Periodic Reporting for period 1 - ENZYME-DCC (Enzyme-Mediated Dynamic Combinatorial Chemistry)
Période du rapport: 2022-07-01 au 2024-12-31
Biomolecular templates define the outcomes of enzymatic reactions in some of the most fundamental of biological processes, such as DNA replication, transcription and translation. In synthetic chemistry, molecular templates have enabled the synthesis of highly complex molecular architectures and interlocked structures. The possibility to use synthetic templates to direct enzymatic reactions and obtain alternative products to those generated in Nature is being investigated in this project. The overall objective of this research programme is to explore a conceptually new approach to the use of enzymes for chemical synthesis and thus establish a new synthetic methodology - Template-Directed Enzyme-Mediated Dynamic Combinatorial Chemistry. We exploit the inherent reversibility of enzymatic reactions to generate dynamic chemical networks, which can be manipulated via supramolecular interactions with artificial template molecules to promote the preferential synthesis of specific products. To implement this novel concept, we are developing an unprecedented, templated, enzymatic approach to oligosaccharide synthesis. We are exploring dynamic systems of interconverting glycans with the goal of accessing unusual or challenging oligosaccharide products.
The specific aims are:
1. To achieve templated enzymatic selective synthesis of large-ring cyclodextrins (cyclodextrins (CDs) with more than 8 glucose units).
2. To explore cyclodextrin glucanotransferase-mediated dynamic mixtures of modified CDs, and, using thiol-functionalised CDs, develop a doubly dynamic system that combines reversible transglycosylation with disulfide exchange.
3. To generate phosphorylase-mediated dynamic mixtures of linear α-1,4-glycans and employ templates to achieve length- and sequence-selective synthesis.
4. To establish a fuelled far-from-equilibrium system for continuous large-ring CD synthesis using templates together with a series of interconnected dynamic enzymatic transformations.
The ultimate success of ENZYME-DCC will be to bring about a paradigm shift in the way that enzymes are employed in biocatalysis, such that reversible reactions, thermodynamic control, and templates come into play. By embracing the complexity of the dynamic mixtures generated, new products, reactivities and recognition motifs will be discovered. In contrast to nucleic acids and proteins, biosynthetic pathways to complex oligosaccharides are not guided by templates. In this research programme, novel methodology will be developed that uses designed, artificial templates to control enzymatic glycosylation, thus capturing the enhanced value of supramolecular chemistry and enzymology working together.
The specific aims are:
1. To achieve templated enzymatic selective synthesis of large-ring cyclodextrins (cyclodextrins (CDs) with more than 8 glucose units).
2. To explore cyclodextrin glucanotransferase-mediated dynamic mixtures of modified CDs, and, using thiol-functionalised CDs, develop a doubly dynamic system that combines reversible transglycosylation with disulfide exchange.
3. To generate phosphorylase-mediated dynamic mixtures of linear α-1,4-glycans and employ templates to achieve length- and sequence-selective synthesis.
4. To establish a fuelled far-from-equilibrium system for continuous large-ring CD synthesis using templates together with a series of interconnected dynamic enzymatic transformations.
The ultimate success of ENZYME-DCC will be to bring about a paradigm shift in the way that enzymes are employed in biocatalysis, such that reversible reactions, thermodynamic control, and templates come into play. By embracing the complexity of the dynamic mixtures generated, new products, reactivities and recognition motifs will be discovered. In contrast to nucleic acids and proteins, biosynthetic pathways to complex oligosaccharides are not guided by templates. In this research programme, novel methodology will be developed that uses designed, artificial templates to control enzymatic glycosylation, thus capturing the enhanced value of supramolecular chemistry and enzymology working together.
We have made significant progress in the development of Enzyme-Mediated Dynamic Combinatorial Chemistry as a synthetic methodology that uniquely combines supramolecular chemistry with enzymology. In particular, the focus, so far, has been on forming dynamic combinatorial libraries of cyclic and linear oligosaccharides. We are investigating the use of different enzymes capable of generating dynamic glycan mixtures and exploring their substrate selectivity to test their tolerance of unnatural functional groups appended to oligosaccharides. Analytical methods are being developed to characterise complex glycan mixtures and to explore the recognition capabilities of different oligosaccharide products. A number of templates have been synthesised with the goal of controling the degree of polymerisation of oligosaccharides formed.
One of our most important achievements, thus far, has been the development of a method for the templated enzymatic synthesis of δ-cyclodextrin. Cyclodextrins (CDs) are industrially important molecular containers formed from linked glucose monomers. α-, β-, and γ-CD, formed from 6, 7, and 8 glucose units, are all produced on a tonne scale for applications in the pharmaceutical, food and cosmetics industries. They are employed to solubilise, stabilise and deliver bioactive guests, such as drugs, flavours and aromas.
Large-ring cyclodextrins have been very little explored in the past, due to synthetic inaccessibility. Previously, only a few hundred milligrams of δ-CD, formed from 9 glucose units, had ever been synthesised or isolated. We have developed a scalable method for the production of δ-CD that gives high yield, high purity, easy isolation of the product and uses a cheap starting material. It is our hope that by making this larger cyclodextrin accessible, it will find many applications, and one day, δ-CD will be as ubiquitous as the well-known α-, β-, and γ-cyclodextrins.
Large-ring cyclodextrins have been very little explored in the past, due to synthetic inaccessibility. Previously, only a few hundred milligrams of δ-CD, formed from 9 glucose units, had ever been synthesised or isolated. We have developed a scalable method for the production of δ-CD that gives high yield, high purity, easy isolation of the product and uses a cheap starting material. It is our hope that by making this larger cyclodextrin accessible, it will find many applications, and one day, δ-CD will be as ubiquitous as the well-known α-, β-, and γ-cyclodextrins.