Structural Elucidation of Agrochemical Metabolic Transformation Products Based on Infrared Ion Spectroscopy to Improve In Silico Toxicity Assessment.

Toxicological assessments of newly developed agrochemical agents consider chemical modifications and their metabolic and biotransformation products. To carry out an in silico hazard assessment, understanding the type of chemical modification and its location on the original compound can greatly enhance the reliability of the evaluation. Here, we present and apply a method based on liquid chromatography-mass spectrometry (LC-MS) enhanced with infrared ion spectroscopy (IRIS) to better delineate the molecular structures of transformation products before in silico toxicology evaluation. IRIS facilitates the recording of IR spectra directly in the mass spectrometer for features selected by retention time and mass-to-charge ratio. By utilizing quantum-chemically predicted IR spectra for candidate molecular structures, one can either derive the actual structure or significantly reduce the number of (isomeric) candidate structures. This approach can assist in making informed decisions. We apply this method to a plant growth stimulant, digeraniol sinapoyl malate (DGSM), that is currently under development. Incubation of the compound in Caco-2 and HepaRG cell lines in multiwell plates and analysis by LC-MS reveals oxidation, glucuronidation, and sulfonation metabolic products, whose structures were elucidated by IRIS and used as input for an in silico toxicology assessment. The toxicity of isomeric metabolites predicted by in silico tools was also assessed, which revealed that assigning the right metabolite structure is an important step in the overall toxicity assessment of the agrochemical. We believe this identification approach can be advantageous when specific isomers are significantly more hazardous than others and can help better understand metabolic pathways.

Exploring the Scaling Factors for Infrared Modes of PANHs - A Case Study on Cationic 3-Azafluoranthene⋠+ and Protonated 3-Azafluoranthene.

Infrared (IR) emission bands by interstellar polycyclic aromatic hydrocarbons (PAHs) and polycyclic aromatic nitrogen heterocycles (PANHs) are observed towards a large variety of interstellar objects and offer detailed insights into the chemistry and physics of the interstellar medium. The analysis of the emission bands, and thus the interpretation of the molecular characteristics of the carriers, heavily relies on the use of density functional theory (DFT) calculated IR spectra. However, there are significant challenges in accurately predicting the experimental IR band positions, particularly for PANH emission vibrational modes around 6 µm. In this work, we present gas-phase mid-infrared (mid-IR) spectra of cationic 3-azafluoranthene (3AF⋅+) and protonated 3-azafluoranthene (3AFH+) to investigate their experimental IR band positions in relation to DFT calculated bands. The experimental spectra are compared to DFT simulated spectra, where different approaches were followed to correct for anharmonicities. The best agreement is achieved by scaling frequencies of modes with large nitrogen displacements with a different factor. Even though our findings might be limited to a small number of PANH structures, they indicate, that nitrogen atom incorporation needs to be accounted for by carefully adjusting the corresponding scaling factors while computing IR spectra of PANHs on DFT level.

Anomeric Triflates versus Dioxanium Ions: Different Product-Forming Intermediates from 3-Acyl Benzylidene Mannosyl and Glucosyl Donors.

Minimal structural differences in the structure of glycosyl donors can have a tremendous impact on their reactivity and the stereochemical outcome of their glycosylation reactions. Here, we used a combination of systematic glycosylation reactions, the characterization of potential reactive intermediates, and in-depth computational studies to study the disparate behavior of glycosylation systems involving benzylidene glucosyl and mannosyl donors. While these systems have been studied extensively, no satisfactory explanations are available for the differences observed between the 3-O-benzyl/benzoyl mannose and glucose donor systems. The potential energy surfaces of the different reaction pathways available for these donors provide an explanation for the contrasting behavior of seemingly very similar systems. Evidence has been provided for the intermediacy of benzylidene mannosyl 1,3-dioxanium ions, while the formation of the analogous 1,3-glucosyl dioxanium ions is thwarted by a prohibitively strong flagpole interaction of the C-2-O-benzyl group with the C-5 proton in moving toward the transition state, in which the glucose ring adopts a B2,5-conformation. This study provides an explanation for the intermediacy of 1,3-dioxanium ions in the mannosyl system and an answer to why these do not form from analogous glucosyl donors.

Development of Nontargeted Workflow of Occupational Exposure by Infrared Ion Spectroscopy and Silicone Wristbands' Passive Sampling.

