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.

We present the successful application of MRR spectroscopy in the microwave region to monitor the output of a continuous pharmaceutical synthesis. Microwave spectroscopy has an excellent capability to distinguish isomers and other structurally similar compounds, and techniques have been developed recently that are also sensitive to enantiomeric excess. A Balle-Flygare-style Fourier transform microwave spectrometer was employed as the detector in this study, along with a new solutions sampling interface that injects crude product solution directly from the reactor, bakes off the solvent, and volatilizes the analyte mixture for analysis. The reaction under study was the catalytic asymmetric hydrogenation of artemisinic acid to produce a stable intermediate in the synthesis of artemisinin, an important antimalarial. The instrument is fully automated and consumes less than 1 mg of analyte in order to analyze the composition of 4 species with a detection limit of approximately 75 ppmw in the solution: the starting material, desired product, epimer of the product, and an overreduction byproduct that is not readily detectable by HPLC or NMR. This talk will describe the results of this study and prospects for future application in pharmaceutical process development.

Continuing our efforts in characterizing small molecules for use as potential chiral tags for the conversion of enantiomeric molecules into spectroscopically distinct diasteromeric complexes for chiral analysis, we examine the microwave spectrum and molecular structure of 3,3-difluoro-1,2-epoxypropane. This compound is available as a high vapor pressure liquid, both in enantiomerically pure form and as a racemic mixture, and it is easily incorporated into a free jet expansion for complex formation and spectroscopic analysis. Like the structurally similar 3,3,3-trifluoro-1,2-epoxypropane, it has a simple, hyperfine-free rotational spectrum. This spectrum has been obtained for the most abundant and four singly-substituted isotopologues, all in natural abundance, and the structure of the molecule determined. In addition, the spectrum and structure of the 3,3-difluoro-1,2-epoxypropane-argon complex are obtained.

We are investigating the suitability of 3,3,3-trifluoro-1,2-epoxypropane [2-(trifluoromethyl)-oxirane, or TFO] as a tag for chiral analysis through conversion of enantiomers into structurally distinct diastereomers via the formation of non-covalently bound heterodimers. This method can determine the absolute stereochemistry and enantiomeric composition of an analyte and shows promise for great impact in analytical chemistry. Using density functional theory, we examine the possible conformations of both homochiral (RR or SS) and heterochiral (RS or SR) homodimers of TFO to guide the search for the microwave spectra of these species. Several conformers are found for each, but the lowest energy heterochiral TFO dimer is a microwave silent one with an inversion center. However, a spectrum is observed that can be assigned to the lowest energy geometry of the homochiral TFO dimer.

Terpenes are the main constituents of essential oils and are responsible for their chemical and biological activities. Additionally, terpenes found in essential oils are often structurally similar, like thymol, carvacrol, p-cymene and terpinen-4-ol in the case of thyme essential oil. These oils are likely to be enantio-enriched because of their natural origin [1]. Broadband rotational spectroscopy offers unparalleled features that make it a unique tool to analyze complex molecular mixtures as those present in essential oils. Even structurally similar molecules as diastereoisomers can be easily detected as they would show a different rotational spectrum. Additionally, the combination of the microwave three-wave mixing (M3WM) technique with the broadband capabilities allow one to distinguish enantiomers within a mixture of chiral molecules and to determine their enantiomeric excess in the gas phase. Here, we present recent results on the analysis of two thyme essential oils from Spain obtained from the leaves of Thymus vulgaris and using the above-mentioned techniques. It is important to bear in mind that the chemical composition of the essential oils coming from the same plant species shows variations according to the environment, growth region and cultivation practices. In our case, terpene compositions of the two studied thyme oils change, even coming from the same country. Linalool is mainly present in one of the oils, whereas thymol is present in the other. The analyses of essential oils with these techniques could extent the use of rotational spectroscopy as a chemical analytical tool (a new application still to be explored in more detail). [1] V. A. Shubert, D. Schmitz, C. Pérez, C. Medcraft, A. Krin, S. R. Domingos, D. Patterson, M. Schnell, J. Phys. Chem. Lett. 7 (2016) 341−350.

