Application of Laser-Induced Acoustic Desorption for Molecular Rotational Resonance Spectroscopy.

Molecular Rotational Resonance (MRR) spectroscopy is a uniquely precise tool for the determination of molecular structures of volatile compounds in mixtures, as the characteristic rotational transition frequencies of a molecule are extremely sensitive to its 3D structure through the moments of inertia in a three-dimensional coordinate system. This enables identification of the compounds based on just a few parameters that can be calculated, as opposed to, for example, mass spectrometric data, which often require expert analysis of 10-20 different signals and the use of many standards/model compounds. This paper introduces a new sampling technique for MRR, laser-induced acoustic desorption (LIAD), to allow the vaporization of nonvolatile and thermally labile analytes without the need for excessive heating or derivatization. In this proof-of-concept study, LIAD was successfully coupled to an MRR instrument to conduct measurements on seven compounds with differing polarities, molecular weights, and melting and boiling points. Identification of three isomers in a mixture was also successfully performed using LIAD/MRR. Based on these results, LIAD/MRR is demonstrated to provide a powerful approach for the identification of nonvolatile and/or thermally labile analytes with molecular weights up to 600 Da in simple mixtures, which does not require the use of reference compounds. In the future, applications to more complex mixtures, such as those relevant to pharmaceutical research, and quantitative aspects of LIAD/MRR will be reported.

Rapid Enantiomeric Excess Measurements of Enantioisotopomers by Molecular Rotational Resonance Spectroscopy.

Recent work in drug discovery has shown that selectively deuterated small molecules can improve the safety and efficacy for active pharmaceutical ingredients. The advantages derive from changes in metabolism resulting from the kinetic isotope effect when deuterium is substituted for a hydrogen atom at a structural position where rate limiting C-H bond breaking occurs. This application has pushed the development of precision deuteration strategies in synthetic chemistry that can install deuterium atoms with high regioselectivity and with stereocontrol. Copper-catalyzed alkene transfer hydrodeuteration chemistry has recently been shown to have high stereoselectivity for deuteration at the metabolically important benzyl C-H position. In this case, stereocontrol results in the creation of enantioisotopomers-molecules that are chiral solely by virtue of the deuterium substitution-and chiral analysis techniques are needed to assess the reaction selectivity. It was recently shown that chiral tag molecular rotational resonance (MRR) spectroscopy provides a routine way to measure the enantiomeric excess and establish the absolute configuration of enantioisotopomers. High-throughput implementations of chiral tag MRR spectroscopy are needed to support optimization of the chemical synthesis. A measurement methodology for high-throughput chiral analysis is demonstrated in this work. The high-throughput ee measurements are performed using cavity-enhanced MRR spectroscopy, which reduces measurement times and sample consumption by more than an order-of-magnitude compared to the previous enantioisotopomer analysis using a broadband MRR spectrometer. It is also shown that transitions for monitoring the enantiomers can be selected from a broadband rotational spectrum without the need for spectroscopic analysis. The general applicability of chiral tag MRR spectroscopy is illustrated by performing chiral analysis on six enantioisotopomer reaction products using a single molecule as the tag for chiral discrimination.

Essential Oil Composition and Enantioselective Profile of Agastache urticifolia (Lamiaceae) and Monardella odoratissima (Lamiaceae) from Utah.

Two species within the Lamiaceae (mint) family, Agastache urticifolia and Monardella odoratissima, are aromatic plants that are native to the Intermountain Region (USA). Essential oil produced through steam distillation was examined to establish the essential oil yield and both the achiral and chiral aromatic profiles of both plant species. The resulting essential oils were analyzed by GC/MS, GC/FID, and MRR (molecular rotational resonance). For A. urticifolia and M. odoratissima, achiral essential oil profiles were largely composed of limonene (71.0%, 27.7%), trans-ß-ocimene (3.6%, 6.9%), and pulegone (15.9%, 4.3%), respectively. Between the two species, eight chiral pairs were analyzed and, interestingly, the dominant enantiomer (calculated as ee%) of limonene and pulegone switched between the two species. Where enantiopure standards were not commercially available, MRR was used as a reliable analytical technique for chiral analysis. This study verifies the achiral profile for A. urticifolia and, for the first time to the authors' knowledge, establishes the achiral profile for M. odoratissima and chiral profile for both species. Additionally, this study confirms the utility and practicality of using MRR for determining chiral profiles in essential oils.

