Investigation of the Proton-Bound Dimer of Dihydrogen Phosphate and Formate Using Infrared Spectroscopy in Helium Droplets.

Understanding the structural and dynamic properties of proton-bound complexes is crucial for elucidating fundamental aspects of chemical reactivity and molecular interactions. In this work, the proton-bound complex between dihydrogen phosphate and formate, and its deuterated counterparts, is investigated using IR action spectroscopy in helium droplets. Contrary to the initial expectation that the stronger phosphoric acid would donate a proton to formate, both experiment and theory show that all exchangeable protons are located in the phosphate moiety. The experimental spectra show good agreement with both scaled harmonic and VPT2 anharmonic calculations, indicating that anharmonic effects are small. Some H-bending modes of the nondeuterated complex are found to be sensitive to the helium environment. In the case of the partially deuterated complexes, the experiments indicate that internal dynamics leads to isomeric interconversion upon IR excitation.

Predicting Structural Motifs of Glycosaminoglycans using Cryogenic Infrared Spectroscopy and Random Forest.

In recent years, glycosaminoglycans (GAGs) have emerged into the focus of biochemical and biomedical research due to their importance in a variety of physiological processes. These molecules show great diversity, which makes their analysis highly challenging. A promising tool for identifying the structural motifs and conformation of shorter GAG chains is cryogenic gas-phase infrared (IR) spectroscopy. In this work, the cryogenic gas-phase IR spectra of mass-selected heparan sulfate (HS) di-, tetra-, and hexasaccharide ions were recorded to extract vibrational features that are characteristic to structural motifs. The data were augmented with chondroitin sulfate (CS) disaccharide spectra to assemble a training library for random forest (RF) classifiers. These were used to discriminate between GAG classes (CS or HS) and different sulfate positions (2-O-, 4-O-, 6-O-, and N-sulfation). With optimized data preprocessing and RF modeling, a prediction accuracy of >97% was achieved for HS tetra- and hexasaccharides based on a training set of only 21 spectra. These results exemplify the importance of combining gas-phase cryogenic IR ion spectroscopy with machine learning to improve the future analytical workflow for GAG sequencing and that of other biomolecules, such as metabolites.

Characterization and Fate of a Septanosyl Ferrier Cation in the Gas and Solution Phases.

Ferrier reactions follow a mechanistic pathway whereby Lewis acid activation of a cyclic enol ether facilitates departure of an allylic leaving group to form a glycosyl Ferrier cation. Attack on the Ferrier cation provides a new acetal linkage concurrent with the transposition of the alkene moiety. The idiosyncratic outcomes of Ferrier reactions of seven-membered ring carbohydrate-based oxepines prompted an investigation of its corresponding septanosyl Ferrier cation. Experiments that characterized the ion, including gas-phase cryogenic IR spectroscopy matched with density functional theory-calculated spectra of candidate cation structures, as well as product analysis from solution-phase Ferrier reactions, are reported here. Results from both approaches revealed an inclination of the seven-membered ring cation to contract to five-membered ring structures. Gas-phase IR spectra matched best to calculated spectra of structures in which five-membered dioxolenium formation opened the oxepine ring. In the solution phase, an attack on the ion by water led to an acyclic enal that cyclized to a C-methylene-aldehydo arabinofuranoside species. Attack by allyl trimethylsilane, on the other hand, was diastereoselective and yielded a C-allyl septanoside.

Infrared action spectroscopy of the deprotonated formic acid trimer, trapped in helium nanodroplets.

Hydrogen bonding interactions are essential in the structural stabilization and physicochemical properties of complex molecular systems, and carboxylic acid functional groups are common participants in these motifs. Consequently, the neutral formic acid (FA) dimer has been extensively investigated in the past, as it represents a useful model system to investigate proton donor-acceptor interactions. The analogous deprotonated dimers, in which two carboxylate groups are bound by a single proton, have also served as informative model systems. In these complexes, the position of the shared proton is mainly determined by the proton affinity of the carboxylate units. However, very little is known about the nature of the hydrogen bonding interactions in systems containing more than two carboxylate units. Here we report a study on the deprotonated (anionic) FA trimer. IR spectra are recorded in the 400-2000 cm-1 spectral range by means of vibrational action spectroscopy of FA trimer ions embedded in helium nanodroplets. Characterization of the gas-phase conformer and assignment of the vibrational features is achieved by comparing the experimental results with electronic structure calculations. To assist in the assignments, the 2H and 18O FA trimer anion isotopologues are also measured under the same experimental conditions. Comparison between the experimental and computed spectra, especially the observed shifts in spectral line positions upon isotopic substitution of the exchangeable protons, suggests that the prevalent conformer, under the experimental conditions, exhibits a planar structure that resembles the crystalline structure of formic acid.

