THE APPLICATION OF MOLECULAR ROTATIONAL SPECTROSCOPY TO ANALYZE REGIO- AND STEREOISOMERS OF CYCLOHEXENE PRODUCED FROM REACTIONS OF A TUNGSTEN BENZENE COMPLEX

Chemical reactions of transition metal complexes can provide precise stereocontrol of the products. A recent development in the Harman lab at the University of Virginia has demonstrated full control of the stereochemistry in the deuteration of benzene to cyclohexene using a benzene-tungsten metal complex. Complete stereocontrol of the deuteration is important in the development of deuterium substituted active pharmaceutical ingredients that offer improved safety through reduced metabolism. The goal of this project is to quantify any regioisomer and enantiomer impurities in one of the possible reaction products, 3-d-cyclohexene, after it has been removed from the metal and functionalized as cyclohexene oxide. Regioisomers can be identified using molecular rotational spectroscopy by simple analysis of the rotational spectrum of cyclohexene oxide. To validate that quantum chemistry provides a structure of cyclohexene oxide with sufficient accuracy to unambiguously identify all ten possible singly-deuterated isomers, these regioisomers have been identified in natural abundance in a broadband rotational spectrum (6-18 GHz) and the rotational constants compared to theoretical predictions. The more challenging analysis is the determination of the enantiomeric excess of the deuteration chemistry. The chiral tagging method is proposed for this analysis and two candidate tag molecules, propylene oxide (PO) and trifluoropropylene oxide (TFPO), have been evaluated for use in this application. Although chiral tag complexes are formed with both tags, TFPO offers two important advantages over PO. First, the signal levels for the chiral tag complex with TFPO are about a factor of four stronger – a major advantage since there is a limited amount of reaction product for testing (100 mg). Second, tagging with TFPO leads to cooling of the two ring pucker isomers of deuterated cyclohexene oxide and this simplifies the analysis of the enantiomeric composition of the sample

DIRECT ANALYSIS OF CRUDE REACTION MIXTURE OF A PHOTOCATALYTIC CH-ARYLATION REACTION VIA MOLECULAR ROTATIONAL RESONANCE SPECTROSCOPY

Analysis of the crude reaction mixture of the arylation product of cyclohexanone with 5-bromo-2-(trifluoromethyl)pyridine was performed by molecular rotational resonance (MRR) spectroscopy. This reaction was part of a recent study to investigate the use of decatungstate photocatalysis to perform direct arylation of aliphatic C-H bonds in order to provide a single-step access to multiple pharmaceutically relevant molecules. [1] Although this approach enables a simpler approach of molecular construction, the prevalence of C-H bonds in any given molecule often results in several arylation regioisomers. For the reaction presented here, prior analysis identified the main reaction product as the 3-substituted arylation product, with the minor component being the 4-substiuted arylation product. In the initial analysis, the isomers were identified using nuclear magnetic resonance (NMR) spectroscopy and separated using ultra performance liquid chromatography (UPLC) in order to determine the relative composition. The crude reaction mixture was subsequently analyzed by broadband molecular rotational resonance spectroscopy. In order to identify the reaction products in the crude reaction mixture using MRR, low energy conformers of the reaction products were identified and the MRR structural parameters were calculated. Species were identified by agreement between theoretical and experimental rotational constants. In addition to the detection of the known reaction species and staring material, several other previously unknown impurities were identified in the sample, including the 2-substituted reaction product and a solvent derived byproduct. These species were verified in the analysis by NMR and UPLC data after identification by MRR spectroscopy. Quantitation of the relative abundance of the regioisomer products by MRR was performed by comparing the line intensity of the experimental transitions to the predictions from theory. The quantitative MRR results were in good agreement with the chromatographic results. This work demonstrates the capability of MRR to perform analysis of complex mixtures, simplifying the workflow of analysis. [1] Perry, I. B.; Brewer, T. F.; Sarver, P. J.; Schultz, D. M.; DiRocco, D. A.; MacMillan, D. W. C., Direct arylation of strong aliphatic C–H bonds. Nature 2018, 560 (7716), 70-75

