DX600, Ac-GDYSHCSPLRYYPWWKCTYPDPEGGG-amide is an ACE-2-specific peptide inhibitor with a Ki of 2.8 nM.

DX600 TFA是血管緊張素轉(zhuǎn)換酶2（ACE2）的抑制劑，不與ACE發(fā)生交叉反應(yīng)。

DX600 TFA is an inhibitor of angiotensin-converting enzyme 2 (ACE2) and does not cross-react with ACE.

二硫鍵廣泛存在與蛋白結(jié)構(gòu)中，對穩(wěn)定蛋白結(jié)構(gòu)具有非常重要的意義，二硫鍵一般是通過序列中的2個Cys的巰基，經(jīng)氧化形成。
 

形成二硫鍵的方法很多：空氣氧化法，DMSO氧化法，過氧化氫氧化法等。
 

二硫鍵的合成過程，  可以通過Ellman檢測以及HPLC檢測方法對其反應(yīng)進程進行監(jiān)測。  
   

如果多肽中只含有1對Cys，那二硫鍵的形成是簡單的。多肽經(jīng)固相或液相合成，然后在pH8-9的溶液中進行氧化。      
 

當需要形成2對或2對以上的二硫鍵時，合成過程則相對復(fù)雜。盡管二硫鍵的形成通常是在合成方案的最后階段完成，但有時引入預(yù)先形成的二硫化物是有利于連合或延長肽鏈的。通常采用的巰基保護基有trt, Acm, Mmt, tBu, Bzl, Mob, Tmob等多種基團。我們分別列出兩種以2-Cl樹脂和Rink樹脂為載體合成的多肽上多對二硫鍵形成路線：
 

二硫鍵反應(yīng)條件選擇    
 

 二硫鍵即為蛋白質(zhì)或多肽分子中兩個不同位點Cys的巰基（-SH）被氧化形成的S-S共價鍵。 一條肽鏈上不同位置的氨基酸之間形成的二硫鍵，可以將肽鏈折疊成特定的空間結(jié)構(gòu)。多肽分 子通常分子量較大，空間結(jié)構(gòu)復(fù)雜，結(jié)構(gòu)中形成二硫鍵時要求兩個半胱氨酸在空間距離上接近。 此外，多肽結(jié)構(gòu)中還原態(tài)的巰基化學(xué)性質(zhì)活潑，容易發(fā)生其他的副反應(yīng)，而且肽鏈上其他側(cè)鏈 也可能會發(fā)生一系列修飾，因此，肽鏈進行修飾所選取的氧化劑和氧化條件是反應(yīng)的關(guān)鍵因素， 反應(yīng)機理也比較復(fù)雜，既可能是自由基反應(yīng)，也可能是離子反應(yīng)。      

反應(yīng)條件有多種選擇，比如空氣氧化，DMSO氧化等溫和的氧化過程，也可以采用H2O2，I2， 汞鹽等激烈的反應(yīng)條件。
 

空氣氧化法： 空氣氧化法形成二硫鍵是多肽合成中最經(jīng)典的方法，通常是將巰基處于還原態(tài)的多肽溶于水中，在近中性或弱堿性條件下（PH值6.5-10），反應(yīng)24小時以上。為了降低分子之間二硫鍵形成的可能，該方法通常需要在低濃度條件下進行。
 

碘氧化法：將多肽溶于25%的甲醇水溶液或30%的醋酸水溶液中，逐滴滴加10-15mol/L的碘進行氧化，反應(yīng)15-40min。當肽鏈中含有對碘比較敏感的Tyr、Trp、Met和His的殘基時，氧化條件要控制的更精確，氧化完后，立即加入維生素C或硫代硫酸鈉除去過量的碘。 當序列中有兩對或多對二硫鍵需要成環(huán)時，通常有兩種情況：
 

