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N-terminal sequence of dynorphin A and the neoendorphins.

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.

脑啡肽(Enkephalins)的定义

脑啡肽是在大脑和内分泌组织中高水平发现的阿片类肽。新出现的含有脑啡肽的肽的主要种类似乎是完整的前体，前脑啡肽【1】。

Enkephalins are opioid peptides that are found at high levels in the brain and endocrine tissues. The major species of newly appearing enkephalin-containing peptide appears to be the intact precursor, proenkephalin 【1】.

脑啡肽(Enkephalins)相关肽

阿片肽构成了一大类小蛋白，它们与鸦片生物碱，吗啡和海洛因相似，能与细胞膜受体相互作用。阿片生物碱衍生物广泛用于镇痛和麻醉。最初的阿片肽家族是脑啡肽，强啡肽和内啡肽。在心脏中已经发现了来自这三个阿片肽家族的代表性肽。已克隆并测序了三种不同的阿片受体：mu（µ），delta（d）和kappa（？）【2】。

Opioid peptides constitute a large group of small proteins that interact with cell membrane receptors similarly to opiate alkaloids, morphine and heroin. Opiate alkaloid derivatives are extensively used for analgesia and anesthesia. The original opioid peptide families are enkephalins, dynorphins, and endorphins. Representative peptides from these three opioid peptide families have been found in the heart. Three different opiate receptors have been cloned and sequenced: mu (µ), delta (d), and kappa (?) 【2】.

脑啡肽(Enkephalins)的发现

Kosterlitz和Hughes在1975年发现了脑啡肽和内啡肽【3】。

Kosterlitz and Hughes discovered enkephalins and endorphins in 1975 【3】.

脑啡肽(Enkephalins)的结构特征

蛋氨酸-脑啡肽（Met-Enk）的氨基酸序列为酪氨酸-甘氨酸-甘氨酸-苯丙氨酸-蛋氨酸。前脑啡肽序列包含五肽Met-Enk的四个拷贝，leu-脑啡肽之一和Met- enk的两个扩展形式（Met- enk -arg 6 -phe 7和met- enk -arg 6 -gly 7 -leu 8）。成对的碱性氨基酸标记这些小肽从前体裂解。原脑啡肽由称为原激素转化酶的内蛋白水解酶加工，该酶在二元氨基酸位点识别并切割。最初的前脑啡肽处理开始于传输到高尔基体网络之前，并且过程很快。后续处理需要在高尔基体网络远端的酸性环境中进行。前脑啡肽对肽B具有快速裂解，而较慢的裂解产生其他中等大小的产物，其最终被裂解成五肽至八肽。在各种组织（肌肉，神经，内分泌）中发现的不同分子量的最终产物可能是由于切割序列的差异和加工所需的局部酶促条件所致【2】。

The amino acid sequence of methionine-enkephalin (Met-Enk) is tyrosine-glycine-glycine-phenylalanine-methionine. The proenkephalin sequence contains four copies of the pentapeptide Met-Enk, one of leu-enkephalin, and two extended forms of Met-enk (Met-enk-arg6-phe7 and met-enk-arg6-gly7-leu8). Pairs of basic amino acids mark these small peptides for cleavage from the precursor. Proenkephalin is processed by endoproteolytic enyzmes termed prohormone convertases, which recognize and cleave at dibasic amino acid sites. Initial proenkephalin processing starts before transport to the golgi network and are rapid. Later processing requires an acidic environment distal to the golgi network. Proenkephalin has a fast cleavage to peptide B, and slower cleavages yield other intermediate sized products that are cleaved ultimately to the penta to octapeptides. The different molecular-weight end products found in diverse tissues (muscle, neural, endocrine) may be due to variations in the cleavage sequence and local enzymatic conditions for processing【2】.

脑啡肽(Enkephalins)的作用方式

肽Met-Enk和Leu-Enk（阿片受体的内源性配体）起神经调节剂或神经递质的作用。脑啡肽在哺乳动物脑中最显着的作用是神经元放电速率的降低，并且已经表明这些肽是抑制性递质。脑啡肽抑制或增强了中枢神经元对几种假定的递质的反应，表明突触后的作用。还显示脑啡肽抑制K +诱导的去甲肾上腺素，多巴胺和乙酰胆碱从大鼠脑片的释放，表明突触前的作用。脑啡肽抑制豚鼠回肠中的肌间神经元的发射。这种抑制可能是由于脑啡肽的直接突触后作用导致神经元膜超极化【4】。为了实现其生物学功能，必须将脑啡肽从水相转运至其膜结合受体蛋白的富含脂质的环境。现已知道，Met-enk通过三种主要的亚型受体起作用，分别称为μ，d和β。-受体。虽然前两个受体亚型介导了Met-enk的经典阿片样物质作用，但据报道β受体参与了该肽的非阿片样物质作用，即对细胞生长的抑制作用【5】。

