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.

Precision Deuteration Using Cu-Catalyzed Transfer Hydrodeuteration to Access Small Molecules Deuterated at the Benzylic Position.

A highly regio- and chemoselective Cu-catalyzed aryl alkyne transfer hydrodeuteration to access a diverse scope of aryl alkanes precisely deuterated at the benzylic position is described. The reaction benefits from a high degree of regiocontrol in the alkyne hydrocupration step, leading to the highest selectivities reported to date for an alkyne transfer hydrodeuteration reaction. Only trace isotopic impurities are formed under this protocol, and analysis of an isolated product by molecular rotational resonance spectroscopy confirms that high isotopic purity products can be generated from readily accessible aryl alkyne substrates.

Assignment of the absolute configuration of molecules that are chiral by virtue of deuterium substitution using chiral tag molecular rotational resonance spectroscopy.

Chiral tag molecular rotational resonance (MRR) spectroscopy is used to assign the absolute configuration of molecules that are chiral by virtue of deuterium substitution. Interest in the improved performance of deuterated active pharmaceutical ingredients has led to the development of precision deuteration reactions. These reactions often generate enantioisotopomer reaction products that pose challenges for chiral analysis. Chiral tag rotational spectroscopy uses noncovalent derivatization of the enantioisotopomer to create the diastereomers of the 1:1 molecular complexes of the analyte and a small, chiral molecule. Assignment of the absolute configuration requires high-confidence determinations of the structures of these weakly bound complexes. A general search method, CREST, is used to identify candidate geometries. Subsequent geometry optimization using dispersion corrected density functional theory gives equilibrium geometries with sufficient accuracy to identify the isomers of the chiral tag complexes produced in the pulsed jet expansion used to introduce the sample into the MRR spectrometer. Rotational constant scaling based on the fact that the diastereomers have the same equilibrium geometry gives accurate predictions allowing identification of the homochiral and heterochiral tag complexes and, therefore, assignment of absolute configuration. The method is successfully applied to three oxygenated substrates from enantioselective Cu-catalyzed alkene transfer hydrodeuteration reaction chemistry.

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.

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.

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.

Preparation of cyclohexene isotopologues and stereoisotopomers from benzene.

The hydrogen isotopes deuterium (D) and tritium (T) have become essential tools in chemistry, biology and medicine1. Beyond their widespread use in spectroscopy, mass spectrometry and mechanistic and pharmacokinetic studies, there has been considerable interest in incorporating deuterium into drug molecules1. Deutetrabenazine, a deuterated drug that is promising for the treatment of Huntington's disease2, was recently approved by the United States' Food and Drug Administration. The deuterium kinetic isotope effect, which compares the rate of a chemical reaction for a compound with that for its deuterated counterpart, can be substantial1,3,4. The strategic replacement of hydrogen with deuterium can affect both the rate of metabolism and the distribution of metabolites for a compound5, improving the efficacy and safety of a drug. The pharmacokinetics of a deuterated compound depends on the location(s) of deuterium. Although methods are available for deuterium incorporation at both early and late stages of the synthesis of a drug6,7, these processes are often unselective and the stereoisotopic purity can be difficult to measure7,8. Here we describe the preparation of stereoselectively deuterated building blocks for pharmaceutical research. As a proof of concept, we demonstrate a four-step conversion of benzene to cyclohexene with varying degrees of deuterium incorporation, via binding to a tungsten complex. Using different combinations of deuterated and proteated acid and hydride reagents, the deuterated positions on the cyclohexene ring can be controlled precisely. In total, 52 unique stereoisotopomers of cyclohexene are available, in the form of ten different isotopologues. This concept can be extended to prepare discrete stereoisotopomers of functionalized cyclohexenes. Such systematic methods for the preparation of pharmacologically active compounds as discrete stereoisotopomers could improve the pharmacological and toxicological properties of drugs and provide mechanistic information related to their distribution and metabolism in the body.

Molecular Structure of 1-Isocyano-1-silacyclopent-3-ene: A Combined Microwave Spectral and Theoretical Study.