自然隨機成環(huán):       序列中的Cys之間隨機成環(huán)，與一對二硫鍵成環(huán)條件相似；
 

定點成環(huán)：       定點成環(huán)即序列中的Cys按照設(shè)計要求形成二硫鍵，反應(yīng)過程相對復(fù)雜。在 固相合成多肽之前，需要提前設(shè)計幾對二硫鍵形成的順序和方法路線，選擇不同的側(cè)鏈 巰基保護基，利用其性質(zhì)差異，分步氧化形成兩對或多對二硫鍵。       通常采用的巰基保護 基有trt, Acm, Mmt, tBu, Bzl, Mob, Tmob等多種基團。

定義
酶是用于生化反應(yīng)的非常有效的催化劑。它們通過提供較低活化能的替代反應(yīng)途徑來加快反應(yīng)速度。酶作用于底物并產(chǎn)生產(chǎn)物。一些物質(zhì)降低或什至停止酶的催化活性被稱為抑制劑。
發(fā)現(xiàn)
1965年，Umezawa H分析了微生物產(chǎn)生的酶抑制劑，并分離出了抑制亮肽素和抗痛藥的胰蛋白酶和木瓜蛋白酶，乳糜蛋白酶抑制的胰凝乳蛋白酶，胃蛋白酶抑制素抑制胃蛋白酶，泛磷酰胺抑制唾液酸酶，烏藤酮抑制酪氨酸羥化酶，多巴汀抑制多巴胺3-羥硫基嘧啶和多巴胺3-羥色胺酶酪氨酸羥化酶和多巴胺J3-羥化酶。最近，一種替代方法已應(yīng)用于預(yù)測新的抑制劑：合理的藥物設(shè)計使用酶活性位點的三維結(jié)構(gòu)來預(yù)測哪些分子可能是抑制劑1。已經(jīng)開發(fā)了用于識別酶抑制劑的基于計算機的方法，例如分子力學(xué)和分子對接。
結(jié)構(gòu)特征
已經(jīng)確定了許多抑制劑的晶體結(jié)構(gòu)。已經(jīng)確定了三種與凝血酶復(fù)合的高效且選擇性的低分子量剛性肽醛醛抑制劑的晶體結(jié)構(gòu)。這三種抑制劑全部在P3位置具有一個新的內(nèi)酰胺部分，而對胰蛋白酶選擇性最高的兩種抑制劑在P1位置具有一個與S1特異性位點結(jié)合的胍基哌啶基。凝血酶的抑制動力學(xué)從慢到快變化，而對于胰蛋白酶，抑制的動力學(xué)在所有情況下都快。根據(jù)兩步機理2中穩(wěn)定過渡態(tài)絡(luò)合物的緩慢形成來檢驗動力學(xué)。
埃米爾•菲舍爾（Emil Fischer）在1894年提出，酶和底物都具有特定的互補幾何形狀，彼此恰好契合。這稱為“鎖和鑰匙”模型3。丹尼爾·科什蘭（Daniel Koshland）提出了誘導(dǎo)擬合模型，其中底物和酶是相當靈活的結(jié)構(gòu)，當?shù)孜锱c酶4相互作用時，活性位點通過與底物的相互作用不斷重塑。
在眾多生物活性肽的成熟過程中，需要由其谷氨酰胺（或谷氨酰胺）前體形成N末端焦谷氨酸（pGlu）。游離形式并與底物和三種咪唑衍生抑制劑結(jié)合的人QC的結(jié)構(gòu)揭示了類似于兩個鋅外肽酶的α/β支架，但有多個插入和缺失，特別是在活性位點區(qū)域。幾種活性位點突變酶的結(jié)構(gòu)分析為針對QC相關(guān)疾病5的抑制劑的合理設(shè)計提供了結(jié)構(gòu)基礎(chǔ)。
作用方式
酶是催化化學(xué)反應(yīng)的蛋白質(zhì)。酶與底物相互作用并將其轉(zhuǎn)化為產(chǎn)物。抑制劑的結(jié)合可以阻止底物進入酶的活性位點和/或阻止酶催化其反應(yīng)。抑制劑的種類繁多，包括：非特異性，不可逆，可逆-競爭性和非競爭性?？赡嬉种苿?nbsp;以非共價相互作用（例如疏水相互作用，氫鍵和離子鍵）與酶結(jié)合。非特異性抑制方法包括最終使酶的蛋白質(zhì)部分變性并因此不可逆的任何物理或化學(xué)變化。特定抑制劑 對單一酶發(fā)揮作用。大多數(shù)毒藥通過特異性抑制酶發(fā)揮作用。競爭性抑制劑是任何與底物的化學(xué)結(jié)構(gòu)和分子幾何結(jié)構(gòu)非常相似的化合物。抑制劑可以在活性位點與酶相互作用，但是沒有反應(yīng)發(fā)生。非競爭性抑制劑是與酶相互作用但通常不在活性位點相互作用的物質(zhì)。非競爭性抑制劑的凈作用是改變酶的形狀，從而改變活性位點，從而使底物不再能與酶相互作用而產(chǎn)生反應(yīng)。非競爭性抑制劑通常是可逆的。不可逆抑制劑與酶形成牢固的共價鍵。這些抑制劑可以在活性位點附近或附近起作用。
