Interband cascade lasers grown simultaneously on GaSb, GaAs and Si substrates.

We report on Sb-based interband cascade lasers simultaneously grown on GaSb, GaAs and Si substrates. 8 µm x 2 mm devices exhibited similar threshold currents around 40 mA at 20°C and achieved continuous-wave (CW) operation up to 65°C on GaSb, GaAs and Si substrates despite a dislocation density of ∼ 4.108 cm-2 for both mismatched substrates. In the CW regime the output power of the devices emitting at 3.3 µm exceeded 30 mW/facet at 20°C. ICLs on GaAs and Si were subsequently aged at 50°C with an injection current of 200 mA, i.e. five times the laser-threshold current. No degradation was observed after 500 h of CW operation, demonstrating the high performance of ICLs and their tolerance to dislocations.

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.

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.

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.

Etched-cavity GaSb laser diodes on a MOVPE GaSb-on-Si template.

We report on 2.3-µm etched-cavity GaSb-based laser diodes (LDs) epitaxially integrated on on-axis (001)Si and benchmarked against their cleaved facet counterparts. The LDs were grown in two steps. First, a GaSb-on-Si template was grown by metal-organic vapor phase epitaxy (MOVPE) before the growth of the LD heterostructure by molecular-beam epitaxy. Different etched-facet geometries operate in continuous wave well above room temperature, and their performance are similar to those of cleaved-cavity LDs. These results show that etching mirrors is a viable route to form laser cavities in the GaSb technology and that MOVPE GaSb-on-Si templates are a suitable platform for optoelectronic devices overgrowth.

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.

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.

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.

Characterization of a trans-trans Carbonic Acid-Fluoride Complex by Infrared Action Spectroscopy in Helium Nanodroplets.

The high Lewis basicity and small ionic radius of fluoride promote the formation of strong ionic hydrogen bonds in the complexation of fluoride with protic molecules. Herein, we report that carbonic acid, a thermodynamically disfavored species that is challenging to investigate experimentally, forms a complex with fluoride in the gas phase. Intriguingly, this complex is highly stable and is observed in abundance upon nanoelectrospray ionization of an aqueous sodium fluoride solution in the presence of gas-phase carbon dioxide. We characterize the structure and properties of the carbonic acid-fluoride complex, F-(H2CO3), and its deuterated isotopologue, F-(D2CO3), by helium nanodroplet infrared action spectroscopy in the photon energy range of 390-2800 cm-1. The complex adopts a C2 v symmetry structure with the carbonic acid in a planar trans-trans conformation and both OH groups forming ionic hydrogen bonds with the fluoride. Substantial vibrational anharmonic effects are observed in the infrared spectra, most notably a strong blue shift of the symmetric hydrogen stretching fundamental relative to predictions from the harmonic approximation or vibrational second-order perturbation theory. Ab initio thermostated ring-polymer molecular dynamics simulations indicate that this blue shift originates from strong coupling between the hydrogen stretching and bending vibrations, resulting in an effective weakening of the OH···F- ionic hydrogen bonds.

Fragment-Based Covalent Ligand Screening Enables Rapid Discovery of Inhibitors for the RBR E3 Ubiquitin Ligase HOIP.

Modification of proteins with polyubiquitin chains is a key regulatory mechanism to control cellular behavior and alterations in the ubiquitin system are linked to many diseases. Linear (M1-linked) polyubiquitin chains play pivotal roles in several cellular signaling pathways mediating immune and inflammatory responses and apoptotic cell death. These chains are formed by the linear ubiquitin chain assembly complex (LUBAC), a multiprotein E3 ligase that consists of 3 subunits, HOIP, HOIL-1L, and SHARPIN. Herein, we describe the discovery of inhibitors targeting the active site cysteine of the catalytic subunit HOIP using fragment-based covalent ligand screening. We report the synthesis of a diverse library of electrophilic fragments and demonstrate an integrated use of protein LC-MS, biochemical ubiquitination assays, chemical synthesis, and protein crystallography to enable the first structure-based development of covalent inhibitors for an RBR E3 ligase. Furthermore, using cell-based assays and chemoproteomics, we demonstrate that these compounds effectively penetrate mammalian cells to label and inhibit HOIP and NF-κB activation, making them suitable hits for the development of selective probes to study LUBAC biology. Our results illustrate the power of fragment-based covalent ligand screening to discover lead compounds for challenging targets, which holds promise to be a general approach for the development of cell-permeable inhibitors of thioester-forming E3 ubiquitin ligases.

