Engineering of unspecific peroxygenases for the β-hydroxylation of amines on a preparative scale.

我们相信，非特异性过氧酶(UPO)是C-H功能化的优秀酶，具有非凡的合成潜力。通过将蛋白质和过程工程相结合，UPOS的潜力将通过在克级规模上合成具有重要药学意义的构建块来展示。UPO是一种真菌酶，能将一种过氧化氢携带的氧气转移到SP3-碳上。UPO催化的反应展示了令人印象深刻的底物范围，有400多个已知例子。它们表现出良好的对映体选择性和惊人的总成交量高达300,000的苄基羟基化。大约目前已有4000个UPO基因被注释，但由于其具有挑战性的异源表达，对不同UPO酶的详细研究不到20个。这些生产限制也大大阻碍了有针对性的蛋白质工程努力，因此目前的底物范围主要包括野生型活动。将UPO的催化机械用于新的工业相关基材将是至关重要的。特别是，携带脂肪胺的底物在活性药物成分(API)中普遍存在，但UPO对这些化合物进行羟基化的例子很少。邻氨基醇的分子类特别令人感兴趣，因为这些基团可以由胺类UPO合成，是制药工业的令人兴奋的支架。拟议的研究项目直接解决了目前UPO对胺底物的限制，目的是利用生物化学和过程工程的综合方法来开发和应用工程UPO。在蛋白质工程领域，该方法包括开发一种检测氨基醇的快速分析系统，并对活性部位进行重塑，包括。用于设计高效转胺UPO的胺锚定区域。在工艺工程方面，将为控制过氧化氢以及离子交换和结晶技术的集成量身定做在线方法，以适应UPO催化反应的要求。实施开发的生物催化剂和方法将在高底物宽度和克级范围内进行明确的调查。研究项目分为八个目标和四个里程碑，将由两个项目合作伙伴以结构化的方式解决这一问题。它从分析、蛋白质、实时分析和选择性产物去除的发展和工程开始，并最终在克级的制备合成中实现。

We are convinced that unspecific peroxygenases (UPOs) represent outstanding enzymes for C–H functionalizations with an extraordinary synthetic potential. By combining protein and process engineering, UPOs’ potential will be demonstrated by synthesizing pharmaceutically important building blocks on a gram-scale. UPOs are fungal enzymes that transfer one peroxide-borne oxygen to sp3-carbons. UPO-catalyzed reactions display an impressive substrate scope with more than 400 known examples. They exhibit excellent enantioselectivities and striking total turnover numbers of up to 300,000 for benzylic hydroxylations. Approx. four thousand putative UPO genes have been annotated, but less than 20 different UPO enzymes were studied in detail due to their challenging heterologous expression. These production limitations also significantly hampered targeted protein engineering efforts, and hence the current substrate scope consists primarily of wild-type activities. It would be of utmost importance to utilize UPOs’ catalytic machinery towards new industrially relevant substrates. In particular, substrates carrying aliphatic amines are ubiquitous in active pharmaceutical ingredients (API), but there are only scarce examples of UPOs hydroxylating these compounds. The molecular class of vicinal amino alcohols is of special interest as these groups could be synthesized by UPOs from amines and represent exciting scaffolds for the pharmaceutical industry. The proposed research project directly addresses the current limitations of UPOs towards amine substrates and aims to utilize an integrated approach of biochemical and process engineering towards the development and application of engineered UPOs. In the field of protein engineering, the methodology includes developing a rapid analysis system for the detection of amino alcohols and a re-shaping of the active site incl. amine anchored regions for the design of potent amine-converting UPOs. On the process engineering side, on-line methodologies for the control of hydrogen peroxide and integration of ion exchange and crystallization techniques will be tailor-made to fit the requirements of UPO-catalyzed reactions. Implementing the developed biocatalysts and methods will be investigated explicitly concerning a high substrate width and in gram-scale.The research project is organized into eight aims and four milestones, which will address the issue in a structured manner by both project partners. It starts from the developments and engineering of assays, proteins, real-time analysis, and selective product removals and culminates in preparative syntheses in gram-scale.