功能
工業(yè)應(yīng)用中， 酶在商業(yè)上被廣泛使用，例如在洗滌劑，食品和釀造工業(yè)中。蛋白酶用于“生物”洗衣粉中，以加速蛋白質(zhì)在諸如血液和雞蛋等污漬中的分解。商業(yè)上使用酶的問題包括：它們是水溶性的，這使得它們難以回收，并且一些產(chǎn)物可以抑制酶的活性（反饋抑制）。
藥物分子，許多藥物分子都是酶抑制劑，藥用酶抑制劑通常以其特異性和效力為特征。高度的特異性和效力表明該藥物具有較少的副作用和較低的毒性。酶抑制劑在自然界中發(fā)現(xiàn)，并且也作為藥理學(xué)和生物化學(xué)的一部分進行設(shè)計和生產(chǎn)6。
天然毒物 通常是酶抑制劑，已進化為保護植物或動物免受天敵的侵害。這些天然毒素包括一些已知最劇毒的化合物。
神經(jīng)氣體（ 例如二異丙基氟磷酸酯（DFP））通過與絲氨酸的羥基反應(yīng)生成酯，從而抑制了乙酰膽堿酯酶的活性位點。
參考
1、Scapin G (2006). Structural biology and drug discovery. Curr. Pharm. Des.,      12(17):2087–2097.
2、Krishnan R, Zhang E, Hakansson K, Arni RK, Tulinsky A, Lim-Wilby MS, Levy OE, Semple JE, Brunck TK (1998). Highly selective mechanism-based thrombin inhibitors:  structures of thrombin and trypsin inhibited with rigid peptidyl aldehydes. Biochemistry, 37 (35):12094-12103.
3、Fischer E (1894). Einfluss der configuration auf die wirkung der enzyme. Ber. Dt. Chem. Ges., 27:2985–2993.
4、Koshland DE (1958). Application of a theory of enzyme specificity to protein synthesis. PNAS., 44 (2):98–104.
5、Huang KF, Liu YL, Cheng WJ, Ko TP, Wang AH (2005). Crystal structures of human glutaminyl cyclase, an enzyme responsible for protein N-terminal pyroglutamate formation. PNAS., 102(37):13117-13122.
6、Holmes CF, Maynes JT, Perreault KR, Dawson JF, James MN (2002). Molecular enzymology underlying regulation of protein phosphatase-1 by natural toxins. Curr Med Chem., 9(22):1981-1989.

Definition
Enzymes are very efficient catalysts for biochemical reactions. They speed up reactions by providing an alternative reaction pathway of lower activation energy. Enzyme acts on substrate and gives rise to a product. Some substances reduce or even stop the catalytic activities of enzymes are called inhibitors.

