A set of unnatural sugar molecules has been generated that have been equipped with a chemical identification tag allowing us to study the target sugar in a biological context. We published a review on the concept of using unnatural sugars to study biological processes in Biochemical Society Transactions (2021). Additionally, as part of the work towards these molecules, we developed a new chemical synthesis methodology which has been published in Chemical Science (2023).
Secondly, the proposal aimed to study a set of human proteins that are involved in the production of the sugar of interest by biochemical analysis and 3-D structure analysis. Because the production and particulary the isolation, in stable form, of these proteins is very challenging, most of our work has focused on a set of homologous bacterial proteins (TarI, TarK, TarL) that perform similar functions to the human targets. We have established a working pipeline for the successful isolation of each of these proteins and developed assays that enable us to test their functional integrity with the use of synthetic substrates produced in house. One of these assays has also been applied to inhibition studies to test if the molecules produced below can perform their desired blocking effect on the protein TarI. As part of this work, we have additionally developed a new enzymatic method for the generation of one of the protein substrates.
Furthermore, two different sets of synthetic molecules have been delivered that are designed to block and label the human proteins mentioned above. These will find use as research tools to dissect the consequences of interfering with sugar production in cells without the need for genetic manipulation, as well as for the detection of protein levels under normal vs disease-mimicking conditions. We published a review on the topic of activity-based protein profiling in Molecules (2020). Initial proof of concept analysis using the TarI assay described above has revealed that some of these molecules do indeed act as blockers of their anticipated target protein.
Finally, a cellular assay has been established for the testing of the unnatural sugars that have been generated above. The results show that three of these molecules label a number of proteins inside cells. The identity of these unknown proteins will be the topic of future research. Additionally, we have demonstrated that one of the molecules can label the intended sugar when attached to its target protein. A manuscript on this work has been published in RSC Chemical Biology (2025). This new platform will enable investigations into the levels and localisation of the sugar of interest upon manipulation of the sugar production pathway or in response to disease-causing mutations.
Aside from the publications mentioned above, the results of the project have also been disseminated through presentations at a number of scientific conferences in the UK (e.g. the RSC Carbohydrate Group Winter meeting, RSC Organic Division North East regional meeting, Biochemical Society meeting) and the US (Gordon Conference on Carbohydrates, ACS Spring National Meeting).
