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Differential mobility spectrometry is a separation technique that may be applied to a variety of analytes ranging from small molecule drugs to peptides and proteins. Although rudimentary theoretical models of differential mobility exist, these models are often only applied to small molecules and atomic ions without considering the effects of dynamic microsolvation. Here, we advance our theoretical description of differential ion mobility in pure N

This article highlights the fundamentals of ion-solvent clustering processes that are pertinent to understanding an ion's behaviour during differential mobility spectrometry (DMS) experiments. We contrast DMS with static-field ion mobility, where separation is affected by mobility differences under the high-field and low-field conditions of an asymmetric oscillating electric field. Although commonly used in mass spectrometric (MS) workflows to enhance signal-to-noise ratios and remove isobaric contaminants, the chemistry and physics that underpins the phenomenon of differential mobility has yet to be fully fleshed out. Moreover, we are just now making progress towards understanding how the DMS separation waveform creates a dynamic clustering environment when the carrier gas is seeded with the vapour of a volatile solvent molecule (

The presence of solvent vapor in a differential mobility spectrometry (DMS) cell creates a microsolvating environment that can mitigate complications associated with field-induced heating. In the case of peptides, the microsolvation of protonation sites results in a stabilization of charge density through localized solvent clustering, sheltering the ion from collisional activation. Seeding the DMS carrier gas (N

Although there has been a surge in popularity of differential mobility spectrometry (DMS) within analytical workflows, determining separation conditions within the DMS parameter space still requires manual optimization. A means of accurately predicting differential ion mobility would benefit practitioners by significantly reducing the time associated with method development. Here, we report a machine learning (ML) approach that predicts dispersion curves in an N2 environment, which are the compensation voltages (CVs) required for optimal ion transmission across a range of separation voltages (SVs) between 1500 to 4000 V. After training a random-forest based model using the DMS information of 409 cationic analytes, dispersion curves were reproduced with a mean absolute error (MAE) of ≤ 2.4 V, approaching typical experimental peak FWHMs of ±1.5 V. The predictive ML model was trained using only m/z and ion-neutral collision cross section (CCS) as inputs, both of which can be obtained from experimental databases before being extensively validated. By updating the model via inclusion of two CV datapoints at lower SVs (1500 V and 2000 V) accuracy was further improved to MAE ≤ 1.2 V. This improvement stems from the ability of the "guided" ML routine to accurately capture Type A and B behaviour, which was exhibited by only 2% and 17% of ions, respectively, within the dataset. Dispersion curve predictions of the database's most common Type C ions (81%) using the unguided and guided approaches exhibited average errors of 0.6 V and 0.1 V, respectively.

The effects of kinetic vs thermodynamic control on endo/exo stereoisomer ratios can be observed in a simple Diels–Alder reaction between N-phenylmaleimide and furan. The use of microwave-promoted synthesis affords the cycloadducts in yields ranging from 65–100%, employing reaction times of 1–10 min at temperatures of 55–130 °C. Short reaction times enable screening of numerous reaction conditions (time and temperature) within a single lab period, where endo:exo product ratios follow the primary facets of kinetic and thermodynamic control. Analysis of product ratios obtained under kinetic control allows for the evaluation of activation parameters (ΔΔH⧧ (≈ ΔEa), ΔΔS⧧, and ΔΔG⧧) by means of the Arrhenius and Eyring equations. Values of ΔΔH⧧, ΔΔS⧧, and ΔΔG⧧ were found to be 6.4 ± 0.3 kJ mol−1, 16.5 ± 0.9 J mol−1 K−1, and 1.5 ± 0.4 kJ mol–1 (ΔΔ: exo – endo), respectively. Experimentally determined activation parameters correlate well with quantum-chemical calculations. Several key teaching points are also addressed, including frontier molecular orbital analysis, reversibility of the Diels–Alder reaction, column chromatography, and the use of 1H nuclear magnetic resonance spectroscopy to assess both stereoisomeric yield and purity.

