新酪啡肽Fmoc固相合成及其活性的检测
摘要
阿片肽(Opioid peptide)是一种神经免疫肽,可分为内源性阿片肽和外源性阿片肽,对中枢神经系统及外周器官均起作用。从已发现的外源性阿片肽的数量来看,很大一部分都来源于酪蛋白。它们以无活性的状态存在于酪蛋白中,当通过酶解才会从酪蛋白前体物中水解出来。本研究中的所要合成的酪啡肽来源于酪蛋白Alpha-S1 -casein的144-158序列,其序列为YFYPELFRQFYQLDA,分子量为:2252.48。
由于该酪啡肽序列较短,利用Fmoc固相合成法可以方便的制备,确定合成的工艺为:以Wang树脂为固相载体,从C端向N端合成,Fmoc-氨基酸为原料,二氯甲烷(DCM)为反应溶剂,O-苯并氮唑-N,N,N’N’-四甲基脲四氟硼酸酯/1-羟基苯并三氮唑(HOBt)/N,N-二异丙基乙胺(DIEA)为缩合体系,进行逐步固相合成。合成完成后进行切割反应,试剂为三氟乙酸/水/苯甲硫醚/石碳酸(TFA/H2O/thioanisole/Phenol)体系(85: 5: 5: 5);切割时间2h;切割温度为24℃。得到的滤液用无水醚沉淀,冷冻干燥,可得到酪啡肽的白色粗产品,得率为57.7%。粗产品经Sephadex G-15进行分离纯化,得到两个峰,经检测发现,第一个峰具有活性,再将第一个峰经高效液相色谱-质谱联用,进行分子量测定。从色谱分析图中可知纯度达43%。合成肽的分子量为2253.4,与酪啡肽的理论值基本一致。为了检测合成的酪啡肽是否具有生物学活性,实验中采用了热板法和醋酸扭体实验分别检测其镇痛活性。结果表明合成的酪啡肽不仅具有中枢镇痛活性,还具有外周镇痛的活性。小鼠负重游泳实验也证明了该肽具有抗疲劳的作用。经spss软件t检验分析,合成的酪啡肽与水解获得的酪啡肽对小鼠痛阈都有明显的提高,两者在显著水平p=0.05下,中枢镇痛和抗疲劳效果没有显著差异,说明它们都有镇痛与抗疲劳的作用,且效果无显著差异。
Opioids, a kind of neuromimune peptides, can be divided into Endogenous Opioid Peptides and Exogenous Opioid Peptides. They work in the central nervous system and peripheral organs. Lots of opioid peptides had been discovered comes from casein. Hidden or inactive in the amino-acid sequence of casein, they can be released or activated via specific, enzyme-mediated proteolysis. In this paper, the synthesized casomorphin is from the 144-158 sequence of Alpha-S1–casein: YFYPELFRQFYQLDA, its molecular weight is 2252.48.
Because of its short sequence, this novel casomorphin can be synthetized conveniently by Fmoc solid-phase peptide synthesis method (SPPS method). This casomorphin was synthesized successfully from C-terminal to N-terminal according to its amino acids sequence with raw material of Fmoc- amino acids and Wang resin as the vector. The optimized technology of this opioid synthesis was as follows: using DCM as the main coupling solvent, TBTU/Hobt/DIEA as the coupling reagent, the synthesis reaction time 3h, and TFA/H2O/thioanisole/Phenol (Volune ratio of 85: 5: 5: 5) as resin cleavage condition, the cleavage reaction time 2h, 24℃as the cleavage temperature. Then the white power of novel casomorphin was precipitated using absolute ether. And then the crude peptide was frozen-dried. The yield of crude product was 57.7%. Sephadex G-15 was used to separate and purify the novel opioid, then collected peaks were analyzed by HPLC-MS. From the chromatogram map, the purity of this peptide was 43%, and the mass spectrogram told the opioids molecular weight was 2253.4. In order to test activities of the opioid, the hot plate test and the writhing method were used. Results showed that this synthesis opioid not only has the active of center analgesia, but also has active of peripheral analgesia. As well as it had obvious anti-fatigue effects since it could prolonged the swimming time of mice. From the t-test results, it was also found that the bio-activities of synthesized casomorphin was corresponded with the ones of nature casomorphin.
