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

开发生成所需酶功能的方法是非常重要的。这为治疗学、生物技术和有机合成提供了非天然酶的途径，并使我们能够解决现有方法无法解决的挑战。目前的奖学金申请支持产生非天然酶功能的新策略。这种方法通过重新利用现有酶的催化机制来发挥新的功能。这项工作的目的是实验室进化的混杂P450单加氧酶催化抗马尔可夫尼科夫氧化烯烃。而在自然界中p450催化烯烃的环氧化反应，我们的目标是重定向氧转移，从烯烃氧化生成相应的羰基。这将通过研究一种新的金属酶工程策略来实现，该策略利用电子效应来调整金属以进行新的催化循环。基于电子效应在小分子过渡金属催化中的重要性，我们期望在金属酶设计中产生重大影响。特别是，我们希望更好地理解如何利用电子效应来获得非天然酶的活性。我们将对进化的变体进行详细的表征，这将对新催化功能的机制起源产生深刻的见解。烯烃催化抗马尔可夫尼科夫氧化靶反应在化学合成中具有重要意义，但目前尚无较好的小分子催化剂溶液。由于生成的酶功能可以很容易地与其他已建立的生物催化剂结合，我们期望这种新酶在一锅酶级联反应和新的生物合成途径的产生中进一步使用。对于这里提出的新酶功能来说尤其如此，因为生成的羰基是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 - spengler酶）的优秀官能团。因此，一些反马尔可夫尼科夫官能化，如正式的反马尔可夫尼科夫水化和氢胺化可以通过酶级联来实现，这是本工作的一个非常有吸引力的应用。

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.