Discovery
In 1965, Umezawa H analysed enzyme inhibitors produced by microorganisms and isolated leupeptin and antipain inhibiting trypsin and papain, chymostatin inhibiting chymotrypsin, pepstatin inhibiting pepsin, panosialin inhibiting sialidases, oudenone inhibiting tyrosine hydroxylase, dopastin inhibiting dopamine 3-hydroxylase, aquayamycin and chrothiomycin inhibiting tyrosine hydroxylase and dopamine J3-hydroxylase . Recently, an alternative approach has been applied to predict new inhibitors: rational drug design uses the three-dimensional structure of an enzyme's active site to predict which molecules might be inhibitors 1. Computer-based methods for identifying inhibitor for an enzyme have been developed, such as molecular mechanics and molecular docking.

Structural Characteristics
The crystal structures of many inhibitors have been determined. The crystal structures of three highly potent and selective low-molecular weight rigid peptidyl aldehyde inhibitors complexed with thrombin have been determined. All the three inhibitors have a novel lactam moiety at the P3 position, while the two with greatest trypsin selectivity have a guanidinopiperidyl group at the P1 position that binds in the S1 specificity site. The kinetics of inhibition vary from slow to fast with thrombin and are fast in all cases with trypsin. The kinetics are examined in terms of the slow formation of a stable transition-state complex in a two-step mechanism 2.

Emil Fischer in 1894 suggested that both the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another.This is known as "the lock and key" model 3. Daniel Koshland suggested induced fit model where substrate and enzymes are rather flexible structures, the active site is continually reshaped by interactions with the substrate as the substrate interacts with the enzyme 4.

N-terminal pyroglutamate (pGlu) formation from its glutaminyl (or glutamyl) precursor is required in the maturation of numerous bioactive peptides. The structure of human QC in free form and bound to a substrate and three imidazole-derived inhibitors reveals an alpha/beta scaffold akin to that of two-zinc exopeptidases but with several insertions and deletions, particularly in the active-site region. The structural analyses of several active-site-mutant enzymes provide a structural basis for the rational design of inhibitors against QC-associated disorders 5.

Mode of Action
Enzymes are proteins that catalyze chemical reactions. Enzymes interact with substrate and convert them into products. Inhibitor binding can stop a substrate from entering the enzyme's active site and/or hinder the enzyme from catalyzing its reaction. There are a variety of types of inhibitors including: nonspecific, irreversible, reversible - competitive and noncompetitive. Reversible inhibitors bind to enzymes with non-covalent interactions like hydrophobic interactions, hydrogen bonds, and ionic bonds. Non-specific methods of inhibition include any physical or chemical changes which ultimately denature the protein portion of the enzyme and are therefore irreversible. Specific Inhibitors exert their effects upon a single enzyme. Most poisons work by specific inhibition of enzymes. A competitive inhibitor is any compound which closely resembles the chemical structure and molecular geometry of the substrate. The inhibitor may interact with the enzyme at the active site, but no reaction takes place. A noncompetitive inhibitor is a substance that interacts with the enzyme, but usually not at the active site.  The net effect of a non competitive inhibitor is to change the shape of the enzyme and thus the active site, so that the substrate can no longer interact with the enzyme to give a reaction. Non competitive inhibitors are usually reversible. Irreversible Inhibitors form strong covalent bonds with an enzyme.  These inhibitors may act at, near, or remote from the active site .

Functions
Industrial application, enzymes are widely used commercially, for example in the detergent, food and brewing industries. Protease enzymes are used in 'biological' washing powders to speed up the breakdown of proteins in stains like blood and egg. Problems using enzymes commercially include: they are water soluble which makes them hard to recover and some products can inhibit the enzyme activity (feedback inhibition) .

Drug molecules, many drug molecules are enzyme inhibitors and a medicinal enzyme inhibitor is usually characterized by its specificity and its potency. A high specificity and potency suggests that a drug will have fewer side effects and less toxic. Enzyme inhibitors are found in nature and are also designed and produced as part of pharmacology and biochemistry 6.

Natural poisons are often enzyme inhibitors that have evolved to defend a plant or animal against predators. These natural toxins include some of the most poisonous compounds known.