Resin and Magnetic Nanoparticle-Based Free Radical Probes for Glycan Capture, Isolation, and Structural Characterization.

By combining the merits of solid supports and free radical activated glycan sequencing (FRAGS) reagents, we develop a multifunctional solid-supported free radical probe (SS-FRAGS) that enables glycan enrichment and characterization. SS-FRAGS comprises a solid support, free radical precursor, disulfide bond, pyridyl, and hydrazine moieties. Thio-activated resin and magnetic nanoparticles (MNPs) are chosen as the solid support to selectively capture free glycans via the hydrazine moiety, allowing for their enrichment and isolation. The disulfide bond acts as a temporary covalent linkage between the solid support and the captured glycan, allowing the release of glycans via the cleavage of the disulfide bond by dithiothreitol. The basic pyridyl functional group provides a site for the formation of a fixed charge, enabling detection by mass spectrometry and avoiding glycan rearrangement during collisional activation. The free radical precursor generates a nascent free radical upon collisional activation and thus simultaneously induces systematic and predictable fragmentation for glycan structure elucidation. A radical-driven glycan deconstruction diagram (R-DECON) is developed to visually summarize the MS2 results and thus allow for the assembly of the glycan skeleton, making the differentiation of isobaric glycan isomers unambiguous. For application to a real-world sample, we demonstrate the efficacy of the SS-FRAGS by analyzing glycan structures enzymatically cleaved from RNase-B.

The role of the mobile proton in fucose migration.

Fucose migration reactions represent a substantial challenge in the analysis of fucosylated glycan structures by mass spectrometry. In addition to the well-established observation of transposed fucose residues in glycan-dissociation product ions, recent experiments show that the rearrangement can also occur in intact glycan ions. These results suggest a low-energy barrier for migration of the fucose residue and broaden the relevance of fucose migration to include other types of mass spectrometry experiments, including ion mobility-mass spectrometry and ion spectroscopy. In this work, we utilize cold-ion infrared spectroscopy to provide further insight into glycan scrambling in intact glycan ions. Our results show that the mobility of the proton is a prerequisite for the migration reaction. For the prototypical fucosylated glycans Lewis x and blood group antigen H-2, the formation of adduct ions or the addition of functional groups with variable proton affinity yields significant differences in the infrared spectra. These changes correlate well with the promotion or inhibition of fucose migration through the presence or absence of a mobile proton.

Selectively Targeting the Kinome-Conserved Lysine of PI3Kδ as a General Approach to Covalent Kinase Inhibition.

Selective covalent inhibition of kinases by targeting poorly conserved cysteines has proven highly fruitful to date in the development of chemical probes and approved drugs. However, this approach is limited to ∼200 kinases possessing such a cysteine near the ATP-binding pocket. Herein, we report a novel approach to achieve selective, irreversible kinase inhibition, by targeting the conserved catalytic lysine residue. We have illustrated our approach by developing selective, covalent PI3Kδ inhibitors that exhibit nanomolar potency in cellular assays, and a duration of action >48 h in CD4+ T cells. Despite conservation of the lysine residue throughout the kinome, the lead compound shows high levels of selectivity over a selection of lipid and protein kinases in biochemical assays, as well as covalent binding to very few off-target proteins in live-cell proteomic studies. We anticipate this approach could offer a general strategy, as an alternative to targeting non-conserved cysteines, for the development of selective covalent kinase inhibitors.

