Vibrational Spectroscopy of Glycans in Helium Nanodroplets

A central theme among the glycosciences is the Janus-faced nature ofglycans. Their tremendous structural diversity enables a myriad ofbiological functions ranging from energy storage to molecular recogni-tion processes. But at the same time, this structural diversity poses aformidable challenge for glycan analysis that impedes the full develop-ment of structural glycobiology. In contrast to genomics and proteomics,glycomics lacks generic sequencing methods that allow reliable, high-throughput analyses with low sample consumption. Instead, a variety ofsophisticated methods is used for glycan analysis, including mass spec-trometry. A general challenge using mass spectrometry alone, however,is the unambiguous identification of isomeric glycans. Therefore, it isoften coupled to orthogonal techniques, such as liquid chromatography.In the last two decades, the combination of mass spectrometry andgas-phase action spectroscopy emerged for glycan analysis. Variouschallenges, however, limited gas-phase spectroscopy to smaller glycans.In particular, the thermal activation of ions during the measurement ininfrared multiple-photon dissociation spectroscopy leads to significantline-broadening, which limits the amount of structural information thatcan be obtained by this method.This work overcomes these limitations by combining mass spectrom-etry and cryogenic vibrational spectroscopy using superfluid helium nanodroplets. The unique low-temperature environment of heliumdroplets allows the acquisition of vibrational spectra in the absenceof significant thermal contributions. A systematic study of isomericglycans demonstrates the outstanding resolving power that provides avariety of well-resolved absorption bands that are unique to each isomer:a true spectral fingerprint. The unique optical signatures allow theresolution of even minute structural details, such as the stereochemicalorientation of a single hydroxy group.The exceptional ability of this method to resolve structural detailswas used to investigate an elusive rearrangement reaction, called fu-cose migration, which frequently leads to the detection of misleadingfragment ions and erroneous sequence assignments in tandem massspectrometry. Because fucose migration was only observed in fragmentions, it was strictly associated with the fragmentation process. In thiswork, cryogenic vibrational spectroscopy reveals that fucose migrationis not restricted to fragment ions, and instead occurs in intact ions aswell. These results generalize fucose migration to a universal issue inmass spectrometry as a whole.In another study, the combination of cryogenic vibrational spec-troscopy and first-principles theory was used to unravel the structureof glycosyl cations, the key intermediates during chemical glycosyla-tion reactions. Various reaction pathways were postulated many yearsago, but the exact structure of glycosyl cations remained obscure dueto their short-lived and reactive nature in the condensed phase. Inthis work, the fragmentation of precursor ions was used to generateglycosyl cations in the gas phase, which provides a unique clean-roomenvironment that stabilizes these transient intermediates. The highlyresolved vibrational spectra obtained for various glycosyl cations allowan in-depth structural analysis that reveals detailed insights into the two fundamental structural motifs that enable stereoselective glycosylationreactions: neighboring group participation and remote participation.These results facilitate the mechanistic understanding of glycosylationreactions and will eventually lead to a more rational design of buildingblocks that is based on structural rather than anecdotal evidence.Een centraal thema in de glyco-wetenschappen is het dubbele karaktervan glycanen. Enerzijds maakt de enorme structurele diversiteit vanglycanen een groot aantal biologische functies mogelijk, variërend vanenergieopslag tot moleculaire herkenningsprocessen. Maar tegelijkertijdvormt deze structurele diversiteit een enorme uitdaging voor de ana-lyse van glycanen, wat de ontwikkeling van structurele glycobiologiebelemmert. In tegenstelling tot genomics en proteomics, ontbreekthet glycomics aan een generieke sequentiemethode die betrouwbare,high-throughput analyses van kleine hoeveelheden glycanen mogelijkmaakt. In plaats daarvan wordt een verscheidenheid aan geavanceerdemethoden gebruikt, waaronder massaspectrometrie. Met uitsluitendmassa-spectrometrische methoden, is een eenduidige identificatie vanverschillende isomeren van glycanen echter slechts beperkt mogelijk.Daarom wordt massaspectrometrie vaak gekoppeld aan orthogonale tech-nieken, zoals vloeistofchromatografie. In de laatste twee decennia is ookde combinatie van massaspectrometrie met gasfase-actiespectroscopieopgekomen voor glycaan analyse. Deze combinatie is tot dusver metname gebruikt voor de analyse van kleinere glycanen. In het bijzonderleidt de thermische activering van ionen gedurende infrarood multifotondissociatie spectroscopie tot een significante lijnverbreding, hetgeen de hoeveelheid structurele informatie beperkt die met deze methodeverkregen kan worden.Het onderzoek beschreven in dit proefschrift lost deze beperkingenop door massaspectrometrie te combineren met cryogene vibratiespec-troscopie, gebruikmakend van superfluïde helium nanodruppels. Delage temperatuur in de heliumdruppels maakt de opname van vibratie-spectra mogelijk in afwezigheid van significante thermische bijdragen.Een systematische studie van isomere glycanen toont het uitstekendeoplossend vermogen van deze techniek. De spectra bevatten volledigopgeloste absorptiebanden die uniek zijn voor een specifiek isomeer:een echte spectrale vingerafdruk. Deze spectra maken een identificatiemogelijk van zeer subtiele structurele details, zoals de stereochemie vaneen enkele hydroxygroep.Het uitzonderlijke vermogen van deze methode om structurele de-tails te onderscheiden is gebruikt om een lastige migratie-reactie, dezogenaamde fucose migratie, te onderzoeken. Deze migratie-reactie leidtvaak tot de detectie van misleidende fragmentionen en foutieve sequen-tietoewijzingen in tandem massaspectrometrie. Omdat fucose migratiealleen werd waargenomen in fragmentionen, werd het enkel geassoci-eerd met het fragmentatieproces. Zoals beschreven in dit proefschrift,onthult cryogene vibratiespectroscopie dat fucose migratie niet beperktis tot fragmentionen maar dat dit ook voorkomt in intacte ionen. Dezeresultaten maken duidelijk dat fucose migratie een universele kwestie isin de massaspectrometrie.In een andere studie is de combinatie van cryogene vibratiespectro-scopie en ab initio theorie gebruikt om de structuur van glycosylkationen,de belangrijkste tussenproducten tijdens chemische glycosyleringsreac-ties, te ontrafelen. Verschillende reactiepaden zijn vele jaren geledengepostuleerd, maar de exacte structuur van glycosylkationen bleef onduidelijk vanwege hun kortstondige en reactieve aard in de gecondenseerdefase. In dit werk is de fragmentatie van precursor-ionen gebruikt omglycosylkationen te genereren in de gasfase. Dit creëert een unieke“clean-room” omgeving die deze tijdelijke tussenproducten stabiliseert.De vibratiespectra verkregen voor verschillende glycosylkationen makeneen diepgaande structurele analyse mogelijk. Dit onthult gedetailleerdeinzichten in de twee fundamentele structurele motieven die stereoselec-tieve glycosyleringsreacties mogelijk maken: participatie van naburigegroepen en participatie op afstand. Deze resultaten maken een mecha-nistisch begrip van glycosyleringsreacties mogelijk en zullen uiteindelijkleiden tot een rationeler ontwerp van de benodigde bouwstenen, geba-seerd op structureel in plaats van anekdotisch bewijs

