Exploiting Halogenase Enzymes: New Reaction Pathways via Enzymatic CH Activation

卤素氟、氯和溴是元素周期表中最活泼的元素，当它们以天然的元素形式使用时，会与其他分子剧烈结合。然而，作为其他分子的一部分，它们扮演着完全不同的角色，赋予有价值的稳定性并引入有利的功能特性。许多药物（包括对人类健康至关重要的药物，如抗生素万古霉素、氯霉素和环丙沙星）、提高作物产量的农用化学品、聚合物和其他有价值的材料中都含有氟、氯和较少程度的溴。具有卤素取代基的分子（有机卤素）也被广泛用于制造药物和其他产品的中间体，因为卤素可以很容易地取代一系列其他官能团。目前，有机卤素化合物的生产涉及多步合成化学方法，使用有害的溶剂、苛刻的化学卤化试剂和昂贵的催化剂，以及不可再生的石化前驱体，对环境有严重的不利影响。在这个项目中，我们的目标是开发基于生物技术的替代工艺，以更经济和环境可持续地生产卤化分子。为了做到这一点，我们将利用自然界进化出来的酶（卤素酶），在具有工业重要性的目标分子内的特定位置选择性地安装卤素。自然界的卤化酶已经进化到在生命系统中以非常低的浓度卤化天然产物。因此，它们通常不具有所需的活性和选择性，以有效的规模进行药物或农用化学品目标分子的卤化。鉴于此，我们将使用x射线晶体学来确定有前途的卤素酶酶的结构，以获得酶活性位点的3D图像，其中底物结合。我们将使用这张图片来改变活性位点的3D结构，使用一种称为诱变的技术，这将使酶能够以更高的效率容纳非天然目标分子。除了有针对性的方法外，我们还将使用更多的随机诱变技术来创建突变的卤化酶酶库（数百万），然后使用新的质谱成像技术和荧光分析来选择具有所需活性和选择性的突变体。这些新的、优化的卤化酶酶将被用于生产关键的卤化基础材料，这些材料是制造药物所必需的，包括抗病毒药物、抗癌药物和其他对人类健康至关重要的药物，以及迫切需要提高作物产量和为不断增长的全球人口提供更多食物的农用化学品。我们的酶还可以用于在生物活性分子的新位置引入卤素，创造出传统化学卤化方法难以或不可能获得的新类似物。这种卤化酶选择性地将氯或溴原子放入分子中的能力，为在进一步的化学转化中操纵卤化产物提供了令人兴奋的机会。卤素是非常通用的官能团，使我们能够将卤素酶与其他化学催化步骤结合起来，以创建具有（例如）碳氮，碳碳或碳氟键的新实体。在每种情况下，卤素酶的精细选择性为制造有价值的产品提供了一条化学捷径，否则将通过漫长而昂贵的合成路线来制造。

The halogens fluorine, chlorine, and bromine are amongst the most reactive elements of the periodic table, combining vigorously with other molecules when used in their native, elemental form. As part of other molecules, however, they play a quite different role, imparting valuable stability and introducing favourable functional properties. Fluorine, chlorine, and to a lesser extent bromine, are found in numerous pharmaceuticals (including drugs critical to human health such as the antibiotics vancomycin, chloramphenicol, and ciproflaxin), agrochemicals that boost crop yields, polymers and other valuable materials. Molecules possessing halogen substituents (organohalogens) are also widely used intermediates for making drugs and other products, as the halogens can be readily substituted for a range of other functional groups.Currently the manufacture of organohalogen compounds involves multistep synthetic chemical methods which use deleterious solvents, harsh chemical halogenating reagents and expensive catalysts, as well as non-renewable petrochemical precursors, which have serious detrimental environmental impact. In this project we aim to develop alternative biotechnology based processes for more economic and environmentally sustainable production of halogenated molecules. To do this we will exploit enzymes that nature has evolved (halogenases) to selectively install halogens at specific positions within target molecules of industrial importance. Nature's halogenating enzymes have evolved to halogenate natural products at very low concentrations in living systems. As a result, they do not generally have the required activity and selectivity for halogenation of pharmaceutical or agrochemical target molecules on a useful scale. In light of this, we will determine the structures of promising halogenase enzymes using X-ray crystallography to obtain a 3D image of the enzyme's active site where the substrate binds. We will use this picture to change the 3D structure of the active site using a technique called mutagenesis, which will enable the enzyme to accommodate non-natural target molecules with enhanced efficiencies. In addition to targeted approaches we will also use more random mutagenesis techniques to create libraries (many millions) of mutant halogenase enzymes, followed by new mass spectrometry imaging technology and fluorescence assays to select mutants with the required activity and selectivity.These new, optimised, halogenase enzymes will then be used to produce the key halogenated building blocks that are required for the manufacture of pharmaceuticals including antiviral agents, anticancer agents and other drugs essential for human healthcare, as well as agrochemicals that urgently are required to boost crops yields and provide more food for the growing global population. Our enzymes can also be used to introduce halogens at new positions in biologically active molecules, creating new analogs that would be difficult or impossible to access by conventional chemical halogenation methods. This ability of halogenase enzymes to selectively place chlorine or bromine atoms into molecules opens up exciting opportunities to manipulate the halogenated products in further chemical transformations. Halogens are extremely versatile functional groups, enabling us to combine halogenases with other chemical catalysis steps to create new entities featuring (for example) carbon-nitrogen, carbon-carbon, or carbon-fluorine bonds. In each case, the exquisite selectivity of the halogenase enzyme provides a chemical shortcut to making valuable products that would otherwise be made via long-winded and expensive synthetic routes.