Upon development of a workflow to analyze (±)-Verapamil and its metabolites using differential mobility spectrometry (DMS), we noticed that the ionogram of protonated Verapamil consisted of two peaks. This was inconsistent with its metabolites, as each exhibited only a single peak in the respective ionograms. The unique behaviour of Verapamil was attributed to protonation at its tertiary amino moiety, which generated a stereogenic quaternary amine. The introduction of additional chirality upon N-protonation of Verapamil renders four possible stereochemical configurations for the protonated ion: (R,R), (S,S), (R,S), or (S,R). The (R,R)/(S,S) and (R,S)/(S,R) enantiomeric pairs are diastereomeric and thus exhibit unique conformations that are resolvable by linear and differential ion mobility techniques. Protonation-induced chirality appears to be a general phenomenon, as N-protonation of 12 additional chiral amines generated diastereomers that were readily resolved by DMS.

Ions can experience significant field-induced heating in a differential mobility cell. To investigate this phenomenon, the fragmentation of several para-substituted benzylpyridinium "thermometer" ions (R = OMe, Me, F, Cl, H, CN) was monitored in a commercial differential mobility spectrometer (DMS). The internal energy of each benzylpyridinium derivative was characterized by monitoring the degree of fragmentation to obtain an effective temperature, Teff, which corresponds to a temperature consistent with treating the observed fragmentation ratio using a unimolecular dissociation rate weighted by a Boltzmann distribution at a temperature T. It was found that ions are sufficiently thermalized after initial activation from the ESI process to the temperature of the bath gas, Tbath. Once a critical field strength was surpassed, significant fragmentation of the benzylpyridinium ions was detected. At the maximum bath gas temperature (450 K) and separation voltage (SV; 4400 V) for our instrument, Teff for the benzylpyridinium derivatives ranged from 664 ± 9 K (p-OMe) to 759 ± 17 K (p-H). The extent of activation at a given SV depends on the ion's mass, degrees of freedom, (NDoF), and collision frequency as represented by the ion's collision cross section. Plots of Teff vs the product of ion mass and NDoF and the inverse of collision cross section produce strong linear relationships. This provides an attractive avenue to estimate ion temperatures at a given SV using only intrinsic properties. Moreover, experimentally determined Teff correlate with theoretically predicted Teff using with a self-consistent method based on two-temperature theory. The various instrumental and external parameters that influence Teff are additionally discussed.

Benzylpyridinium analogs are effective thermometer ions since monitoring the formation of the benzylium fragment produced from heterolytic cleavage of the C-N bond can be linked to the ion's internal energy. In this study, three para-substituted benzylpyridinium ions containing ethoxy (OEt), isopropoxy (OiPr) and tert-butoxy (OtBu) substitutents were synthesized and evaluated as chemical thermometers. Intriguingly, the product ion spectra of the three benzylpyridinium ions were dominated by m/z 107 instead of the anticipated benzylium species. Deuterium labeling suggested that the m/z 107 fragment resulted from an intramolecular elimination (Ei), which formed via a four-membered transition state (TS). The fragmentation pathway appears to be an anomaly within the mass spectrometry literature, as four-membered pericyclic TSs are usually accompanied by the formation of an exceptionally stable neutral molecule (e.g., CO2). Quantum-chemical calculations confirmed our hypothesis that stabilization of the strained TS is afforded by hyperconjugation (ΔG‡tert-butoxy < isopropyoxy < ethoxy).

The experimental determination of ion-neutral collision cross sections (CCSs) is generally confined to ion mobility spectrometry (IMS) technologies that operate under the so-called low-field limit or those that enable empirical calibration strategies (e.g., traveling wave IMS; TWIMS). Correlation of ion trajectories to CCS in other non-linear IMS techniques that employ dynamic electric fields, such as differential mobility spectrometry (DMS), has remained a challenge since its inception. Here, we describe how an ion's CCS can be measured from DMS experiments using a machine learning (ML)-based calibration. The differential mobility of 409 molecular cations (m/z: 86-683 Da and CCS 110-236 A2) was measured in a N2 environment to train the ML framework. Several open-source ML routines were tested and trained using DMS-MS data in the form of the parent ion's m/z and the compensation voltage required for elution at specific separation voltages between 1500 and 4000 V. The best performing ML model, random forest regression, predicted CCSs with a mean absolute percent error of 2.6 ± 0.4% for analytes excluded from the training set (i.e., out-of-the-bag external validation). This accuracy approaches the inherent statistical error of ∼2.2% for the MobCal-MPI CCS calculations employed for training purposes and the