Nerve gases such as diisopropylfluorophosphate (DFP) inhibit the active site of acetylcholine esterase by reacting with the hydroxyl group of serine to make an ester.

References

Scapin G (2006). Structural biology and drug discovery. Curr. Pharm. Des.,      12(17):2087–2097.

Krishnan R, Zhang E, Hakansson K, Arni RK, Tulinsky A, Lim-Wilby MS, Levy OE, Semple JE, Brunck TK (1998). Highly selective mechanism-based thrombin inhibitors:  structures of thrombin and trypsin inhibited with rigid peptidyl aldehydes. Biochemistry, 37 (35):12094-12103.

Fischer E (1894). Einfluss der configuration auf die wirkung der enzyme. Ber. Dt. Chem. Ges., 27:2985–2993.

Koshland DE (1958). Application of a theory of enzyme specificity to protein synthesis. PNAS., 44 (2):98–104.

Huang KF, Liu YL, Cheng WJ, Ko TP, Wang AH (2005). Crystal structures of human glutaminyl cyclase, an enzyme responsible for protein N-terminal pyroglutamate formation. PNAS., 102(37):13117-13122.

Holmes CF, Maynes JT, Perreault KR, Dawson JF, James MN (2002). Molecular enzymology underlying regulation of protein phosphatase-1 by natural toxins. Curr Med Chem., 9(22):1981-1989.

多肽Ac-Gly-Asp-Tyr-Ser-His-Cys-Ser-Pro-Leu-Arg-Tyr-Tyr-Pro-Trp-Trp-Lys-Cys-Thr-Tyr-Pro-Asp-Pro-Glu-Gly-Gly-Gly-NH2的合成步驟：

1、合成MBHA樹脂：取若干克MBHA樹脂（如初始取代度為0.5mmol/g）和1倍樹脂摩爾量的Fmoc-Linker-OH加入到反應(yīng)器中，加入DMF，攪拌使氨基酸完全溶解。再加入樹脂2倍量的DIEPA，攪拌混合均勻。再加入樹脂0.95倍量的HBTU，攪拌混合均勻。反應(yīng)3-4小時后，用DMF洗滌3次。用2倍樹脂體積的10%乙酸酐/DMF 進行封端30分鐘。然后再用DMF洗滌3次，甲醇洗滌2次，DCM洗滌2次，再用甲醇洗滌2次。真空干燥12小時以上，得到干燥的樹脂{Fmoc-Linker-MHBA Resin}，測定取代度。這里測得取代度為 0.3mmol/g。結(jié)構(gòu)如下圖：

2、脫Fmoc：取2.01g的上述樹脂，用DCM或DMF溶脹20分鐘。用DMF洗滌2遍。加3倍樹脂體積的20%Pip/DMF溶液，鼓氮氣30分鐘，然后2倍樹脂體積的DMF 洗滌5次。得到 H2N-Linker-MBHA Resin 。（此步驟脫除Fmoc基團，茚三酮檢測為藍色，Pip為哌啶）。結(jié)構(gòu)圖如下：

3、縮合：取1.81mmol Fmoc-Gly-OH 氨基酸，加入到上述樹脂里，加適當DMF溶解氨基酸，再依次加入3.62mmol DIPEA，1.72mmol HBTU。反應(yīng)30分鐘后，取小樣洗滌，茚三酮檢測為無色。用2倍樹脂體積的DMF 洗滌3次樹脂。（洗滌樹脂，去掉殘留溶劑，為下一步反應(yīng)做準備）。得到Fmoc-Gly-Linker-MBHA Resin。氨基酸：DIPEA：HBTU：樹脂=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-Gly-Linker-MBHA Resin