Cryogenic infrared spectroscopy reveals remarkably short NH+⋯F hydrogen bonds in fluorinated phenylalanines.

In past decades, hydrogen bonds involving organic fluorine have been a highly disputed topic. Obtaining clear evidence for the presence of fluorine-specific interactions is generally difficult because of their weak nature. Today, the existence of hydrogen bonds with organic fluorine is widely accepted and supported by numerous studies. However, strong bonds with short H⋯F distances remain scarce and are primarily found in designed model compounds. Using a combination of cryogenic gas-phase infrared spectroscopy and density functional theory, we here analyze a series of conformationally unrestrained fluorinated phenylalanine compounds as protonated species. The results suggest proximal NH+⋯F hydrogen bonds with an exceptionally close H⋯F distance (1.79 Å) in protonated ortho-fluorophenylalanine.

Decoding the Fucose Migration Product during Mass-Spectrometric analysis of Blood Group Epitopes.

Fucose is a signaling carbohydrate that is attached at the end of glycan processing. It is involved in a range of processes, such as the selectin-dependent leukocyte adhesion or pathogen-receptor interactions. Mass-spectrometric techniques, which are commonly used to determine the structure of glycans, frequently show fucose-containing chimeric fragments that obfuscate the analysis. The rearrangement leading to these fragments-often referred to as fucose migration-has been known for more than 25 years, but the chemical identity of the rearrangement product remains unclear. In this work, we combine ion-mobility spectrometry, radical-directed dissociation mass spectrometry, cryogenic IR spectroscopy of ions, and density-functional theory calculations to deduce the product of the rearrangement in the model trisaccharides Lewis x and blood group H2. The structural search yields the fucose moiety attached to the galactose with an α(1→6) glycosidic bond as the most likely product.

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.

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.

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 ß-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.

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

Over the course of the COVID-19 pandemic, mRNA-based vaccines have gained tremendous importance. The development and analysis of modified RNA molecules benefit from advanced mass spectrometry and require sufficient understanding of fragmentation processes. Analogous to the degradation of RNA in solution by autohydrolysis, backbone cleavage of RNA strands was equally observed in the gas phase; however, the fragmentation mechanism remained elusive. In this work, autohydrolysis-like intermediates were generated from isolated RNA dinucleotides in the gas phase and investigated using cryogenic infrared spectroscopy in helium nanodroplets. Data from both experiment and density functional theory provide evidence for the formation of a five-membered cyclic phosphate intermediate and rule out linear or six-membered structures. Furthermore, the experiments show that another prominent condensed-phase reaction of RNA nucleotides can be induced in the gas phase: the tautomerization of cytosine. Both observed reactions are therefore highly universal and intrinsic properties of the investigated molecules.

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.

Non-covalent double bond sensors for gas-phase infrared spectroscopy of unsaturated fatty acids.

The position and configuration of carbon-carbon double bonds in unsaturated fatty acids is crucial for their biological functions and influences health and disease. However, double bond isomers are not routinely distinguished by classical mass spectrometry workflows. Instead, they require sophisticated analytical approaches usually based on chemical derivatization and/or instrument modification. In this work, a novel strategy to investigate fatty acid double bond isomers (18:1) without prior chemical treatment or modification of the ion source was implemented by non-covalent adduct formation in the gas phase. Fatty acid adducts with sodium, pyridinium, trimethylammonium, dimethylammonium, and ammonium cations were characterized by a combination of cryogenic gas-phase infrared spectroscopy, ion mobility-mass spectrometry, and computational modeling. The results reveal subtle differences between double bond isomers and confirm three-dimensional geometries constrained by non-covalent ion-molecule interactions. Overall, this study on fatty acid adducts in the gas phase explores new avenues for the distinction of lipid double bond isomers and paves the way for further investigations of coordinating cations to increase resolution.

Helium Nanodroplet Infrared Action Spectroscopy of the Proton-Bound Dimer of Hydrogen Sulfate and Formate: Examining Nuclear Quantum Effects.