Ion mobility-based separation prior to mass spectrometry has become an invaluable tool in the structural elucidation of gas-phase ions and in the characterization of complex mixtures. Application of ion mobility to structural studies requires an accurate methodology to bridge theoretical modelling of chemical structure with experimental determination of an ion's collision cross section (CCS). Herein, we present a refined methodology for calculating ion CCS using parallel computing architectures that makes use of atom specific parameters, which we have called MobCal-MPI. Tuning of ion-nitrogen van der Waals potentials on a diverse calibration set of 162 molecules returned a RMSE of 2.60% in CCS calculations of molecules containing the elements C, H, O, N, F, P, S, Cl, Br, and I. External validation of the ion-nitrogen potential was performed on an additional 50 compounds not present in the validation set, returning a RMSE of 2.31% for the CCSs of these compounds. Owing to the use of parameters from the MMFF94 forcefield, the calibration of the van der Waals potential can be extended to additional atoms defined in the MMFF94 forcefield (i.e., Li, Na, K, Si, Mg, Ca, Fe, Cu, Zn). We expect that the work presented here will serve as a foundation for facile determination of molecular CCSs, as MobCal-MPI boasts up to 64-fold speedups over traditional calculation packages.

The physicochemical properties of [Trpn–H]− and [Trpn⋯Cl]− (n = 1, 2) have been investigated in a combined computational and experimental infrared multiple dissociation (IRMPD) study. IRMPD spectra within the 850–1900 cm−1 region indicate that deprotonation is localized on the carboxylic acid moiety in [Trpn–H]− clusters. A combination of hydrogen bonding and higher order charge–quadrupole interactions appear to influence cluster geometries for all investigated systems. Calculated global minimum and low energy geometries of [Trp⋯Cl]− and [Trp2⋯Cl]− clusters favour coordination of the halide by the indole NH. [Trp2–H]− and [Trp2⋯Cl]− exhibit additional π–π interactions between the heterocyclic side chains.

The microsolvated state of a molecule, represented by its interactions with only a small number of solvent molecules, can play a key role in determining the observable bulk properties of the molecule. This is especially true in cases where strong local hydrogen bonding exists between the molecule and the solvent. One method that can probe the microsolvated states of charged molecules is differential mobility spectrometry (DMS), which rapidly interrogates an ion’s transitions between a solvated and desolvated state in the gas phase (i.e., few solvent molecules present). However, can the results of DMS analyses of a class of molecules reveal information about the bulk physicochemical properties of those species? Our findings presented here show that DMS behaviors correlate strongly with the measured solution phase pKa and pKb values, and cell permeabilities of a set of structurally related drug molecules, even yielding high-resolution discrimination between isomeric forms of these drugs. This is due to DMS’s ability to separate species based upon only subtle (yet predictable) changes in structure: the same subtle changes that can influence isomers’ different bulk properties. Using 2-methylquinolin-8-ol as the core structure, we demonstrate how DMS shows promise for rapidly and sensitively probing the physicochemical properties of molecules, with particular attention paid to drug candidates at the early stage of drug development. This study serves as a foundation upon which future drug molecules of different structural classes could be examined., Differential mobility spectrometry can be used to probe the microsolvated forms of molecules, providing insights into their bulk physicochemical properties, including passive cell permeability.

Tris(pentafluorophenyl)borane, B(C6F5)3, has been found to be an effective catalyst to access the hydridoborate anion, [N(CH2CH2CH2)3Sn][HB(C6F5)3], via hydride abstraction from the hypercoordinated tin reagent, iPr-tricarbastannatrane. This process has been applied to the B(C6F5)3-catalyzed transfer 1,4-hydrostannylation of electron-deficient olefins, namely benzylidene barbituric acids. Insights into the mechanism have been obtained via a series of 1H, 2H, 11B, 13C, and 119Sn NMR spectroscopy, mass spectrometry, and labeling experiments.

We herein describe a flexible synthesis of a small library of 68Ga-labeled CAIX-targeted molecules via an orthogonal 2-cyanobenzothiazole (CBT)/1,2-aminothiol click reaction. Three novel CBT-functionalized chelators (1&ndash, 3) were successfully synthesized and labeled with the positron emitter gallium-68. Cross-ligation between the pre-labeled bifunctional chelators (BFCs) and the 1,2-aminothiol-acetazolamide derivatives (8 and 9) yielded six new 68Ga-labeled CAIX ligands with high radiochemical yields. The click reaction conditions were optimized to improve the reaction rate for applications with short half-life radionuclides. Overall, our methodology allows for a simple and efficient radiosynthetic route to produce a variety of 68Ga-labeled imaging agents for tumor hypoxia.