Analysis of isomeric mixtures by molecular rotational resonance spectroscopy.

Recent developments in molecular rotational resonance (MRR) spectroscopy that have enabled its use as an analytical technique for the precise determination of molecular structure are reviewed. In particular, its use in the differentiation of isomeric compounds-including regioisomers, stereoisomers and isotopic variants-is discussed. When a mixture of isomers, such as resulting from a chemical reaction, is analyzed, it is highly desired to be able to unambiguously identify the structures of each of the components present, as well as quantify them, without requiring complex sample preparation or reference standards. MRR offers unique capabilities for addressing this analytical challenge, owing to two factors: its high sensitivity to a molecule's structure and its high spectral resolution, allowing mixtures to be resolved without separation of components. This review introduces core theoretical principles, an introduction to MRR instrumentation and the methods by which spectra can be interpreted with the aid of computational chemistry to correlate the observed patterns to molecular structures. Recent articles are discussed in which this technique was applied to help chemists complete challenging isomer analyses. Developments in the use of MRR for chiral analysis and in the measurement of isotopically labeled compounds are also highlighted.

Direct Construction of Peaks from Free Induction Decay Curves for Gas Chromatography-Molecular Rotational Resonance Spectroscopy without Fourier Transforms.

The concept of coupling gas chromatography with molecular rotational resonance spectroscopy (GC-MRR) was introduced in 2020, combining the separation capabilities of GC with the unparalleled specificity of MRR. In this study, we address the challenge of the high data throughput of MRR spectrometers, as GC-MRR spectrometers can generate thousands to millions of data points per second. In the previous GC-MRR studies, a free induction decay (FID) measurement was Fourier transformed to generate each point on the chromatogram. Such extensive calculations limit the performance, sensitivity, and speed of GC-MRR. A direct approach is proposed here to extract peak intensity from FID using the Gram-Schmidt vector orthogonalization method. First, analyte-free FIDs are used to construct a basis set representing the instrument's background noise, and then the remaining FIDs are orthogonalized to this fixed basis set. Each FID yields a single intensity value after Gram-Schmidt orthogonalization. The magnitude of the orthogonalized analyte FID is the signal intensity plotted in the chromatogram. This approach is computationally much faster (up to 10 times) than the conventional Fourier transform algorithm, is at least as sensitive as the FT algorithm, and maintains or improves the chromatographic peak shape. We compare the sensitivity, linearity, and chromatographic peak shapes for the Fourier transform and Gram-Schmidt approaches using both synthetically generated FIDs and instrumental data. This approach would allow the summed peak intensity to be displayed essentially in real-time, following which identified peaks can be further investigated to identify and quantify the species associated with each.

Enantioselective Synthesis of Enantioisotopomers with Quantitative Chiral Analysis by Chiral Tag Rotational Spectroscopy.

Fundamental to the synthesis of enantioenriched chiral molecules is the ability to assign absolute configuration at each stereogenic center, and to determine the enantiomeric excess for each compound. While determination of enantiomeric excess and absolute configuration is often considered routine in many facets of asymmetric synthesis, the same determinations for enantioisotopomers remains a formidable challenge. Here, we report the first highly enantioselective metal-catalyzed synthesis of enantioisotopomers that are chiral by virtue of deuterium substitution along with the first general spectroscopic technique for assignment of the absolute configuration and quantitative determination of the enantiomeric excess of isotopically chiral molecules. Chiral tag rotational spectroscopy uses noncovalent chiral derivatization, which eliminates the possibility of racemization during derivatization, to perform the chiral analysis without the need of reference samples of the enantioisotopomer.

Chiral analysis of pantolactone with molecular rotational resonance spectroscopy.