The proton-bound dimer of hydrogen sulfate and formate is an archetypal structure for ionic hydrogen-bonding complexes that contribute to biogenic aerosol nucleation. Of central importance for the structure and properties of this complex is the location of the bridging proton connecting the two conjugate base moieties. The potential energy surface for bridging proton translocation features two local minima, with the proton localized at either the formate or hydrogen sulfate moiety. However, electronic structure methods reveal a shallow potential energy surface governing proton translocation, with a barrier on the order of the zero-point energy. This shallow potential complicates structural assignment and necessitates a consideration of nuclear quantum effects. In this work, we probe the structure of this complex and its isotopologues, utilizing infrared (IR) action spectroscopy of ions captured in helium nanodroplets. The IR spectra indicate a structure in which a proton is shared between the hydrogen sulfate and formate moieties, HSO4-···H+···-OOCH. However, because of the nuclear quantum effects and vibrational anharmonicities associated with the shallow potential for proton translocation, the extent of proton displacement from the formate moiety remains unclear, requiring further experiments or more advanced theoretical treatments for additional insight.

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.

Plate-height model of ion mobility-mass spectrometry: Part 2-Peak-to-peak resolution and peak capacity.

In a previous work, we explored zone broadening and the achievable plate numbers in linear drift tube ion mobility-mass spectrometry through developing a plate-height model [1]. On the basis of these findings, the present theoretical study extends the model by exploring peak-to-peak resolution and peak capacity in ion mobility separations. The first part provides a critical overview of chromatography-influenced resolution equations, including refinement of existing formulae. Furthermore, we present exact resolution equations for drift tube ion mobility spectrometry based on first principles. Upon implementing simple modifications, these exact formulae could be readily extended to traveling wave ion mobility separations and to cases when ion mobility spectrometry is coupled to mass spectrometry. The second part focuses on peak capacity. The well-known assumptions of constant plate number and constant peak width form the basis of existing approximate solutions. To overcome their limitations, an exact peak capacity equation is derived for drift tube ion mobility spectrometry. This exact solution is rooted in a suitable physical model of peak broadening, accounting for the finite injection pulse and subsequent diffusional spreading. By borrowing concepts from the theoretical toolbox of chromatography, we believe that the present study will help in integrating ion mobility spectrometry into the unified language of separation science.

Cryogenic Infrared Spectroscopy Reveals Structural Modularity in the Vibrational Fingerprints of Heparan Sulfate Diastereomers.

Heparan sulfate and heparin are highly acidic polysaccharides with a linear sequence, consisting of alternating glucosamine and hexuronic acid building blocks. The identity of hexuronic acid units shows a variability along their sequence, as d-glucuronic acid and its C5 epimer, l-iduronic acid, can both occur. The resulting backbone diversity represents a major challenge for an unambiguous structural assignment by mass spectrometry-based techniques. Here, we employ cryogenic infrared spectroscopy on mass-selected ions to overcome this challenge and distinguish isomeric heparan sulfate tetrasaccharides that differ only in the configuration of their hexuronic acid building blocks. High-resolution infrared spectra of a systematic set of synthetic heparan sulfate stereoisomers were recorded in the fingerprint region from 1000 to 1800 cm-1. The experiments reveal a characteristic combination of spectral features for each of the four diastereomers studied and imply structural modularity in the vibrational fingerprints. Strong spectrum-structure correlations were found and rationalized by state-of-the-art quantum chemical calculations. The findings demonstrate the potential of cryogenic infrared spectroscopy to extend the mass spectrometry-based toolkit for the sequencing of heparan sulfate and structurally related biomolecules.

The Impact of Leaving Group Anomericity on the Structure of Glycosyl Cations of Protected Galactosides.

It has been reported that fragments produced by glycosidic bond breakage in mass spectrometry-based experiments can retain a memory of their anomeric configuration, which has major implications for glycan sequencing. Herein, we use cryogenic vibrational spectroscopy and ion mobility-mass spectrometry to study the structure of B-type fragments of protected galactosides. Cationic fragments were generated from glycosyl donors carrying trichloroacetimidate or thioethyl leaving groups of different anomeric configuration. The obtained infrared signatures indicate that the investigated fragments exhibit an identical structure, which suggests that there is no anomeric memory in B-type ions of fully protected monosaccharides.

Plate-height model of ion mobility-mass spectrometry.

