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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.

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.

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.

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.

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.

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 I

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.

Direct analyses of crude reaction mixtures have been carried out using molecular rotational resonance (MRR) spectroscopy, allowing identification and quantification of major and minor components without sample purification or reference standards., 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.

A molecular rotational resonance (MRR) spectrometer designed to monitor the product composition of an asymmetric continuous flow reaction online is presented. The MRR technique is highly sensitive to small changes in molecular structure and, as such, is capable of rapidly quantifying isomers as well as other impurities in a complex mixture, without chromatographic separation or chemometrics. The spectrometer in this study operates by automatically drawing a portion of the reaction solution into a reservoir, volatizing it by heating, and measuring the highly resolved MRR spectra of each of the components of interest in order to determine their relative quantity in the mixture. The reaction under study was the hydrogenation of artemisinic acid, an intermediate step in the semisynthesis of the antimalarial drug artemisinin. Four analytes were characterized in each measurement: the starting material, the product, a diastereomer of the product, and an overreduction byproduct that was not directly quantifiable by either HPLC or NMR methods. The MRR instrument has a measurement cycle time of approximately 17 min for this analysis and can run for several hours without any user interaction.

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.

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)Cβ spin pairs in the amino acids ILV, leading to the absence of Cβ 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.