Non-natural anti-Markovnikov oxidation of olefins by redirecting the oxygen transfer in P450 monooxygenases

开发产生所需酶功能的方法是非常重要的。这提供了获得非天然酶的治疗，生物技术以及有机合成，并使我们能够解决超出目前的approaches.The奖学金申请的范围支持一个新的战略，产生非天然酶功能的挑战。这种方法利用现有的酶，通过重新利用它们的催化机制来承担新的功能。这项工作的目的是实验室的一个混杂的P450单加氧酶的催化抗马尔可夫尼科夫氧化烯烃的演变。在自然界中P450催化烯烃的环氧化，我们的目标是重新定向氧转移，以产生相应的羰基从烯烃氧化。这将通过研究一种新的金属酶工程策略来进行，该策略利用电子效应来调整金属以进行新的催化循环。基于电子效应在过渡金属小分子催化中的重要性，我们期待着对金属酶设计产生重大影响。特别是，我们希望更好地了解如何利用电子效应来获得非天然酶活性。我们将对这些衍生物进行详细的表征，从而深入了解新催化功能的机理起源。烯烃的催化反马尔可夫尼科夫氧化目标反应在化学合成中非常重要，但它没有很好的小分子催化剂解决方案。由于所产生的酶的功能可以很容易地与其他已建立的生物催化剂相结合，我们希望进一步使用这种新的酶在一锅酶级联反应，并在新的生物合成途径的产生。这对于本文提出的新酶功能尤其如此，因为所产生的羰基对于已建立的生物催化剂（例如，酮还原酶、转氨酶、醛缩酶、ThDP依赖性裂解酶、羟基腈裂解酶和Pictet-Spenglerases）。因此，几个反马尔可夫官能化，如正式的反马尔可夫水合和加氢胺化可以实现与酶级联，这是一个非常有吸引力的应用这项工作。

Developing methods to generate desired enzyme function is of high importance. This provides access to non-natural enzymes for therapeutics, biotechnology as well as organic synthesis and allows us to address challenges beyond the reach of current approaches.The present fellowship application supports a new strategy for generating non-natural enzyme function. This approach takes advantage of existing enzymes by repurposing their catalytic machinery to take on new functions. The aim of this work is the laboratory evolution of a promiscuous P450 monooxygenase for the catalytic anti-Markovnikov oxidation of olefins. While in nature P450s catalyze the epoxidation of olefins, we target to redirect oxygen transfer to generate the corresponding carbonyls from olefin oxidation. This will be performed by studying a new metalloenzyme engineering strategy, which utilizes electronic effects to tune the metal for novel catalytic cycles. Based on the significance of electronic effects in transition metal catalysis using small molecules, we expect a substantial impact in metalloenzyme design. In particular, we suppose to gain a better understanding of how electronic effects can be utilized to access non-natural enzyme activity. We will perform a detailed characterization of the evolved variants which will yield insights into the mechanistic origin of the new catalytic function.The target reaction of catalytic anti-Markovnikov oxidation of olefins is highly important in chemical synthesis, yet it has no good small-molecule catalyst solution. Since the generated enzyme function can be readily combined with other established biocatalysts, we expect further use of this novel enzyme in one-pot enzymatic cascades and in the generation of novel biosynthetic pathways. This is especially true for the new enzyme function proposed here, since the generated carbonyls are excellent functional groups for established biocatalysts (e.g., ketoreductases, transaminases, aldolases, ThDP-dependent lyases, hydroxynitrile lyases and Pictet-Spenglerases). Therefore, several anti-Markovnikov functionalizations such as formal anti-Markovnikov hydration and hydroamination could be realized with enzyme cascades, which is a highly attractive application of this work.