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TFLLR-NH2是选择性的PAR1激动剂，EC50值为1.9 μM。

TFLLR-NH2 is a selective PAR1 agonist with an EC50 of 1.9 μM.

TFLLR-NH2 2TFA来源于蛋白酶激活受体-1（PAR1），是一种选择性PAR1激动剂，EC50为1.9μM。

TFLLR-NH2 2TFA, derived from the protease-activated receptor-1 (PAR1), is a selective PAR1 agonist with an EC50 of 1.9 μM.

"Protease activated receptors (PARs) are a distinctive four-member family of seven transmembrane G protein-coupled receptors (GPCRs) widely expressed in inflammatory cells. PARs are cleaved by certain serine proteases to expose a tethered ligand domain, this ligand domain then binds to and activates the receptors to initiate multiple signalling cascades. These PAR-activating proteases therefore represent PAR agonists. This PAR-1 agonist peptide represents the sequence of the tethered ligand and is therefore capable of activating the receptor independently of N-terminal proteolysis. TFLLR-NH2 is more selective to PAR-1 than the PAR-1 agonist SFLLRN-NH2.Activation of PAR-1 induces platelet aggregation and IL-6 release from monocytes and T cells, as well as several other cellular pathways including those involved in allergic inflammation, neurogenic inflammation and the potentiation of NMDA receptor activity in the hippocampus."

The endothelium is a layer of cells that lines the inner surface of blood vessels and lymphatic vessels, forming a barrier between circulating blood or lymph and the rest of the body. It is involved in maintaining vascular homeostasis as well as in inflammation. The endothelium regulates vascular tone and blood pressure through release of nitric oxide (NO) and other substances, such as prostacyclin, vasoactive peptides, endothelin-1, tumor necrosis factor-α, interleukin-1β, and thromboxane A2. Endothelial cells are activated by various means such as increased intracellular Ca2+ concentration or basic fibroblast growth factor (bFGF). This activation can be inhibited by receptor antagonists such as neurokinin-1 receptor antagonists.

H-Thr-Phe-Leu-Leu-Arg-NH2 is a peptide that has been shown to reduce the severity of lung inflammation and injury in rats with pulmonary hypertension. It also prevents the release of proinflammatory cytokines such as tumor necrosis factor alpha (TNFα) and interleukin 6 (IL6). This peptide also induces an antibody response against collagen gel, which is used to treat fibrotic diseases and wounds. The peptide binds to the soybean trypsin inhibitor, preventing it from inhibiting protease enzymes, thereby allowing for protein synthesis. H-Thr-Phe-Leu-Leu-Arg-NH2 has been shown to have antiapoptotic effects by restoring mitochondrial membrane potential in cancer cells. This peptide can be used as a therapeutic agent for various diseases including primary pulmonary hypertension, cancer tissues, and mitochondrial disorders.

多肽H2N-Thr-Phe-Leu-Leu-Arg-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2.63g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2.37mmol 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4.73mmol DIPEA，2.25mmol 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-Arg(Pbf)-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-Arg(Pbf)-Linker-MBHA Resin

Fmoc-Leu-Arg(Pbf)-Linker-MBHA Resin

H2N-Leu-Arg(Pbf)-Linker-MBHA Resin

Fmoc-Leu-Leu-Arg(Pbf)-Linker-MBHA Resin

H2N-Leu-Leu-Arg(Pbf)-Linker-MBHA Resin

Fmoc-Phe-Leu-Leu-Arg(Pbf)-Linker-MBHA Resin

H2N-Phe-Leu-Leu-Arg(Pbf)-Linker-MBHA Resin

Fmoc-Thr(tBu)-Phe-Leu-Leu-Arg(Pbf)-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-Thr(tBu)-Phe-Leu-Leu-Arg(Pbf)-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-Thr-Phe-Leu-Leu-Arg-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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