酵母菌部位及立體選擇性催化酮類還原反應方法與高效液相層析電噴灑離子化質譜線上分析技術之發展

以生物催化劑進行不對稱合成具有高鏡像選擇性的優點，本研究以苯基正丙基酮為模型化合物，探討在水相及正己烷–水二相溶液系統中，酵母菌中醇去氫酶的活性及反應機制。研究發現，在沒有添加鋅離子的二相溶液系統，低正己烷體積比可以產生14.5%到46.5%的S-鏡像異構物過剩值，而中到高正己烷體積比則產生53.7%到約100%的R-鏡像異構物過剩值。若二相溶液系統中含有鋅離子，鏡像選擇性均為S-1-苯基-1-丁醇過剩，其鏡像異構物過剩值從27.5%到約100%。因此，對於苯基正丙基酮還原反應，控制是否添加輔因子鋅離子及改變二相溶液中正己烷體積比，便能得到想要的鏡像選擇性。本研究也以酵母菌催化雌固酮及雄烯二酮還原反應，為確認反應是否僅選擇性地產生β-雌二醇或睪固酮，便使用高靈敏度的電噴灑離子化質譜儀分析鑑定反應液。然而酵母菌反應液中存在著複雜的基質，必需先以線上固相萃取技術對樣品線上去雜質及前濃縮分析物，再結合液相層析電噴灑離子化質譜儀分析類固醇。同時也發展出新型的分析方法，管柱後連續注入內標準品黃體酮，此分析方法在分析過程中使分析物與內標準品同時間離子化，故可提高分析的精確度及準確度，並可減少仰賴昂貴的同位素內標準品的需求。對於雌激素及雄激素的分析，電噴灑離子化模式分別以負離子及正離子模式進行，而內標準品黃體酮的最佳濃度分別為 3.0 mg L-1及1.0 μg L-1。此分析系統對於所有分析物有高靈敏度，定量極限值在0.4到1.2 μg L-1的範圍，分析準確度及精確度分別大於94%及小於8.8%的相對標準偏差值。藉由電噴灑離子化質譜儀的鑑定，雌固酮還原反應僅產生β-雌二醇，而雄烯二酮還原反應則同時產生睪固酮及表睪酮。酵母菌催化雌固酮及雄烯二酮還原反應是以創新的雙槽連續反應系統進行研究，用以解決批式反應中受質抑制及低產物回收量的問題。在雙槽系統中，其中一槽是用於培養酵母菌細胞，並連續補充活細胞至進行連續還原反應的反應槽中。相較於批式反應，此種連續反應系統於雌固酮還原反應中，可將產率從54.8%增加到64.8%，β-雌二醇回收量從3.0 mg增加至12.9 mg。於雄烯二酮還原反應中，則將睪固酮產率從9.1%提升至26.2%，回收量從0.47 mg增加到5.4 mg。β-雌二醇及睪固酮之非鏡像異構物過剩值分別是大於99%及80%，顯示此還原反應具優異的部位及立體選擇性。

關鍵字

醇去氫酵素； R-及S-1-苯基-1-丁醇；苯基正丙基酮；正己烷–水二相溶液系統；雌固酮； β-雌二醇；睪固酮；雄烯二酮；電噴灑離子化質譜儀；線上固相萃取；管柱後連續注入內標準品法；雙槽連續反應；部位及立體選擇性