Pentapeptides Met-Enk and Leu-Enk, the endogenous ligands for the opiate receptor, function as neuromodulators or neurotransmitters. The most prominent action of enkephalins in the mammalian brain is depression of neuronal firing rate and it has been suggested that these peptides are inhibitory transmitters. The response of central neurones to several putative transmitter substances is depressed or enhanced by enkephalins, suggesting a postsynaptic action. It has also been shown that enkephalins suppress the K+-induced release of noradrenaline, dopamine and acetylcholine from rat brain slices, indicating a presynaptic effect. The firing of myenteric neurones in the guinea-pig ileum is inhibited by enkephalins. This inhibition is probably due to a direct postsynaptic action of the enkephalins resulting in a hyperpolarisation of the neuronal membrane 【4】. To achieve their biological function, enkephalins must be transported from an aqueous phase to the lipid-rich environment of their membrane bound receptor proteins. It is now known that Met-enk acts via three main subtypes of receptors referred to as µ, d and ? - receptors. While the first two receptor subtypes mediate the classic opioid effects of Met-enk, ?-receptors are reported to be involved in the non-opioid actions of the peptide, i.e. the inhibitory effect on the cell growth【5】.

脑啡肽(Enkephalins)的功能

前脑啡肽是神经肽的前体，在神经内分泌和神经系统中具有多种功能。激活后，发现T辅助淋巴细胞表达高水平的前脑啡肽mRNA，并分泌大量的Met-Enk神经肽，这可能表明了免疫系统和神经系统相互作用的轴【6】。脑啡肽引起抗伤害感受和增强的吗啡镇痛作用，但它们也阻碍了耐受性和身体依赖性的发展。除了其中枢和外周镇痛作用外，阿片类药物还可以调节免疫活性和细胞增殖。此外，众所周知，它们在不同的生理过程中具有重要作用，例如细胞分化和再生，炎症，癌症和血管生成以及镇痛作用【5】。

Proenkephalin is a precursor for neuropeptides with a variety of functions in the neuroendocrine and nervous systems. Upon activation, T-helper lymphocytes were found to express high levels of proenkephalin mRNA and to secrete large amounts of the Met-Enk neuropeptide, perhaps indicating an axis by which the immune and nervous systems interact 【6】. Enkephalins cause antinociception and potentiated morphine analgesia but they also block the development of tolerance and physical dependence. In addition to their central and peripheral antinociceptive function, opioids can modulate immune activity and cell proliferation. Moreover it is known that they have significant role in different physiological processes like cell differentiation and regeneration, inflammation, cancer and angiogenesis and analgesia effects 【5】.

脑啡肽(Enkephalins)的参考文献

1、Fleminger G, Lahm HW, Udenfriend S (1984).Changes in rat adrenal catecholamines and proenkephalin metabolism after denervation. PNAS., 81(11):3587-3590.
2、Barbara A. Barron. 2000. Cardiac Opioids. Proceedings of the Society for Experimental Biology and Medicine, 224:1-7.
2、Fratta W, Yang HY, Hong J, Costa E (1977). Stability of Met-enkephalin content in brain structures of morphine-dependent or foot shock-stressed rats. Nature, 268(5619):452-453.
4、Wouters W, Den Bercken JV (1979). Hyperpolarisation and depression of slow synaptic inhibition by enkephalin in frog sympathetic ganglion. Nature, 277:53-54.
5、Tsanova A, Dacheva D, Penchev V, Georgiev G, Pajpanova T, Golovinski E, Lalchev Z (2009). Comparative study of the interaction between synthetic methionine-enkephalin and monolayers of zwitterionic and negatively 6、charged phospholipids. Biotechnol & Biotechnol., 23:463-466.
7、Rattner A, Korner M, Rosen H, Baeuerle PA, Citri Y (1991). Nuclear factor Kappa B activates proenkephalin transcription in T lymphocytes. Molecular and Cellular Biology, 11(2):1017-1022.

多肽H2N-Tyr-Ala-Gly-Phe-Leu-COOH的合成步骤：

1、合成CTC树脂：称取1.63g CTC Resin（如初始取代度约为0.7mmol/g）和1.37mmol Fmoc-Leu-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3.42mmol 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-Leu-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-Leu-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3.42mmol Fmoc-Phe-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6.85mmol DIPEA，3.25mmol 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-Phe-Leu-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-Phe-Leu-CTC Resin

Fmoc-Gly-Phe-Leu-CTC Resin

H2N-Gly-Phe-Leu-CTC Resin

Fmoc-Ala-Gly-Phe-Leu-CTC Resin

H2N-Ala-Gly-Phe-Leu-CTC Resin

Fmoc-Tyr(tBu)-Ala-Gly-Phe-Leu-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)-Ala-Gly-Phe-Leu-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-Ala-Gly-Phe-Leu-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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