IRIS characterization of reactive intermediates involved in the production of epilepsy biomarkers
Epilepsy is one of the most common neurological disorders, affecting almost 50 million people worldwide. It has a large impact on social and mental functioning and therefore the quality of life of patients. A major challenge in epilepsy research is the identification of diagnostic biomarkers. N-acyliminium ions (NAIs), positively charged carbocations that are stabilized by an adjacent nitrogen atom, are useful intermediates for the synthesis of nitrogen containing chemical compounds such as alkaloids and amino acids. The characterization of NAIs is crucial to predict how exactly they react with other molecules. As part of a longstanding collaboration between the Synthetic Organic Chemistry group within the Institute for Molecules and Materials (IMM) and HFML-FELIX, NAIs were characterized using infrared ion spectroscopy (IRIS). For example, some of the NAIs characterized in this study were used to synthesize amino acid-type biomarkers associated with epilepsy in a stereoselective manner. These reaction intermediates are challenging to characterize as they are highly reactive and hence not stable.
From reactive intermediates to biomarker discovery
The structure of reactive intermediates formed during (bio)chemical reactions impacts their behaviour in chemical reactions. However, since these intermediates are highly reactive they are not stable and therefore it is difficult to characterize their structure. Over the past few years, Floris Rutjes and Thomas Boltje (Synthetic Organic Chemistry, IMM) have collaborated with Jos Oomens and Jonathan Martens (HFML-FELIX) to address this challenge. Using the unique laser facilities of HFML-FELIX, reactive intermediates can be generated and studied in the gas-phase using Infrared Ion Spectroscopy (IRIS). In the past few years, this methodology has been used to characterize intermediates formed during glycosylation reactions in the gas phase 1 and could be translated to the solution phase using NMR more recently.2 “For many years, we have been studying IR spectra of ionized carbonaceous molecules, including carbocations, often from an astrochemical perspective. We knew of course that carbocations are important intermediates in organic chemistry, but through this collaboration we can really start to answer relevant questions”, Jos Oomens says.
Comparison of calculated (colour-filled) and IRIS-generated spectra of NAIs
Another important area of collaboration between these groups is biomarker discovery. Using IRIS, biomarkers from clinical samples can be characterized with unprecedented sensitivity. The synthetic organic chemistry group can enable the ultimate confirmation of biomarker structure by the preparation of synthetic standards thereby also enabling the quantitation of biomarkers in clinical samples. Using this workflow, biomarkers were characterized for clinical indications such as inborn errors of amino acid metabolism and epilepsy.3“Our most recent report is a combination of these two main research lines. We characterized reactive intermediates to better understand how we can prepare epilepsy biomarkers in a stereoselective manner”, Floris Rutjes explains.
While Thomas Boltje’s expertise is the use of oxygen-stabilized carbocations to synthesize new sugars, Rutjes has been focusing his synthesis research over the years on nitrogen-stabilized carbocations to create biologically active nitrogen heterocycles and natural products. Since oxygen and nitrogen are next to each other in the Periodic Table, both cations have some similarities in terms of reactivity, but also very clear differences. Nitrogen has stronger electron-donating properties than oxygen, making oxygen-stabilized cations more reactive and therefore more susceptible to stabilizing effects of other functional groups in the molecule. This then leads to a different stereochemical outcome of the reaction.
Floris Rutjes, smiling: “Despite the fact that NAIs have been known for a long time, intricate details of the stereodirecting effects so far could not always be explained. The IRIS measurements provide relevant new insights, in fact, they in hindsight explain the stereochemical outcome of reactions that I did during my own PhD research thirty years ago. Isn’t that wonderful?”
Characterization of Cyclic N-Acyliminium Ions by Infrared Ion Spectroscopy, Jona Merx, Kas Houthuijs, Hidde Elferink, Eva Witlox, Jasmin Mecinovic, Jos Oomens, Jonathan Martens, Thomas Boltje and Floris P. J. T. Rutjes, Chemistry a European Journal, 2021
Characterization of glycosyl dioxolenium ions and their role in glycosylation reactions. Thomas Hansen, Hidde Elferink, Jacob M. A. van Hengst, Kas J. Houthuijs, Wouter A. Remmerswaal, Alexandra Kromm, Giel Berden, Stefan van der Vorm, Anouk M. Rijs, Hermen S. Overkleeft, Dmitri V. Filippov, Floris P. J. T. Rutjes, Gijsbert A. van der Marel, Jonathan Martens, Jos Oomens, Jeroen D. C. Codée, Thomas J. Boltje. Nature Communications, 11, 2020, https://doi.org/10.1038/s41467-020-16362-x.
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Direct experimental characterization of glycosyl cations by infrared ion spectroscopy. Elferink H., Severijnen M.E., Martens J., Mensink R.A., Berden G., Oomens J., Rutjes F.P.J.T., Rijs A.M.*, Boltje T.J.*, J. Am. Chem. Soc., 2018, 140, 6034-6038. 10.1021/jacs.8b01236
Characterization of Mannosyl Dioxanium Ions in Solution Using Chemical Exchange Saturation Transfer NMR. Frank F. J. de Kleijne, Hidde Elferink, Sam J. Moons, Paul B. White & Thomas J. Boltje. Angew. Chem. Int. Ed., 2021, https://doi.org/10.1002/anie.202109874
Untargeted metabolomics and infrared ion spectroscopy identify biomarkers for pyridoxine-dependent epilepsy. Engelke UF, van Outersterp RE, Merx J, van Geenen FA, van Rooij A, Berden G, Huigen MC, Kluijtmans LA, Peters TM, Al-Shekaili HH & Leavitt BR. The Journal of Clinical Investigation, 2021, https://doi.org/10.1172/JCI148272
Metabolite Identification Using Infrared Ion Spectroscopy─Novel Biomarkers for Pyridoxine-Dependent Epilepsy. Rianne E. van Outersterp, Udo F.H. Engelke, Jona Merx, Giel Berden, Mathias Paul, Thomas Thomulka, Albrecht Berkessel, Marleen C.D.G. Huigen, Leo A.J. Kluijtmans, Jasmin Mecinović, Floris P.J.T. Rutjes, Clara D.M. van Karnebeek, Ron A. Wevers, Thomas J. Boltje, Karlien L.M. Coene, Jonathan Martens& Jos Oomens. Anal. Chem, 2021, https://doi/10.1021/acs.analchem.1c02896
Amadori rearrangement product as potential biomarkers for inborn errors of amino-acid metabolism. Rianne E. van Outersterp, Sam J. Moons, Udo F. H. Engelke, Herman Bentlage, Tessa M. A. Peters, Arno van Rooij, Marleen C. D. G. Huigen, Siebolt de Boer, Ed van der Heeft, Leo A. J. Kluijtmans, Clara D. M. van Karnebeek, Ron A. Wevers , Giel Berden, Jos Oomens, Thomas J. Boltje, Karlien L. M. Coene & Jonathan Martens. Communications Biology, 2021, https://doi.org/10.1038/s42003-021-01909-5