Fluorovinyl thioethers as stereoelectronic mimetics of acyl co-enzyme-A enol/ates

酶是进行新陈代谢反应的生物催化剂。它们存在于所有生物中，如高等哺乳动物、植物和细菌。如果植物或细菌中的酶反应能被选择性地抑制，那么如果我们能抑制植物酶，就有可能开发除草剂等农用化学药剂，如果我们能抑制细菌酶，就有开发抗生素的前景。酶抑制是农用化学品和制药研究中的一种既定策略。设计酶抑制剂的一种方法是在分子水平上模拟化学过程中不稳定的中间体，因为酶擅长与不稳定的中间体结合，并有效地处理它们。这就是他们进行催化的方式。这位研究人员的独创性在于试图设计一种合适的、稳定的对不稳定中间体的模拟。这是一个挑战，但如果它能够实现，那么一种酶抑制的策略就打开了。这项提议的目的是模拟烯醇或/和烯醇中间体，当它们执行催化功能时，这些中间体被认为是在酶的表面上短暂的。烯酸根有一个氧原子与一个双键相连。这是非常不稳定的，因为它可以重排成更稳定的羰基形式。然而，作为其正常功能的一种手段，烯基通过与酶表面的相互作用来稳定。在这个提议中，附着在烯基双键上的氧将被氟取代。这是一种聚偏氟乙烯。这是稳定的，我们已经通过计算机模拟表明，它具有与烯醇体大致相同的电子轮廓。这是一个新的想法，该提案将致力于在酶的水平上进行探索。所选的三种酶中的两种对农化工业非常重要，这些酶一直是制备除草剂的抑制重点。这些酶是乙酰辅酶A羧基酶(ACC)，反式烯醇辅酶A还原酶。这些酶利用辅酶-A酯。辅酶-A是一种相对复杂的生物大分子，以其预期的方式制备其衍生物具有挑战性。然而，在前期工作中，我们开发了一种化学方法来制备所需的氟乙烯基硫醚，以及一种生化(酶)方法来将合成的基序制备成完全形成的辅酶-A衍生物。现在令人兴奋的是制备完整的氟乙烯基硫醚基序的辅酶-A衍生物，并评估它们与适当的酶结合并抑制适当酶的能力。该提议的一个方面是评估氟原子在模拟氧原子中的重要性。因此，类似物将用氟制备，然后不加氟，用氢取代它。工作假说预计会有显著的氟效应。有几种方法可以评估辅酶-A衍生物是否会与酶结合，也可以评估它们的相对亲和力。这涉及到酶的分析，以及评估基序是否是一个好的抑制剂(强结合蛋白)。这也可以通过量热分析(ITC)进行评估，在这种情况下，良好的结合会导致放热，从而放出热量。该实验室拥有很好的仪器，可以进行详细的酶分析和量热分析。我们还计划将我们精心设计的辅酶-A衍生物与酶共结晶。这些是活性中间体(烯醇类)的模拟物，它们应该与酶表面紧密结合。X射线分析将使我们看起来非常清楚这种模拟如何结合到酶口袋中。在节目结束时，我们将能够演示如何引入和操作这种新的含氟基序，以及它在酶抑制方面的潜力。这里的重点是针对农用化学品的研究，然而，出现的原理将同样适用于药物研究，以及更普遍的酶抑制的合理方法。

Enzymes are biological catalysts that carry out the reactions of metabolism. They are found in all living forms such as higher mammals, plants and bacteria. If an enzyme reaction in a plant or a bacterium can be selectively inhibited, then there are prospects of developing agrochemical agents such as herbicides if we can inhibit plant enzymes, or antibiotics if we inhibit bacterial enzymes.Enzyme inhibition is an established strategy in agrochemicals and pharmaceuticals research.One way of designing enzyme inhibitors is to mimic, at the molecular level, an unstable intermediate in the chemical process, because the enzyme is good at binding to unstable intermediates, and processing them efficiently. That is how they carry out their catalysis. The ingenuity for the researcher comes in trying to design a suitable stable mimic of an unstable intermediate. This is a challenge, but if it can be achieved, then a strategy for enzyme inhibition opens up.This proposal aims to mimic enol or/and enolate intermediates, that are thought to be transient on the surface of enzymes, when they are carrying out their catralytic function. An enolate has an oxygen atom attached to a double bond. This is very unstable as it can rearrange to a more stable carbonyl form. The enolate however is stabilised by interactions with the enzyme surface, as a means of its proper functioning.In this proposal, the oxygen attached to the enolate double bond will be replaced by a fluorine. This is a vinylfluoride. This is stable, and we have shown by computer modelling that it has approximately the same electronic profile as an enolate. This is a new idea, and the proposal will aim to explore this at the enzyme level.Two of the three enzymes selected are important to the agrochemical industry, enzymes that have been the focus of inhibition to prepare herbicides. Thes enzymes are acetyl CoA carboxylase (ACC), trans enoyl Co-A-reductase. The enzymes utilise co-enzyme-A esters. Co-enzyme-A is a relatively complex biomolecule, and it challenging to prepare derivatives of it in the way that is envisaged. However in preliminary work we have developed a chemical method to prepare the required fluorovinyl thioether, and a biochemical (enzyme) method to eleborate the synthesised motif into a fully formed co-enzyme-A derivative. The excitement now is to prepare full co-enzyme-A derivatives of the fluorovinyl thioether motif, and assess their ability to bind to and also inhibit appropriate enzymes.One aspect of the proposal is to assess how important the fluorine atom is in mimicking the oxygen atom. Therefore analogues will be prepared with fluorine, and then without fluorine, replacing it for a hydrogen. The working hypothesis anticipates that there will be a significant fluorine effect. There are several methods for assessing if the co-enzyme-A derivatives will bind to the enzymes, and also for assessing their relative affinities. This involves enzymes assays, and assessing if the motif is a good inhibitor (strong binder). This can also be assessed by calorimetry (ITC), where good binding leads to an exotherm, and heat is evolved. The lab has good instrumentation for detailed enzyme assay and calorimetry analysis.We also plan to co-crystallise our elaborate co-enzyme-A derivatives with the enzymes. These are mimetics of reactive intermediates (enolates) and they should bind tightly to the enzyme surface. X-ray analysis will enable us to look very closley as to how this mimetic binds into the enzyme pocket.At the end of the programme we will be able demonstrate how to introduce and manipulate this new fluorine containing motif, and its potential in enzyme inhibition. The focus here is orientated towards agrochemicals reserach, however the principles that emerge will be equally applicable to pharmaceuticals research, and rational approachedsto enzyme inhibition more generally.