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Deltorphin，YmFHLMD酰胺，一种首先从南美蛙Phyllomedusa sauvagei的皮肤提取物中分离出来的肽，对应于预测的前吗啡七肽。Deltorphin是δ-阿片受体的一种高效且完全选择性的激动剂。

Deltorphin, YmFHLMD-amide, a peptide first isolated from skin extracts of the South American frog Phyllomedusa sauvagei, corresponds to the predicted prodermorphin heptapepeptide. Deltorphin acts as a highly potent and fully selective agonist for the δ-opioid receptor.

含蛋氨酸 2 的 deltorphin 在关键位置进行蛋氨酸取代，影响多肽核心疏水相互作用。该修饰细微改变受体亲和力与结构动力学。其理化特征适用于阿片受体通路与构象偏好探究。研究者将其用于探究多肽折叠与配体特异性信号事件。

(Met2)-Deltorphin features a methionine substitution at a key position influencing hydrophobic interactions within the peptide core. The modification subtly alters receptor affinity and structural dynamics. Its physicochemical traits allow investigation of opioid receptor pathways and conformational preferences. Researchers employ it to explore peptide folding and ligand-specific signaling events.

Deltorphin (Deltorphin A; Dermenkephalin) 是一种有生物活性的肽。Deltorphin A 肽是从南美青蛙 (Phyllomedusa sauvagei) 的皮肤提取物中分离出来的。 Deltorphin A 是δ-阿片受体的有效选择性激动剂。
 

皮啡肽(Dermorphin)的定义

在属于Phyllomedusinae亚科的南美蛙类皮肤中发现了一组阿片肽。从这些蛙类的几个物种中分离出的第一种肽是dermorphin，该肽对µ型阿片受体表现出高亲和力和高选择性【1】。

A group of opioid peptides has been discovered in the skin of South American frogs belonging to the subfamily Phyllomedusinae. The first peptide isolated from several species of these frogs was dermorphin which was shown to have high affinity and selectivity for µ-type opioid receptors 【1】.

皮啡肽(Dermorphin)的相关多肽

在发现dermorphins后，研究人员随后从双色叶尾蜥的皮肤中分离出了另外两种对δ受体亲和力更高的肽。与dermorphins类似，这些肽的第二位氨基酸也是D-丙氨酸，因此被命名为[D-Ala2]deltorphins I和II 【1】。Dermorphins和deltorphins都是七肽，其共同的氨基端序列为Tyr-D-Ala-Phe【2】。

After the discovery of dermorphins, two additional peptides with even higher affinity for the d receptor were subsequently isolated from the skin of Phyllomedusa bicolor. Like dermorphin, these peptides contain D-alanine as the second amino acid and they have been termed [D-Ala2] deltorphins I and II 【1】.  Dermorphins and deltorphins are heptapeptides with the common amino terminal sequence Tyr-D-Ala-Phe 【2】.

皮啡肽(Dermorphin)的发现

1981年，Montecucchi等人从阿根廷青蛙Phyllomedusa sauvagei的皮肤中提取了一种名为dermorphin的七肽，该七肽优先结合µ型阿片受体。Erspamer等人于1989年报道了从P. sauvagei的皮肤中分离出deltorphins【2】。

In 1981, Montecucchi et al., extracted from the skin of the Argentinian frog Phyllomedusa sauvagei a heptapeptide named dermorphin, which preferentially binds to µ-type opioid receptors. Erspamer et al., in 1989 reported the isolation of deltorphins from the skin of P. sauvagei 【2】.

皮啡肽(Dermorphin)的结构特征

这些是具有共同氨基末端序列Tyr-D-Ala-Phe的七肽。由于这一初始序列是保守的，因此推测它对于与阿片受体的µ和d位点结合是必需的。与地吗啡不同，地吗啡肽在位置4上有一个带电氨基酸，在地吗啡的情况下是组氨酸，在[D-Ala2]-地吗啡的情况下是天冬氨酸或谷氨酸。该位置上带正电或负电氨基酸的存在对结合特性几乎没有影响。因此，地吗啡肽C末端区域的其他特征很可能对观察到的受体选择性至关重要。地吗啡肽是灵活的线性肽。它们对d位点的亲和力比合成脑啡肽类似物2高10至200倍。地吗啡肽含有氨基末端序列Tyr-Ala-Phe，该序列前有一个典型的前激素加工信号Lys-Arg。地吗啡肽序列之后是更复杂的加工序列Gly-Glu-Ala-Lys-Lys，其中甘氨酸是形成羧基末端酰胺所必需的。在来自Ph. sauvagei的地吗啡前体中也发现了相同的侧翼序列【1】。

