[D-Ala2]leucine-enkephalin 是一種 δ 阿片類(lèi) (delta opioid) 激動(dòng)劑，是一種能長(zhǎng)效抗降解的亮氨酸腦非肽。
[D-Ala2]leucine-enkephalin, a delta opioid agonist, is a degradation resistant long-acting Leu-enkephalin.

很多蛋白在細(xì)胞中非常容易被降解，或被標(biāo)記，進(jìn)而被選擇性地破壞。但含有部分D型氨基酸的多肽則顯示了很強(qiáng)的抵抗蛋白酶降解能力。

腦啡肽和前腦啡肽

定義
腦啡肽是在大腦和內(nèi)分泌組織中高水平發(fā)現(xiàn)的阿片類(lèi)肽。新出現(xiàn)的含有腦啡肽的肽的主要種類(lèi)似乎是完整的前體，前腦啡肽1。

相關(guān)肽
阿片肽構(gòu)成了一大類(lèi)小蛋白，它們與鴉片生物堿，嗎啡和海洛因相似，能與細(xì)胞膜受體相互作用。阿片生物堿衍生物廣泛用于鎮(zhèn)痛和麻醉。最初的阿片肽家族是腦啡肽，強(qiáng)啡肽和內(nèi)啡肽。在心臟中已經(jīng)發(fā)現(xiàn)了來(lái)自這三個(gè)阿片肽家族的代表性肽。已克隆并測(cè)序了三種不同的阿片受體：mu（µ），delta（d）和kappa（？）2。

發(fā)現(xiàn)
Kosterlitz和Hughes在1975年發(fā)現(xiàn)了腦啡肽和內(nèi)啡肽3。

結(jié)構(gòu)特征
蛋氨酸-腦啡肽（Met-Enk）的氨基酸序列為酪氨酸-甘氨酸-甘氨酸-苯丙氨酸-蛋氨酸。前腦啡肽序列包含五肽Met-Enk的四個(gè)拷貝，leu-腦啡肽之一和Met- enk的兩個(gè)擴(kuò)展形式（Met- enk -arg 6 -phe 7和met- enk -arg 6 -gly 7 -leu 8）。成對(duì)的堿性氨基酸標(biāo)記這些小肽從前體裂解。原腦啡肽由稱(chēng)為原激素轉(zhuǎn)化酶的內(nèi)蛋白水解酶加工，該酶在二元氨基酸位點(diǎn)識(shí)別并切割。最初的前腦啡肽處理開(kāi)始于傳輸?shù)礁郀柣w網(wǎng)絡(luò)之前，并且過(guò)程很快。后續(xù)處理需要在高爾基體網(wǎng)絡(luò)遠(yuǎn)端的酸性環(huán)境中進(jìn)行。前腦啡肽對(duì)肽B具有快速裂解，而較慢的裂解產(chǎn)生其他中等大小的產(chǎn)物，其最終被裂解成五肽至八肽。在各種組織（肌肉，神經(jīng)，內(nèi)分泌）中發(fā)現(xiàn)的不同分子量的最終產(chǎn)物可能是由于切割序列的差異和加工所需的局部酶促條件2所致。

作用方式
肽Met-Enk和Leu-Enk（阿片受體的內(nèi)源性配體）起神經(jīng)調(diào)節(jié)劑或神經(jīng)遞質(zhì)的作用。腦啡肽在哺乳動(dòng)物腦中最顯著的作用是神經(jīng)元放電速率的降低，并且已經(jīng)表明這些肽是抑制性遞質(zhì)。腦啡肽抑制或增強(qiáng)了中樞神經(jīng)元對(duì)幾種假定的遞質(zhì)的反應(yīng)，表明突觸后的作用。還顯示腦啡肽抑制K +誘導(dǎo)的去甲腎上腺素，多巴胺和乙酰膽堿從大鼠腦片的釋放，表明突觸前的作用。腦啡肽抑制豚鼠回腸中的肌間神經(jīng)元的發(fā)射。這種抑制可能是由于腦啡肽的直接突觸后作用導(dǎo)致神經(jīng)元膜超極化4。。為了實(shí)現(xiàn)其生物學(xué)功能，必須將腦啡肽從水相轉(zhuǎn)運(yùn)至其膜結(jié)合受體蛋白的富含脂質(zhì)的環(huán)境。現(xiàn)已知道，Met-enk通過(guò)三種主要的亞型受體起作用，分別稱(chēng)為μ，d和β。-受體。雖然前兩個(gè)受體亞型介導(dǎo)了Met-enk的經(jīng)典阿片樣物質(zhì)作用，但據(jù)報(bào)道β受體參與了該肽的非阿片樣物質(zhì)作用，即對(duì)細(xì)胞生長(zhǎng)的抑制作用5。