CHIRAL ANALYSIS OF THUJONE IN ESSENTIAL OIL SAMPLES

Thujone is a natural product present in several common plants, such as sage, cedar leaf, and wormwood.[1] Thujone is a neurotoxin that can cause serious health complications in high concentrations, with different stereoisomers having different levels of toxicity.[1] This work extends on previous work to analyze thujone by molecular spectroscopy,[2-3] and presents new efforts to determine the enantiomeric excess (ee) of the alpha- and beta-thujone in several essential oil (EO) samples by chiral tagging. There are four stereoisomers of thujone which arise from the different orientation of the methyl and isopropyl group on the bicyclo[3,1,0]hexan-3-one structure. Alpha-thujone has the methyl and isopropyl group trans and beta-thujone has the two groups cis. Each of these diastereomers have three conformers from the rotational of the isopropyl group. Of the three conformers, all three of alpha-thujone and the lowest two of beta-thujone were observed experimentally. In order to determine the enantiomeric excess of alpha- and beta-thujone in various samples, the homochiral and heterochiral complexes with propylene oxide were assigned using quantum chemistry calculations at the B3LYP D3BJ / def2tzvp level of theory. There was $^{13}$C-level sensitivity to determine carbon framework structures of the strongest homochiral and heterochiral complex of alpha-thujone. Several sage and cedar leaf essential oils were analyzed. There was high enantiopurity of alpha- and beta-thujone in all samples. Additionally, we were able to determine the ee of fenchone, which is present in in cedar leaf EO samples, and camphor, which is present in opposite enantiopurity in sage and cedar leaf.[4] [1] Williams, J. D. et al. (2016). Detection of the Previously Unobserved Stereoisomers of Thujone in the Essential Oil and Consumable Products of Sage (Salvia officinalis L.) Using Headspace Solid-Phase Microextraction-Gas Chromatography-Mass Spectrometry. Journal of Agricultural and Food Chemistry, 64(21), 4319-4326. [2] Kisiel, Z.; Legon, A.C. (1978). Conformations of Some Bicyclic Monoterpenes Based on Bicyclo[3.1.0]hexane from Their Low-Resolution Microwave Spectra. Journal of the American Chemical Society, 100, 8166-8169. [3] Kisiel, Z. Chirped pulse rotational spectroscopy of a single thujone+water sample. In International Symposium of Molecular Spectroscopy, http://hdl.handle.net/2142/91165: 2016. [4] Tateo, F. et al. (1999). Update on enantiomeric composition of (1R)-(+)- and (1S)-(-)-camphor in essential oils by enantioselective gas chromatography. Anal. Commun., 36, 149-151

REACTION FLASK ANALYSIS OF THE ASYMMETRIC HYDROGENATION OF ARTEMISINIC ACID

There is currently a search for a reliable, low cost, synthetic or semi-synthetic method of production for artemisinin – a potent antimalarial drug with limited natural supply. Synthesis of artemisinin from artemisinic acid can be broken down into two key steps: the asymmetric hydrogenation of AA to dihydroartemisinic acid (DHAA) and the oxidation and complex rearrangement of DHAA to form artemisinin. This work reports the reaction flask analysis of the stereospecific conversion of AA to DHAA using chirped-pulse Fourier transform microwave spectroscopy (CP-FTMW). Successful monitoring of this reaction requires resolution of multiple species: artemisinic acid (AA), (R,R)-dihydroartemisinic acid (DHAA), (R,S)-dihydroartemisinic acid (epiDHAA), and the over-reduced form tetrahydroartemisinic acid (THAA). The rotational spectra of these compounds have been obtained through measurements on purified samples with quantities in the 20-100 mg level. For two species (AA and (R,R)-DHAA) the broadband rotational spectrum had 13C-level sensitivity permitting a carbon framework structure determination. For the analysis of the reaction mixture a 70 mg sample was provided. We were able to identify all species in the reaction mixture without further purification. Using dipole moments from quantum chemistry, the relative abundance of each species in the reaction mixture was determined: 14.85\% AA, 57.28\% DHAA, 9.19\% epiDHAA, and 18.67\% THAA