These are heptapeptides with the common aminoterminal sequence Tyr-D-Ala-Phe. Since this initial sequence is conserved, it is presumed that it is necessary for binding to both µ and d sites of opioid receptors. Unlike dermorphin, the deltorphins have a charged amino acid in position 【4】, namely histidine in the case of deltorphin and aspartic or glutamic acid in the case of [D-Ala2]-deltorphins. The presence of positively or negatively charged amino acids in this position has little influence on the binding characteristics. It thus seems likely that other features in the C-terminal regions of deltorphins are essential for the observed receptor selectivity. Deltorphins are flexible linear peptides. They show an affinity for d sites 10 to 200 times higher than that of the synthetic enkephalin analogue 【2】. Deltorphins contain the amino terminal sequence Tyr-Ala-Phe, which is preceded by the typical prohormone processing signal Lys-Arg. Following the deltorphin sequence is the more complex processing sequence Gly-Glu-Ala-Lys-Lys, the glycine being required for the formation of the carboxyl-terminal amide. The same flanking sequences have also been found in the dermorphin precursors from Ph. sauvagei 【1】.

类似物：D-丙氨酸2，谷氨酸4] - 和 [D-丙氨酸2，天冬氨酸4] 德尔托啡酰胺，其L异构体，以及1-(3,5-二碘酪氨酰基)[D-丙氨酸2] 德尔托啡是采用固相合成法制备的德尔托啡的合成类似物【2】。

Analogs: D-Ala2, Glu4] - and [D-Ala2, Asp4] deltorphinamides, their L isomers, and 1-(3, 5-diiodotyrosyl) [D-Ala2] deltorphins are synthetic analogs of deltorphins prepared by solid-phase synthesis 【2】.

地吗啡类似物，包括[Lys7-NH2]地吗啡、[Arg7-NH2]地吗啡和[Asn7-NH2]地吗啡，均表现出强效的镇痛作用。[Lys7-NH2]地吗啡的镇痛效果持续时间比地吗啡本身更长【3】。

Dermorphin analogs, [Lys7-NH2] dermorphin, [Arg7-NH2] dermorphin, and [Asn7-NH2] dermorphin behave as potent analgesic agents. The antinociception effect of [Lys7-NH2] dermorphin lasts longer than that of dermorphin itself 【3】.

在另一项研究中，分析了在冷（4–7°C）、热中性（27–28°C）和热（31–33°C）环境下，以0.5 mg/kg剂量腹腔注射经立体化学修饰的dermorphin类似物（Tyr-D-Ala-Phe-Gly-Tyr-Hyp-Ser-NH2、Tyr-D-Ala-Phe-Gly-Tyr-[D-Pro]-Ser-NH2、Tyr-D-Ala-Phe-Gly-Tyr-[dehydro-Pro]-Ser-NH2和Tyr-D-Ala-Phe-Gly-Tyr-[D-dehydro-Pro]-Ser-NH2）的影响。氨基酸残基脯氨酸（位置6，Pro6）的立体化学修饰被证明会诱导肽的体温调节效应发生特定变化。用DPro6替代Pro6会产生最显著的结果：在冷环境下，它显著减弱了dermorphin的体温调节效应；在热环境下，则完全抵消了dermorphin的体温调节效应；在热中性条件下，则逆转了dermorphin特有的体温调节反应。由此获得的数据表明，Pro6残基在实现dermorphin的这种生理活性中起着重要作用[4]。

In another study, the influence of dermorphin analogs with stereochemical modification of the amino acid residue proline in position 6 (Pro6), Tyr-D-Ala-Phe-Gly-Tyr-Hyp-Ser-NH2, Tyr-D-Ala-Phe-Gly-Tyr-[D-Pro]-Ser-NH2, Tyr-D-Ala-Phe-Gly-Tyr-[dehydro-Pro]-Ser-NH2, and Tyr-D-Ala-Phe-Gly-Tyr-[D-dehydro-Pro]-Ser-NH2, was analysed after their intraperitoneal injection at 0.5 mg/kg dose in the cold (4–7°C), thermoneutral (27–28°C), and hot (31–33°C) environment. Stereochemical modifications of amino acid residue Pro6 proved to induce specific changes in the thermoregulatory effect of the peptide. Substitution of DPro6 for Pro6 has the most dramatic consequences: it considerably attenuated the thermoregulatory effect of dermorphin in the cold environment, cancelled it in the hot environment, and inverted the dermorphin-specific thermoregulatory response in thermoneutral conditions. The data thus obtained indicate the important role of Pro6 residue in realization of this physiological activity of dermorphins 【4】.

皮啡肽(Dermorphin)的作用方式

皮肤素和德尔托啡分别对µ型和d型阿片受体具有高亲和力和高选择性【2】。

Dermorphin and deltorphins have high affinity and selectivity for µ- and d-type opioid receptors, respectively 【2】.

