Nachweis des entfernten Nachbargruppeneffekts in Glykosylierungen durch kalte Infrarotspektroskopie

Controlling the stereochemistry during glycosynthesis is usually achieved by using elaborate protecting group techniques. For example, 1,2-trans-glycosidic bonds are formed by the introduction of participating protecting groups at the neighboring C2-position. This reaction is believed to proceed via a glycosyl cation, where the protecting group interacts with the positive charge at the anomeric carbon, effectively shielding the cis-side from nucleophilic attack, leading to the concept of neighboring group participation. The formation of 1,2-cis-glycosidic bonds remains, however, a major hurdle to date. Empirical studies have shown that their formation can be aided by the introduction of participating protecting groups at the remote C4- or C6-positions, leading to the phenomenon of remote participation. Due to the short-lived nature of glycosyl cations, they cannot be studied readily in the condensed phase. The gas-phase environment inside a mass spectrometer, however, can be used to isolate them. In a recent publication, cryogenic infrared spectroscopy was used to unravel the structure of glycosyl cations. Evidence for the existence of neighboring group participation was found in various monosaccharide building blocks, as the acetyl protecting group at the C2-position forms a covalent bond with the anomeric carbon. While these results enable a better understanding of neighboring group participation, the structural motifs underlying remote participation remain poorly understood. Their study would help optimizing reaction conditions aiding the formation of 1,2-cis-glycosidic bonds. Here, a combination of first-principles theory and cryogenic infrared spectroscopy in the low-temperature environment of superfluid helium droplets (0.4 K) is used to decipher the structure of galactose building blocks exhibiting remote participation. Galactose building blocks carrying an acetyl protecting group at the C4-position form α-selective dioxolenium-type structures, exhibiting a covalent bond between the anomeric carbon and the oxygen atom of the carbonyl group of the acetyl protecting group. Contrary, galactose carrying an acetyl group solely at the C6-position does not exhibit remote participation and forms non-selective oxocarbenium-type structures. Furthermore, a novel type of interaction between benzyl protecting groups and the anomeric carbon, leading to α-selective oxonium-type structures, is observed. The recorded data can be used by algorithms based on artificial intelligence to predict the best reaction conditions for performing glycosylation reactions with a defined stereochemical outcome

Remote participation during glycosylation reactions of galactose building blocks: Direct evidence from cryogenic vibrational spectroscopy

The stereoselective formation of 1,2‐cis‐glycosidic bonds is challenging. However, 1,2‐cis‐selectivity can be induced by remote participation of C4 or C6 ester groups. Reactions involving remote participation are believed to proceed via a key ionic intermediate, the glycosyl cation. Although mechanistic pathways were postulated many years ago, the structure of the reaction intermediates remained elusive owing to their short‐lived nature. Herein, we unravel the structure of glycosyl cations involved in remote participation reactions via cryogenic vibrational spectroscopy and first principles theory. Acetyl groups at C4 ensure α‐selective galactosylations by forming a covalent bond to the anomeric carbon in dioxolenium‐type ions. Unexpectedly, also benzyl ether protecting groups can engage in remote participation and promote the stereoselective formation of 1,2‐cis‐glycosidic bonds

Mass Spectrometry-Based Techniques to Elucidate the Sugar Code

Cells encode information in the sequence of biopolymers, such as nucleic acids, proteins, and glycans. Although glycans are essential to all living organisms, surprisingly little is known about the “sugar code” and the biological roles of these molecules. The reason glycobiology lags behind its counterparts dealing with nucleic acids and proteins lies in the complexity of carbohydrate structures, which renders their analysis extremely challenging. Building blocks that may differ only in the configuration of a single stereocenter, combined with the vast possibilities to connect monosaccharide units, lead to an immense variety of isomers, which poses a formidable challenge to conventional mass spectrometry. In recent years, however, a combination of innovative ion activation methods, commercialization of ion mobility–mass spectrometry, progress in gas-phase ion spectroscopy, and advances in computational chemistry have led to a revolution in mass spectrometry-based glycan analysis. The present review focuses on the above techniques that expanded the traditional glycomics toolkit and provided spectacular insight into the structure of these fascinating biomolecules. To emphasize the specific challenges associated with them, major classes of mammalian glycans are discussed in separate sections. By doing so, we aim to put the spotlight on the most important element of glycobiology: the glycans themselves

