結合性雙液相層析管柱系統分析酵母菌催化前掌性酮類於二相反應液中之掌性二級醇

Analysis of Chiral Secondary Alcohol in Biphasic Culture of Yeast Mediated Prochiral Ketone Reduction by Coupled Dual Column Liquid Chromatography System

蔡向榮(Shiang-Rong Tsai)

指導教授 ： 鄭建業

中原大學/理學院/化學研究所/碩士(2003年)

https://doi.org/10.6840/cycu200300551

摘要

酵母菌Saccharomyces cerevisiae或Candida utilis以搖瓶方式在七種不同體積比正己烷/水二相溶液系統下對苯基正丙基酮進行還原反應，並用Chiralcel OB掌性液相層析管柱搭配流動相正己烷/異丙醇/冰醋酸=99.2/0.7/0.1 (v/v/v)分離分析掌性產物R-及S-1-苯基-1-丁醇；在低正己烷體積比二相溶液系統中，產生S-1-苯基-1-丁醇過剩之反應；在中到高正己烷體積比二相溶液系統中，只有R-1-苯基-1-丁醇產生。
酵母菌S. cerevisiae或C. utilis 在2 L發酵槽之二相溶液系統還原反應中，R-及S-1-苯基-1-丁醇所受到的間質干擾無法以單一Chiralcel OB管柱或Chiralpak AD管柱之掌性高效能液相層析消除，並且有分析時間過長的問題。連結Chiralcel OB管柱及Chiralpak AD管柱配合管柱轉換技術之應用可同時解決間質的干擾及縮短分析時間。此雙管柱結合之高效能液相層析系統具有高準確度(回收率>98.6%)及高精確度(RSD<1.6%)，並且所使用之UV偵測器對於苯基正丙基酮、R-及S-1-苯基-1-丁醇三種分析物的偵測極限值分別為9 ppb、48 ppb及84 ppb。此技術之發展可成功的應用於線上樣品清潔及分析反應物苯基正丙基酮及產物R-與S-1-苯基-1-丁醇，並有利於未來液相層析-質譜儀分析掌性化合物之分析技術發展。
酵母菌於2 L發酵槽中對前掌性酮類之掌性選擇性還原反應受到許多因素影響，如反應溶液之正己烷及水體積比不同會產生不同形式之掌性異構物過剩，反應液中添加Zn2+離子不但會增加鏡相異構物過剩值也會增加產率，反應液水層pH值對酵母菌生長及苯基正丙基酮還原反應之影響也各有不同；已發現酵母菌生長時於pH 7.0為最佳，於反應時則以pH 5.0具最佳之鏡相過剩值及最佳產率，此結果於單一水相反應或於正己烷/水體積比為40/60之二相溶液中反應均適用。酵母菌S. cerevisiae之選擇性還原反應也應用於由雌素酮至雌二醇之類固醇生產，目前定性分析的結果顯示反應良好，至於產率及鏡像選擇則有待後續研究。

關鍵字

高效能液相層析 ； 掌性化合物 ； Saccharomyces cerevisiae ； candida utilis ； Chiralpak AD管柱 ； 雌二醇 ； 雌素酮 ； Chiralcel OB管柱 ； 酵母菌 ； R-及S-1-苯基-1-丁醇 ； 管柱轉換技術 ； 正己烷/水二相溶液系統 ； 苯基正丙基酮

並列摘要

A shaker-flask type reduction of phenyl n-propyl ketone with yeast Saccharomyces cerevisiae or Candida utilis are performed in seven different kinds of volume ratio n-hexane/water biphasic solution. A commercialized Chiralcel OB column and a mobile phase of n-hexane/isopropanol/acetic acid (99.2/0.7/0.1, v/v/v) has been used for the separation and analysis of chiral products R- and S-1-phenyl- 1-butanol. In the biphasic culture with a low volume percentage of n-hexane, the product is S-1-phenyl-1-butanol enantiomeric excess. In the biphasic cultures of moderate to high volume percentage of n-hexane, the product is exclusive R-1-phenyl-1-butanol.
In S. cerevisiae or C. utilis mediated biphasic culture reduction with a 2 L bench scale fermentor, the matrix interference subjected in the analysis of R- and S-1-phenyl-1-butanol cannot be eliminated by the chiral high-performance liquid chromatography with a single column of either the Chiralcel OB column or the Chiralpak AD column. The analysis of the chiral products in the fermentation system with single column also exhibits a long analysis time. However, the application of column switching technique can hyphenate the Chiralcel OB column and the Chiralpak AD column together to resolve not only the matrix interference but a reduction of the analysis time. This coupled column system for the chiral products analysis possesses high accuracy (a recovery rate > 98.6%) and high precision (RSD > 1.6%). The limits of detection of phenyl n-propyl ketone, R- and S-1-phenyl-1-butanol determined by the coupled column system for the UV detector are 9 ppb, 48 ppb and 84 ppb, respectively. This technique for the on-line sample clean-up and analysis of phenyl n-propyl ketone, R- and S-1-phenyl-1-butanol in the biphasic culture is successful. This technique is also helpful in the development of an analytical technique with liquid chromatography-mass spectrometry for chiral compounds.
There are many factors affect the yeast mediated enantioselective prochiral ketone reduction with a 2 L bench scale fermentor such as that the yeast mediated reduction performed with the biphasic n-hexane/water culture of different volume ratio produces different enantiomeric excess chiral products, the addition of zinc ions in the aqueous medium can raise not only the enantiomeric excess value but the reaction yield, the change of pH for the reaction culture also influences the growth of yeast and the reduction of phenyl n-propyl ketone. We found that the yeast can be grown best at pH 7.0 and the reaction has a best enantiomeric excess value and a best yield at pH 5.0. The effect of pH is the same for both the reactions in single phase aqueous culture or the n-hexane/water biphasic culture of volume ratio 40/60. The S. cerevisiae mediated estrone reduction has been successfully proceeded to produce estradiol, however, the yield and the enantioselectivity of the reaction should be surveyed further.

並列關鍵字

R- and S-1-phenyl-1-butanol ； estradiol ； biphasic n-hexane/water culture ； column switching technique ； estrone ； high-performance liquid chromatography ； chiral compounds ； S. cerevisiae ； the Chiralpak AD column ； phenyl n-propyl ketone ； Chiralcel OB column ； Yeast ； C. utilis

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