功能
前腦啡肽是神經(jīng)肽的前體，在神經(jīng)內(nèi)分泌和神經(jīng)系統(tǒng)中具有多種功能。激活后，發(fā)現(xiàn)T輔助淋巴細(xì)胞表達(dá)高水平的前腦啡肽mRNA，并分泌大量的Met-Enk神經(jīng)肽，這可能表明了免疫系統(tǒng)和神經(jīng)系統(tǒng)相互作用的軸6。腦啡肽引起抗傷害感受和增強(qiáng)的嗎啡鎮(zhèn)痛作用，但它們也阻礙了耐受性和身體依賴(lài)性的發(fā)展。除了其中樞和外周鎮(zhèn)痛作用外，阿片類(lèi)藥物還可以調(diào)節(jié)免疫活性和細(xì)胞增殖。此外，眾所周知，它們?cè)诓煌纳磉^(guò)程中具有重要作用，例如細(xì)胞分化和再生，炎癥，癌癥和血管生成以及鎮(zhèn)痛作用5。

參考

1、Fleminger G, Lahm HW, Udenfriend S (1984).Changes in rat adrenal catecholamines and proenkephalin metabolism after denervation. PNAS., 81(11):3587-3590.
2、Barbara A. Barron. 2000. Cardiac Opioids. Proceedings of the Society for Experimental Biology and Medicine, 224:1-7.
2、Fratta W, Yang HY, Hong J, Costa E (1977). Stability of Met-enkephalin content in brain structures of morphine-dependent or foot shock-stressed rats. Nature, 268(5619):452-453.
4、Wouters W, Den Bercken JV (1979). Hyperpolarisation and depression of slow synaptic inhibition by enkephalin in frog sympathetic ganglion. Nature, 277:53-54.
5、Tsanova A, Dacheva D, Penchev V, Georgiev G, Pajpanova T, Golovinski E, Lalchev Z (2009). Comparative study of the interaction between synthetic methionine-enkephalin and monolayers of zwitterionic and negatively 6、charged phospholipids. Biotechnol & Biotechnol., 23:463-466.
7、Rattner A, Korner M, Rosen H, Baeuerle PA, Citri Y (1991). Nuclear factor Kappa B activates proenkephalin transcription in T lymphocytes. Molecular and Cellular Biology, 11(2):1017-1022.

Enkephalins and Proenkephalins

Definition
Enkephalins are opioid peptides that are found at high levels in the brain and endocrine tissues. The major species of newly appearing enkephalin-containing peptide appears to be the intact precursor, proenkephalin 1.

Related Peptides

Opioid peptides constitute a large group of small proteins that interact with cell membrane receptors similarly to opiate alkaloids, morphine and heroin. Opiate alkaloid derivatives are extensively used for analgesia and anesthesia. The original opioid peptide families are enkephalins, dynorphins, and endorphins. Representative peptides from these three opioid peptide families have been found in the heart. Three different opiate receptors have been cloned and sequenced: mu (µ), delta (d), and kappa (?) 2.

Discovery

Kosterlitz and Hughes discovered enkephalins and endorphins in 1975 3.

Structural Characteristics

The amino acid sequence of methionine-enkephalin (Met-Enk) is tyrosine-glycine-glycine-phenylalanine-methionine. The proenkephalin sequence contains four copies of the pentapeptide Met-Enk, one of leu-enkephalin, and two extended forms of Met-enk (Met-enk-arg6-phe7 and met-enk-arg6-gly7-leu8). Pairs of basic amino acids mark these small peptides for cleavage from the precursor. Proenkephalin is processed by endoproteolytic enyzmes termed prohormone convertases, which recognize and cleave at dibasic amino acid sites. Initial proenkephalin processing starts before transport to the golgi network and are rapid. Later processing requires an acidic environment distal to the golgi network. Proenkephalin has a fast cleavage to peptide B, and slower cleavages yield other intermediate sized products that are cleaved ultimately to the penta to octapeptides. The different molecular-weight end products found in diverse tissues (muscle, neural, endocrine) may be due to variations in the cleavage sequence and local enzymatic conditions for processing 2.

