Enzymatic Control over Reactive Intermediates Enables Direct Oxidation of Alkenes to Carbonyls by a P450 Iron-Oxo Species

The aerobic oxidation of alkenes to carbonyls is an important and challenging transformation in synthesis. Recently, a new P450-based enzyme (aMOx) has been evolved in the laboratory to directly oxidize styrenes to their corresponding aldehydes with high activity and selectivity. The enzyme utilizes a heme-based, high-valent iron-oxo species as a catalytic oxidant that normally epoxidizes alkenes, similar to other catalysts. How the evolved aMOx enzyme suppresses the commonly preferred epoxidation and catalyzes direct carbonyl formation is currently not well understood. Here, we combine computational modelling together with mechanistic experiments to study the reaction mechanism and unravel the molecular basis behind the selectivity achieved by aMOx. Our results describe that although both pathways are energetically accessible diverging from a common covalent radical intermediate, intrinsic dynamic effects determine the strong preference for epoxidation. We discovered that aMOx overrides these intrinsic preferences by controlling the accessible conformations of the covalent radical intermediate. This disfavors epoxidation and facilitates the formation of a carbocation intermediate that generates the aldehyde product through a fast 1,2-hydride migration. Electrostatic preorganization of the enzyme active site also contributes to the stabilization of the carbocation intermediate. Computations predicted that the hydride migration is stereoselective due to the enzymatic conformational control over the intermediate species. These predictions were corroborated by experiments using deuterated styrene substrates, which proved that the hydride migration is cis- and enantioselective. Our results demonstrate that directed evolution tailored a highly specific active site that imposes strong steric control over key fleeting biocatalytic intermediates, which is essential for accessing the carbonyl forming pathway and preventing competing epoxidation.烯烃的需氧氧化制备羰基化合物反应，是合成领域中一类兼具重要性与挑战性的转化过程。近期，科研人员通过实验室进化手段获得了一种新型P450基酶（aMOx），可高活性、高选择性地将苯乙烯直接氧化为对应的醛类产物。该酶以基于血红素的高价铁氧活性物种作为催化氧化剂，与其他同类催化剂类似，其通常可催化烯烃发生环氧化反应。目前，对于经进化改造的aMOx酶如何抑制常规条件下更易发生的环氧化路径，并催化直接生成羰基化合物的具体机制，学界仍未完全阐明。本研究结合计算建模与机理实验，对该反应的机理进行探究，阐明了aMOx实现选择性催化的分子基础。研究结果显示，尽管两条反应路径均源自同一共价自由基中间体且在能量上均可行，但内在的动态效应决定了酶对环氧化路径的强烈偏好。我们发现，aMOx通过调控共价自由基中间体的可及构象，打破了这类固有反应偏好。这一调控机制不利于环氧化路径的进行，同时促进了碳正离子中间体的形成，该中间体可通过快速的1,2-氢迁移生成醛类产物。酶活性位点的静电预组织效应，同样有助于稳定该碳正离子中间体。计算模拟预测，由于酶对中间体构象的调控作用，氢迁移过程具有立体选择性。这一预测通过采用氘代苯乙烯底物开展的实验得到了验证，实验结果证实氢迁移过程兼具顺式立体选择性与对映选择性。本研究结果表明，定向进化技术精准塑造了一个具有高度特异性的活性位点，该位点可对关键的瞬时生物催化中间体施加严格的空间位阻调控，这对于实现羰基生成路径、避免竞争性环氧化反应至关重要。