Novel Method to Produce Beta-Amino Acids

生产 β-氨基酸的新方法

• 批准号: 7124220
• 负责人: SPIROS KAMBOURAKIS
• 金额: $ 38.36万
• 依托单位: BIOCATALYTICS, INC.
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7124220
• 关键词: Escherichia coli; aminoacid; carboxyltransferase /carbamoyltransferase; catalyst; chemical synthesis; directed evolution; enzyme mechanism; enzyme substrate complex; method development; molecular cloning; peptide chemical synthesis; uracil analog

DESCRIPTION (provided by applicant): Beta-Amino acids are rapidly growing in importance as a key class of pharmaceutical intermediates, with applications in a number of current and future drugs. A cost-effective process for the production of optically pure beta-amino acids will be developed in this project to accommodate the commercial needs for beta-amino acids. The method involves two enzymatic reactions starting from conveniently prepared racemic substituted dihydrouracils. The 5,6-dihydrouracil, which may be substituted at either the 5 or 6 positions with various functional groups, is contacted with a dihydrouracilase enzyme to catalyze a stereoselective hydrolysis reaction, producing an enantiomerically-enriched chiral N-carbamoyl-beta-amino acid and an enantiomerically enriched, unreacted chiral 5,6-dihydrouracil. The chiral N-carbamoyl-(-amino acid was then decarbamoylated enzymatically to produce an optically pure (-amino acid. The opposite enantiomer of the beta-amino acid can be produced in pure form by recovering and hydrolyzing the unreacted chiral 5,6- dihydrouracil using the enzymes with opposite stereoselectivity. An important feature of this method is that the two enzyme reaction can be carried out in one pot and it is not necessary to isolate the intermediate N-carbamoyl-beta-amino acids. This proof of concept has been demonstrated in Phase 1 using 5,6-dihydrouracil and 5-methyl-5,6-dihydrouracil. In Phase II, the top priority will be searching for carbamoylase enzymes with a broader substrate range, particularly including aryl-substituted N-carbamoyl-(-amino acids using directed evolution technology. Dihydrouracilases with opposite enantioselectivity (i.e. enantioselectively hydrolyzing the R-enantiomer of dihydrouracils) will also be found and produced. The cost-effective one-pot process to convert racemic substituted dihydrouracils to the corresponding enantiomerically pure beta-amino acids via the coupled dihydrouracilase/carbamoylase two-enzyme system in the non-immobilized and immobilized forms will be developed further using the enzymes acquired in Phase 2. Finally, the production of enantiomerically-pure beta-amino acids will be demonstrated on the 100 gram scale for important commercial targets.

描述（由申请人提供）： β-氨基酸作为一类重要的药物中间体，其重要性正在迅速增长，并在许多当前和未来的药物中应用。本项目将开发一种具有成本效益的生产光学纯β-氨基酸的工艺，以满足β-氨基酸的商业需求。该方法包括两个酶促反应，从方便制备的外消旋取代的二氢尿嘧啶开始。5，6-二氢尿嘧啶，其可以在5或6位被各种官能团取代，与二氢尿嘧啶酶接触以催化立体选择性水解反应，产生对映体富集的手性N-氨基甲酰基-β-氨基酸和对映体富集的未反应的手性5，6-二氢尿嘧啶。然后将手性N-氨基甲酰基-β-氨基酸酶促脱氨甲酰化以产生光学纯的β-氨基酸。β-氨基酸的相反对映异构体可以通过使用具有相反立体选择性的酶回收和水解未反应的手性5，6-二氢尿嘧啶而以纯的形式产生。该方法的一个重要特点是两个酶反应可以在一锅中进行，并且不需要分离中间体N-氨基甲酰基-β-氨基酸。已在第1阶段使用5，6-二氢尿嘧啶和5-甲基-5，6-二氢尿嘧啶证明了这一概念验证。 在第二阶段，首要任务将是寻找具有更广泛底物范围的氨甲酰化酶，特别是使用定向进化技术的芳基取代的N-氨基甲酰基-（-）氨基酸。还将发现并产生具有相反对映体选择性（即对映体选择性水解二氢尿嘧啶的R-对映体）的二氢尿嘧啶酶。将使用第2阶段获得的酶进一步开发通过非固定化和固定化形式的偶联二氢尿嘧啶酶/氨甲酰化酶双酶系统将外消旋取代二氢尿嘧啶转化为相应的对映体纯β-氨基酸的具有成本效益的一锅法。最后，对映体纯β-氨基酸的生产将在100克规模的重要商业目标上得到证明。