Mode of Action

Pentapeptides Met-Enk and Leu-Enk, the endogenous ligands for the opiate receptor, function as neuromodulators or neurotransmitters. The most prominent action of enkephalins in the mammalian brain is depression of neuronal firing rate and it has been suggested that these peptides are inhibitory transmitters. The response of central neurones to several putative transmitter substances is depressed or enhanced by enkephalins, suggesting a postsynaptic action. It has also been shown that enkephalins suppress the K+-induced release of noradrenaline, dopamine and acetylcholine from rat brain slices, indicating a presynaptic effect. The firing of myenteric neurones in the guinea-pig ileum is inhibited by enkephalins. This inhibition is probably due to a direct postsynaptic action of the enkephalins resulting in a hyperpolarisation of the neuronal membrane 4. To achieve their biological function, enkephalins must be transported from an aqueous phase to the lipid-rich environment of their membrane bound receptor proteins. It is now known that Met-enk acts via three main subtypes of receptors referred to as µ, d and ? - receptors. While the first two receptor subtypes mediate the classic opioid effects of Met-enk, ?-receptors are reported to be involved in the non-opioid actions of the peptide, i.e. the inhibitory effect on the cell growth 5.

Functions

Proenkephalin is a precursor for neuropeptides with a variety of functions in the neuroendocrine and nervous systems. Upon activation, T-helper lymphocytes were found to express high levels of proenkephalin mRNA and to secrete large amounts of the Met-Enk neuropeptide, perhaps indicating an axis by which the immune and nervous systems interact 6. Enkephalins cause antinociception and potentiated morphine analgesia but they also block the development of tolerance and physical dependence. In addition to their central and peripheral antinociceptive function, opioids can modulate immune activity and cell proliferation. Moreover it is known that they have significant role in different physiological processes like cell differentiation and regeneration, inflammation, cancer and angiogenesis and analgesia effects 5.

References

Fleminger G, Lahm HW, Udenfriend S (1984).Changes in rat adrenal catecholamines and proenkephalin metabolism after denervation. PNAS., 81(11):3587-3590.
Barbara A. Barron. 2000. Cardiac Opioids. Proceedings of the Society for Experimental Biology and Medicine, 224:1-7.
Fratta W, Yang HY, Hong J, Costa E (1977). Stability of Met-enkephalin content in brain structures of morphine-dependent or foot shock-stressed rats. Nature, 268(5619):452-453.
Wouters W, Den Bercken JV (1979). Hyperpolarisation and depression of slow synaptic inhibition by enkephalin in frog sympathetic ganglion. Nature, 277:53-54.
Tsanova A, Dacheva D, Penchev V, Georgiev G, Pajpanova T, Golovinski E, Lalchev Z (2009). Comparative study of the interaction between synthetic methionine-enkephalin and monolayers of zwitterionic and negatively charged phospholipids. Biotechnol & Biotechnol., 23:463-466.
Rattner A, Korner M, Rosen H, Baeuerle PA, Citri Y (1991). Nuclear factor Kappa B activates proenkephalin transcription in T lymphocytes. Molecular and Cellular Biology, 11(2):1017-1022.

多肽H2N-Tyr-DAla-Gly-Phe-Leu-COOH的合成步驟：

1、合成CTC樹(shù)脂：稱(chēng)取0.61g CTC Resin（如初始取代度約為1.01mmol/g）和0.74mmol Fmoc-Leu-OH于反應(yīng)器中，加入適量DCM溶解氨基酸（需要注意，此時(shí)CTC樹(shù)脂體積會(huì)增大好幾倍，避免DCM溶液過(guò)少），再加入1.85mmol DIPEA（Mw：129.1,d：0.740g/ml），反應(yīng)2-3小時(shí)后，可不抽濾溶液，直接加入1ml的HPLC級(jí)甲醇，封端半小時(shí)。依次用DMF洗滌2次，甲醇洗滌1次，DCM洗滌一次，甲醇洗滌一次，DCM洗滌一次，DMF洗滌2次（這里使用甲醇和DCM交替洗滌，是為了更好地去除其他溶質(zhì)，有利于后續(xù)反應(yīng)）。得到  Fmoc-Leu-CTC Resin。結(jié)構(gòu)圖如下：