A new approach using orthogonal analytical techniques is developed for chemical identification. High resolution mass spectrometry and infrared ion spectroscopy are applied through a 5-level confidence paradigm to demonstrate the effectiveness of nontargeted workflow for the identification of hazardous organophosphates. Triphenyl phosphate is used as a surrogate organophosphate for occupational exposure, and silicone wristbands are used to represent personal samplers. Spectral data of a target compound is combined with spectral data of the sodium adduct and quantum chemical calculations to achieve a confirmed identification. Here, we demonstrate a nontargeted workflow that identifies organophosphate exposure and provides a mechanism for selecting validated methods for quantitative analyses.

Fluorinated Propionic Acids Unmasked: Puzzling Fragmentation Phenomena of the Deprotonated Species.

Environmental contamination by per- and polyfluorinated substances (PFAS) is an emerging concern for the public. In this study, short-chain PFAS such as deprotonated per- and polyfluorinated propionic acids are investigated using a combination of infrared multiple-photon dissociation (IRMPD) spectroscopy, collision-induced dissociation (CID), and density functional theory calculations. IRMPD and CID proceed via multiple competing pathways: (1) production of fluoroformate (FCO2-) and the associated ethylene derivative, (2) production of HF and the associated carbanion, or (3) loss of CO2 and the associated carbanion. Fluorinated propionic acids with at least one fluorine atom bound to the terminal carbon yield FCO2-, whereas loss of HF is observed in polyfluorinated species with at least one fluorine atom bound to the α-carbon. To explore the reaction pathways of the various fluorinated propionic acids, the nudged elastic band method is employed. The relative energy of the four-membered ring transition state leading to FCO2- dictates which product channel is observed in dissociation.

Taming Conformational Heterogeneity on Ion Racetrack to Unveil Principles that Drive Membrane Permeation of Cyclosporines.

Our study reveals the underlying principles governing the passive membrane permeability in three large N-methylated macrocyclic peptides (N-MeMPs): cyclosporine A (CycA), Alisporivir (ALI), and cyclosporine H (CycH). We determine a series of conformers required for robust passive membrane diffusion and those relevant to other functions, such as binding to protein targets or intermediates, in the presence of solvent additives. We investigate the conformational interconversions and establish correlations with the membrane permeability. Nuclear magnetic resonance (NMR) and cyclic ion-mobility spectrometry-mass spectrometry (cIMS-MS) are employed to characterize conformational heterogeneity and identify cis-amides relevant for good membrane permeability. In addition, ion mobility selected cIMS-MS and infrared (IR) multiple-photon dissociation (IRMPD) spectroscopy experiments are conducted to evaluate the energy barriers between conformations. We observe that CycA and ALI, both cyclosporines with favorable membrane permeabilities, display multiple stable and well-defined conformers. In contrast, CycH, an epimer of CycA with limited permeability, exhibits fewer and fewer stable conformers. We demonstrate the essential role of the conformational shift from the aqueous cis MeVal11-MeBmt1 state (A1) to the closed conformation featuring cis MeLeu9-MeLeu10 (C1) in facilitating membrane permeation. Additionally, we highlight that the transition from A1 to the all-trans open conformation (O1) is specifically triggered by the presence of CaCl2. We also capture a set of conformers with cis Sar3-MeLeu4, MeLeu9-MeLeu10, denoted as I. Conformationally selected cIMS-MS and IRMPD data of [CycA+Ca]2+ show immediate repopulation of the original population distribution, suggesting that CaCl2 smooths out the energy barriers. Finally, our work presents an improved sampling molecular dynamics approach based on a refined force field that not only consistently and accurately captures established conformers of cyclosporines but also exhibits strong predictive capabilities for novel conformers.

Experimental comparison of cylindrical and plane parallel ionization chambers for reference dosimetry in continuous and pulsed scanned proton beams.

Objective. In this experimental work we compared the determination of absorbed dose to water using four ionization chambers (ICs), a PTW-34045 Advanced Markus, a PTW-34001 Roos, an IBA-PPC05 and a PTW-30012 Farmer, irradiated under the same conditions in one continuous- and in two pulsed-scanned proton beams.Approach. The ICs were positioned at 2 cm depth in a water phantom in four square-field single-energy scanned-proton beams with nominal energies between 80 and 220 MeV and in the middle of 10 × 10 × 10 cm3dose cubes centered at 10 cm or 12.5 cm depth in water. The water-equivalent thickness (WET) of the entrance window and the effective point of measurement was considered when positioning the plane parallel (PP) ICs and the cylindrical ICs, respectively. To reduce uncertainties, all ICs were calibrated at the same primary standards laboratory. We used the beam quality (kQ) correction factors for the ICs under investigation from IAEA TRS-398, the newly calculated Monte Carlo (MC) values and the anticipated IAEA TRS-398 updated recommendations.Main results. Dose differences among the four ICs ranged between 1.5% and 3.7% using both the TRS-398 and the newly recommendedkQvalues. The spread among the chambers is reduced with the newlykQvalues. The largest differences were observed between the rest of the ICs and the IBA-PPC05 IC, obtaining lower dose with the IBA-PPC05.Significance. We provide experimental data comparing different types of chambers in different proton beam qualities. The observed dose differences between the ICs appear to be related to inconsistencies in the determination of thekQvalues. For PP ICs, MC studies account for the physical thickness of the entrance window rather than the WET. The additional energy loss that the wall material invokes is not negligible for the IBA-PPC05 and might partially explain the lowkQvalues determined for this IC. To resolve this inconsistency and to benchmark MC values,kQvalues measured using calorimetry are needed.

