Unnatural amino acids by deracemization

通过去消旋作用产生非天然氨基酸

• 批准号: 7903301
• 负责人: Paul Phillip Taylor
• 金额: $ 37.94万
• 依托单位: RICHMOND CHEMICAL CORPORATION
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7903301
• 关键词: Affect; Agrochemicals; Amino Acids; Antidiabetic Drugs; Antineoplastic Agents; Area; Basic Amino Acids; Biological Sciences; Chemical Industry; Chemicals; D-Amino Acid Dehydrogenase; Data; Development; Drug Formulations; Drug Industry; Drug Kinetics; Economic Development; Economics; Enzymes; Evolution; Family; Fermentation; Foundations; Glycine; Goals; HIV Protease Inhibitors; Immobilization; In Vitro; Industry; Isomerism; Leucine; Marketing; Metals; Methods; Modeling; Mutagenesis; Optics; Oxidases; Oxygen; Performance; Pharmacologic Substance; Phase; Preparation; Process; Production; Property; Reaction; Recovery; Refuse Disposal; Resolution; Route; Side; Solid; Stream; Synechococcus; Techniques; Technology; Validation; Variant; Water; Work; base; bioprocess; catalyst; cost; cost effective; directed evolution; enantiomer; high throughput screening; member; norvaline; oncology; public health relevance; success; thermostability; virology; wasting

DESCRIPTION (provided by applicant): Unnatural amino acids represent one of the largest and growing classes of intermediates used by the pharmaceutical, agrochemical and fine chemical industries. Demand for these compounds at large scale and high optical purity is now very significant, due to their application and success in single enantiomer pharmaceuticals, particularly for the areas of virology and oncology. No synthetic or biosynthetic method however, has proven sufficiently versatile to prepare these compounds broadly at scale. Richmond Chemical Corporation (RC Corp.) has recently developed an efficient and general chemo-enzymatic process to prepare enantiomerically pure L- and D-amino acids in high yield by deracemization of racemic starting materials. This method involves the concerted action of an enantioselective amino acid oxidase biocatalyst and a non-selective chemical reducing catalyst to affect the stereo-inversion of one enantiomer, and can result in an enantiomeric excess (ee) of > 99 % from the starting racemate, with product yields of over 90 %. This approach, conducted entirely in water, (thereby minimizing waste streams) compares very favorably to resolution processes, which have a maximum single pass yield of 50 %. We have successfully developed methods to optimize the efficiency of the process and establish competitive economics at scale. However, the breadth of application of this process is limited by the substrate range of the native oxidase biocatalysts. In the Phase I work of this project, we successfully applied powerful methods of in vitro enzyme evolution to demonstrate the adaptability of the oxidase biocatalysts, by successfully isolating variants of D-amino acid oxidase with high activity and enantioselectivity towards sterically bulky amino acids of high commercial importance. These amino acids are of high relevance due to their favorable pharmacokinetic properties but are difficult to prepare by traditional methods. In addition, the stability of the natural D-amino acid oxidase was greatly enhanced using the same approach. In parallel we successfully identified conditions in which the reduction step can be carried out with near quantitative yield under mild conditions. These results validate key aspects required for deracemization as a platform bioprocess and we now propose in Phase II to develop the scalable chemo-enzymatic deracemization processes to prepare these and additional unnatural amino acids for industry. We will continue the directed evolution of the oxidase biocatalyst and optimize critical reaction parameters including substrate load, biocatalyst production, formulation and re-use, as well as product isolation and recovery. The optimized deracemization processes will be implemented in the commercial manufacture of unnatural amino acids at large-scale. This work plan will result in new general bioprocess technology for the fine chemical industry, to produce chiral synthetic intermediates for pharmaceutical applications at commercial scale. PUBLIC HEALTH RELEVANCE: This project is aimed towards the development of an economical enzymatic process for the synthesis of optically pure unnatural amino acids. These amino acids are in great demand for the synthesis of a wide range of important pharmaceutical compounds such HIV protease inhibitors, anti-cancer agents and anti-diabetic drugs.

描述（由申请人提供）：非天然氨基酸是制药、农业化学和精细化学工业中使用的最大和不断增长的中间体类别之一。由于这些化合物在单一对映体药物中的应用和成功，特别是在病毒学和肿瘤学领域，现在对这些化合物的大规模和高光学纯度的需求非常显著。然而，没有合成或生物合成方法已被证明具有足够的通用性来大规模制备这些化合物。里士满化学公司（RC公司）最近开发了一种有效和通用的化学-酶促方法，通过外消旋起始原料的去外消旋化以高产率制备对映体纯的L-和D-氨基酸。该方法涉及对映选择性氨基酸氧化酶生物催化剂和非选择性化学还原催化剂的协同作用，以影响一种对映体的立体转化，并且可以导致起始外消旋物的对映体过量（ee）> 99%，产物收率超过90%。这种完全在水中进行的方法（从而最大限度地减少废物流）与最大单程产率为50%的拆分方法相比非常有利。我们已经成功开发出优化工艺效率的方法，并在规模上建立了具有竞争力的经济性。然而，该方法的应用范围受到天然氧化酶生物催化剂的底物范围的限制。在该项目的第一阶段工作中，我们成功地应用了体外酶进化的强大方法来证明氧化酶生物催化剂的适应性，通过成功地分离具有高活性和对映体选择性的D-氨基酸氧化酶的变体，对具有高度商业重要性的空间体积大的氨基酸。这些氨基酸由于其有利的药代动力学性质而具有高度相关性，但难以通过传统方法制备。此外，天然D-氨基酸氧化酶的稳定性大大提高，使用相同的方法。同时，我们成功地确定了在温和条件下可以以接近定量产率进行还原步骤的条件。这些结果验证了去消旋化作为平台生物过程所需的关键方面，我们现在建议在第二阶段开发可扩展的化学-酶促去消旋化过程，以制备这些和其他非天然氨基酸用于工业。我们将继续氧化酶生物催化剂的定向进化，并优化关键反应参数，包括底物负荷，生物催化剂生产，配方和重复使用，以及产品分离和回收。优化的去外消旋化工艺将在非天然氨基酸的大规模商业化生产中实施。该工作计划将为精细化工行业带来新的通用生物工艺技术，以商业规模生产用于制药应用的手性合成中间体。公共卫生相关性：该项目旨在开发一种经济的酶促方法，用于合成光学纯的非天然氨基酸。这些氨基酸在合成多种重要的药物化合物如HIV蛋白酶抑制剂、抗癌剂和抗糖尿病药物方面有很大的需求。