A molecular rotational resonance spectroscopy method for measuring the enantiomeric excess of pantolactone, an intermediate in the synthesis of panthenol and pantothenic acid, is presented. The enantiomers are distinguished via complexation with a small chiral tag molecule, which produces diastereomeric complexes in the pulsed jet expansion used to inject the sample into the spectrometer. These complexes have distinct moments of inertia, so their spectra are resolved by MRR spectroscopy. Quantitative enantiomeric excess (EE) measurements are made by taking the ratio of normalized complex signal levels when a chiral tag sample of high, known EE is used, while the absolute configuration of the sample can be determined from electronic structure calculations of the complex geometries. These measurements can be performed without the need for reference samples with known enantiopurity. Two instruments were used in the analysis. A broadband, chirped-pulse spectrometer is used to perform structural characterization of the complexes. The broadband spectrometer is also used to determine the EE; however, this approach requires relatively long measurement times. A targeted MRR spectrometer is also used to demonstrate EE analysis with approximately 15-min sample-to-sample cycle time. The quantitative accuracy of the method is demonstrated by comparison with chiral gas chromatography and through the measurement of a series of reference samples prepared from mixtures of (R)-pantolactone and (S)-pantolactone samples of known EE.

Enhancing Sensitivity for High-Selectivity Gas Chromatography-Molecular Rotational Resonance Spectroscopy.

A next-generation gas chromatograph-molecular rotational resonance (MRR) spectrometer (GC-MRR) with instrumental improvements and higher sensitivity is described. MRR serves as a structural information-rich detector for GC with extremely narrow linewidths and capabilities surpassing 1H nuclear magnetic resonance/Fourier transform infrared spectroscopy/mass spectrometry (MS) while offering unparalleled specificity in regard to a molecule's three-dimensional structure. With a Fabry-Pérot cavity and a supersonic jet incorporated into a GC-MRR, dramatic improvements in sensitivity for molecules up to 244 Da were achieved in the microwave region compared to the only prior work, which demonstrated the GC-MRR idea for the first time with millimeter waves. The supersonic jet cools the analytes to ∼2 K, resulting in a limited number of molecular rotational and vibrational levels and enabling us to obtain stronger GC-MRR signals. This has allowed the limits of detection of the GC-MRR to be comparable to a GC thermal conductivity detector with an optimized choice of gases. The performance of this GC-MRR system is reported for a range of molecules with permanent dipole moments, including alcohols, nitrogen heterocyclics, halogenated compounds, dioxins, and nitro compounds in the molecular mass range of 46-244 Da. The lowest amount of any substance yet detected by MRR in terms of mass is reported in this work. A theoretically unexpected finding is reported for the first time about the effect of the GC carrier gas (He, Ne, and N2) on the sensitivity of the analysis in the presence of the gas driving the supersonic jet (He, Ne, and N2) in the GC-MRR. Finally, the idea of total molecule monitoring in the GC-MRR analogous to selected ion monitoring in GC-MS is illustrated. Structural isomers and isotopologues of bromobutanes and bromonitrobenzenes are used to demonstrate this concept.

σ-Hole activation and structural changes upon perfluorination of aryl halides: direct evidence from gas phase rotational spectroscopy.

Enhancement of the σ-hole on the halogen atom of aryl halides due to perfluorination of the ring is demonstrated by use of the Extended Townes-Dailey (ETD) model coupled to a Natural Atomic Orbital Bond analysis on two perfluorinated aryl halides (C6F5Cl and C6F5Br) and their hydrogenated counterparts. The ETD analysis, which quantifies the halogen p-orbitals populations, relies on the nuclear quadrupole coupling constants which in this work are accurately determined experimentally from the rotational spectra. The rotational spectra investigated by Fourier-transform microwave spectroscopy performed in supersonic expansion are reported for the parent species of C6F5Cl and C6F5Br and their 13C, 37Cl or 81Br substituted isotopologues observed in natural abundance. The experimentally determined rotational constants combined with theoretical data at the MP2/aug-cc-pVTZ level provide precise structural information from which an elongation of the ring along its symmetry axis due to perfluorination is proved.