Structure and fragmentation chemistry of the peptide radical cations of glycylphenylalanylglycine (GFG).

Herein, we explore the generation and characterization of the radical cations of glycylphenylalanylglycine, or [GFG]•+, formed via dissociative electron-transfer reaction from the tripeptide to copper(II) within a ternary complex. A comprehensive investigation employing isotopic labeling, infrared multiple-photon dissociation (IRMPD) spectroscopy, and density functional theory (DFT) calculations elucidated the details and energetics in formation of the peptide radical cations as well as their dissociation products. Unlike conventional aromatic-containing peptide radical cations that primarily form canonical π-radicals, our findings reveal that 75% of the population of the experimentally produced [GFG]•+ precursors are [GFα•G]+, where the radical resides on the middle α-carbon of the phenylalanyl residue. This unexpected isomeric ion has an enthalpy of 6.8 kcal/mol above the global minimum, which has an N-terminal captodative structure, [Gα•FG]+, comprising 25% of the population. The [b2-H]•+ product ions are also present in a ratio of 75/25 from [GFα•G]+/ [Gα•FG]+, the results of which are obtained from matches between the IRMPD action spectrum and predicted IR absorption spectra of the [b2-H]•+ candidate structures, as well as from IRMPD isomer population analyses.

Characterization of elusive rhamnosyl dioxanium ions and their application in complex oligosaccharide synthesis.

Attaining complete anomeric control is still one of the biggest challenges in carbohydrate chemistry. Glycosyl cations such as oxocarbenium and dioxanium ions are key intermediates of glycosylation reactions. Characterizing these highly-reactive intermediates and understanding their glycosylation mechanisms are essential to the stereoselective synthesis of complex carbohydrates. Although C-2 acyl neighbouring-group participation has been well-studied, the reactive intermediates in more remote participation remain elusive and are challenging to study. Herein, we report a workflow that is utilized to characterize rhamnosyl 1,3-bridged dioxanium ions derived from C-3 p-anisoyl esterified donors. First, we use a combination of quantum-chemical calculations and infrared ion spectroscopy to determine the structure of the cationic glycosylation intermediate in the gas-phase. In addition, we establish the structure and exchange kinetics of highly-reactive, low-abundance species in the solution-phase using chemical exchange saturation transfer, exchange spectroscopy, correlation spectroscopy, heteronuclear single-quantum correlation, and heteronuclear multiple-bond correlation nuclear magnetic resonance spectroscopy. Finally, we apply C-3 acyl neighbouring-group participation to the synthesis of complex bacterial oligosaccharides. This combined approach of finding answers to fundamental physical-chemical questions and their application in organic synthesis provides a robust basis for elucidating highly-reactive intermediates in glycosylation reactions.

A spectroscopic test suggests that fragment ion structure annotations in MS/MS libraries are frequently incorrect.

Modern untargeted mass spectrometry (MS) analyses quickly detect and resolve thousands of molecular compounds. Although features are readily annotated with a molecular formula in high-resolution small-molecule MS applications, the large majority of them remains unidentified in terms of their full molecular structure. Collision-induced dissociation tandem mass spectrometry (CID-MS2) provides a diagnostic molecular fingerprint to resolve the molecular structure through a library search. However, for de novo identifications, one must often rely on in silico generated MS2 spectra as reference. The ability of different in silico algorithms to correctly predict MS2 spectra and thus to retrieve correct molecular structures is a topic of lively debate, for instance in the CASMI contest. Underlying the predicted MS2 spectra are the in silico generated product ion structures, which are normally not used in de novo identification, but which can serve to critically assess the fragmentation algorithms. Here we evaluate in silico generated MSn product ion structures by comparison with structures established experimentally by infrared ion spectroscopy (IRIS). For a set of three dozen product ion structures from five precursor molecules, we find that virtually all fragment ion structure annotations in three major in silico MS2 libraries (HMDB, METLIN, mzCloud) are incorrect and caution the reader against their use for structure annotation of MS/MS ions.