生物转化法合成R-PAC及其衍生物和类似物的研究
发布时间：2018-10-09 13:52
【摘要】：R-苯基乙酰基甲醇(R-PAC)是一种重要的手性有机合成中间体,在制药业中常作为前体物质来合成苯丙胺类药物,如麻黄碱,去甲麻黄碱及安非他命等等。本研究利用生物转化的方法,选择海栖热袍菌乙酰羟酸合酶的催化亚基(TmAHAS-CSU)为生物催化剂,以丙酮酸及苯甲醛为反应底物,在硫胺素焦磷酸及其它辅因子的存在下,以最优化的实验条件制备得到了 R-PAC,分离后产率为83.6%,纯度为98.3%(HPLC检测结果)。同时,我们还测定了该生物催化剂转化苯甲醛衍生物及类似物的可能性,结果显示其有较宽的底物谱。首先,我们利用本实验室前期制备的能够过表达海栖热袍菌乙酰羟酸合酶催化亚基(TmAHAS-CSU)的重组工程菌株EcR(DE3)-pET28a-TmAHAS-CSU,采取自诱导方式制备了 TmAHAS-CSU,并经过Ni-NTA亲和层析得到了纯化后的目的蛋白。然后我们分别利用纯化后的TmAHAS-CSU蛋白及重组工程菌株全菌为催化剂探讨了生物转化法制备R-PAC的方法。在酶TmAHAS-CSU催化的反应中,为了使R-PAC产率最大化,对其合成体系进行优化,最终确定最佳反应体系为50 mM的磷酸盐(PBS)缓冲液,pH 8.0,反应时间40min,反应温度为80℃,助溶剂DMSO含量1%,底物丙酮酸(Pyr)和苯甲醛(BA)的最佳比例为1.25:1,最佳浓度分别为50 mM和40 mM。在实验中我们还发现,以重组工程菌株的全菌催化该反应时能够获得更加理想的产率。此外,还以苯甲醛的衍生物以及一些芳杂环基甲醛化合物作为底物,与另一底物Pyr在重组工程菌全菌的催化下进行反应,分别利用HPLC、LC-MS以及1H-NMR对部分反应结果进行检测和鉴定。结果发现,甲氧基取代及单羟基取代的苯甲醛衍生物等共计13种化合物能够有效进行该反应,这不但扩充了 TmAHAS-CSU的底物谱范围,更为进一步研究TmAHAS-CSU打下了良好的基础。
[Abstract]:R- phenylacetyl methanol (R-PAC) is an important chiral organic synthesis intermediate, which is often used as a precursor in pharmaceutical industry to synthesize amphetamines, such as ephedrine, norephedrine and amphetamines. In this study, the catalytic subunit (TmAHAS-CSU) of acetyl hydroxy acid synthase (TmAHAS-CSU) was selected as the biocatalyst, pyruvate and benzaldehyde were used as the reaction substrates in the presence of thiamine pyrophosphoric acid and other cofactors in the presence of thiamine pyrophosphoric acid and other cofactors, in the presence of thiamine pyrophosphoric acid and other cofactors. R-PAC was prepared under the optimum experimental conditions. The yield of R-PAC was 83.6% and the purity was 98.3% (HPLC result). At the same time, the possibility of conversion of benzaldehyde derivatives and analogues by this biocatalyst has been determined. The results show that the biocatalyst has a wide substrate spectrum. Firstly, the recombinant engineering strain EcR (DE3) -pET28a-TmAHAS-CSU, which was prepared in our laboratory to over-express the catalytic subunit of Acetyl hydroxylase synthase (TmAHAS-CSU), was used to prepare TmAHAS-CSU, by self-induction and the purified target protein was obtained by Ni-NTA affinity chromatography. Then we used purified TmAHAS-CSU protein and recombinant engineering strain as catalyst to study the preparation of R-PAC by biotransformation. In the reaction catalyzed by enzyme TmAHAS-CSU, in order to maximize the yield of R-PAC, the synthesis system was optimized. The optimum reaction system was determined as pH 8.0, reaction time 40 min, reaction temperature 80 鈩，
本文编号：2259575
[Abstract]:R- phenylacetyl methanol (R-PAC) is an important chiral organic synthesis intermediate, which is often used as a precursor in pharmaceutical industry to synthesize amphetamines, such as ephedrine, norephedrine and amphetamines. In this study, the catalytic subunit (TmAHAS-CSU) of acetyl hydroxy acid synthase (TmAHAS-CSU) was selected as the biocatalyst, pyruvate and benzaldehyde were used as the reaction substrates in the presence of thiamine pyrophosphoric acid and other cofactors in the presence of thiamine pyrophosphoric acid and other cofactors, in the presence of thiamine pyrophosphoric acid and other cofactors. R-PAC was prepared under the optimum experimental conditions. The yield of R-PAC was 83.6% and the purity was 98.3% (HPLC result). At the same time, the possibility of conversion of benzaldehyde derivatives and analogues by this biocatalyst has been determined. The results show that the biocatalyst has a wide substrate spectrum. Firstly, the recombinant engineering strain EcR (DE3) -pET28a-TmAHAS-CSU, which was prepared in our laboratory to over-express the catalytic subunit of Acetyl hydroxylase synthase (TmAHAS-CSU), was used to prepare TmAHAS-CSU, by self-induction and the purified target protein was obtained by Ni-NTA affinity chromatography. Then we used purified TmAHAS-CSU protein and recombinant engineering strain as catalyst to study the preparation of R-PAC by biotransformation. In the reaction catalyzed by enzyme TmAHAS-CSU, in order to maximize the yield of R-PAC, the synthesis system was optimized. The optimum reaction system was determined as pH 8.0, reaction time 40 min, reaction temperature 80 鈩，
本文编号：2259575
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