Copper-Catalyzed Transfer Hydrodeuteration of Aryl Alkenes with Quantitative Isotopomer Purity Analysis by Molecular Rotational Resonance Spectroscopy.

A copper-catalyzed alkene transfer hydrodeuteration reaction that selectively incorporates one hydrogen and one deuterium atom across an aryl alkene is described. The transfer hydrodeuteration protocol is selective across a variety of internal and terminal alkenes and is also demonstrated on an alkene-containing complex natural product analog. Beyond using 1H, 2H, and 13C NMR analysis to measure reaction selectivity, six transfer hydrodeuteration products were analyzed by molecular rotational resonance (MRR) spectroscopy. The application of MRR spectroscopy to the analysis of isotopic impurities in deuteration chemistry is further explored through a measurement methodology that is compatible with high-throughput sample analysis. In the first step, the MRR spectroscopy signatures of all isotopic variants accessible in the reaction chemistry are analyzed using a broadband chirped-pulse Fourier transform microwave spectrometer. With the signatures in hand, measurement scripts are created to quantitatively analyze the sample composition using a commercial cavity enhanced MRR spectrometer. The sample consumption is below 10 mg with analysis times on the order of 10 min using this instrument-both representing order-of-magnitude reduction compared to broadband MRR spectroscopy. To date, these measurements represent the most precise spectroscopic determination of selectivity in a transfer hydrodeuteration reaction and confirm that product regioselectivity ratios of >140:1 are achievable under this mild protocol.

Direct regioisomer analysis of crude reaction mixtures via molecular rotational resonance (MRR) spectroscopy.

Direct analyses of crude reaction mixtures have been carried out using molecular rotational resonance (MRR) spectroscopy. Two examples are presented, a demonstration application in photocatalytic CH-arylation as well as generation of an intermediate in a natural product synthesis. In both cases, the reaction can proceed at more than one site, leading to a mixture of regioisomers that can be challenging to distinguish. MRR structural parameters were calculated for the low lying conformers for the desired compounds, and then compared to the experimental spectra of the crude mixtures to confirm the presence of these species. Next, quantitation was performed by comparing experimentally measured line intensities with simulations based on computed values for the magnitude and direction of the molecular dipole moment of each species. This identification and quantification was performed without sample purification and without isolated standards of the compounds of interest. The values obtained for MRR quantitation were in good agreement with the chromatographic values. Finally, previously unknown impurities were discovered within the photocatalytic CH-arylation work. This paper demonstrates the utility of MRR as a reaction characterization tool to simplify analytical workflows.

Rapid quantification of isomeric and dehalogenated impurities in pharmaceutical raw materials using MRR spectroscopy.

We demonstrate the application of molecular rotational resonance (MRR) spectroscopy to quantify regioisomeric, dehalogenated, and enantiomeric impurities in two raw materials used in the synthesis of a HIV integrase inhibitor, cabotegravir. Characterization of these raw material impurities is important due to their ability to introduce structurally similar impurities into the final drug product. MRR, due to its high resolution and selectivity to small changes in molecular structure, can perform these measurements rapidly and without the need for developing a chromatographic separation method. For 2,4-difluorobenzylamine, four impurities were quantified (benzylamine, 2-fluorobenzylamine, 4-fluorobenzylamine, and 2,6-difluorobenzylamine), while for (S)-alaninol (2-amino-1-propanol), its enantiomer, (R)-alaninol, was measured using a chiral tagging method. For both samples, the resonance frequencies of each compound of interest were first determined using a broadband spectrometer before evaluating analytical performance metrics on a faster targeted spectrometer. For the fluorobenzylamines, quantitative performance was demonstrated over the range 0.05-5 % (v/v) for each impurity, while for alaninol, measurements were performed over the range of 70-99 % enantiomeric excess. This study suggests that MRR can be useful for validating the purity of pharmaceutical raw materials.

