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N-末端乙酰化内吗啡肽-1是经修饰的Endomorphin-1。
N-terminally acetylated Endomorphin-1 is a modified Endomorphin-1.

Definition
Endomorphin (EM)-1 and EM-2 are opioid tetrapeptides located in the central nervous system and immune tissues with high selectivity and affinity for the µ-opioid receptor 1.

Related Peptides
Opioid peptides and their G-protein-coupled receptors (d, ? and µ) are located in the central nervous system and peripheral tissues. The opioid system has been studied to determine the intrinsic mechanism of modulation of pain and to develop uniquely effective pain-control substances with minimal abuse potential and side effects. Two types of endogenous opioid peptides exist, one containing Try-Gly-Gly-Phe as the message domain (enkephalins, endorphins, dynorphins) and the other containing the Tyr-Pro-Phe/Trp sequence (endomorphins-1 and -2) 2.

Discovery
In 1997, Zadina et al., isolated Endomorphin 1 (EM1) and endomorphin 2 (EM2) from bovine brain, and reported them to be tetrapeptides having the highest specificity and affinity for the µ receptor of any endogenous substance so far described 3.

Structural Characteristics
Opioidmimetics and opioid peptides containing the amino acid sequence of the message domain of endomorphins (EMs), Tyr-Pro-Phe/Trp, have been found to exhibit unique binding activity. Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) have high µ­ receptor affinity and remarkable selectivity 2. The proper spatial orientation and conformational restriction of the third aromatic ring is supposed to be crucial for the interaction of EMs with MOR (µ­ opioid receptor) 4.

Mode of Action
The endomorphins have the highest specificity and affinity to the µ receptor among all endogenous substance so far described. EM1 is more effective than the µ- selective analogue DAMGO in vitro and produces potent and prolonged analgesia in mice. EM2 (H-Tyr-Pro-Phe-Phe-NH2) also has a high affinity and selectivity to the µ receptor 2. The µ-opioid receptors are G protein-coupled receptors that play a pivotal role in the analgesic effects of opioid receptor agonists used clinically. Endomorphin-induced antinociception is mediated by spinal µ-opioid receptors 5.

Functions
Endomorphins have been implicated in a broad range of physiological functions including antinociceptive, cardiovascular, respiratory, digestive, rewarding, and endocrine responses 5. EM 1 and EM2 have significant naloxone-sensitive, vasodepressor activity 6. They modulate phagocytosis, chemotaxis and superoxide anion production by microglia 7. The analogues designed based on endomorphins may have therapeutic potential 8.

References

1. Coventry TL, Jessop DS, Finn DP, Crabb MD, Kinoshita H, Harbuz MS (2001). Endomorphins and activation of the hypothalamo-pituitary-adrenal axis. J Endocrinol., 169(1):185-193.
2. Okada Y, Tsuda Y, Bryant SD, Lazarus LH (2002). Endomorphins and related opioid peptides. Vitam Horm., 65:257-279.
3. Zadina JE, Hackler L, Ge LJ, Kastin AJ (1997). A potent and selective endogenous agonist for the mu-opiate receptor. Nature, 386(6624):499–502
4. Yu Y, Shao X, Cui Y, Liu HM, Wang CL, Fan YZ, Liu J, Dong SL, Cui YX, Wang R (2007).Structure-activity study on the spatial arrangement of the third aromatic ring of endomorphins 1 and 2 using an atypical constrained C terminus. ChemMedChem., 2(3):309-317.
5. Xie H, Woods JH, Traynor JR, Ko MC (2008). The Spinal Antinociceptive Effects of Endomorphins in Rats: Behavioral and G Protein Functional Studies. Anesth Analg., 106(6):1873-1881.
6. Champion HC, Zadina JE, Kastin AJ, Hackler L, Ge LJ, Kadowitz PJ (1997).. The Endogenous Mu-Opioid Receptor Agonists Endomorphins 1 and 2 Have Novel Hypotensive Activity in the Rabbit. Biochemi Biophysl Res Commun., 235(3) 567-570
7. Azuma Y, Ohura K, Wang PL, Shinohara M (2001). Endomorphins 1 and 2 modulate chemotaxis, phagocytosis and superoxide anion production by microglia. J Neuroimmunol., 119(1):51-56.
8. Huo XF, Ren WH, Wu N, Wang R (1998).The design and synthesis of endomorphins and their analogues. Chinese Science Bulletin., 46(13):1096-1099.  

多肽Ac-Tyr-Pro-Trp-Phe-NH2的合成步骤：

1、合成MBHA树脂：取若干克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1.55g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1.4mmol 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2.79mmol DIPEA，1.33mmol 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-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-Phe-Linker-MBHA Resin

Fmoc-Trp(Boc)-Phe-Linker-MBHA Resin

H2N-Trp(Boc)-Phe-Linker-MBHA Resin

Fmoc-Pro-Trp(Boc)-Phe-Linker-MBHA Resin

H2N-Pro-Trp(Boc)-Phe-Linker-MBHA Resin

Fmoc-Tyr(tBu)-Pro-Trp(Boc)-Phe-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)-Pro-Trp(Boc)-Phe-Linker-MBHA 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1.4mmol乙酸酐到树脂中，再加入2.79mmol 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-Tyr(tBu)-Pro-Trp(Boc)-Phe-Linker-MBHAResin。 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-Tyr-Pro-Trp-Phe-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。）

7、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。

8、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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