Periodic Reporting for period 2 - GlycoSpec (Unravelling Glycochemistry with Ion Mobility Spectrometry and Gas-Phase Spectroscopy)
Période du rapport: 2022-02-01 au 2023-07-31
Oligosaccharides or glycans are essential in nature and central participants in virtually every biological process. The extensive structural diversity enables glycans to encode rich information in biological functions; however, it also creates major challenges in almost all aspects of the glycosciences. The synthetic formation of glycosidic bonds during glycan assembly for example, is mechanistically still not fully understood. This is largely a result of highly-reactive, but short-lived glycosyl cation intermediates, which are difficult to study using established techniques. However, the structure of these intermediates dictates the stereochemistry of the resulting glycosidic bond, the control of which is absolutely crucial for a successful synthesis. An equally important aspect is the sequencing of glycans, which is generally complicated by the frequent occurrence of isomers. This is further complicated by poorly understood rearrangement reactions during mass spectrometric analysis, which often lead to erroneous structural assignments. Here as well, cationic intermediates are the key species that determine the outcome of the rearrangement.
The aim of GlycoSpec is to unravel fundamental aspects of oligosaccharide reaction and fragmentation mechanisms by probing cationic glycan intermediates. To achieve that, a unique combination of instrument and method development (ion mobility-mass spectrometry and cold-ion spectroscopy), chemical synthesis and theory will be used. The gain in mechanistic understanding will provide the basis to tailor building blocks and reaction conditions and is expected to lead to a major advancement in glycosynthesis. The mechanistic understanding of fragmentation reactions will furthermore lead to general rules for the prediction of tandem mass spectra. Just like in the early years of proteomics, this will make the technology automatable and accessible for broader applications. Therefore, GlycoSpec will help to initiate the pursuit of glycomics.
The aim of GlycoSpec is to unravel fundamental aspects of oligosaccharide reaction and fragmentation mechanisms by probing cationic glycan intermediates. To achieve that, a unique combination of instrument and method development (ion mobility-mass spectrometry and cold-ion spectroscopy), chemical synthesis and theory will be used. The gain in mechanistic understanding will provide the basis to tailor building blocks and reaction conditions and is expected to lead to a major advancement in glycosynthesis. The mechanistic understanding of fragmentation reactions will furthermore lead to general rules for the prediction of tandem mass spectra. Just like in the early years of proteomics, this will make the technology automatable and accessible for broader applications. Therefore, GlycoSpec will help to initiate the pursuit of glycomics.
During the first half of GlycoSpec two major aspects were in focus: 1) the purchase and assessment of a commercially available IMS instrument that will later be modified for spectroscopic experiments and 2) the analysis of reaction intermediates from glycosylations and other chemical reactions using existing instrumentation. Both have been achieved successfully. The instrument has been purchased and was extensively tested using various molecular classes. In order to implement tagging spectroscopy, we teamed up with external collaborators who provide the required technology. The first prototype cold ion trap is currently in production and will be tested within the next couple of months. In parallel, a variety of experiments on reactive species has been performed on using an existing instrumental setup that utilized droplets of superfluid helium to achieve cryogenic temperatures for spectroscopy experiments. In particular, we elucidated the molecular structure of fluorinated glycosidic (Eur. J. Org. Chem. 2022) and Ferrier-type (Org. Lett. 2020) reaction intermediates. More importantly, we recently succeeded for the first time to link the knowledge on the intermediate structure with the chemical properties of the leaving groups and use this information to tune glycosylation reactions (J. Am. Chem. Soc. 2023). This proof-of-principle experiment is one of the major aims of GlycoSpec and will now be used as a blueprint for the tuning of other glycosylation reactions. Finally, the technique was used to explore reactive intermediates and rearrangement reactions in other molecular classes such as the autohydrolysis of RNA (Angew. Chem. Int. Ed. 2022) or the fragmentation of lipids (Anal. Bioanal. Chem. 2022) and lead to the identification of the final product of fucose migration in glycan tandem MS experiments (Angew. Chem. Int. Ed. 2023)
An important aspect of GlycoSpec that clearly goes beyond state-of-the art is the development of an easy-to-use IMS-IR-MS instrument that is based on a commercially available backbone. The work on the prototype is progressing well and on the long run it will help to democratize gas-phase IR spectroscopy technology for a broader use, not only in glycomics, but also in lipidomics and metabolomics applications. In addition, we recently assessed the potential of artificial intelligence for the structural annotation of gas-phase IR spectra of unknowns (J. Am. Chem. Soc. 2023). The data impressively shows that it is possible to unambiguously identify unknown molecules using database spectra of known related compounds. As a consequence, it is not necessary to cover the full chemical space of all analytes with synthetic standards, which is difficult if not impossible for certain molecular classes such as glycosaminoglycans or metabolites.