A Gas Chromatography-Molecular Rotational Resonance Spectroscopy Based System of Singular Specificity.

We designed and demonstrated the unique abilities of the first gas chromatography-molecular rotational resonance spectrometer (GC-MRR). While broadly and routinely applicable, its capabilities can exceed those of high-resolution MS and NMR spectroscopy in terms of selectivity, resolution, and compound identification. A series of 24 isotopologues and isotopomers of five organic compounds are separated, identified, and quantified in a single run. Natural isotopic abundances of mixtures of compounds containing chlorine, bromine, and sulfur heteroatoms are easily determined. MRR detection provides the added high specificity for these selective gas-phase separations. GC-MRR is shown to be ideal for compound-specific isotope analysis (CSIA). Different bacterial cultures and groundwater were shown to have contrasting isotopic selectivities for common organic compounds. The ease of such GC-MRR measurements may initiate a new era in biosynthetic/degradation and geochemical isotopic compound studies.

Direct Measurements of Small Polar Impurities in Gasoline Mixtures Using Molecular Rotational Resonance Spectroscopy.

This paper reports our efforts to determine whether rotational spectroscopy is a useful tool for petroleum analysis. These efforts include the use of a BrightSpec molecular rotational resonance (MRR) spectrometer, which operates in the 260-290 GHz frequency range, to record rotational spectra of small polar contaminants in commercial gasoline. The observed rotational spectra showed rich, but assignable, patterns due to the sensitivity of the MRR to only small polar compounds. Any interference from a complex hydrocarbon matrix, which in conventional chromatographic methods obscures signals from small polar contaminants, is nearly eliminated. In addition to the evident rotational spectrum of ethanol, the spectra of toluene, ethyl cyanide, and acetaldehyde have also been detected. A quantitative method for ethanol has been developed and demonstrated in this paper, whereas the specific analyses of the other polar impurities will be reported in the future. The validity of MRR to be used as an analytical instrument has been examined by constructing a standard linear curve using dilutions of ethanol in water. The linearity and percentage recovery parameters are satisfactory.

Molecular polymorphism: microwave spectra, equilibrium structures, and an astronomical investigation of the HNCS isomeric family.

The rotational spectra of thioisocyanic acid (HNCS), and its three energetic isomers (HSCN, HCNS, and HSNC) have been observed at high spectral resolution by a combination of chirped-pulse and Fabry-Pérot Fourier-transform microwave spectroscopy between 6 and 40 GHz in a pulsed-jet discharge expansion. Two isomers, thiofulminic acid (HCNS) and isothiofulminic acid (HSNC), calculated here to be 35-37 kcal mol(-1) less stable than the ground state isomer HNCS, have been detected for the first time. Precise rotational, centrifugal distortion, and nitrogen hyperfine coupling constants have been determined for the normal and rare isotopic species of both molecules; all are in good agreement with theoretical predictions obtained at the coupled cluster level of theory. On the basis of isotopic spectroscopy, precise molecular structures have been derived for all four isomers by correcting experimental rotational constants for the effects of rotation-vibration interaction calculated theoretically. Formation and isomerization pathways have also been investigated; the high abundance of HSCN relative to ground state HNCS, and the detection of strong lines of SH using CH3CN and H2S, suggest that HSCN is preferentially produced by the radical-radical reaction HS + CN. A radio astronomical search for HSCN and its isomers has been undertaken toward the high-mass star-forming region Sgr B2(N) in the Galactic Center with the 100 m Green Bank Telescope. While we find clear evidence for HSCN, only a tentative detection of HNCS is proposed, and there is no indication of HCNS or HSNC at the same rms noise level. HSCN, and tentatively HNCS, displays clear deviations from a single-excitation temperature model, suggesting weak masing may be occurring in some transitions in this source.

Chiral recognition and atropisomerism in the sevoflurane dimer.