Chiral analysis was performed on samples of isopulegol and its isomers using chiral tag rotational spectroscopy. Isopulegol, with three chiral centers, has 8 stereoisomers. There are four diastereomers with distinct geometries and the diastereomer ratio can be determined using traditional rotational spectroscopy. To determine the enantiomeric ratio for each diastereomer the chiral tagging method was used to convert these enantiomers into distinguishable diastereomer complexes. Isopulegol was placed into the nozzles of a chirped-pulsed Fourier transform microwave spectrometer and was heated to 323K. The isopulegol was complexed with a 0.1% mixture of propylene oxide in neon as the carrier gas. The measurement methodology for EE determinations is: 1) a 400K average spectrum is measured using the enantiopure S-propylene oxide, 2) the tag is purged by flowing pure neon over the sample and heating, and 3) a 400K average spectrum using racemic propylene oxide is measured. Enantiopure samples of (-)-isopulegol and (+)-isopuelgol were purchased from Sigma Aldrich and used to create standards of 0, 5, 10, 30, 55, 80, and 90 enantiomeric excess of (-)-isopulegol. The calibration curve was fit using a linear expression with zero offset giving a slope of 1.005 ± 0.007 (R² = 0.99935). These results demonstrate that the method has linear performance over the full EE scale. The reference solution with EE=80 was measured in six separate runs to assess reproducibility. The average of the measurements was 80.595% with a standard deviation of 0.274. A sample of isopulegol provided as a mixture of isomers (Alfa Aesar) was analyzed using the chiral tag method. The enantiomeric excess for the two most abundant diastereomers were determined: isopulegol: EE=4.1(4) and neoisopulegol: EE=4.8(4). The similar enantiomeric excess values for these isomers is consistent with the usual production method for isopulegol where the EE of the reagent (citronellal) sets the EE for all four diastereomer products.

Rotational spectroscopy has been extended for use in chiral analysis by both the chiral tagging and three-wave mixing methods. The chiral tagging method uses complex formation between two species in a pulsed jet: the first, a small chiral tag of known absolute configuration and the second, an unknown analyte. The complexing of the tag with the unknown analyte converts the enantiomers to diastereomers. Cedrol ((1S,2R,5S,7R,8R)-2,6,6,8-tetramethyltricyclo[5.3.1.0^1.5]undecan-8-ol, C₁₆H₂₆O, MW 222.37) was chosen as a test molecule for the chiral tagging method to assess the potential for extending the approach to larger molecules. In addition to its large size, cedrol has conformational flexibility from both the internal rotation of the hydroxyl group and a ring pucker coordinate. As the analyte molecules become larger, there is the potential that the difference in the rotational constants of the diastereomer complexes differ by amounts too small to reliably predict by quantum chemistry making difficult to produce a high-confidence assignment of the absolute configuration. The chiral tag measurement of cedrol used propylene oxide as the tag. The estimates of the rotational constants and dipole moments of the complexes were obtained using the B3LYP-D3BJ method and def2TZVP basis set. Six isomers for the cedrol / propylene oxide complex were observed using an enantiopure tag. The challenges in identifying these isomers in the quantum chemistry calculations will be discussed. The agreement between the experimental and theoretical rotational constants was at the  ∼ 1% level and this makes it possible to assign the absolute configuration. The quality of the theoretical structure of the cedrol / propylene oxide complex was assessed by determining the experimental carbon atom framework geometry through 13C isotopic substitution using natural abundance.

Microwave three-wave mixing was demonstrated in 2013 by Patterson, Schnell, and Doyle (D. Patterson, M. Schnell, and J.M Doyle, Nature 497, 475- 478 (2013)) to distinguish a pair of enantiomers by rotational spectroscopy. This is possible because the product of the three electric dipole components in the principal axis system has opposite sign for enantiomers. The three-wave measurement produces a coherent emission signal proportional to the product of the dipole moment components making it possible to distinguish enantiomers through the phase of the signal. The enantiomeric excess (EE) is then proportional to the signal amplitude. Microwave three-wave mixing experiments were performed to quantify the EE and assess the limits of low EE measurements of the molecules propylene oxide, 1,2-propanediol, and isopulegol. Challenges in these measurements will be discussed including the need for low-frequency coherence transfer pulses due to phase matching requirements in generation of the chiral signal. Other measurement issues like the possibility of off-resonance direct excitation of the chiral transition that can limit instrument performance will be described. Lastly, a test of the linearity of the three-wave mixing signals as a function of EE using isopulegol reference mixtures will be presented.

