初始取代度约为0.62mmol/g）和0.39mmol Fmoc-Ile-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0.97mmol 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-Ile-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-Ile-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0.97mmol Fmoc-Arg(Pbf)-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1.93mmol DIPEA，0.92mmol 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-Arg(Pbf)-Ile-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-Arg(Pbf)-Ile-CTC Resin
Fmoc-Arg(Pbf)-Arg(Pbf)-Ile-CTC Resin
H2N-Arg(Pbf)-Arg(Pbf)-Ile-CTC Resin
Fmoc-Leu-Arg(Pbf)-Arg(Pbf)-Ile-CTC Resin
H2N-Leu-Arg(Pbf)-Arg(Pbf)-Ile-CTC Resin
Fmoc-Phe-Leu-Arg(Pbf)-Arg(Pbf)-Ile-CTC Resin
H2N-Phe-Leu-Arg(Pbf)-Arg(Pbf)-Ile-CTC Resin
Fmoc-Gly-Phe-Leu-Arg(Pbf)-Arg(Pbf)-Ile-CTC Resin
H2N-Gly-Phe-Leu-Arg(Pbf)-Arg(Pbf)-Ile-CTC Resin
Fmoc-Gly-Gly-Phe-Leu-Arg(Pbf)-Arg(Pbf)-Ile-CTC Resin
H2N-Gly-Gly-Phe-Leu-Arg(Pbf)-Arg(Pbf)-Ile-CTC Resin
Fmoc-Tyr(tBu)-Gly-Gly-Phe-Leu-Arg(Pbf)-Arg(Pbf)-Ile-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-Tyr(tBu)-Gly-Gly-Phe-Leu-Arg(Pbf)-Arg(Pbf)-Ile-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-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-COOH。结构图见产品结构图。
切割液选择：1）TFA：H2O=95%：5%、TFA：H2O=97.5%：2.5%
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
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