We have examined the stereoselectivity of molecular recognition between two molecules of the anesthetic sevoflurane using broadband rotational spectroscopy. The transient axial chirality of sevoflurane is revealed upon the formation of the dimer, as two different diastereoisomers made of either homo- or heterochiral species are detected in a supersonic jet expansion. The conformational assignment was confirmed by the observation of eighteen different isotopologues in natural abundance (all possible (13)C's and two (18)O species of the homochiral form). The two clusters are formed in practically equal proportions (1.1 : 1), probably due to their similar hydrogen bonding topologies. In both clusters the complex is stabilized by a primary C-H···O hydrogen bond, assisted by weak C-HF interactions. This intermolecular binding regime is characterized by a mixture of electrostatic and dispersive interactions, midway between classical hydrogen bonds and van der Waals clusters.

Water-water and water-solute interactions in microsolvated organic complexes.

A structural study of microsolvated clusters of ß-propiolactone (BPL) formed in a pulsed molecular jet expansion is presented. The rotational spectra of BPL-(H2O)n (n=1-5) adducts have been analyzed by broadband microwave spectroscopy. Unambiguous identification of the structures has been achieved using isotopic substitution and experimental measurements of the cluster dipole moment. The observed structures are discussed in terms of the different intermolecular interactions between water molecules and between water and BPL, which include n-π* interactions involving the lone pairs of electrons on water oxygen atoms and the antibonding orbital of the BPL carbonyl group. The changes induced in the structures of the water hydrogen-bonding network by complexation to BPL indicate that water clusters adopt specific configurations to maximize their links to solute molecules.

Probing the C-H⋠⋠⋠π weak hydrogen bond in anesthetic binding: the sevoflurane-benzene cluster.

Cooperativity between weak hydrogen bonds can be revealed in molecular clusters isolated in the gas phase. Here we examine the structure, internal dynamics, and origin of the weak intermolecular forces between sevoflurane and a benzene molecule, using multi-isotopic broadband rotational spectra. This heterodimer is held together by a primary C-H⋅⋅⋅π hydrogen bond, assisted by multiple weak C-H⋅⋅⋅F interactions. The multiple nonbonding forces hinder the internal rotation of benzene around the isopropyl C-H bond in sevoflurane, producing detectable quantum tunneling effects in the rotational spectrum.

Segmented chirped-pulse Fourier transform submillimeter spectroscopy for broadband gas analysis.

Chirped-pulse Fourier transform spectroscopy has recently been extended to millimeter wave spectroscopy as a technique for the characterization of room-temperature gas samples. Here we present a variation of this technique that significantly reduces the technical requirements on high-speed digital electronics and the data throughput, with no reduction in the broadband spectral coverage and no increase in the time required to reach a given sensitivity level. This method takes advantage of the frequency agility of arbitrary waveform generators by utilizing a series of low-bandwidth chirped excitation pulses paired in time with a series of offset single frequency local oscillators, which are used to detect the molecular free induction decay signals in a heterodyne receiver. A demonstration of this technique is presented in which a 67 GHz bandwidth spectrum of methanol (spanning from 792 to 859 GHz) is acquired in 58 µs.

The interplay of hydrogen bonding and dispersion in phenol dimer and trimer: structures from broadband rotational spectroscopy.

The structures of the phenol dimer and phenol trimer complexes in the gas phase have been determined using chirped-pulse Fourier transform microwave spectroscopy in the 2-8 GHz band. All fourteen (13)C and (18)O phenol dimer isotopologues were assigned in natural abundance. A full heavy atom experimental substitution structure was determined, and a least-squares fit ground state r0 structure was determined by proper constraint of the M06-2X/6-311++g(d,p) ab initio structure. The structure of phenol dimer features a water dimer-like hydrogen bond, as well as a cooperative contribution from inter-ring dispersion. Comparisons between the experimental structure and previously determined experimental structures, as well as ab initio structures from various levels of theory, are discussed. For phenol trimer, a C3 symmetric barrel-like structure is found, and an experimental substitution structure was determined via measurement of the six unique (13)C isotopologues. The least-squares fit rm((1)) structure reveals a similar interplay between hydrogen bonding and dispersion in the trimer, with water trimer-like hydrogen bonding and C-H···π interactions.