Fmoc-Gly-Gly-Linker-MBHA Resin

H2N-Gly-Gly-Linker-MBHA Resin

Fmoc-Gly-Gly-Gly-Linker-MBHA Resin

H2N-Gly-Gly-Gly-Linker-MBHA Resin

Fmoc-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

H2N-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

Fmoc-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

H2N-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

Fmoc-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

H2N-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

Fmoc-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

H2N-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

Fmoc-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

H2N-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

Fmoc-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

H2N-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

Fmoc-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

H2N-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

Fmoc-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

H2N-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

Fmoc-Trp(Boc)-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

H2N-Trp(Boc)-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

Fmoc-Trp(Boc)-Trp(Boc)-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

H2N-Trp(Boc)-Trp(Boc)-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

Fmoc-Pro-Trp(Boc)-Trp(Boc)-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

H2N-Pro-Trp(Boc)-Trp(Boc)-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

Fmoc-Tyr(tBu)-Pro-Trp(Boc)-Trp(Boc)-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

H2N-Tyr(tBu)-Pro-Trp(Boc)-Trp(Boc)-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

Fmoc-Tyr(tBu)-Tyr(tBu)-Pro-Trp(Boc)-Trp(Boc)-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

H2N-Tyr(tBu)-Tyr(tBu)-Pro-Trp(Boc)-Trp(Boc)-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

Fmoc-Arg(Pbf)-Tyr(tBu)-Tyr(tBu)-Pro-Trp(Boc)-Trp(Boc)-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

H2N-Arg(Pbf)-Tyr(tBu)-Tyr(tBu)-Pro-Trp(Boc)-Trp(Boc)-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

Fmoc-Leu-Arg(Pbf)-Tyr(tBu)-Tyr(tBu)-Pro-Trp(Boc)-Trp(Boc)-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

H2N-Leu-Arg(Pbf)-Tyr(tBu)-Tyr(tBu)-Pro-Trp(Boc)-Trp(Boc)-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

Fmoc-Pro-Leu-Arg(Pbf)-Tyr(tBu)-Tyr(tBu)-Pro-Trp(Boc)-Trp(Boc)-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

H2N-Pro-Leu-Arg(Pbf)-Tyr(tBu)-Tyr(tBu)-Pro-Trp(Boc)-Trp(Boc)-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

Fmoc-Ser(tBu)-Pro-Leu-Arg(Pbf)-Tyr(tBu)-Tyr(tBu)-Pro-Trp(Boc)-Trp(Boc)-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

H2N-Ser(tBu)-Pro-Leu-Arg(Pbf)-Tyr(tBu)-Tyr(tBu)-Pro-Trp(Boc)-Trp(Boc)-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

Fmoc-Cys(Trt)-Ser(tBu)-Pro-Leu-Arg(Pbf)-Tyr(tBu)-Tyr(tBu)-Pro-Trp(Boc)-Trp(Boc)-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

H2N-Cys(Trt)-Ser(tBu)-Pro-Leu-Arg(Pbf)-Tyr(tBu)-Tyr(tBu)-Pro-Trp(Boc)-Trp(Boc)-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

Fmoc-His(Trt)-Cys(Trt)-Ser(tBu)-Pro-Leu-Arg(Pbf)-Tyr(tBu)-Tyr(tBu)-Pro-Trp(Boc)-Trp(Boc)-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

H2N-His(Trt)-Cys(Trt)-Ser(tBu)-Pro-Leu-Arg(Pbf)-Tyr(tBu)-Tyr(tBu)-Pro-Trp(Boc)-Trp(Boc)-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

Fmoc-Ser(tBu)-His(Trt)-Cys(Trt)-Ser(tBu)-Pro-Leu-Arg(Pbf)-Tyr(tBu)-Tyr(tBu)-Pro-Trp(Boc)-Trp(Boc)-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

H2N-Ser(tBu)-His(Trt)-Cys(Trt)-Ser(tBu)-Pro-Leu-Arg(Pbf)-Tyr(tBu)-Tyr(tBu)-Pro-Trp(Boc)-Trp(Boc)-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

Fmoc-Tyr(tBu)-Ser(tBu)-His(Trt)-Cys(Trt)-Ser(tBu)-Pro-Leu-Arg(Pbf)-Tyr(tBu)-Tyr(tBu)-Pro-Trp(Boc)-Trp(Boc)-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

