Novel Method to Produce Beta-Amino Acids

生产 β-氨基酸的新方法

• 批准号: 6880694
• 负责人: JAMES L KILGORE
• 金额: $ 44万
• 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/6880694
• 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-氨基酸作为一类关键的药物中间体的重要性正在迅速增长，在许多当前和未来的药物中都有应用。该项目将开发一种经济有效的光学纯β-氨基酸生产工艺，以满足β-氨基酸的商业需求。该方法涉及从方便制备的外消旋取代的二氢尿嘧啶开始的两个酶促反应。 5,6-二氢尿嘧啶可在 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氨基酸。 5,6-二氢尿嘧啶。然后将手性 N-氨基甲酰基-(-氨基酸) 进行酶促脱氨基甲酰化，产生光学纯的 (-氨基酸)。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，可以以纯形式生产 β-氨基酸的相反对映体。该方法的一个重要特征是， 酶反应可一锅进行，无需分离中间体N-氨基甲酰基-β-氨基酸。这一概念验证已在第一阶段使用 5,6-二氢尿嘧啶和 5-甲基-5,6-二氢尿嘧啶进行了演示。 第二阶段的首要任务是利用定向进化技术寻找具有更广泛底物范围的氨甲酰酶，特别是包括芳基取代的N-氨甲酰-(-氨基酸)。还将发现并生产具有相反对映选择性（即对映选择性水解二氢尿嘧啶的R-对映体）的二氢尿嘧啶酶。 将使用第二阶段获得的酶进一步开发一种经济有效的一锅法，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的二氢尿嘧啶转化为相应的对映体纯β-氨基酸。 β-氨基酸将以 100 克规模展示用于重要的商业目标。