Spectral features due to inter-Landau-level transitions in the Raman spectrum of bilayer graphene

We investigate the contribution of the low-energy electronic excitations towards the Raman spectrum of bilayer graphene for the incoming photon energy Omega >> 1eV. Starting with the four-band tight-binding model, we derive an effective scattering amplitude that can be incorporated into the commonly used two-band approximation. Due to the influence of the high-energy bands, this effective scattering amplitude is different from the contact interaction amplitude obtained within the two-band model alone. We then calculate the spectral density of the inelastic light scattering accompanied by the excitation of electron-hole pairs in bilayer graphene. In the absence of a magnetic field, due to the parabolic dispersion of the low-energy bands in a bilayer crystal, this contribution is constant and in doped structures has a threshold at twice the Fermi energy. In an external magnetic field, the dominant Raman-active modes are the n_{-} to n_{+} inter-Landau-level transitions with crossed polarisation of in/out photons. We estimate the quantum efficiency of a single n_{-} to n_{+} transition in the magnetic field of 10T as I_{n_{-} to n_{+}}~10^{-12}.Comment: 7 pages, 3 figures, expanded version published in PR

On the existence of traveling waves in the 3D Boussinesq system

We extend earlier work on traveling waves in premixed flames in a gravitationally stratified medium, subject to the Boussinesq approximation. For three-dimensional channels not aligned with the gravity direction and under the Dirichlet boundary conditions in the fluid velocity, it is shown that a non-planar traveling wave, corresponding to a non-zero reaction, exists, under an explicit condition relating the geometry of the crossection of the channel to the magnitude of the Prandtl and Rayleigh numbers, or when the advection term in the flow equations is neglected.Comment: 15 pages, to appear in Communications in Mathematical Physic

Energy solutions to one-dimensional singular parabolic problems with BVBV data are viscosity solutions

We study one-dimensional very singular parabolic equations with periodic boundary conditions and initial data in $BV$, which is the energy space. We show existence of solutions in this energy space and then we prove that they are viscosity solutions in the sense of Giga-Giga.Comment: 15 page

Directional approach to spatial structure of solutions to the Navier-Stokes equations in the plane

We investigate a steady flow of incompressible fluid in the plane. The motion is governed by the Navier-Stokes equations with prescribed velocity $u_\infty$ at infinity. The main result shows the existence of unique solutions for arbitrary force, provided sufficient largeness of $u_\infty$. Furthermore a spacial structure of the solution is obtained in comparison with the Oseen flow. A key element of our new approach is based on a setting which treats the directino of the flow as \emph{time} direction. The analysis is done in framework of the Fourier transform taken in one (perpendicular) direction and a special choice of function spaces which take into account the inhomogeneous character of the symbol of the Oseen system. From that point of view our technique can be used as an effective tool in examining spatial asymptotics of solutions to other systems modeled by elliptic equations

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

Spectroscopy of Small and Large Biomolecular Ions in Helium-Nanodroplets

A vast number of experiments have now shown that helium nanodroplets are an exemplary cryogenic matrix for spectroscopic investigations. The experimental techniques are well established and involve in most cases the pickup of evaporated neutral species by helium droplets. These techniques have been extended within our research group to enable nanodroplet pickup of anions or cations stored in an ion trap. By using electrospray ionization (ESI) in combination with modern mass spec- trometric methods to supply ions to the trap, an immense variety of mass-to-charge selected species can be doped into the droplets and spectroscopically investigated. We have combined this droplet doping methodology with IR action spectroscopy to investigate anions and cations ranging in size from a few atoms to proteins that consist of thousands of atoms. Herein, we show examples of small complexes of fluoride anions (F-) with CO2 and H2O and carbohydrate molecules. In the case of the small complexes, novel compounds could be identified, and quantum chemistry can in some instances quantitatively explain the results. For biologically relevant complex carbohydrate molecules, the IR spectra are highly diagnostic and allow the differentiation of species that would be difficult or impossible to identify by more conventional methods

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