由于该酪啡肽序列较短,利用Fmoc固相合成法可以方便的制备,确定合成的工艺为:以Wang树脂为固相载体,从C端向N端合成,Fmoc-氨基酸为原料,二氯甲烷(DCM)为反应溶剂,O-苯并氮唑-N,N,N’N’-四甲基脲四氟硼酸酯/1-羟基苯并三氮唑(HOBt)/N,N-二异丙基乙胺(DIEA)为缩合体系,进行逐步固相合成。合成完成后进行切割反应,试剂为三氟乙酸/水/苯甲硫醚/石碳酸(TFA/H2O/thioanisole/Phenol)体系(85: 5: 5: 5);切割时间2h;切割温度为24℃。得到的滤液用无水醚沉淀,冷冻干燥,可得到酪啡肽的白色粗产品,得率为57.7%。粗产品经Sephadex G-15进行分离纯化,得到两个峰,经检测发现,第一个峰具有活性,再将第一个峰经高效液相色谱-质谱联用,进行分子量测定。从色谱分析图中可知纯度达43%。合成肽的分子量为2253.4,与酪啡肽的理论值基本一致。为了检测合成的酪啡肽是否具有生物学活性,实验中采用了热板法和醋酸扭体实验分别检测其镇痛活性。结果表明合成的酪啡肽不仅具有中枢镇痛活性,还具有外周镇痛的活性。小鼠负重游泳实验也证明了该肽具有抗疲劳的作用。经spss软件t检验分析,合成的酪啡肽与水解获得的酪啡肽对小鼠痛阈都有明显的提高,两者在显著水平p=0.05下,中枢镇痛和抗疲劳效果没有显著差异,说明它们都有镇痛与抗疲劳的作用,且效果无显著差异。
Opioids, a kind of neuromimune peptides, can be divided into Endogenous Opioid Peptides and Exogenous Opioid Peptides. They work in the central nervous system and peripheral organs. Lots of opioid peptides had been discovered comes from casein. Hidden or inactive in the amino-acid sequence of casein, they can be released or activated via specific, enzyme-mediated proteolysis. In this paper, the synthesized casomorphin is from the 144-158 sequence of Alpha-S1–casein: YFYPELFRQFYQLDA, its molecular weight is 2252.48.
Because of its short sequence, this novel casomorphin can be synthetized conveniently by Fmoc solid-phase peptide synthesis method (SPPS method). This casomorphin was synthesized successfully from C-terminal to N-terminal according to its amino acids sequence with raw material of Fmoc- amino acids and Wang resin as the vector. The optimized technology of this opioid synthesis was as follows: using DCM as the main coupling solvent, TBTU/Hobt/DIEA as the coupling reagent, the synthesis reaction time 3h, and TFA/H2O/thioanisole/Phenol (Volune ratio of 85: 5: 5: 5) as resin cleavage condition, the cleavage reaction time 2h, 24℃as the cleavage temperature. Then the white power of novel casomorphin was precipitated using absolute ether. And then the crude peptide was frozen-dried. The yield of crude product was 57.7%. Sephadex G-15 was used to separate and purify the novel opioid, then collected peaks were analyzed by HPLC-MS. From the chromatogram map, the purity of this peptide was 43%, and the mass spectrogram told the opioids molecular weight was 2253.4. In order to test activities of the opioid, the hot plate test and the writhing method were used. Results showed that this synthesis opioid not only has the active of center analgesia, but also has active of peripheral analgesia. As well as it had obvious anti-fatigue effects since it could prolonged the swimming time of mice. From the t-test results, it was also found that the bio-activities of synthesized casomorphin was corresponded with the ones of nature casomorphin.
