Selective methylation and alkylation using methyl transferases

背景：甲基对生物活性化合物的理化和生物学性质具有显著的有益影响。这种所谓的“甲基效应”在药物化学中被广泛利用，以增加亲脂性和生物利用度，以及保护化合物在体内免受酶降解，从而提供了一种微调药物候选物的代谢和治疗应用功效的方法。1甲基化还可以通过立体电子和空间效应改变小分子的构象。然而，使用传统的合成方法很难实现选择性甲基化反应，通常使用有毒试剂如甲基碘。酶促甲基化提供了一种非常有价值的替代方法，使用甲基转移酶（MT），迄今为止在生物催化合成中受到的关注相对较少。2它们是特别有用的酶，用于化合物的区域选择性甲基化以及化合物文库的多样化。在最近的工作中，我们已经开始研究MT的克隆和使用，以及其他酶，以驱动平衡朝向所需产物，以及在原位产生昂贵的生态因子S-腺苷甲硫氨酸（SAM）。3，4在初步研究中，我们使用了不同的MT与多巴胺和类似物以及5 '-甲硫腺苷/S-腺苷高半胱氨酸核苷酶（MTAN）进行选择性甲基化。此外，我们已经使用了甲硫氨酸腺苷转移酶（MAT）形成SAM和SAM类似物，以扩大该方法，使其他选择性烷基化。2，3目的：在这个跨学科的项目中，我们将开发MT和辅因子供应/回收系统的酶促反应级联与一系列化合物的选择性甲基化的O-，N-和C-基团。此外，我们的目标是整合通过其他酶生成的羟基或氨基的原位烷基化，以确保使用SAM和SAM类似物构建2个高度集成的新反应级联，以形成杂环化合物。项目：在化学培训轮换期间，（与海伦·海尔斯）和生物化学工程（与共同监督人约翰沃德）学生将开发的技能在酶的表达，酶筛选，底物合成，分析，产品分离和表征。他们还将在生物催化反应中使用可用的MT与生物碱5或醇和胺，以建立初始区域和立体选择性。这将包括使用MAT原位形成SAM和使用MTAN驱动反应向产物方向发展。然后，他们将专注于建立初始转氨酶（TAm）+ MT级联反应，以期通过甲基化/乙基化步骤和MAT和MTAN的掺入来优化级联反应。此外，他们将使用宏基因组学和生物信息学策略鉴定新的O-，N-，C-MT，并筛选这些以建立底物范围。在博士学位的后半部分，他们将使用MT与其他SAM类似物，例如由MTAN和烯丙基甲硫氨酸原位合成的烯丙基-SAM，将探索与可以原位制备的生物碱进行3-酶一锅烯丙基化反应。如前所述。5此外，使用氨基醇进行烯丙基化和随后的化学环化以产生化学酶级联反应。如果需要，将进行MT诱变以增强酶的性能。在最后一年，建立成功的结果，扩展到其他酶级联，

Background: Methyl groups can have a significant beneficial effect on the physicochemical andbiological properties of bioactive compounds. This so called "methyl group effect", has beenwidely exploited in medicinal chemistry to increase lipophilicity and bioavailability, as well asprotecting compounds in vivo from enzymatic degradation, thus offering a method for finetuning adrug candidate's metabolism and efficacy in therapeutic applications.1 Methylation can also alterthe conformations of small molecules, through stereoelectronic and steric effects. However, it isvery difficult to achieve selective methylation reactions using traditional synthetic approaches andtypically, toxic reagents such as methyl iodide are used. Enzymatic methylation provides a veryvaluable alternative method using methyl transferases (MTs) which have received comparativelylittle attention to date in biocatalytic syntheses.2 They are particularly useful enzymes for theregioselective methylation of compounds as well as the diversification of compound libraries.In recent work we have started to investigate the cloning and use of MTs, together with otherenzymes to drive the equilibrium towards the desired product, as well as generating the expensiveco-factor S-adenosyl methionine (SAM) in situ.3,4 In preliminary studies we have used differentMTs with dopamine and analogues for selective methylations, together with 5'-methylthioadenosine/S-adenosyl homocysteine nucleosidase (MTAN). Furthermore, we haveused a methionine adenosyltransferase (MAT) for the formation of SAM and SAM analogues toexpand the approach, enabling other selective alkylations.2,3Aims: In this interdisciplinary project we will develop MTs and the cofactor supply/recyclingsystems in enzymatic reaction cascades with a range of compounds for the selective methylationof O-, N- and C-groups. In addition, we aim to integrate the in situ alkylation of enzymaticallygenerated hydroxyl or amino groups formed via other enzymes to ensure the construction of2highly integrated new reaction cascades towards heterocyclic compounds using SAM and SAManalogues.Project: During the training rotations in Chemistry (with Helen Hailes) and BiochemicalEngineering (with the co-supervisor John Ward) the student will develop skills in enzymeexpression, enzyme screening, substrate synthesis, assays, product isolation andcharacterisation. They will also use available MTs with alkaloids5 or alcohols and amines inbiocatalytic reactions to establish initial regio and stereoselectivities. This will include the use ofMAT for the formation of SAM in situ and MTAN to drive the reaction towards the product Theywill then focus on establishing an initial transaminase (TAm) + MT cascade with a view tooptimising the cascade with a methylation/ethylation step and incorporation of MAT and MTAN. Inaddition they will identify new O-, N-, C-MTs using metagenomic and bioinformatics strategies andscreen screen these to establish the substrate scope. In the later half of the PhD they will use ofMTs with other-SAM analogues such as allyl-SAM, synthesised in situ from MTAN and allylmethionine,will be explored to give a 3- enzyme one pot allylation reaction with alkaloids that canbe prepared in situ as previously described.5 Also, the use of amino alcohols for allylation andsubsequent chemical cyclisations to produce a chemoenzymatic cascade. If required MTmutagenesis will be carried out to enhance enzyme performance. In the final year, building uponsuccessful results, extension to other enzyme cascades and