並列摘要

The asymmetric synthesis mediated with biocatalyst possesses the advantage of high enantioselectivity. In this research, phenyl n-propyl ketone was used as a model compound to examine the activity and the reaction mechanism of yeast alcohol dehydrogenase in aqueous culture and hexane-water biphasic culture. We found the reaction without Zn2+ ion in the biphasic culture of low hexane volume ratio produces an S-enantiomeric excess of 14.5% to 46.5% and in the biphasic culture of middle to high hexane volume ratio the reaction exhibits an R-enantiomeric excess of 53.7% to about 100%. For the biphasic culture with Zn2+ ion, the enantioselectivity is in S-1-phenyl-1-butanol excess and the enantiomeric excess is from 27.5% to about 100%. Therefore, for the reduction of phenyl n-propyl ketone the enantioselectivity can be controlled by the addition of cofactor Zn2+ ion in the cell culture and the change of the hexane volume ratio in the biphasic culture. In our research, yeast mediated reduction of estrone and androstenedione was performed. In order to confirm if only β-estradiol or testosterone is selectively produced in the reaction, the electrospray ionization/mass spectrometry with high sentitivity was used to analyze and identify the cell culture. However, the matrix in cell culture was complicated, an on-line solid-phase extraction technique was applied to perform the on-line elimination of impurities and preconcentration of analytes which was then coupled to the liquid chromatography-electrospray ionization/mass spectrometry (LC-ESI/MS) for steroid analysis. For quantitative analysis of steroids, a novel analysis method of continuous postcolumn infusion of internal standard progesterone was also developed. When it wa coupled to the LC-ESI/MS the analysis precision and accuracy was increased by ionizing the analyte and the internal standard at the same time and the need for the expensive isotopic internal standard was reduced. For the analysis of estrogen and androgen, the ionization process was operated at negative and positive mode, separately. The optimized concentration levels of internal standard progesterone were 3.0 mg L-1 and 1.0 μg L-1, respectively. High sensitivity was found for all steroidal analytes to give the limit of quantitation in the range from 0.4 to 1.2 μg L-1. The analysis accuracy was larger than 94% and the analysis precision with a relative standard deviation value was smaller than 8.8%. By the identification of ESI/MS only β-estradiol was produced in the estrone reduction culture and testosterone and epitestosterone were found in the androstenedione reduction culture. Saccharomyces cerevisiae mediated reduction of estrone and androstenedione was studied by an innovative continuous cell culture of dual stirred tank to solve the substrate inhibition and low product recovery of the batch cell culture. For the dual tank system, one was used for incubating yeast cell and the continuously supplying viable cells to the other tank that was used to perform the continuous reduction. With respect to the batch cell culture, this kind of continuous cell culture can increase the accumulated yield from 54.8% to 64.8% and the recovered amount of β-estradiol from 3.0 to 12.9 mg for the estrone reduction. For the androstenedione reduction, the testosterone yield was increased from 9.1% to 26.2% and the recovered amount of testosterone was increased from 0.47 to 5.4 mg. With the continuous dual tank system, the diastereomeric excess value of β-estradiol and testosterone was lager than 99% and 80%, respectively, that indicated an excellent and good regio- and stereo-selectivity of the reaction.

並列關鍵字

alcohol dehydrogenase ； phenyl n-propyl ketone ； R- and S-1-phenyl-1-butanol ； continuous cell culture with dual stirred tank ； hexane-water biphasic culture ； on-line solid-phase extraction ； β-estradiol ； androstenedione ； testosterone ； electrospray ionization/mass spectrometry ； estrone ； regio-selectivity ； continuous postcolumn infusion of internal standard ； stereo-selectivity.

參考文獻

1. H. Gest, “The discovery of microorganisms by Robert Hooke and Antoni van Leeuwenhoek, fellows of the Royal Society,” Notes Rec. R. Soc. Lond. 2004, 58, 187-201.

2. R. Csuk, B.I. Glazer, “Baker’s yeast mediated transformations in organic chemistry,” Chem. Rev. 1991, 91, 49-97.

7. T.J. Leitereg, D.G. Guadagni, J. Harris, T.R. Mon, R. Teranishi, “Chemical and sensory data supporting the difference between the odors of the enantiomeric carvones,” J. Agr. Food Chem. 1971, 19, 785-787.

8. M. Hargittai, I. Hargittai, “Symmetry in chemistry,” Eur. Rev. 2005, 13, 61-75.

9. T. Stephens, R. Brynner, “Dark Remedy: The Impact of Thalidomide and Its Revival as a Vital Medicine,” Perseus Publishing, Massachusetts, USA, 2001, Chapter 1.

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酵母菌部位及立體選擇性催化酮類還原反應方法與高效液相層析電噴灑離子化質譜線上分析技術之發展

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