New microwave spectra of two bicyclic monoterpenols have been observed using CP-FTMW spectroscopy in the 6-18 GHz region of the electromagnetic spectrum. These spectra have given insight into the low energy conformers of the two molecules, with at least one conformer of nopol and multiple conformers of myrtenol having been observed. Rotational constants of the assigned structures will be reported and discussed in comparison to theory. The study of these molecules adds to a growing body of work on the spectroscopy of chiral monoterpenes.J. Mol. Spectrosc. 342 (2017) 109-115^, J. Mol. Spectrosc. 336 (2017) 22-28

The analysis of chiral molecules with multiple chiral centers is a challenging problem in analytical chemistry. The goal of the analysis is to determine the fractional composition for each unique stereoisomer. In the most general case, a molecule with N chiral centers will have 2^N distinct stereoisomers. Half of these, 2^N−1, will be molecules with distinct molecular structures (the diastereomers). The diasteormer composition can be analyzed by normal spectroscopy methods because they have distinct spectra. For each diastereomer, there are the two non-superimposable mirror images (the enantiomers) and additional measurement methodology is required to determine the enantiomeric ratio using spectroscopy. Furthermore, in many applications the “unwanted” isomers (diastereomers and/or enantiomers) will be present as low-abundance impurities placing strong demands on the dynamic range of the spectroscopic technique. A commercial sample of (1R)-endo-(+)-Fenchyl alcohol (C10H18O, four stereoisomers) has been analyzed using nuclear magnetic resonance (NMR), infrared (IR), and rotational spectroscopy. The commercial sample has a small amount ( 3%) of the diastereomer as an impurity. The ability to quantitatively identify the diastereomer impurity using quantum chemistry estimates of the NMR, IR, and rotational spectrum parameters will be discussed. The enantiomer analysis uses chiral resolving agents for NMR spectroscopy, vibrational circular dichroism (VCD) for IR spectroscopy, and chiral tag rotational spectroscopy. The ability of these techniques to verify the stereochemistry of the dominant (1R)-endo-(+)-Fenchyl alcohol will be discussed. The ability to identify the enantiomeric excess of fenchyl alcohol and the possibility of performing enantiomer analysis on the low abundance diastereomer using the direct sample without purification will also be presented.

Determining the absolute configuration (AC) of a chiral analyte is a challenging analytical problem. In 2013, Patterson, Schnell, and Doyle showed that molecular rotational spectroscopy can be used to determine AC through the phase of a coherent emission signal generated by special three-wave mixing cycles. An alternate approach to determine AC is to convert enantiomers into diastereomers. Diastereomers possess distinct geometries, and, therefore, produce distinct rotational spectra. The enantiomer-to-diastereomer approach is accomplished in rotational spectroscopy by creating weakly bound complexes of the analyte with an enantiopure chiral tag molecule in a pulsed jet expansion. The AC of cedrol (C₁₅H₂₆O) has previously been determined by rotational spectroscopy using propylene oxide (C₃H₆O) as the tag. The size difference of cedrol relative to propylene oxide results in diastereomer complexes possessing similar rotational constants. The difference in rotational constants between the diastereomer complexes is small enough that it approaches the confidence limit of the computational methods used (B3LYP D3BJ 6-311G++(d,p)). Therefore, full carbon structures of the complexes were required to confidently assign the AC. The current study exploits a structural motif of the complexes to increase confidence in the AC assignment without needing to experimentally determine the carbon structure. To a good approximation, the change in inertia between the diastereomer complexes arises from changing the position of the propylene oxide methyl group. By modifying propylene oxide, the AC of cedrol is determined without observing ¹³C spectra in natural abundance. Two approaches are tested. The first method involves using a propylene oxide derivative with a -CF₃ group instead of a methyl group, which provides a larger inertia difference between the diastereomer complexes. The second approach involves using an isotopically labeled tag. Quantum calculations can predict isotopic shifts in rotational constants to a higher degree of accuracy than the constants themselves, allowing for higher confidence in the AC assignment.