We report two bifunctional chelators, DFO-Cys and DFO-CBT, to label biovectors with zirconium-89 according to the 2-cyanobenzothiazole/1,2-aminothiol cycloaddition. Their features are high labeling yields, rapid and efficient bioconjugation, metabolically stable luciferin-based end products, and applicability to orthogonal two-step labeling of sensitive biomolecules.

Differential mobility spectrometry (DMS) has shown promise as an analytical tool in the field of lipidomics. However, the underlying mechanism that drives DMS-based lipid separations is still somewhat unclear. Here, we investigate the finer details in the separability of the regioisomeric lipids 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) from 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC), including the effect of cation choice, chemical modifier, and temperature. We conduct DMS-MS studies that are supported by a hybrid molecular dynamics and quantum mechanical approach to explore the conformations and energetics of the [OPPC···X] + and [POPC···X] + (X = Ag, K) constructs. Computational models evaluated using density functional theory reveal structural differences between low energy regioisomeric silver adducts, which translates to unique collision cross sections. Structural differences in regioisomers, as reflected through collision cross section evaluations, are not retained in potassiated adducts. Population weightings suggest coalescence of [OPPC···Ag] + and [POPC···Ag] + collision cross sections as higher energy species become populated at elevated temperatures. This effect presents itself experimentally, revealing diminished resolving power as the temperature of the DMS cell is increased. The results outlined here provides atomistic insight into how dynamic ion collision cross sections affect separations and guidance for future DMS-driven lipidomics applications.

Tris(pentafluorophenyl)borane, B(C

Herein, we describe a synthetic strategy for the regioselective labeling of peptides by using a bioorthogonal click reaction between 2‐cyanobenzothiazole (CBT) and a 1,2‐aminothiol moiety. This methodology allows for the facile and site‐specific modification of peptides with various imaging agents, including fluorophores and radioisotope‐containing prosthetic groups. We investigated the feasibility of an early‐stage incorporation of dipeptide 1 into targeting vectors, such as c[RGDyK(C)] and HER2 pep, during solid‐phase peptide synthesis. Then, the utility of the click reaction to label bioactive peptides with a CBT‐modified imaging agent (FITC–CBT, 9) was assessed. The ligation reaction was found to be highly selective and efficient under various conditions. The fluorescently labeled peptides 2 and 3 were obtained in respective yields of 88 and 82 % under optimized conditions.

The Front Cover shows an innovative methodology for the regioselective labeling of peptides. Early‐stage incorporation of a clickable handle during SPPS provides flexibility for the functionalization of peptide‐based targeting vectors. Once the click handle has been ideally positioned, an imaging probe is conjugated to the targeting vector by 2‐cyanobenzothiazole (CBT)/1,2‐aminothiol cycloaddition. This key reaction is as easy as just “one click”. The labeling reaction is rapid, biocompatible, orthogonal, and highly efficient. It offers a nearly ideal labeling strategy for peptides and a powerful tool for the development of novel imaging agents for biomedical applications. More information can be found in the Communication by K.‐T. Chen et al. on page 256 in Issue 3, 2018 (DOI: 10.1002/open.201700191).

Understanding the mechanisms and energetics of ion solvation is critical in many scientific areas. Here, we present a methodlogy for studying ion solvation using differential mobility spectrometry (DMS) coupled to mass spectrometry. While in the DMS cell, ions experience electric fields established by a high frequency asymmetric waveform in the presence of a desired pressure of water vapor. By observing how a specific ion's behavior changes between the high- and low-field parts of the waveform, we gain knowledge about the aqueous microsolvation of that ion. In this study, we applied DMS to investigate the aqueous microsolvation of protonated quinoline-based drug candidates. Owing to their low binding energies with water, the clustering propensity of 8-substituted quinolinium ions was less than that of the 6- or 7-substituted analogues. We attribute these differences to the steric hinderance presented by subtituents in the 8-position. In addition, these experimental DMS results were complemented by extensive computational studies that determined cluster structures and relative thermodynamic stabilities.