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[Pyr1]-Apelin-13 是一种高效的，选择性的内源性 apelin 受体激动剂。
[Pyr1]-Apelin-13 is a highly potent, selective endogenous apelin receptor (APJ) agonist.

[Pyr1]-Apelin-13 improves locomotor activity and reduces pain symptoms, cavity size and caspase-3 levels in rats.

定义

首先从牛胃组织提取物中分离出的肽Apelin是人G蛋白偶联的孤儿APJ受体的内源性配体。该配体以APJ内源性配体命名为“ Apelin”。

发现

在1993年，O'Dowd等人。确定了G蛋白偶联受体的基因，称为APJ，与血管紧张素受体（AT-1）^1的同源性约为30％。因此，直到1998年Tatemoto等人时，APJ仍是“孤立的”受体。发现了肽apelin。这些作者发现，牛胃提取物中富含肽的级分可促进表达APJ受体的细胞的细胞外酸化速率。通过监视此活性，他们纯化了36个氨基酸的肽，称为apelin ²，并进行了测序。

结构特点

Apelin是源自77-aa前体prepro apelin的36-aa肽，已从人和动物克隆了cDNA。在所有物种 中，C末端区域的23个氨基酸序列都是保守的，对生物学活性至关重要。77-aa prepro apelin包含许多碱性氨基酸对（Arg-Arg，Arg-Lys）是内肽酶的蛋白水解切割位点。检测到的内源性形式（通过色谱分离和免疫测定）主要包括Apelin – 36，– 17，– 13，以及在翻译后通过焦谷氨酸[Pyr1 ]。该修饰赋予对肽酶²降解的抗性^。 

作用机理

apelin信号的转导途径取决于与耦合到APJ受体的G蛋白的相互作用，而与Ras蛋白无关。尽管取决于蛋白激酶C（PKC）。如果PKC，磷脂酶C，Na ⁺ -H ⁺和Na ⁺ -Ca ²⁺交换子被抑制，则apelin的作用会显着降低（正性肌力作用）。

除腺苷酸环化酶抑制途径外，在PKC依赖性过程中，apelin还通过PTX（百日咳毒素）敏感的G蛋白激活ERKs（细胞外调节激酶）途径。apelin还通过两种机制激活内皮细胞的增殖控制：一种是ERK依赖性的，另一种是PI3K依赖性的。PI3K / Akt似乎负责磷酸化和内皮一氧化氮合酶的活化，这对于apelin血管活性作用是必不可少的³。

功能

孤儿受体APJ的新型内源性配体Apelin调节心脏收缩力-Apelin在心脏中显示高水平的mRNA表达。在离体的灌流大鼠心脏中，输注apelin会产生剂量依赖性的正性肌力作用。用U-73122抑制磷脂酶C和用stauzrosporine抑制蛋白激酶C，并显着减弱apelin引起的肌力作用⁴。

新型肽apelin通过一氧化氮依赖性机制降低血压-麻醉大鼠中apelin-13和apelin-36的剂量为10 nmol / kg给药后平均动脉压降低了11 +/- 4，并且5 +/- 4毫米汞柱，分别²。

Apelin水平免疫反应在大鼠下丘脑和垂体- Apelin水平-immunoreactive（IR）神经元是在丘脑核，即，视上（SON），室旁（PVN）核尤其丰富。这表明，Apelin水平含的SON和PVN，胞体如精氨酸加压素（AVP） -和催产素（OXY）含大细胞神经元，向垂体神经叶项目。这两种神经激素从垂体后叶释放到全身循环中，控制体液平衡和乳汁排出/子宫收缩。也有报道说，apelin IR与SON和PVN巨细胞神经元群体中的神经物理素I共定位，表明apelin由OXY神经元⁵表示。

集中施用apelin-13对大鼠的影响-前丘脑素和APJ mRNA在下丘脑区域发现，已知对调节食物和水的摄入量以及垂体激素的释放很重要。大鼠脑室内给予焦谷氨酰化的apelin-13对食物的摄入影响很小，但剂量依赖性地增加了饮酒行为和饮水量。与盐水对照⁶相比，Apelin-13的饮水量增加，血浆ACTH和皮质酮水平显着提高，血浆催乳素，LH和FSH（内分泌轴）降低。

参考：

1.     O’Dowd, Heiber, Chan A, Heng, Tsui, Kennedy JL, Petronis A (1993). A human gene that shows identity with the gene encoding the angiotensin receptor is located on chromosome 11. Gene.,136:355–360.

2.     Tatemoto K, Hosoya, Habata, Fujii R, Kakegawa T, Kawamata Y, Hinuma S, Kitada, Kurokawa T, Onda (1998). Isolation and characterization of a novel endogenous peptide ligand for the human APJ receptor. Biochem. Biophys. Res. Commun., 251:471–476

3.     Ricardo Ladeiras-Lopes, João Ferreira-Martins, Adelino F. Leite-Moreira (2008). The Apelinergic System: The Role Played in Human Physiology and Pathology and Potential Therapeutic Applications. Arq. Bras. Cardiol., 90(5): 343-349.

