127103-11-1
基本信息
戈雷拉肽,THYMOSINΒ4(1-4)
乙?;?絲氨酰-天冬氨酰-賴氨酰-脯氨酸
GORALATIDE
Acetyl-SDKP
SERASPENIDE
AC-SDKP-DELTA
Thymosin β4 (1-4)
Thymosin β4 (1-4)
AC-SER-ASP-LYS-PRO
AC-SER-ASP-LYS-PRO-OH
ACETYL-SER-ASP-LYS-PRO
物理化學性質(zhì)
常見問題列表
1.肽樹脂的合成
(1)制備Fmoc-Pro-樹脂:
稱取5克2-氯-三苯氯甲基樹脂,用二氯甲烷(DCM)100ml浸泡60分鐘,在上述的樹脂中,加入DIEA 4.5ml,3.38g Fmoc-Pro-OH,25℃反應(yīng)2小時,再加入封閉試劑甲醇1.5ml,25℃反應(yīng)2小時,樹脂用35ml異丙醇洗滌兩次、再用35ml N,N-二甲基甲酰胺(DMF)洗兩次,獲得Fmoc-Pro-樹脂;
(2)制備Fmoc-Lys(Boc)-OH樹脂:
在步驟(1)的Fmoc-Pro-樹脂中,加入35ml脫帽試劑,25℃反應(yīng)10分鐘,用真空泵抽干,重新加入35ml脫帽試劑25℃反應(yīng)30分鐘,抽干,用35ml異丙醇洗滌2 次,35mlDMF洗滌2次,重蒸餾的35mlDMF洗滌2次,取少量樹脂加4ml茚三酮試劑, 沸水中加熱3min,檢測結(jié)果呈陽性。加入用重蒸餾的DMF溶解的4.69gFmoc-Lys(Boc) -OH、2mlDIEA、3.22gTBTU、5mlHOBt的混合物,22℃反應(yīng)60分鐘,用真空泵抽干,用異丙醇洗滌2次,DMF洗滌2次,抽干,取少量樹脂加4ml茚三酮試劑,沸水中加熱3min,檢測結(jié)果呈陰性。獲得Fmoc-Lys(Boc)-Pro-樹脂;
所述的脫帽試劑的組分和體積比為:PIP∶DMF=1∶2.5,下同;
(3)制備Fmoc-D-Asp(otBu)-Lys(Boc)-Pro-樹脂:
在步驟(2)的Fmoc-Lys(Boc)-Pro-樹脂中,加入脫帽試劑脫帽兩次,條件 同步驟(2);加入用重蒸餾的DMF溶解的4.12g Fmoc-D-Asp(otBu)-OH、2mlDIEA、3.22gTBTU、5mlHOBt的混合物,22℃反應(yīng)60分鐘,用真空泵抽干,用異丙醇洗滌2 次,DMF洗滌2次,取少量樹脂加4ml茚三酮試劑,沸水中加熱3min,檢測結(jié)果呈陰 性,抽干,取少量樹脂加4ml茚三酮試劑,沸水中加熱3min,檢測結(jié)果呈陰性。獲 得Fmoc-D-Asp(otBu)--Lys(Boc)-Pro-樹脂;
其余操作以及工藝條件同上;
(4)制備Fmoc-Ser(tBu)-D-Asp(otBu)-Lys(Boc)-Pro-樹脂:
在步驟(3)的Fmoc-D-Asp(otBu)-Lys(Boc)-Pro-樹脂中,加入脫帽試劑脫帽兩次,條件同步驟(2);加入用重蒸餾的DMF溶解的3.84g Fmoc-Ser(tBu)-OH、2mlDIEA、 3.22gTBTU、2mlHOBt的混合物,22℃反應(yīng)60分鐘,用真空泵抽干,用異丙醇洗滌2 次,DMF洗滌2次,抽干,取少量樹脂加4ml茚三酮試劑,沸水中加熱3min,檢測 結(jié)果呈陰性,獲得Fmoc-Ser(tBu)-D-Asp(otBu)-Lys(Boc)-Pro-樹脂;
(5)制備Ac-Ser(tBu)-D-Asp(otBu)--Lys(Boc)-Pro-樹脂(肽的乙?;?:
在步驟(4)的Fmoc-Ser(tBu)-D-Asp(otBu)-Lys(Boc)-Pro-樹脂中,加入脫帽試劑脫帽兩次,條件同步驟(2);加入6ml乙酸酐、50mlDCM、3mlDIEA的混合物,22℃反應(yīng)60分鐘,用真空泵抽干,用異丙醇洗滌2次、DMF洗滌2次,甲醇洗3次,甲醇浸泡30-60分鐘,抽干,用氮氣吹干肽樹脂成干顆粒,所得到的肽樹脂為獲得Ac-Ser(tBu)-D-Asp(otBu)-Lys(Boc)-Pro-樹脂;
2.肽樹脂的切割分離
