Development of reaction method for yeast mediated regio- and stereo-selective reduction of ketone and on-line analysis technique of LC-ESI-MS

• Main Authors: Hsiang-Rong Tsai, 蔡向榮
• Other Authors: Cheanyeh Cheng
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/18898511677183861210

博士 === 中原大學 === 化學研究所 === 98 === 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.