H2N-Tyr(tBu)-Ser(tBu)-His(Trt)-Cys(Trt)-Ser(tBu)-Pro-Leu-Arg(Pbf)-Tyr(tBu)-Tyr(tBu)-Pro-Trp(Boc)-Trp(Boc)-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

Fmoc-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-His(Trt)-Cys(Trt)-Ser(tBu)-Pro-Leu-Arg(Pbf)-Tyr(tBu)-Tyr(tBu)-Pro-Trp(Boc)-Trp(Boc)-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

H2N-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-His(Trt)-Cys(Trt)-Ser(tBu)-Pro-Leu-Arg(Pbf)-Tyr(tBu)-Tyr(tBu)-Pro-Trp(Boc)-Trp(Boc)-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

Fmoc-Gly-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-His(Trt)-Cys(Trt)-Ser(tBu)-Pro-Leu-Arg(Pbf)-Tyr(tBu)-Tyr(tBu)-Pro-Trp(Boc)-Trp(Boc)-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin

以上中間結(jié)構(gòu)，均可在專肽生物多肽計算器-多肽結(jié)構(gòu)計算器中，一鍵畫出。

最后再經(jīng)過步驟二得到 H2N-Gly-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-His(Trt)-Cys(Trt)-Ser(tBu)-Pro-Leu-Arg(Pbf)-Tyr(tBu)-Tyr(tBu)-Pro-Trp(Boc)-Trp(Boc)-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHA Resin，結(jié)構(gòu)如下：

5、乙酸酐反應(yīng)連接：在上述樹脂中，加入適當DMF后，再加入1.81mmol乙酸酐到樹脂中，再加入3.62mmol DIPEA，鼓氮氣反應(yīng)30分鐘。用2倍樹脂體積的DMF 洗滌3次樹脂（洗滌樹脂，去掉殘留溶劑，為下一步反應(yīng)做準備）。 得到Ac-Gly-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-His(Trt)-Cys(Trt)-Ser(tBu)-Pro-Leu-Arg(Pbf)-Tyr(tBu)-Tyr(tBu)-Pro-Trp(Boc)-Trp(Boc)-Lys(Boc)-Cys(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Asp(OtBu)-Pro-Glu(OtBu)-Gly-Gly-Gly-Linker-MBHAResin。 結(jié)構(gòu)如下：

6、切割：6倍樹脂體積的切割液（或每1g樹脂加8ml左右的切割液），搖床搖晃 2小時，過濾掉樹脂，用冰無水乙醚沉淀濾液，并用冰無水乙醚洗滌沉淀物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Ac-Gly-Asp-Tyr-Ser-His-Cys-Ser-Pro-Leu-Arg-Tyr-Tyr-Pro-Trp-Trp-Lys-Cys-Thr-Tyr-Pro-Asp-Pro-Glu-Gly-Gly-Gly-NH2。結(jié)構(gòu)圖見產(chǎn)品結(jié)構(gòu)圖。

切割液選擇：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%

（前兩種適合沒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。）

7、純化凍干：使用液相色譜純化，收集目標峰液體，進行凍干，獲得蓬松的粉末狀固體多肽。不過這時要取小樣復(fù)測下純度 是否目標純度。

8、最后總結(jié)：

杭州專肽生物技術(shù)有限公司（ALLPEPTIDE http://amynixphotography.com）主營定制多肽合成業(yè)務(wù)，提供各類長肽，短肽，環(huán)肽，提供各類修飾肽，如：熒光標記修飾（CY3、CY5、CY5.5、CY7、FAM、FITC、Rhodamine B、TAMRA等），功能基團修飾肽（疊氮、炔基、DBCO、DOTA、NOTA等），同位素標記肽（N15、C13），訂書肽（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、一對或者多對二硫鍵環(huán)），生物素標記肽，PEG修飾肽，甲基化修飾肽等。

以上所有內(nèi)容，為專肽生物原創(chuàng)內(nèi)容，請勿發(fā)布到其他網(wǎng)站上。