4.     Szokodi I, Tavi, Foldes G, Voutilainen-Myllyla S, Tokola H (2002). Apelin, the novel endogenous ligand of the orphan receptor APJ, regulates cardiac contractility. Circ Res., 91 (5): 434-40.

5.     Brailoiu C, Dun SL, Yang J, Ohsawa M, & Dun NJ (2002). Apelin-immunoreactivity in the rat hypothalamus and pituitary. Neurosci. Lett. 327 , 193–197.

6.     Taheri S, Murphy K, Cohen M, Sujkovic E, Kennedy A, Dhillo W, Dakin C, Sajedi A, Ghatei M, Bloom S (2002). The effects of centrally administered apelin-13 on food intake, water intake and pituitary hormone release in rats. Biochem. Biophys. Res. Commun., 291: 1208–1212.

下面为英文形式。

Definition

Apelin, a peptide first isolated from bovine stomach tissue extracts, is the endogenous ligand of the human G protein-coupled orphan APJ receptor. The ligand was named “Apelin” after APJ endogenous ligand.

Discovery

In 1993, O’Dowd et al. identified the gene for G-protein coupled receptor, named APJ, which shows about 30 % homology to the angiotensin receptor (AT-1)1. Thus, APJ remained an “orphaned” receptor until 1998 when Tatemoto et al. discovered the peptide apelin. These authors found that peptide-enriched fractions of bovine stomach-extracts promoted extra-cellular acidification rates of cells expressing the APJ receptor. By monitoring this activity, they purified, sequenced a 36 amino acid peptide named apelin2.

Structural characteristics

Apelin is a 36-aa peptide derived from a 77-aa precursor, preproapelin, for which cDNAs have been cloned from humans and animals. In all species a sequence of 23 amino acids in the C-terminal region is conserved, and is critical for biological activity.77-aa preproapelin contains a number of basic amino acid pairs (Arg – Arg, Arg – Lys) are the proteolytic cleavage site for endopeptidase. The endogenous forms detected (by chromatographic separation and immunoassay) comprise predominantly Apelin – 36, – 17, – 13 and following post translation modification by enzymatic conversion of N - terminal glutamate by pyroglutamate [Pyr1 ]. This modification confers resistance to degradation by peptidase2. 

Mechanism of action

The transduction pathways for apelin signal depends on the interaction with a G-protein coupled to the APJ receptor, independently of Ras protein; although dependent on Protein Kinase C (PKC). If PKC, phospholipase C, the Na+-H+ and Na+-Ca2+ exchanger are inhibited, apelin effects significantly reduces (positive inotropic effect).

In addition to adenyl cyclase inhibition pathway, apelin activates ERKs (extracellular regulated kinases) pathways through a PTX (pertussis toxin) sensitive G-protein, in a PKC-dependent process. Endothelial cells proliferation control is also activated by apelin through two mechanisms: one is ERK-dependent and the other is PI3K-dependent. PI3K/Akt seems to be responsible for the phosphorilation and resulting activation of endothelial nitric oxide synthase, which is indispensable for apelin vasoactive effects3. 
Functions

Apelin, the novel endogenous ligand of the orphan receptor APJ, regulates cardiac contractility- apelin exhibit high levels of mRNA expression in the heart. In isolated perfused rat hearts, infusion of apelin induced a dose-dependent positive inotropic effect. Inhibition of phospholipase C with U-73122 and suppression of protein kinase C with stauzrosporine and markedly attenuated the apelin-induced inotropic effect4.

The novel peptide apelin lowers blood pressure via a nitric oxide-dependent mechanism - mean arterial pressure after the administration of apelin-13, and apelin-36 in doses of 10 nmol/kg in anaesthetized rats was reduced by 11+/-4, and 5+/-4 mm Hg, respectively2.

Apelin-immunoreactivity in the rat hypothalamus and pituitary- Apelin-immunoreactive (IR) neurons are particularly abundant in hypothalamic nuclei, i.e., the supraoptic (SON), paraventricular (PVN) nuclei. It suggests that apelin-containing cell bodies of the SON and PVN, like arginine vasopressin (AVP) - and oxytocin (OXY)-containing magnocellular neurons, project toward the neural lobe of the pituitary. These two neurohormones released from posterior-pituitary into systemic circulation, control fluid balance and milk ejection/uterus contractility. Also it has been reported that apelin IR colocalizes with neurophysin-I in a population of SON and PVN magnocellular neurons, indicating that apelin is expressed by OXY neurons5.

The effects of centrally administered apelin-13 on rats - Preproapelin and APJ mRNA are found in hypothalamic regions known to be important in the regulation of food and water intake, and pituitary hormone release. The intracerebroventricular administration of pyroglutamylated apelin-13 on rats had little effect on food intake, but dose-dependently increased drinking behaviour and water intake. Apelin-13 increased water intake and also significantly increased plasma ACTH and corticosterone and decreased plasma prolactin, LH and FSH (endocrine axes), compared to saline control6.