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 oft he enantioisotopomer

ISOTOPOLOGUE AND ISOTOPOMER ANALYSIS OF DEUTERATED CYCLOHEXENE USING MOLECULAR ROTATIONAL RESONANCE SPECTROSCOPY

Molecular rotational resonance (MRR) spectroscopy was used to identify and quantitate the relative abundance of cyclohexene isotopologues prepared from a tungsten-benzene complex. Of the 1024 possible deuterium isomers, 992 of the geometries are pairs of rotationally equivalent geometries producing only 496 isomers with distinguishable rotational spectra. In addition, there are 32 species that yield identical structure for C2-rotation. Therefore, there are 528 different deuterium isomers that have distinguishable rotational spectra. Due the transient chirality associated with the ring pucker of cyclohexene, each isotopomer typically has two rotationally distinct forms in the sample. A rotational spectroscopy methodology was developed to perform automated isotopologue and isotopomer analysis of the synthetic samples. The analysis uses a single reference geometry to identify the isomers. The reference structure is obtained from the singly-substituted $^{13}$C, doubly-substituted $^{13}$C, and singly-substituted deuterium isotopomer rotational constants that are obtained from measurements in natural abundance using a commercial cyclohexene sample. In total, 26 deuterium isomers were identified in various samples of isotopically enriched cyclohexene. Of these 26 isomers represent 15 chemically distinct species. The confidence of the identification is assessed by comparing the root-mean-squared (RMS) error for the three rotational constants of each species. In all cases, the experimental rotational constants can be attributed to a single isotopomer with high confidence. This analysis demonstrates the potential for routine, fast isotopomer identification that provides site-specific information of deuterium incorporation. For the cyclohexene samples prepared using the tungsten complex, rotational spectroscopy verified high stereoselectivity in the synthesis with about 1\% or less over- and under-deuteration in most cases

Chiral Analysis of Linalool, an Important Natural Fragrance and Flavor Compound, by Molecular Rotational Resonance Spectroscopy

The chiral analysis of terpenes in complex mixtures of essential oils, necessary for authentication, has been further developed using chiral tagging molecular rotational resonance (MRR) spectroscopy. One analyte that is of particular interest is linalool (3,7-dimethyl-1,6-octadien-3-ol), a common natural chiral terpene found in botanicals with its enantiomers having unique flavor, fragrance, and aromatherapy characteristics. In this MRR demonstration, resolution of the enantiomers is achieved through the addition of a chiral tag, which creates non-covalent diastereomeric complexes with distinct spectral signatures. The relative stereochemistry of the complexes is identified by the comparison of calculated spectroscopic parameters with experimentally determined parameters of the chiral complexes with high accuracy. The diastereomeric complex intensities are analyzed to determine the absolute configuration (AC) and enantiomeric excess (EE) in each sample. Here, we demonstrate the use of chiral tagging MRR spectroscopy to perform a quantitative routine enantiomer analysis of linalool in complex essential oil mixtures, without the need for reference samples or chromatographic separation

Chiral Analysis of Linalool, an Important Natural Fragrance and Flavor Compound, by Molecular Rotational Resonance Spectroscopy

The chiral analysis of terpenes in complex mixtures of essential oils, necessary for authentication, has been further developed using chiral tagging molecular rotational resonance (MRR) spectroscopy. One analyte that is of particular interest is linalool (3,7-dimethyl-1,6-octadien-3-ol), a common natural chiral terpene found in botanicals with its enantiomers having unique flavor, fragrance, and aromatherapy characteristics. In this MRR demonstration, resolution of the enantiomers is achieved through the addition of a chiral tag, which creates non-covalent diastereomeric complexes with distinct spectral signatures. The relative stereochemistry of the complexes is identified by the comparison of calculated spectroscopic parameters with experimentally determined parameters of the chiral complexes with high accuracy. The diastereomeric complex intensities are analyzed to determine the absolute configuration (AC) and enantiomeric excess (EE) in each sample. Here, we demonstrate the use of chiral tagging MRR spectroscopy to perform a quantitative routine enantiomer analysis of linalool in complex essential oil mixtures, without the need for reference samples or chromatographic separation

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

Online Stereochemical Process Monitoring by Molecular Rotational Resonance Spectroscopy

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