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背景
内皮素-1（ET-1）是内皮素家族主要的多肽，在血管和非血管组织参与多种生物学过程，包括心脏、肾脏和中枢神经系统。

在细胞水平，ET-1的作用是由ETA和ETB两个特定受体亚型介导的，这两者与磷脂酶C（PLC）功能上相耦合。

ET家族多肽的C-末端片段是高度保守序列，因为C末端片段仅激活ETB，可以用它来区分ETA和ETB的两个ET受体亚型。

参考文献：

1. Rubanyi, G. M., and M. A. Polokoff. Endothelins: molecular biology, biochemistry, pharmacology, physiology, and pathophysiology. Pharmacol. Rev. 46: 325–415, 1994

2. Simonson MS. Endothelins: multifunctional renal peptides. Physiol Rev 1993; 73: 375–411.

3. PM Vanhoutte Endothelin-1. A matter of life and breath, Nature, 368 (1994), pp. 693–694

Definition

A peptidergic activity produced in endothelial cells that caused coronary vasoconstriction was described in 1985, and a family of peptides, named the endothelins, was subsequently isolated and identified. The three members of the family — endothelin-1 (ET-1), endothelin-2 (ET-2), and endothelin-3 (ET-3 )— are produced in a variety of tissues, where they act as modulators of vasomotor tone, cell proliferation, and hormone production 1.

Related Peptides

The 21-amino acid peptide ET-1 is the predominant isoform of the endothelin peptide family, which includes ET-2, ET-3, and ET-4. It exerts various biological effects, including vasoconstriction and the stimulation of cell proliferation in tissues both within and outside of the cardiovascular system. ET-1 is synthesized by endothelin-converting enzymes (ECE), chymases, and non-ECE metalloproteases; it is regulated in an autocrine fashion in vascular and nonvascular cells 2.

Discovery

Endothelin, one of the most potent vasoconstrictors, was first discovered by Yanagisawa and co-workers in 1981. It was first isolated, characterized, and cloned in porcine aortic endothelial cells 3.

Structural Characteristics

First of the three isoforms, the ET-1, is a 21-amino acid peptide; it has a molecular weight of 2,492, free carboxyl and amino termini and has two intramolecular disulfide bonds. It is present in many mammalian species, including humans. Other two human endothelin isopeptides, ET-2 and ET-3 are encoded by separate genes. They contain two intramolecular disulfide bonds. They also contain a cluster of polar charged side chains in the hairpin loop and a hydrophobic COOH terminus, containing the aromatic indole side chain at trp21 3.

Mode of Action

Two endothelin receptors have been characterised in the mammals. They are classified according to the relative binding affinities of the 3-endothelin isopeptides to the receptors. The order of affinity of endothelins for 1st receptor type designated ETA is ET-1 > ET-2 > ET- 3. The second receptor subtype designated ETB shows equipotent affinity for all 3 endothelins 3.

Functions

Endothelins appear to act mainly as local paracrine/autocrine peptides, but circulating levels of endothelins also have great biological significance especially in pathological states of increased serum concentration.

Pathophysiology of Endothelins:

1. Renal haemodynamics: In various studies in dogs and rats it has been seen that endothelin peptides have both contractile and promitogenic actions in renal mesangial cells.
2. Renal disease: In various studies it has been shown that ET-1 plays a role in the pathogenesis of acute renal failure after renal ischaemia, i.e., plasma levels of ET-1 are increased in patients with acute renal failure.
3. Hypertension: ET-1 causes potent vasoconstriction and prolonged elevation of blood pressure in experimental models. But the relationship between the plasma levels of ET-1 and severity of hypertension is inconsistent in humans.
4. Heart failure: Plasma endothelin levels are increased in animal models of CHF (Chronic Heart Failure) and in patients with CHF. In patients, increased plasma endothelin levels correlate closely with the degree of haemodynamic and functional impairment, with higher levels predicting a greater likelihood of death or need for cardiac transplantation.
5. Ischaemic heart disease: In human studies, plasma endothelin levels are increased in unstable angina and acute myocardial infarction.
6. Variant angina: Patients with Prinzmetal’s angina are known to have endothelial dysfunction affecting the L-arginine nitric oxide system, and as a potent vasoconstrictor of coronary arteries, endothelin-1 has been implicated in the pathophysiology of this condition.
7. Primary pulmonary hypertension: In primary pulmonary hypertension there is proliferation of pulmonary arterial smooth muscle and endothelial injury. It has been observed that depending on the state of vasomotor tone, endothelin isopeptides can cause either pulmonary vasodilatation or vasoconstriction.
8. Raynaud’s disease: Raynaud’s disease is seen commonly in cold climates and is associated with vasospastic conditions like migraine and Prinzmetal’s angina. There has been exaggerated increase in endothelin levels in venous blood draining from the cold challenged arm, in cases with Raynaud’s disease as compared with responses in healthy volunteers.
9. Subarachnoid haemorrhage (SAH): Endothelin-1 has a causative role in mediating sub-arachnoid hemorrhage induced vasospasm. Plasma and CSF endothelin levels are significantly increased in patients after SAH and plasma levels of endothelins are highest in those who develop vasospasm.
10. Migraine: In the recent studies it has been found that levels of endothelins are increased during migraine headaches 3.

References

1. Levin ER (1995). Endothelins. NEJM., 333:356-363.
2.  Lüscher TF, Barton M (2000). Endothelins and Endothelin Receptor Antagonists Circulation., 102:2434:2440.
3. Jain SK, Yadava RK, Raikar R (2002). Role of Endothelins in Health and Disease. JIACM,  3(1):59-64.

多肽Ac-His-Leu-Asp-Ile-Ile-Trp-COOH的合成步骤：

1、合成CTC树脂：称取1.0g CTC Resin（如初始取代度约为0.31mmol/g）和0.37mmol Fmoc-Trp(Boc)-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0.93mmol 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-Trp(Boc)-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-Trp(Boc)-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0.93mmol Fmoc-Ile-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1.86mmol DIPEA，0.88mmol 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-Ile-Trp(Boc)-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-Ile-Trp(Boc)-CTC Resin

Fmoc-Ile-Ile-Trp(Boc)-CTC Resin

H2N-Ile-Ile-Trp(Boc)-CTC Resin

Fmoc-Asp(OtBu)-Ile-Ile-Trp(Boc)-CTC Resin

H2N-Asp(OtBu)-Ile-Ile-Trp(Boc)-CTC Resin

Fmoc-Leu-Asp(OtBu)-Ile-Ile-Trp(Boc)-CTC Resin

H2N-Leu-Asp(OtBu)-Ile-Ile-Trp(Boc)-CTC Resin

Fmoc-His(Trt)-Leu-Asp(OtBu)-Ile-Ile-Trp(Boc)-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-His(Trt)-Leu-Asp(OtBu)-Ile-Ile-Trp(Boc)-CTC Resin，结构如下：

5、乙酸酐反应连接：在上述树脂中，加入适当DMF后，再加入0.93mmol乙酸酐到树脂中，再加入1.86mmol DIPEA，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Ac-His(Trt)-Leu-Asp(OtBu)-Ile-Ile-Trp(Boc)-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Ac-His-Leu-Asp-Ile-Ile-Trp-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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