The microwave spectrum of 1-isocyano-1-silacyclopent-3-ene has been obtained from broad-band chirped-pulse Fourier transform microwave spectroscopy. The rotational constants (RCs) for the standard abundant isotopic species are A = 3328.4182(23), B = 1017.69404(53), and C = 1012.33297(58) MHz. The symmetric quartic centrifugal distortion constants, using the Ir representation in CS symmetry for ΔJ,ΔJK, ΔK, and δJ, have been evaluated; similarly, the 14N nuclear quadrupole coupling has been determined. Several singly substituted isotopologues observed in natural abundance enabled most of the heavy atom substructure to be determined. The five-membered ring is close to planar, but the orientation of the isocyanate unit, derived from the N13CO spectrum, unexpectedly lies above the ring center in a cis C2,5-Si-N═C conformation. Our initial equilibrium structural searches led to a trans orientation of the C2,5-Si-N═C unit, i.e., bending away from the ring. When the cis conformation was applied, the final equilibrium structure, assuming CS symmetry, gave RC values of 3221.3 ( A), 1037.0 ( B), and 1031.3 ( C) MHz, very close to the MW values. This enabled the full-equilibrium structure to be determined with confidence. The principal bond lengths were 1.7157 (Si-N), 1.8696 (Si-C), 1.1998 (N═C), and 1.1737 (C═O) Å, with angles 163.3 (Si-N═C), 178.1 (N═C═O), 96.5 (C-Si-C), and 118.7° (C-C═C), respectively. The extensive widening of the SiNC angle is particularly notable; the SiNCO unit has a trans dihedral angle. The cis orientation implies a (weak) attractive force between the ring and isocyanate groups by a through-space interaction. An atoms in molecule study, where the local minima of electron density are determined, fails to disclose the exact nature of the interaction; however, a highly polarized skeleton was obtained. A systematic theoretical study of the Si-N═C angle potential energy surface (PES) relative to the ring gave a very shallow double minimum with the barrier being less than 1 cm-1; a polynomial fit to the surface shows major contributions of both harmonic and quartic components. A similar study of the XSiN angle, where X is at the ring center, also gave a PES with considerable quartic character.

Nonuniform sampling by quantiles.

A flexible strategy for choosing samples nonuniformly from a Nyquist grid using the concept of statistical quantiles is presented for broad classes of NMR experimentation. Quantile-directed scheduling is intuitive and flexible for any weighting function, promotes reproducibility and seed independence, and is generalizable to multiple dimensions. In brief, weighting functions are divided into regions of equal probability, which define the samples to be acquired. Quantile scheduling therefore achieves close adherence to a probability distribution function, thereby minimizing gaps for any given degree of subsampling of the Nyquist grid. A characteristic of quantile scheduling is that one-dimensional, weighted NUS schedules are deterministic, however higher dimensional schedules are similar within a user-specified jittering parameter. To develop unweighted sampling, we investigated the minimum jitter needed to disrupt subharmonic tracts, and show that this criterion can be met in many cases by jittering within 25-50% of the subharmonic gap. For nD-NUS, three supplemental components to choosing samples by quantiles are proposed in this work: (i) forcing the corner samples to ensure sampling to specified maximum values in indirect evolution times, (ii) providing an option to triangular backfill sampling schedules to promote dense/uniform tracts at the beginning of signal evolution periods, and (iii) providing an option to force the edges of nD-NUS schedules to be identical to the 1D quantiles. Quantile-directed scheduling meets the diverse needs of current NUS experimentation, but can also be used for future NUS implementations such as off-grid NUS and more. A computer program implementing these principles (a.k.a. QSched) in 1D- and 2D-NUS is available under the general public license.

Enhanced biosynthetically directed fractional carbon-13 enrichment of proteins for backbone NMR assignments.

Routes to carbon-13 enrichment of bacterially expressed proteins include achieving uniform or positionally selective (e.g. ILV-Me, or (13)C', etc.) enrichment. We consider the potential for biosynthetically directed fractional enrichment (e.g. carbon-13 incorporation in the protein less than 100%) for performing routine n-(D)dimensional NMR spectroscopy of proteins. First, we demonstrate an approach to fractional isotope addition where the initial growth media containing natural abundance glucose is replenished at induction with a small amount (e.g. 10%(w/w)u-(13)C-glucose) of enriched nutrient. The approach considered here is to add 10% (e.g. 200mg for a 2g/L culture) u-(13)C-glucose at the induction time (OD600=0.8), resulting in a protein with enhanced (13)C incorporation that gives almost the same NMR signal levels as an exact 20% (13)C sample. Second, whereas fractional enrichment is used for obtaining stereospecific methyl assignments, we find that (13)C incorporation levels no greater than 20%(w/w) yield (13)C and (13)C-(13)C spin pair incorporation sufficient to conduct typical 3D-bioNMR backbone experiments on moderate instrumentation (600 MHz, RT probe). Typical 3D-bioNMR experiments of a fractionally enriched protein yield expected backbone connectivities, and did not show amino acid biases in this work, with one exception. When adding 10% u-(13)C glucose to expression media at induction, there is poor preservation of (13)Cα-(13)Cß spin pairs in the amino acids ILV, leading to the absence of Cß signals in HNCACB spectra for ILV, a potentially useful editing effect. Enhanced fractional carbon-13 enrichment provides lower-cost routes to high throughput protein NMR studies, and makes modern protein NMR more cost-accessible.