Ground-State Structure of the Proton-Bound Formate Dimer by Cold-Ion Infrared Action Spectroscopy.

The proton-bound dicarboxylate motif, RCOO- ⋅H+ ⋅- OOCR, is a prevalent chemical configuration found in many condensed-phase systems. The proton-bound formate dimer HCOO- ⋅H+ ⋅- OOCH was studied utilizing cold-ion IR action spectroscopy in the range 400-1800 cm-1 . The spectrum obtained at ca. 0.4 K of ions captured in He nanodroplets was compared to that measured at ca. 10 K by photodissociation of Ar-ion complexes. Similar band patterns are obtained by the two techniques that are consistent with calculations for a C2 symmetry structure with a proton shared equally between the two formate moieties. Isotopic substitution experiments point to the nominal parallel stretch of the bridging proton appearing as a sharp, dominant feature near 600 cm-1 . Multidimensional anharmonic calculations reveal that the bridging proton motion is strongly coupled to the flanking -COO- framework, an effect that is in line with the expected change in -C=O bond rehybridization upon protonation.

Fucose Migration in Intact Protonated Glycan Ions: A Universal Phenomenon in Mass Spectrometry.

Fucose is an essential deoxysugar that is found in a wide range of biologically relevant glycans and glycoconjugates. A recurring problem in mass spectrometric analyses of fucosylated glycans is the intramolecular migration of fucose units, which can lead to erroneous sequence assignments. This migration reaction is typically assigned to activation during collision-induced dissociation (CID) in tandem mass spectrometry (MS). In this work, we utilized cold-ion spectroscopy and show for the first time that fucose migration is not limited to fragments obtained in tandem MS and can also be observed in intact glycan ions. This observation suggests a possible low-energy barrier for this transfer reaction and generalizes fucose migration to an issue that may universally occur in any type of mass spectrometry experiment.

Glycan Fingerprinting via Cold-Ion Infrared Spectroscopy.

The diversity of stereochemical isomers present in glycans and glycoconjugates poses a formidable challenge for comprehensive structural analysis. Typically, sophisticated mass spectrometry (MS)-based techniques are used in combination with chromatography or ion-mobility separation. However, coexisting structurally similar isomers often render an unambiguous identification impossible. Other powerful techniques such as gas-phase infrared (IR) spectroscopy have been limited to smaller glycans, since conformational flexibility and thermal activation during the measurement result in poor spectral resolution. This limitation can be overcome by using cold-ion spectroscopy. The vibrational fingerprints of cold oligosaccharide ions exhibit a wealth of well-resolved absorption features that are diagnostic for minute structural variations. The unprecedented resolution of cold-ion spectroscopy coupled with tandem MS may render this the key technology to unravel complex glycomes.

Real-Time Studies of Iron Oxalate-Mediated Oxidation of Glycolaldehyde as a Model for Photochemical Aging of Aqueous Tropospheric Aerosols.

The complexation of iron(III) with oxalic acid in aqueous solution yields a strongly absorbing chromophore that undergoes efficient photodissociation to give iron(II) and the carbon dioxide anion radical. Importantly, iron(III) oxalate complexes absorb near-UV radiation (λ > 350 nm), providing a potentially powerful source of oxidants in aqueous tropospheric chemistry. Although this photochemical system has been studied extensively, the mechanistic details associated with its role in the oxidation of dissolved organic matter within aqueous aerosol remain largely unknown. This study utilizes glycolaldehyde as a model organic species to examine the oxidation pathways and evolution of organic aerosol initiated by the photodissociation of aqueous iron(III) oxalate complexes. Hanging droplets (radius 1 mm) containing iron(III), oxalic acid, glycolaldehyde, and ammonium sulfate (pH ∼3) are exposed to irradiation at 365 nm and sampled at discrete time points utilizing field-induced droplet ionization mass spectrometry (FIDI-MS). Glycolaldehyde is found to undergo rapid oxidation to form glyoxal, glycolic acid, and glyoxylic acid, but the formation of high molecular weight oligomers is not observed. For comparison, particle-phase experiments conducted in a laboratory chamber explore the reactive uptake of gas-phase glycolaldehyde onto aqueous seed aerosol containing iron and oxalic acid. The presence of iron oxalate in seed aerosol is found to inhibit aerosol growth. These results suggest that photodissociation of iron(III) oxalate can lead to the formation of volatile oxidation products in tropospheric aqueous aerosols.

