One-Step Synthesis of D-Amino Acids

描述(申请人提供)：许多目前生产的药物，如β-内酰胺类抗生素、凝血酶抑制剂和生育治疗，以及精细化学品，如杀虫剂，含有D-氨基酸。其中一些化合物的生产规模超过5000吨/年。此外，还有一些D-氨基酸药物处于研究或临床试验阶段，用于治疗女性的子宫内膜异位症和子宫肌瘤、男性的良性前列腺肥大和艾滋病毒等疾病。D-氨基酸的优势是通常比L的同类产品更具生物活性，而且它们通常也更稳定，因为它们在肝脏、肾脏和血液中不太可能经历酶降解。显然，对手性纯D-氨基酸的需求很大，预计在不久的将来，随着它们成为新药物的关键成分，这一需求将大幅上升。 虽然目前存在生产D-氨基酸的技术，但它们都有缺点，包括产率低和需要多个反应步骤。需要新的方法来改进现有的技术。本提案中讨论的一种方法是使用D-氨基酸脱氢酶从2-酮酸前体生产D-氨基酸。这将允许从廉价的起始原料一步合成D-氨基酸。为此，建议使用直接进化来产生范围广泛的D-氨基酸脱氢酶。起始酶2，6-二氨基-D-脱氢酶对合成D-氨基酸具有很高的选择性。这项建议的具体目标是建立一个突变DAPDH文库，筛选该文库对重要D-氨基酸的活性，鉴定最好的突变体，并使用这些突变体来进行一种或多种关键D-氨基酸的克级合成。

DESCRIPTION (provided by applicant): Many currently produced drugs such as beta-Iactam antibiotics, thrombin inhibitors and fertility treatments, and fine chemicals such as insecticides contain D-amino acids. Some of these compounds are produced on a scale greater than 5000 tons/yr. In addition, there are a number of D-amino acid containing drugs, either in the research or clinical test stages, used for the treatment of such illnesses as endometriosis and uterine fibroids in women, benign prostatic hypertrophy in men, and HIV. D-Amino acids have the advantage of often being more bioactive than their L-counterpart and they are also frequently more stable as they are less likely to undergo enzymatic degradation in the liver, kidney, and bloodstream. Clearly, there is significant demand for enantiomerically pure D-amino acids and this is expected to rise significantly in the near future as they become critical components of new pharmaceuticals. While current technologies exist for producing D-amino acids they all have drawbacks including poor yields and the requirement of multiple reaction steps. New methods to improve upon current technologies are needed. One method, as discussed in this proposal, is to produce D-amino acids from the 2-keto acid precursor using a D-amino acid dehydrogenase. This will allow for the single-step synthesis of D-amino acid from inexpensive starting materials. To this end, it is proposed to use direct evolution to generate broadrange D-amino acid dehydrogenases. The starting enzyme, meso-2,6-diaminopimelate-D-dehydrogenase (DAPDH) is highly selective for the synthesis of D-amino acids. The specific aims of this proposal are to generate a library of mutant DAPDH, screen this library for activity towards important D-amino acids, characterize the best mutants, and use these mutants to perform a gram scale synthesis of one or more key D-amino acids.