2、脫Fmoc：加3倍樹(shù)脂體積的20%Pip/DMF溶液，鼓氮?dú)?0分鐘，然后2倍樹(shù)脂體積的DMF 洗滌5次。得到 H2N-Leu-CTC Resin 。（此步驟脫除Fmoc基團(tuán)，茚三酮檢測(cè)為藍(lán)色，Pip為哌啶）。結(jié)構(gòu)圖如下：

3、縮合：取1.85mmol Fmoc-Phe-OH 氨基酸，加入到上述樹(shù)脂里，加適當(dāng)DMF溶解氨基酸，再依次加入3.7mmol DIPEA，1.76mmol HBTU。反應(yīng)30分鐘后，取小樣洗滌，茚三酮檢測(cè)為無(wú)色。用2倍樹(shù)脂體積的DMF 洗滌3次樹(shù)脂。（洗滌樹(shù)脂，去掉殘留溶劑，為下一步反應(yīng)做準(zhǔn)備）。得到Fmoc-Phe-Leu-CTC Resin。氨基酸：DIPEA：HBTU：樹(shù)脂=3：6：2.85：1（摩爾比）。結(jié)構(gòu)圖如下：

4、依次循環(huán)步驟二、步驟三，依次得到

H2N-Phe-Leu-CTC Resin

Fmoc-Gly-Phe-Leu-CTC Resin

H2N-Gly-Phe-Leu-CTC Resin

Fmoc-DAla-Gly-Phe-Leu-CTC Resin

H2N-DAla-Gly-Phe-Leu-CTC Resin

Fmoc-Tyr(tBu)-DAla-Gly-Phe-Leu-CTC Resin

以上中間結(jié)構(gòu)，均可在專(zhuān)肽生物多肽計(jì)算器-多肽結(jié)構(gòu)計(jì)算器中，一鍵畫(huà)出。

最后再經(jīng)過(guò)步驟二得到 H2N-Tyr(tBu)-DAla-Gly-Phe-Leu-CTC Resin，結(jié)構(gòu)如下：

5、切割：6倍樹(shù)脂體積的切割液（或每1g樹(shù)脂加8ml左右的切割液），搖床搖晃 2小時(shí)，過(guò)濾掉樹(shù)脂，用冰無(wú)水乙醚沉淀濾液，并用冰無(wú)水乙醚洗滌沉淀物3次，最后將沉淀物放真空干燥釜中，常溫干燥24小試，得到粗品H2N-Tyr-DAla-Gly-Phe-Leu-COOH。結(jié)構(gòu)圖見(jiàn)產(chǎn)品結(jié)構(gòu)圖。

切割液選擇：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%

（前兩種適合沒(méi)有容易氧化的氨基酸，例如Trp、Cys、Met。第三種適合幾乎所有的序列。）

6、純化凍干：使用液相色譜純化，收集目標(biāo)峰液體，進(jìn)行凍干，獲得蓬松的粉末狀固體多肽。不過(guò)這時(shí)要取小樣復(fù)測(cè)下純度 是否目標(biāo)純度。

7、最后總結(jié)：

杭州專(zhuān)肽生物技術(shù)有限公司（ALLPEPTIDE http://amynixphotography.com）主營(yíng)定制多肽合成業(yè)務(wù)，提供各類(lèi)長(zhǎng)肽，短肽，環(huán)肽，提供各類(lèi)修飾肽，如：熒光標(biāo)記修飾（CY3、CY5、CY5.5、CY7、FAM、FITC、Rhodamine B、TAMRA等），功能基團(tuán)修飾肽（疊氮、炔基、DBCO、DOTA、NOTA等），同位素標(biāo)記肽（N15、C13），訂書(shū)肽（Stapled Peptide）,脂肪酸修飾肽（Pal、Myr、Ste），磷酸化修飾肽（P-Ser、P-Thr、P-Tyr），環(huán)肽（酰胺鍵環(huán)肽、一對(duì)或者多對(duì)二硫鍵環(huán)），生物素標(biāo)記肽，PEG修飾肽，甲基化修飾肽

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