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

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.

Who will benefit from this research?
Organofluorine compounds make up 20% of all pharmaceuticals products internationally and 30% or all agrochemical products on the market. The organic materials industry relies very heavily in selectively fluorinated organic compounds and polymers. So innovation in the selective incorporation of fluorine contributes substantially to products that are designed to improve health, well being and the societal challenges of a growing population and developing third world sector (BRIC Nations). That is the global perspective of who will benefit from this research. More tangibly this research prtogramme is focussed on the agrochemical sector and to also to the pharmaceuticals discovery sector. We will explore the introduction of thefluorovinyl thioether motif into enzyme substrate analogues. This research will be partially guided by Syngenta, a global leader in innovation in the agrochemicals sector. So we have aligned directly with an a key member of a relevant industry. This provides us with a platform in which to introduce this new motif and its performance to a wide audience. Enzymes relevant to agrochemistry appears to be an appropriate arena in which to profile the research and if successful there could be much wider applications particularly in specfic enzyme inhibition approrpiate to medicinal chemistry.

How will they benefit?
They will benefit by being able to utilise these new motifs in their own research programmes. They will hopefully be stimuated by the design concepts and researchers in the area will take up this design and modify it to their own purposes. Therefore the programme will stimulate thinking, and new approached to current problems and challenges.

Staff working on this project will become expert in organo-fluorine chemistry and its association with biochemistry and enzyme inhibition. This extends the concepts of using fluorine as a mimetic Those working on the project and those associated with it will become specialists in the design strategy and execution of the preparation of particular molecules of interest (Dial a molecule; Grand Challenge).

The PDRA will develop synthesis skills and know how in organic fluorine chemistry, biochemistry, enzyme assays and will become a specialist in understanding the specific effects of fluorine in terms of modulating properties. They will also become adept at monitoring fluorinated compounds in the context of biochemistry by 19F-NMR. The programme will extend to interactions with structural biologists, and will be underpinned by molecular biology techniques. Such a knowledge, understanding and skills development to meet the modern challenges of chemical and synthetic biology.
St Andrews University are very proactive in developing IPR. This will be developed in consultation with Business Development Managers (BDMs) associated with tResearch Business Development & Contracts.

Beneficiaries within the wider public? Results from such a programme will be used in more general arenas for discussing the relevance of chemistry and molecular design to enzyme inhibition in the service of food security and health. This type of programme is particularly well suited to public interactions because it engages with thse major societal concerns.

The research will be published and disseminated in leading journals and at appropriate international conferences.