In the past decade, ion mobility spectrometry (IMS) in combination with mass spectrometry (IM-MS) became a widely employed technique for the separation and structural characterization of ionic species in the gas phase. Similarly to chromatography, where studies on the mechanism of band broadening and adequate plate-height equations have been aiding method development and promoting advancements in column technology, a suitable resolving power theory of drift tube ion mobility-mass spectrometry (DTIM-MS) is essential to stimulate further progress in this emerging field of separation science. In the present study, therefore, we explore dispersion processes in detail and present a plate-height model of ion mobility-mass spectrometry. We quantify the effects of five major dispersion processes that contribute to zone broadening and determine the resolving power in DTIM-MS: diffusion, Coulomb repulsion, electric field inhomogeneities, the finite initial spread of the ion cloud and dispersion outside the mobility cell. A solution is provided to account for the nonuniform separation field in IM-MS in the presence of multiple compartments. The equations - derived from first principles - serve as the basis for formulating an expression that is similar in nature to van Deemter's plate-height equation for chromatography. A comprehensive set of experiments was performed on a custom-built DTIM-MS instrument to evaluate the accuracy of the plate-height model, resulting in satisfactory agreement between experiment and theory. Building on these findings, the plate-height equation was employed to explore the influence of drift gas pressure, injection pulse-width and the mobility of ions on resolving power from a theoretical point of view. Our findings may aid instrument design and development in the future, as well as the optimization of measurement conditions to improve ion mobility separations. By employing the plate-height concept and the general formalism of differential migration processes to describe zone spreading in IM-MS, we aim to find a common ground between this emerging method and such well-established techniques as HPLC or CZE. We also hope that the work presented here will facilitate a broader acceptance of IMS as a separation method of great potential by the communities of chromatography and electrophoresis, as well as that of mass spectrometry.

IR action spectroscopy of glycosaminoglycan oligosaccharides.

Glycosaminoglycans (GAGs) are a physio- and pharmacologically highly relevant class of complex saccharides, possessing a linear sequence and strongly acidic character. Their repetitive linear core makes them seem structurally simple at first glance, yet differences in sulfation and epimerization lead to an enormous structural diversity with only a few GAGs having been successfully characterized to date. Recent infrared action spectroscopic experiments on sulfated mono- and disaccharide ions show great promise. Here, we assess the potential of two types of gas-phase action spectroscopy approaches in the range from 1000 to 1800 cm-1 for the structural analysis of complex GAG oligosaccharides. Synthetic tetra- and pentasaccharides were chosen as model compounds for this benchmark study. Utilizing infrared multiple photon dissociation action spectroscopy at room temperature, diagnostic bands are largely unresolved. In contrast, cryogenic infrared action spectroscopy of ions trapped in helium nanodroplets yields resolved infrared spectra with diagnostic features for monosaccharide composition and sulfation pattern. The analysis of GAGs could therefore significantly benefit from expanding the conventional MS-based toolkit with gas-phase cryogenic IR spectroscopy. Graphical abstract.

Probing the conformational landscape and thermochemistry of DNA dinucleotide anions via helium nanodroplet infrared action spectroscopy.

Isolation of biomolecules in vacuum facilitates characterization of the intramolecular interactions that determine three-dimensional structure, but experimental quantification of conformer thermochemistry remains challenging. Infrared spectroscopy of molecules trapped in helium nanodroplets is a promising methodology for the measurement of thermochemical parameters. When molecules are captured in a helium nanodroplet, the rate of cooling to an equilibrium temperature of ca. 0.4 K is generally faster than the rate of isomerization, resulting in "shock-freezing" that kinetically traps molecules in local conformational minima. This unique property enables the study of temperature-dependent conformational equilibria via infrared spectroscopy at 0.4 K, thereby avoiding the deleterious effects of spectral broadening at higher temperatures. Herein, we demonstrate the first application of this approach to ionic species by coupling electrospray ionization mass spectrometry (ESI-MS) with helium nanodroplet infrared action spectroscopy to probe the structure and thermochemistry of deprotonated DNA dinucleotides. Dinucleotide anions were generated by ESI, confined in an ion trap at temperatures between 90 and 350 K, and entrained in traversing helium nanodroplets. The infrared action spectra of the entrained ions show a strong dependence on pre-pickup ion temperature, consistent with the preservation of conformer population upon cooling to 0.4 K. Non-negative matrix factorization was utilized to identify component conformer infrared spectra and determine temperature-dependent conformer populations. Relative enthalpies and entropies of conformers were subsequently obtained from a van't Hoff analysis. IR spectra and conformer thermochemistry are compared to results from ion mobility spectrometry (IMS) and electronic structure methods. The implementation of ESI-MS as a source of dopant molecules expands the diversity of molecules accessible for thermochemical measurements, enabling the study of larger, non-volatile species.