Directed Evolution of Halogenases for Small Molecule Functionalization

 说明(申请人提供)：卤化有机化合物被广泛用作构建块、合成中间体和最终产品，用于制药和农用化学品。这些化合物的用途，包括它们的生物活性，源于卤素取代赋予它们的独特的反应性和物理性质。卤化的重要性和当前卤化方法的局限性促使我们开发新的卤化催化剂。本提案概述了黄素依赖卤代酶(FDHS)在一系列涉及选择性卤化的合成方法中的发展。我们的初步结果包括通过迭代突变进行FDH定向进化的第一个例子，我们将使用我们开发的方法进一步提高这些酶的底物范围、选择性和反应活性，用于实际的合成应用。这将需要产生FDH变体，并筛选这些变体在相对于天然底物色氨酸逐渐增大、形状改变和亲核性降低的底物上的活性。然后将使用结构、动力学和计算方法来提供对工程酶的严格表征。这些数据将为后续的工程工作提供信息。具体地说，FDHS将被改造成利用分子识别来超越底物的反应性，并通过后期卤化和串联卤化/交叉偶联来调节芳烃和含烯烃底物的生物活性。还将展示使用FDH催化将发射正电子的卤素掺入到这些化合物中来合成PET探针。总之，这些实验将显著推进现代合成卤化方法，并提高我们为其他C-H键官能化反应设计酶的能力。

 DESCRIPTION (provided by applicant): Halogenated organic compounds are used extensively as building blocks, synthetic intermediates, and end use products for pharmaceutical and agrochemical applications. The utility of these compounds, including their biological activity, arises from the reactivity and physical properties uniquely conferred to them by halogen substitution. The importance of halogenation and limitations associated with current halogenation methods prompted us to develop new halogenation catalysts. This proposal outlines the evolution of flavin dependent halogenases (FDHs) for a range synthetic methods involving selective halogenation. Our preliminary results include the first examples of FDH directed evolution via iterative mutagenesis, and we will use the methods we have developed to further improve the substrate scope, selectivity, and reactivity of these enzymes for practical synthetic applications. This will entail generating FDH variants and screening those variants for activity on substrates with progressively increased sizes, altered shapes, and reduced nucleophilicities, relative to the native substrate, tryptophan. Structural, kinetic, and computational methods will then be used to provide rigorous characterization of the engineered enzymes. These data will inform subsequent engineering efforts. Specifically, FDHs will be engineered to override substrate reactivity using molecular recognition and to tune the biological activity of arene and olefin-containing substrates via late-stage halogenation and tandem halogenation/cross-coupling. Synthesis of PET probes using FDH catalyzed incorporation of positron emitting halogens into these compounds will also be demonstrated. Together, these experiments will significantly advance modern synthetic halogenation methods and improve our ability to engineer enzymes for other C-H bond functionalization reactions.