专肽生物_定制多肽合成服务_多肽合成定制服务_多肽合成厂家_多肽合成公司

背景
吗啡类似物存在于不同物种的大脑或垂体中。在这些肽主要结构的C端序列包含Beta-Lipotropin (beta-LPH)。

在人和猪的垂体及下丘脑神经垂体提取物中，在分离的片段序列和羧基终端部分之间的部分也观察到相应Beta-Lipotropins。

参考文献：

1. Bradbury, A.F., Smyth, D.G. and Snell, C.R. (1976), Biochem. Biophys. Res. Commun. 6!J, 950-956.

2. Ling, N., Burgus, R. and Guillemin, R. (1976). Proc. Natl. Acad. Sci. USA., 73, 3942-3946.

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H2N-Glu-Leu-Ala-Gly-Ala-Pro-Pro-Glu-Pro-Ala-COOH的合成步骤：

1、合成CTC树脂：称取1.44g CTC Resin（如初始取代度约为0.35mmol/g）和0.6mmol Fmoc-Ala-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1.51mmol 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-Ala-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-Ala-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1.51mmol 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3.02mmol DIPEA，1.44mmol 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-Ala-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-Ala-CTC Resin

Fmoc-Glu(OtBu)-Pro-Ala-CTC Resin

H2N-Glu(OtBu)-Pro-Ala-CTC Resin

Fmoc-Pro-Glu(OtBu)-Pro-Ala-CTC Resin

H2N-Pro-Glu(OtBu)-Pro-Ala-CTC Resin

Fmoc-Pro-Pro-Glu(OtBu)-Pro-Ala-CTC Resin

H2N-Pro-Pro-Glu(OtBu)-Pro-Ala-CTC Resin

Fmoc-Ala-Pro-Pro-Glu(OtBu)-Pro-Ala-CTC Resin

H2N-Ala-Pro-Pro-Glu(OtBu)-Pro-Ala-CTC Resin

Fmoc-Gly-Ala-Pro-Pro-Glu(OtBu)-Pro-Ala-CTC Resin

H2N-Gly-Ala-Pro-Pro-Glu(OtBu)-Pro-Ala-CTC Resin

Fmoc-Ala-Gly-Ala-Pro-Pro-Glu(OtBu)-Pro-Ala-CTC Resin

H2N-Ala-Gly-Ala-Pro-Pro-Glu(OtBu)-Pro-Ala-CTC Resin

Fmoc-Leu-Ala-Gly-Ala-Pro-Pro-Glu(OtBu)-Pro-Ala-CTC Resin

H2N-Leu-Ala-Gly-Ala-Pro-Pro-Glu(OtBu)-Pro-Ala-CTC Resin

Fmoc-Glu(OtBu)-Leu-Ala-Gly-Ala-Pro-Pro-Glu(OtBu)-Pro-Ala-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-Glu(OtBu)-Leu-Ala-Gly-Ala-Pro-Pro-Glu(OtBu)-Pro-Ala-CTC 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-Glu-Leu-Ala-Gly-Ala-Pro-Pro-Glu-Pro-Ala-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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