1.     O’Dowd, Heiber, Chan A, Heng, Tsui, Kennedy JL, Petronis A (1993). A human gene that shows identity with the gene encoding the angiotensin receptor is located on chromosome 11. Gene.,136:355–360.

2.     Tatemoto K, Hosoya, Habata, Fujii R, Kakegawa T, Kawamata Y, Hinuma S, Kitada, Kurokawa T, Onda (1998). Isolation and characterization of a novel endogenous peptide ligand for the human APJ receptor. Biochem. Biophys. Res. Commun., 251:471–476

3.     Ricardo Ladeiras-Lopes, João Ferreira-Martins, Adelino F. Leite-Moreira (2008). The Apelinergic System: The Role Played in Human Physiology and Pathology and Potential Therapeutic Applications. Arq. Bras. Cardiol., 90(5): 343-349.

4.     Szokodi I, Tavi, Foldes G, Voutilainen-Myllyla S, Tokola H (2002). Apelin, the novel endogenous ligand of the orphan receptor APJ, regulates cardiac contractility. Circ Res., 91 (5): 434-40.

5.     Brailoiu C, Dun SL, Yang J, Ohsawa M, & Dun NJ (2002). Apelin-immunoreactivity in the rat hypothalamus and pituitary. Neurosci. Lett. 327 , 193–197.

6.     Taheri S, Murphy K, Cohen M, Sujkovic E, Kennedy A, Dhillo W, Dakin C, Sajedi A, Ghatei M, Bloom S (2002). The effects of centrally administered apelin-13 on food intake, water intake and pituitary hormone release in rats. Biochem. Biophys. Res. Commun., 291: 1208–1212.

多肽Pyr-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe-COOH的合成步骤：

1、合成CTC树脂：称取2.48g CTC Resin（如初始取代度约为0.62mmol/g）和1.85mmol Fmoc-Phe-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4.61mmol 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-Phe-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-Phe-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4.61mmol 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9.23mmol DIPEA，4.38mmol 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-Phe-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-Pro-Phe-CTC Resin

Fmoc-Met-Pro-Phe-CTC Resin

H2N-Met-Pro-Phe-CTC Resin

Fmoc-Pro-Met-Pro-Phe-CTC Resin

H2N-Pro-Met-Pro-Phe-CTC Resin

Fmoc-Gly-Pro-Met-Pro-Phe-CTC Resin

H2N-Gly-Pro-Met-Pro-Phe-CTC Resin

Fmoc-Lys(Boc)-Gly-Pro-Met-Pro-Phe-CTC Resin

H2N-Lys(Boc)-Gly-Pro-Met-Pro-Phe-CTC Resin

Fmoc-His(Trt)-Lys(Boc)-Gly-Pro-Met-Pro-Phe-CTC Resin

H2N-His(Trt)-Lys(Boc)-Gly-Pro-Met-Pro-Phe-CTC Resin

Fmoc-Ser(tBu)-His(Trt)-Lys(Boc)-Gly-Pro-Met-Pro-Phe-CTC Resin

H2N-Ser(tBu)-His(Trt)-Lys(Boc)-Gly-Pro-Met-Pro-Phe-CTC Resin

Fmoc-Leu-Ser(tBu)-His(Trt)-Lys(Boc)-Gly-Pro-Met-Pro-Phe-CTC Resin

H2N-Leu-Ser(tBu)-His(Trt)-Lys(Boc)-Gly-Pro-Met-Pro-Phe-CTC Resin

Fmoc-Arg(Pbf)-Leu-Ser(tBu)-His(Trt)-Lys(Boc)-Gly-Pro-Met-Pro-Phe-CTC Resin

H2N-Arg(Pbf)-Leu-Ser(tBu)-His(Trt)-Lys(Boc)-Gly-Pro-Met-Pro-Phe-CTC Resin

Fmoc-Pro-Arg(Pbf)-Leu-Ser(tBu)-His(Trt)-Lys(Boc)-Gly-Pro-Met-Pro-Phe-CTC Resin

H2N-Pro-Arg(Pbf)-Leu-Ser(tBu)-His(Trt)-Lys(Boc)-Gly-Pro-Met-Pro-Phe-CTC Resin

Fmoc-Arg(Pbf)-Pro-Arg(Pbf)-Leu-Ser(tBu)-His(Trt)-Lys(Boc)-Gly-Pro-Met-Pro-Phe-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-Arg(Pbf)-Pro-Arg(Pbf)-Leu-Ser(tBu)-His(Trt)-Lys(Boc)-Gly-Pro-Met-Pro-Phe-CTC Resin，结构如下：

5、焦谷氨酸反应连接：在上述树脂中，加入适当DMF后，再加入4.61mmol 焦谷氨酸到树脂中，再加入9.23mmol DIPEA、4.38mmol HBTU，鼓氮气反应30分钟。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Pyr-Arg(Pbf)-Pro-Arg(Pbf)-Leu-Ser(tBu)-His(Trt)-Lys(Boc)-Gly-Pro-Met-Pro-Phe-CTCResin。 结构如下：

6、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Pyr-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe-COOH。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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