Cryogenic infrared spectroscopy provides mechanistic insight into the fragmentation of phospholipid silver adducts

Tandem mass spectrometry is arguably the most important analytical tool for structure elucidation of lipids and other metabolites. By fragmenting intact lipid ions, valuable structural information such as the lipid class and fatty acyl composition are readily obtainable. The information content of a fragment spectrum can often be increased by the addition of metal cations. In particular, the use of silver ions is deeply rooted in the history of lipidomics due to their propensity to coordinate both electron-rich heteroatoms and C = C bonds in aliphatic chains. Not surprisingly, coordination of silver ions was found to enable the distinction of sn-isomers in glycerolipids by inducing reproducible intensity differences in the fragment spectra, which could, however, not be rationalized. Here, we investigate the fragmentation behaviors of silver-adducted sn- and double bond glycerophospholipid isomers by probing fragment structures using cryogenic gas-phase infrared (IR) spectroscopy. Our results confirm that neutral headgroup loss from silver-adducted glycerophospholipids leads to dioxolane-type fragments generated by intramolecular cyclization. By combining high-resolution IR spectroscopy and computational modelling of silver-adducted fragments, we offer qualitative explanations for different fragmentation behaviors of glycerophospholipid isomers. Overall, the results demonstrate that gas-phase IR spectroscopy of fragment ions can significantly contribute to our understanding of lipid dissociation mechanisms and the influence of coordinating cations

Unveiling Glycerolipid Fragmentation by Cryogenic Infrared Spectroscopy

Mass spectrometry is routinely employed for structure elucidation of molecules. Structural information can be retrieved from intact molecular ions by fragmentation; however, the interpretation of fragment spectra is often hampered by poor understanding of the underlying dissociation mechanisms. For example, neutral headgroup loss from protonated glycerolipids has been postulated to proceed via an intramolecular ring closure but the mechanism and resulting ring size have never been experimentally confirmed. Here we use cryogenic gas-phase infrared (IR) spectroscopy in combination with computational chemistry to unravel the structures of fragment ions and thereby shed light on elusive dissociation mechanisms. Using the example of glycerolipid fragmentation, we study the formation of protonated five-membered dioxolane and six-membered dioxane rings and show that dioxolane rings are predominant throughout different glycerolipid classes and fragmentation channels. For comparison, pure dioxolane and dioxane ions were generated from tailor-made dehydroxyl derivatives inspired by natural 1,2- and 1,3-diacylglycerols and subsequently interrogated using IR spectroscopy. Furthermore, the cyclic structure of an intermediate fragment occurring in the phosphatidylcholine fragmentation pathway was spectroscopically confirmed. Overall, the results contribute substantially to the understanding of glycerolipid fragmentation and showcase the value of vibrational ion spectroscopy to mechanistically elucidate crucial fragmentation pathways in lipidomics

Chondroitin Sulfate Disaccharides in the Gas Phase: Differentiation and Conformational Constraints

Glycosaminoglycans (GAGs) are a family of complex carbohydrates vital to all mammalian organisms and involved in numerous biological processes. Chondroitin and dermatan sulfate, an important class of GAGs, are linear macromolecules consisting of disaccharide building blocks of N-acetylgalactosamine and two different uronic acids. The varying degree and the site of sulfation render their characterization challenging. Here, we combine mass spectrometry with cryogenic infrared spectroscopy in the wavenumber range from 1000 to 1800 cm-1. Fingerprint spectra were recorded for a comprehensive set of disaccharides bearing all known motifs of sulfation. In addition, state-of-the-art quantum chemical calculations were performed to aid the understanding of the differences in the experimental fingerprint spectra. The results show that the degree and position of charged sulfate groups define the size of the conformational landscape in the gas phase. The detailed understanding of cryogenic infrared spectroscopy for acidic and often highly sulfated glycans may pave the way to utilize the technique in fragment-based sequencing approaches

Studying the Key Intermediate of RNA Autohydrolysis by Cryogenic Gas-Phase Infrared Spectroscopy