Mass spectrometric sampling of a liquid surface by nanoliter droplet generation from bursting bubbles and focused acoustic pulses: application to studies of interfacial chemistry.

The complex chemistry occurring at the interface between liquid and vapor phases contributes significantly to the dynamics and evolution of numerous chemical systems of interest, ranging from damage to the human lung surfactant layer to the aging of atmospheric aerosols. This work presents two methodologies to eject droplets from a liquid water surface and analyze them via mass spectrometry. In bursting bubble ionization (BBI), droplet ejection is achieved via the formation of a jet following bubble rupture at the surface of a liquid to yield 250 µm diameter droplets (10 nL volume). In interfacial sampling by an acoustic transducer (ISAT), droplets are produced by focusing pulsed piezoelectric transducer-generated acoustic waves at the surface of a liquid, resulting in the ejection of droplets of 100 µm in diameter (500 pL volume). In both experimental methodologies, ejected droplets are aspirated into the inlet of the mass spectrometer, resulting in the facile formation of gas-phase ions. We demonstrate the ability of this technique to readily generate spectra of surface-active analytes, and we compare the spectra to those obtained by electrospray ionization. Charge measurements indicate that the ejected droplets are near-neutral (<0.1% of the Rayleigh limit), suggesting that gas-phase ion generation occurs in the heated transfer capillary of the instrument in a mechanism similar to thermospray or sonic spray ionization. Finally, we present the oxidation of oleic acid by ozone as an initial demonstration of the ability of ISAT-MS to monitor heterogeneous chemistry occurring at a planar water/air interface.

Hydrogen bonding constrains free radical reaction dynamics at serine and threonine residues in peptides.

Free radical-initiated peptide sequencing (FRIPS) mass spectrometry derives advantage from the introduction of highly selective low-energy dissociation pathways in target peptides. An acetyl radical, formed at the peptide N-terminus via collisional activation and subsequent dissociation of a covalently attached radical precursor, abstracts a hydrogen atom from diverse sites on the peptide, yielding sequence information through backbone cleavage as well as side-chain loss. Unique free-radical-initiated dissociation pathways observed at serine and threonine residues lead to cleavage of the neighboring N-terminal Cα-C or N-Cα bond rather than the typical Cα-C bond cleavage observed with other amino acids. These reactions were investigated by FRIPS of model peptides of the form AARAAAXAA, where X is the amino acid of interest. In combination with density functional theory (DFT) calculations, the experiments indicate the strong influence of hydrogen bonding at serine or threonine on the observed free radical chemistry. Hydrogen bonding of the side-chain hydroxyl group with a backbone carbonyl oxygen aligns the singly occupied π orbital on the ß-carbon and the N-Cα bond, leading to low-barrier ß-cleavage of the N-Cα bond. Interaction with the N-terminal carbonyl favors a hydrogen-atom transfer process to yield stable c and z(•) ions, whereas C-terminal interaction leads to effective cleavage of the Cα-C bond through rapid loss of isocyanic acid. Dissociation of the Cα-C bond may also occur via water loss followed by ß-cleavage from a nitrogen-centered radical. These competitive dissociation pathways from a single residue illustrate the sensitivity of gas-phase free radical chemistry to subtle factors such as hydrogen bonding that affect the potential energy surface for these low-barrier processes.