皮啡肽(Dermorphin)的功能

在啮齿动物和灵长类动物（包括人类）中，δ-内啡肽是一类强效镇痛剂。部分δ-内啡肽可进入血脑屏障，并在外周给药后产生中枢镇痛作用。δ-内啡肽家族还包括μ1-阿片受体选择性激动剂，这些激动剂能产生强烈的阿片类镇痛作用，但会刺激肺通气。对长期暴露于δ-内啡肽的大鼠和小鼠进行的实验表明，它们不仅具有比吗啡更高的镇痛效果和效力，而且与吗啡相比，产生耐受性、依赖性和阿片类药物副作用的可能性更低【5】。[3H][D-Ala2]δ-内啡肽I是结合研究中有价值的探针，因为其亲和力和选择性是迄今为止已知的所有δ-选择性配体中最高的[2]。

Dermorphin peptides are potent analgesics in rodents and primates, including man. Some dermorphins can enter the blood-brain barrier and produce central antinociception after peripheral administration. The dermorphin family also includes µ 1-opioid receptor selective agonists that produce intense opioid analgesia, but stimulate pulmonary ventilation. Experiments in rats and mice chronically exposed to dermorphins have shown that not only do they have higher antinociceptive efficacy and potency than morphine, but they are also less likely than morphine to produce tolerance, dependence and opiate side effects 【5】.   [3H][D-Ala2] deltorphin I is a valuable probe for binding studies, since its affinity and selectivity are the highest of all the d -selective ligands known to date 【2】.

皮啡肽(Dermorphin)的相关文献

1. K Richter K, R Egger R, L Negri L, R Corsi R, C Severini C,  Kreil G (1990). cDNAs encoding [D-Ala2]deltorphin precursors from skin  of Phyllomedusa bicolor also contain genetic information for three dermorphin-related opioid peptides (amphibian skin peptides / precursors). PNAS., 87(12)4836-4839.
2. Erspamer V, Melchiorri P, Falconieri-Erspamer G, Negri L, Corsi R, Severini C, Barra D,  Simmaco M Kreil G (1989). Deltorphins: A family of naturally occurring peptides with high affinity and selectivity for d opioid binding sites (amphibian skin peptides/mouse vas deferens assay/receptor binding assay). PNAS., 86:188-5192.
3. Negri L, Erspamer GF, Severini C, Potenza RL, Melchiorri P, Erspamer V (1992). Dermorphin-related peptides from the skin of Phyllomedusa bicolor and their amidated analogs activate two µ opioid receptor subtypes that modulate antinociception and catalepsy in the rat. PNAS., 89:7203-7207.
4. Emel’yanova TG, Usenko AB, Bonartsev AP,  Kamenskii AA, Guzevatykh LS,  Andreeva LA, Alfeeva L, Myasoedov NF (2002). Effect of Dermorphin Analogs on Thermoregulation of Rats under Various Thermal Conditions. Biology Bulletin, 29(3):284–289.
5. Melchiorri P, Negri L. (1996). The dermorphin peptide family. Gen Pharmacol., 27(7):1099-1107.

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.

很多蛋白在细胞中非常容易被降解，或被标记，进而被选择性地破坏。但含有部分D型氨基酸的多肽则显示了很强的抵抗蛋白酶降解能力。

多肽H2N-Tyr-DMet-Phe-His-Leu-Met-Asp-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2.92g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2.63mmol Fmoc-Asp(OtBu)-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5.26mmol DIPEA，2.5mmol 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-Asp(OtBu)-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-Asp(OtBu)-Linker-MBHA Resin

Fmoc-Met-Asp(OtBu)-Linker-MBHA Resin

H2N-Met-Asp(OtBu)-Linker-MBHA Resin

Fmoc-Leu-Met-Asp(OtBu)-Linker-MBHA Resin

H2N-Leu-Met-Asp(OtBu)-Linker-MBHA Resin

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

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

Fmoc-Phe-His(Trt)-Leu-Met-Asp(OtBu)-Linker-MBHA Resin

H2N-Phe-His(Trt)-Leu-Met-Asp(OtBu)-Linker-MBHA Resin

Fmoc-DMet-Phe-His(Trt)-Leu-Met-Asp(OtBu)-Linker-MBHA Resin

H2N-DMet-Phe-His(Trt)-Leu-Met-Asp(OtBu)-Linker-MBHA Resin

Fmoc-Tyr(tBu)-DMet-Phe-His(Trt)-Leu-Met-Asp(OtBu)-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-Tyr(tBu)-DMet-Phe-His(Trt)-Leu-Met-Asp(OtBu)-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-Tyr-DMet-Phe-His-Leu-Met-Asp-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

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