把吹干的Ac-Ser(tBu)-D-Asp(otBu)-Lys(Boc)-Pro-樹脂倒入玻璃的切割瓶中,加入預(yù)冷至-20℃的切肽試劑(TFA∶H2O=95∶5,體積比)80ml,20℃反應(yīng)1.5小時,過濾除去樹脂,加-20℃的冰乙醚沉淀,離心收集沉淀物,用冷乙醚洗滌1次,低溫冷凍干燥16小時,獲得四肽異構(gòu)體粗品2.1g;
3.對四肽異構(gòu)體粗品的分離純化
(1)樣品準備:將四肽異構(gòu)體粗品溶于水溶液中,過濾,備用。
(2)四肽異構(gòu)體粗品HPLC分析:取濾液20μl用HPLC分析,色譜條件:C18色譜柱 (Hypersil-ODS2,Φ5um 4.6×250mm),流動相:A:0.1%三氟乙酸加99.9%水;B: 0.1%三氟乙酸加99.9%乙腈;梯度洗脫;流速為1ml/min;檢測波長為:215nm;
(3)四肽異構(gòu)體粗品HPLC純化:
取濾液3ml用HPLC純化,選用C18色譜柱(Hypersil-ODS2,Φ5um 10×250mm), 流動相:A:0.1%三氟乙酸加99.9%水;B:0.1%三氟乙酸加99.9%乙腈;梯度洗脫; 流速為10ml/min;檢測波長為:215nm;收集目標峰液,樣品峰合并后去鹽,凍干, 獲得四肽異構(gòu)體純品1.56g。
(4)四肽異構(gòu)體純品分析:
取少量樣品,配成0.5mg/ml,過濾,取20μl用HPLC分析,條件同四肽異構(gòu)體 粗品HPLC分析條件。
本實施例中,戈雷拉肽粗品的純度為91.8670%,收率為 74.3%,純度為98%(HPLC歸一化法)。
N-Acetyl-Ser-Asp-Lys-Pro is degraded specifically by ACE, and its plasma level rises substantially during ACE inhibitor therapy. Flow cytometry of rat cardiac fibroblasts treated with N-Acetyl-Ser-Asp-Lys-Pro shows significant inhibition of the progression of cells from G0/G1 phase to S phase of the cell cycle. Moreover, phosphorylation and nuclear translocation of Smad2 is decreased in cardiac fibroblasts treated with N-Acetyl-Ser-Asp-Lys-Pro. N-acetyl-seryl-aspartyl-lysyl-proline appears to exert this function by blocking the action of a stem cell-specific proliferation stimulator and acts selectively on quiescent progenitors. N-Acetyl-Ser-Asp-Lys-Pro inhibits collagenase expression and activation is associated with increased expression of TIMP-1 and TIMP-2. N-Acetyl-Ser-Asp-Lys-Pro normalizes the IL-1β-mediated increase in MMP-2 and MMP-9 activities and MMP-13 expression.
N-Acetyl-Ser-Asp-Lys-Pro prevents hypertension-induced inflammatory cell infiltration, collagen deposition, nephrin downregulation and albuminuria, which could lead to renoprotection in hypertensive mice.