引文
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[2] Reinscheid RK, Nothacker HP, Bourson A, et al. Orphanin FQ: A neuropeptide that activates an opioidlike G protein-coupled receptor[J]. Science, 1995, 270(5337): 792-794.
[3] Zadina JE, Hackler L, Ge LJ, et al. A potent and selective endogenous agonist for the mu-opiate receptor[J]. Nature, 1997, 386(6624): 499-502.
[4] Perpetuo EA, Juliano L, Lebrun I. Biochemical and pharmacological aspects of two bradykinin- potentiating peptides obtained from tryptic hydrolysis of casein[J]. Journal of Protein Chemistry, 2003, 22(7-8): 601–606.
[5]韩飞,施用晖,刘勇,等.一种新阿片肽的分离纯化[J].中国生物工程杂志, 2005, 25(2): 31-34.
[6]张建辉.阿片肽的制备纯化及其测定方法的研究[D].天津,天津商业大学, 2007.
[7]韩翔,王萍,董强.阿片肽、阿片受体及其拮抗剂在中枢神经系统中的作用[J].神经损伤与功能重建, 2006, 1(2): 112 -113.
[8] Honda T, Shirasu N, Isozaki K, et al. Differential receptor binding characteristics of consecutive phenylalanines inμ-opioid specific peptide ligand endomorphin-2[J]. Bioorganic&Medicinal Chemistry, 2007, 15(11): 3883- 3888.
[9] Szatmari I, Biyashev D, T?mb?ly C, et al. Influence of degradation on binding properties and biological activity of endomorphin-1[J]. Biochemical and Biophysical Research Communications, 2001, 284(3): 771-776.
[10] Fichna J, Janecka A, Bailly L, et al. In vitro characterization of novel peptide inhibitors of endomorphin-degrading enzymes in the rat brain[J]. Chemical Biology & Drug Design, 2006, 68(3):173-175.
[11] Podlogar BL, Germana PM, Ferguson DM, et al. Conformational analysis of the endogenous mu-opioid agonist endomorphin-1 using NMR spectroscopy and molecular modeling[J]. FEBS Letters, 1998, 439(1-2): 13-20.
[12] Fiori S, Renner C, Cramer J, et al. Preferred conformation of endomorphin-1 in aqueous and membrane-mimetic environments[J]. Journal of Molecular Biology, 1999, 291(1):163-175.
[13] Leitgeb B, Szekeres A, Tóth G. Conformational analysis of endomorphin-1 by molecular dynamics methods[J]. Journal of Peptide Research, 2003, 62(4): 145-157.
[14] Leitgeb B, ?tv?s F, Tóth G. Conformational analysis of endomorphin-2 by molecular dynamics methods[J]. Biopolymers, 2003, 68(4): 497-511.
[15] Leitgeb B, Tóth G. Aromatic-aromatic and proline-aromatic interactions in endormorphin-1 and endomorphin-2[J]. European Journal of Medicinal Chemistry, 2005, 40(7): 674-686.
[16] Uezono T, Toraya S, Obata M, et al. Structure and orientation of dynorphin bound to lipid bilayers by 13C solid-state NMR[J]. Journal of Molecular Structure, 2005, 749(1-3): 13-19.
[17]王玉秀,童昭岗,胡海青.内源性阿片肽系统免疫调节功能的研究进展[J].中国临床康复, 2003, 7(10): 1558-1559.
[18] Anton B, Phillipe L, Calva JC, et al. Endomorphin 1 and endomorphin 2 suppress in vitro antibody formation at ultra-low concentrations: Anti-peptide antibodies but not opioid antagonists block the activity[J]. Brain, Behavior, and Immunity, 2008, 22(6): 824-832.
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