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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.

Peptide H-YGGFLRRI-OH is a Research Peptide with significant interest within the field academic and medical research. Recent citations using H-YGGFLRRI-OH include the following: Lethal factor active-site mutations affect catalytic activity in vitro SE Hammond, PC Hanna - Infection and immunity, 1998 - Am Soc Microbiolhttps://journals.asm.org/doi/abs/10.1128/iai.66.5.2374-2378.1998 Improved 3D triple resonance experiments, HNN and HN (C) N, for HN and 15N sequential correlations in (13C, 15N) labeled proteins: application to unfolded SC Panchal, NS Bhavesh , RV Hosur - Journal of biomolecular NMR, 2001 - Springerhttps://link.springer.com/article/10.1023/A:1011239023422 In vivo detection of optically-evoked opioid peptide release R Al-Hasani , JMT Wong , OS Mabrouk , JG McCall - Elife, 2018 - elifesciences.orghttps://elifesciences.org/articles/36520 Application of HN (C) N to rapid estimation of 1J (N-Calpha) coupling constants correlated to Ã\x8fË\x86 torsion angles in proteins: implication to structural genomics NS Bhavesh , A Chatterjee, SC Panchal - and biophysical research , 2003 - Elsevierhttps://www.sciencedirect.com/science/article/pii/S0006291X03020898 Neuroprotective peptides and new strategies for ischemic stroke drug discoveries LV Dergunova, IB Filippenkov, SA Limborska - Genes, 2023 - mdpi.comhttps://www.mdpi.com/2073-4425/14/5/953 Identification of synaptic metabolites of dynorphin A (1-8) by electrospray ionization and tandem mass spectrometry L Prokai , AD Zharikova - Rapid communications in mass , 1998 - Wiley Online Libraryhttps://analyticalsciencejournals.onlinelibrary.wiley.com/doi/abs/10.1002/(SICI)1097-0231(19981130)12:22%3C1796::AID-RCM362%3E3.0.CO;2-7 Comparative distribution of neurons containing FLFQPQRFamide-like (morphine-modulating) peptide and related neuropeptides in the rat brain L Kivipelto, P Panula - European Journal of Neuroscience, 1991 - Wiley Online Libraryhttps://onlinelibrary.wiley.com/doi/abs/10.1111/j.1460-9568.1991.tb00078.x Secondary structure transitions and aggregation induced in dynorphin neuropeptides by the detergent sodium dodecyl sulfate L Hugonin, A Barth, A Graslund - Biochimica et Biophysica , 2008 - Elsevierhttps://www.sciencedirect.com/science/article/pii/S0005273608002198 12 Ion Mobility MALDI AS Woods , JA Schultz, SN Jackson - Ion Mobility Spectrometry , 2010 - books.google.comhttps://books.google.com/books?hl=en&lr=&id=jodz0-V-lUAC&oi=fnd&pg=PA257&dq=(%22YGGFLRR%5EI%22+OR+%22H-YGGFLRR%5EI-OH%22+OR+%22H-YGGFLRRI-OH%22+OR+%22YGGFLRRI%22)+AND+peptide&ots=3UR3ve9ptF&sig=AssaA5YRDtkVuJHhVzxHb8fEhrQ Sulfation, the Up-and-Coming Post-Translational Modification: Its Role and Mechanism in Protein-Protein Interaction AS Woods , HYJ Wang, SN Jackson - Journal of proteome , 2007 - ACS Publicationshttps://pubs.acs.org/doi/abs/10.1021/pr060529g Dynorphin A (1-8) in human placenta: amino acid sequence determined by tandem mass spectrometry A Agbas , MS Ahmed , W Millington, B Cemerikic - Peptides, 1995 - Elsevierhttps://www.sciencedirect.com/science/article/pii/019697819500013A

强啡肽的定义

强啡肽是一类内源性阿片肽，在大脑的许多不同部位产生，包括下丘脑、海马和脊髓，根据产生部位的不同，具有许多不同的生理作用。

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.

强啡肽的相关多肽

Dynorphins来源于前体蛋白proynorphin。当前强啡肽在加工过程中被前蛋白转化酶2（PC2）切割时，会释放出多种活性肽：强啡肽A、强啡肽B、“大强啡肽”和A/β-新强啡肽1。

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-endorphin【1】.

强啡肽的发现 

Dynophin于20世纪70年代中期在阿片受体和内源性阿片肽领域最重要的研究人员之一Avram Goldstein的实验室中被发现。Goldstein与日本生物化学家Shinro Tachibana合作进行了分子鉴定，以进行纯化，M.Hunkapiller和L.Hood进行了微测序。

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.

强啡肽的结构特点

从猪垂体中分离出一种4000道尔顿的强啡肽（也称为“大强啡肽”）。它有32个氨基酸，氨基末端有一个称为强啡肽a的十七肽（17个氨基酸序列），羧基末端有一种相关的十三肽（13个氨基酸序列，强啡肽B）。这两种肽由“处理信号”Lys-Arg【2】分离。

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-Arg【2】.  

强啡肽的作用机制

Dynorphins主要通过一种名为？的G蛋白偶联受体发挥作用？-阿片受体（KOR）【3】。尽管KOR是所有强啡肽的主要受体，但这些肽确实对µ-阿片受体（MOR）、d-阿片受体、N-甲基-d-天冬氨酸（NMDA）型谷氨酸受体和缓激肽受体有一定的亲和力。不同的强啡肽在受体上表现出不同的受体选择性和效力。强啡肽和强啡肽A都比强啡肽B更有效、更具选择性。强啡肽通过与多巴胺神经末梢上的KOR结合来减少多巴胺的释放，从而导致药物耐受和戒断症状。

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.

强啡肽的功能

Dynorphins调节疼痛反应。它们可以显著抑制吗啡或β-内啡肽诱导的镇痛作用【4】。Dynorphins抑制多巴胺的释放，这会抵消可卡因的愉悦作用【5】。它们通过控制食欲和昼夜节律来维持体内平衡【6】。除了在控制体重方面的作用外，还发现强啡肽可以调节体温【7】。

Dynorphins modulate pain response. They can significantly inhibit morphine- or beta-endorphin-induced analgesia【4】. Dynorphins inhibit dopamine release that would counter the pleasurable effects of cocaine【5】.  They are important in maintaining homeostasis through appetite control and circadian rhythms【6】. In addition to their role in weight control, dynorphins have also been found to regulate body temperature【7】.

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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