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70967-97-4,組織蛋白酶G基質I：Suc-Ala-Ala-Pro-Phe-pNA,Suc-Ala-Ala-Pro-Phe-pNA,Suc-AAPF-pNA,杭州專肽生物的產品

組織蛋白酶G基質I：Suc-Ala-Ala-Pro-Phe-pNA

A readily soluble, specific and sensitive substrate for chymotrypsin and human pancreatic elastase. It is also hydrolyzed by cathepsin G and chymase. Furthermore it is the standard substrate for FK-506 binding proteins (FKBPs, also called macrophilins) an

編號：111237

CAS號：70967-97-4

單字母：Suc-AAPF-pNA

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  • 編號:111237
    中文名稱:組織蛋白酶G基質I：Suc-Ala-Ala-Pro-Phe-pNA
    CAS號:70967-97-4
    單字母:Suc-AAPF-pNA
    三字母:Suc

    N端丁二酸修飾

    -Ala

    丙氨酸

    -Ala

    丙氨酸

    -Pro

    脯氨酸

    -Phe

    苯丙氨酸

    -pNA

    對硝基苯胺

    氨基酸個數:4
    分子式:C30H36O9N6
    平均分子量:624.64
    精確分子量:624.25
    等電點(PI):-
    pH=7.0時的凈電荷數:-
    平均親水性:-1.1666666666667
    疏水性值:1.2
    外觀與性狀:白色粉末狀固體
    消光系數:-
    來源:人工化學合成，僅限科學研究使用，不得用于人體。
    純度:95%、98%
    鹽體系:可選TFA、HAc、HCl或其它
    儲存條件:負80℃至負20℃
    標簽:pNA修飾肽    Suc修飾肽   

  • A readily soluble, specific and sensitive substrate for chymotrypsin and human pancreatic elastase. It is also hydrolyzed by cathepsin G and chymase. Furthermore it is the standard substrate for FK-506 binding proteins (FKBPs, also called macrophilins) and cyclophilins, which belong to the group of peptidyl prolyl cis-trans isomerases (PPIases). Thus, Suc-AAPF-pNA has been used for an uncoupled protease-free assay of PPIase activity.

  • DOI名稱
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  • 多肽Suc-Ala-Ala-Pro-Phe-pNA的合成步驟：

    1、合成CTC樹脂：稱取2.56g CTC Resin（如初始取代度約為0.57mmol/g）和1.75mmol Fmoc-Phe-OH于反應器中，加入適量DCM溶解氨基酸（需要注意，此時CTC樹脂體積會增大好幾倍，避免DCM溶液過少），再加入4.38mmol DIPEA（Mw：129.1,d：0.740g/ml），反應2-3小時后，可不抽濾溶液，直接加入1ml的HPLC級甲醇，封端半小時。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，有利于后續(xù)反應）。得到  Fmoc-Phe-CTC Resin。結構圖如下：

    2、脫Fmoc：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-Phe-CTC Resin 。（此步驟脫除Fmoc基團，茚三酮檢測為藍色，Pip為哌啶）。結構圖如下：

    3、縮合：取4.38mmol Fmoc-Pro-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8.76mmol DIPEA，4.16mmol HBTU。反應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，為下一步反應做準備）。得到Fmoc-Pro-Phe-CTC Resin。氨基酸：DIPEA：HBTU：樹脂=3：6：2.85：1（摩爾比）。結構圖如下：

    4、依次循環(huán)步驟二、步驟三，依次得到

    H2N-Pro-Phe-CTC Resin

    Fmoc-Ala-Pro-Phe-CTC Resin

    H2N-Ala-Pro-Phe-CTC Resin

    Fmoc-Ala-Ala-Pro-Phe-CTC Resin

    以上中間結構，均可在專肽生物多肽計算器-多肽結構計算器中，一鍵畫出。

    最后再經過步驟二得到 H2N-Ala-Ala-Pro-Phe-CTC Resin，結構如下：

    5、丁二酸反應連接：在上述樹脂中，加入適當DMF后，再加入4.38mmol丁二酸到樹脂中，再加入8.76mmol DIPEA，鼓氮氣反應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，為下一步反應做準備）。 得到Suc-Ala-Ala-Pro-Phe-CTC Resin。 結構如下：

    6、全保護切割：配置0.5%TFA/DCM溶液，溶液體積約為樹脂體積的3倍。再次用DCM洗滌樹脂2遍（去除殘留DMF），后將配置好的溶液倒入到反應器中，反應30分鐘。抽濾樹脂，收集濾液（此時多肽已經從樹脂上分離，存在于濾液中）。多肽序列為 Suc-Ala-Ala-Pro-Phe-CTC Resin。 在濾液中添加DIEPA，調PH至7-8。用飽和NaHCO3洗滌濾液，分離出DCM層溶液?？蛇m當旋蒸DCM層溶液，減少有機溶劑。再次加入1或2倍體積的乙酸乙酯，用稀HCl溶液調PH至微酸性，將多肽從DCM層萃取到乙酸乙酯層。用飽和NaCl洗滌2次乙酸乙酯層。用無水硫酸鎂吸收乙酸乙酯層的水分。通過減壓旋蒸，直接將乙酸乙酯完全旋蒸掉，得到晶體狀固體多肽，用于下一步C端反應。或通過減壓旋蒸保留適量乙酸乙酯的溶液體積，加入冰乙醚析出 多肽，然后對多肽進行烘干操作即可用于下一步C端反應。Suc-Ala-Ala-Pro-Phe-COOH的結構圖如下。

    7、4-硝基苯胺反應連接：在上述樹脂中，加入適當DMF后，再加入4.38mmol 4-硝基苯胺到樹脂中，再加入8.76mmol DIPEA、4.16mmol HBTU，鼓氮氣反應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，為下一步反應做準備）。 得到 Suc-Ala-Ala-Pro-Phe-pNA。 結構如下：

    8、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Suc-Ala-Ala-Pro-Phe-pNA。結構圖見產品結構圖。

    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。）

    9、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測下純度 是否目標純度。

    10、最后總結：

    杭州專肽生物技術有限公司（ALLPEPTIDE http://amynixphotography.com）主營定制多肽合成業(yè)務，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。

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