Over the course of the COVID-19 pandemic, mRNA-basedvaccineshave gained tremendous importance. The development and analysis of modified RNA moleculesbenefit from advanced mass spectrometry and require sufficient understanding of fragmentation processes.Analogous tothe degradation of RNA in solution by autohydrolysis,backbone cleavage of RNA strands wasequally observedin the gas phase; however, the fragmentation mechanism remained elusive.In this work,autohydrolysis-like intermediates weregenerated from isolated RNA dinucleotidesin the gas phaseand investigatedusing cryogenic infrared spectroscopy in helium nanodroplets.Data from both experiment and density functional theory provide evidence forthe formation of a five-membered cyclic phosphateintermediateand rule outlinear orsix-membered structures. Furthermore, the experiments show that another prominent condensed-phase reactionof RNA nucleotides can be induced in the gas phase: the tautomerization of cytosine.Both observed reactions aretherefore highlyuniversal and intrinsic properties of the investigated molecules

The Influence of the Electron Density in Acyl Protecting Groups on the Selectivity of Galactose Formation

The stereoselective formation of 1,2-cis-glycosidic bonds is a major bottleneck in the synthesis of carbohydrates. We here investigate how the electron density in acyl protecting groups influences the stereoselectivity by fine-tuning the efficiency of remote participation. Electron-rich C4-pivaloylated galactose building blocks show an unprecedented α-selectivity. The trifluoroacetylated counterpart with electron-withdrawing groups, on the other hand, exhibits a lower selectivity. Cryogenic infrared spectroscopy in helium nanodroplets and density functional theory calculations revealed the existence of dioxolenium-type intermediates for this reaction, which suggests that remote participation of the pivaloyl protecting group is the origin of the high α-selectivity of the pivaloylated building blocks. According to these findings, an α-selective galactose building block for glycosynthesis is developed based on rational considerations and is subsequently employed in automated glycan assembly exhibiting complete stereoselectivity. Based on the obtained selectivities in the glycosylation reactions and the results from infrared spectroscopy and density functional theory, we suggest a mechanism by which these reactions could proceed

Untersuchung des reaktiven Intermediats der RNA Autohydrolyse mittels kryogener Infrarotspektroskopie in der Gasphase

Im Laufe der COVID-19 Pandemie haben mRNA-basierte Impfstoffe an immenser Bedeutung gewonnen. Massenspektrometrie ist für die Entwicklung und Analyse von modifizierten RNA Molekülen unerlässlich, setzt jedoch ein grundlegendes Verständnis über Fragmentierungsprozesse voraus. Analog zu der Zersetzung von RNA in Lösung durch Autohydrolyse, kann die Spaltung des RNA Rückgrats ebenso in der Gasphase stattfinden. Bislang sind die Fragmentierungsmechanismen jedoch unzureichend untersucht. In dieser Arbeit wurden Intermediate aus isolierten RNA Dinukleotiden in der Gasphase generiert und mittels kryogener Infrarotspektroskopie in Helium-Nanotröpfchen untersucht. Die experimentellen Daten, unterstützt durch Dichtefunktionaltheorie, liefern Hinweise dafür, dass die Bildung eines fünfgliedrigen zyklischen Phosphat-Intermediats begünstigt ist, während lineare oder sechsgliedrige Strukturen ausgeschlossen werden können. Weiterhin zeigen die Experimente, dass eine zusätzliche, bekannte Reaktion von RNA Nukleotiden in Lösung auch in der Gasphase induziert werden kann: die Tautomerisierung von Cytosin. Die beiden beobachteten Reaktionen spiegeln daher universelle und intrinsische Eigenschaften der untersuchten Moleküle wider

Neighboring Group Participation of Benzoyl Protecting Groups in C3- and C6-Fluorinated Glucose

Fluorination is a potent method to modulate chemical properties of glycans. Here, we study how C3- and C6-fluorination of glucosyl building blocks influence the structure of the intermediate of the glycosylation reaction, the glycosyl cation. Using a combination of gas-phase infrared spectroscopy and first-principles theory, glycosyl cations generated from fluorinated and non-fluorinated monosaccharides are structurally characterized. The results indicate that neighboring group participation of the C2-benzoyl protecting group is the dominant structural motif for all building blocks, correlating with the β-selectivity observed in glycosylation reactions. The infrared signatures indicate that participation of the benzoyl group in enhanced by resonance effects. Participation of remote acyl groups such as Fmoc or benzyl on the other hand is unfavored. The introduction of the less bulky fluorine leads to a change in the conformation of the ring pucker, whereas the structure of the active dioxolenium site remains unchanged