【摘要】：R-苯基乙�；状�(R-PAC)是一種重要的手性有機(jī)合成中間體,在制藥業(yè)中常作為前體物質(zhì)來(lái)合成苯丙胺類(lèi)藥物,如麻黃堿,去甲麻黃堿及安非他命等等。本研究利用生物轉(zhuǎn)化的方法,選擇海棲熱袍菌乙酰羥酸合酶的催化亞基(TmAHAS-CSU)為生物催化劑,以丙酮酸及苯甲醛為反應(yīng)底物,在硫胺素焦磷酸及其它輔因子的存在下,以最優(yōu)化的實(shí)驗(yàn)條件制備得到了 R-PAC,分離后產(chǎn)率為83.6%,純度為98.3%(HPLC檢測(cè)結(jié)果)。同時(shí),我們還測(cè)定了該生物催化劑轉(zhuǎn)化苯甲醛衍生物及類(lèi)似物的可能性,結(jié)果顯示其有較寬的底物譜。首先,我們利用本實(shí)驗(yàn)室前期制備的能夠過(guò)表達(dá)海棲熱袍菌乙酰羥酸合酶催化亞基(TmAHAS-CSU)的重組工程菌株EcR(DE3)-pET28a-TmAHAS-CSU,采取自誘導(dǎo)方式制備了 TmAHAS-CSU,并經(jīng)過(guò)Ni-NTA親和層析得到了純化后的目的蛋白。然后我們分別利用純化后的TmAHAS-CSU蛋白及重組工程菌株全菌為催化劑探討了生物轉(zhuǎn)化法制備R-PAC的方法。在酶TmAHAS-CSU催化的反應(yīng)中,為了使R-PAC產(chǎn)率最大化,對(duì)其合成體系進(jìn)行優(yōu)化,最終確定最佳反應(yīng)體系為50 mM的磷酸鹽(PBS)緩沖液,pH 8.0,反應(yīng)時(shí)間40min,反應(yīng)溫度為80℃,助溶劑DMSO含量1%,底物丙酮酸(Pyr)和苯甲醛(BA)的最佳比例為1.25:1,最佳濃度分別為50 mM和40 mM。在實(shí)驗(yàn)中我們還發(fā)現(xiàn),以重組工程菌株的全菌催化該反應(yīng)時(shí)能夠獲得更加理想的產(chǎn)率。此外,還以苯甲醛的衍生物以及一些芳雜環(huán)基甲醛化合物作為底物,與另一底物Pyr在重組工程菌全菌的催化下進(jìn)行反應(yīng),分別利用HPLC、LC-MS以及1H-NMR對(duì)部分反應(yīng)結(jié)果進(jìn)行檢測(cè)和鑒定。結(jié)果發(fā)現(xiàn),甲氧基取代及單羥基取代的苯甲醛衍生物等共計(jì)13種化合物能夠有效進(jìn)行該反應(yīng),這不但擴(kuò)充了 TmAHAS-CSU的底物譜范圍,更為進(jìn)一步研究TmAHAS-CSU打下了良好的基礎(chǔ)。
[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，

本文編號(hào)：2259575