Chiral tag rotational spectroscopy can be used for quantitative determination of the ratio of the two enantiomers of a chiral molecule. The strategy for chiral tag rotational spectroscopy is to convert the enantiomers of the analyte into diastereomers through non-covalent attachment of a small, chiral tag molecule. The analyte enantiomer ratio, which is used to determine the enantiomeric excess (EE), is determined by comparing the transition intensities of rotational transitions for the homochiral and heterochiral complexes when both a racemic and enantiopure tag sample is used. A calibration curve for EE determination of 3-methylcyclohexanone tagged with 3-butyn-2-ol will be presented. The role that intensity fluctuations in back-to-back measurements of the rotational spectra of the chiral tag complexes play in determining the EE measurement accuracy will be described. In applications to pharmaceutical chemistry the main need is the ability make quantitative EE determinations in the high enantiopurity limit of the analyte. This requirement poses challenges for chiral tag rotational spectroscopy from both the measurement sensitivity and the availability of high enantiopurity tag samples. Two analysis methods for high EE measurements will be discussed. In one case, the enantioimpurity detection limit is decreased by the co-adding of multiple rotational transitions of the homochiral and heterochiral tag complex. The second strategy uses a lower enantioimpurity tag to speed the EE determination of high enantioimpurity samples. In this case, the ability to accurately determine the tag EE is crucial and the functional dependence of EE measurement precision in chiral tag rotational spectroscopy provides the limit on measurement accuracy that can be achieved.

The enantiomeric excess (EE) of a chiral sample can be determined from molecular clusters of the analyte and a chiral tag in a pulsed-jet expansion. The complexation converts analyte enantiomers into rotationally distinct diastereomer complexes. The EE is determined by comparing rotational transition intensities resulting from diastereomer complexes when a racemic tag is used and when an enantiopure tag is used. For a pair of diastereomer transitions, the connection between the normalized intensity ratio (R) and the analyte EE is: (R-1)/(R+1) = (EEanalyte)(EEtag). Therefore, accurately determining an analyte EE requires an accurate “enantiopure” tag EE. The EE calibration of three frequently used tags (3-butyn-2-ol, propylene oxide, and 2-(trifluoromethyl)oxirane) is described. For butynol, EE determinations are possible using homochiral and heterochiral dimers, known as “auto tagging.” Optimal sensitivity is achieved between 6-18 GHz due to the size of the dimers. A pulse generation system utilizing a frequency doubler with low harmonic generation is employed to improve spurious signal performance compared to schemes requiring an external reference clock. Several butynol dimer isomers are formed in the jet expansion, and EE determinations are equivalent regardless of the isomers used in the analysis. Determination of EE by auto tagging was validated by chiral GC. Also, a series of butynol samples with EE between 50 and 90 were prepared gravimetrically by mixing high enantiopurity butynol (EE = 98% by chiral GC and auto tag) with racemic butynol. Chiral tagging measured the EEs in this range with greater accuracy than chiral GC. The EEs of propylene oxide and 2-(trifluoromethyl)oxirane were determined by tagging with the calibrated butynol. Neither molecule reports an EE in their Certificate of Analysis, possibly because their high volatility precludes separation by chiral GC. To validate the EE determinations, both tags were used to measure the EE of an analyte and were found to be equivalent within measurement accuracy.

Microwave three-wave mixing is a rotational spectroscopy technique that can measure the difference between a pair or enantiomers and quantify a ratio between them where traditional rotational spectroscopy cannot. The measurement principle was shown in 2013 by Patterson, Schnell, and Doyle using a DC field to achieve the necessary state mixing [1] and was extended by Patterson and Doyle later that year to the three-wave mixing approach most commonly used now [2]. Grabow, also in 2013, has provided a description of the measurement in the intuitive language of NMR spectroscopy [3]. Despite the introduction of the technique in 2013, there has been little work to develop the method into a quantitative analytical chemistry tool. In this talk we focus on using this method for enantiomeric excess (EE) determinations. Because the chiral signal produced in this technique is proportional to the difference in the enantiomer populations (a value that is proportional to the total amount of sample present), it is necessary to reference the amplitude of the chiral signal to a quantity proportional to the total sample concentration – available from a “normal” rotational signal measurement. Furthermore, the method requires a sample of known EE for scale calibration – a significant limitation in analytical chemistry. The results from two measurement pulse sequences that provides total population calibration will be presented. Calibration curves for samples that cover a range of EE (gravimetrically calibrated) will be presented for isopulegol and menthone. The ability to obtain accurate EE determinations in a simple mixture using the calibrated measurement scheme is tested using two commercial samples of isopulegol that contain all eight stereoisomers produced in the citronellal cyclization reaction. The three-wave mixing technique can provide EE accuracy to about 5%, and this performance, especially for high enantiopurity samples, limits its applicability in many industry applications. [1] D. Patterson, M. Schnell, and J.M Doyle, “Enantiomer-specific detection of chiral molecules via microwave spectroscopy”, Nature 497, 475- 478 (2013). [2] D. Patterson and J.M. Doyle, “Sensitive Chiral Analysis via Microwave Three-Wave Mixing”, Phys. Rev. Lett. 111, 023008 (2013). [3] J. Grabow, “Fourier Transform Microwave Spectroscopy: Handedness Caught by Rotational Coherence”, Angew. Chem. Int. Ed. 52, 11698-11700 (2013).

The method of microwave three-wave mixing can be used to perform chiral analysis by molecular rotational spectroscopy.[1-3] We have developed pulse sequences that can be used to determine the enantiomeric excess (EE) of a component that is present in a complex mixture. To make an EE determination, the measurement needs both a chiral three-wave mixing measurement and a signal that is proportional to the total amount of the molecules. The measurement provides both the EE and the identity of the dominant enantiomer (Based on the phase). Furthermore, a sample of known EE must be available to generate a calibration curve. Validation measurements of the technique indicate that EE determinations with about 5% accuracy are possible using this technique. The major strength of three-wave mixing is its ability to perform EE measurements directly in complex mixtures. Unlike chiral tag rotational spectroscopy, three-wave mixing does not add to spectral congestion resulting from the formation of isomers with the chiral tag. The performance of the calibrated EE schemes presented previously at ISMS [4] and expanded in the previous talk will be presented for EE determinations of molecular components in essential oils. Primary validation comes from spiking a new chiral molecule with known EE into an essential oil. EE determinations of menthone and isomenthone in a series of commercial samples that have also been analyzed by the chiral tag method will be reported. These measurements show that both menthone and isomenthone are found in high enantiopurity in natural samples. [1] D. Patterson, M. Schnell, and J.M Doyle, “Enantiomer-specific detection of chiral molecules via microwave spectroscopy”, Nature 497, 475- 478 (2013). [2] D. Patterson and J.M. Doyle, “Sensitive Chiral Analysis via Microwave Three-Wave Mixing”, Phys. Rev. Lett. 111, 023008 (2013). [3]J. Grabow, “Fourier Transform Microwave Spectroscopy: Handedness Caught by Rotational Coherence”, Angew. Chem. Int. Ed. 52, 11698-11700 (2013). [4] M.S. Holdren, B. Pate, C. Embly, A. Wu, K.J. Mayer, J. Dittman, P. Buonicotti, G. Haghtalab, B. Mitchell, “Enantiomeric Excess Measurements using Microwave Three-Wave Mixing”, Talk TC08, ISMS 73rd Meeting Archive, https://dx.doi.org/10.15278/isms.2018.TC08.

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 ¹³C-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.

Molecular rotational spectroscopy is well suited for the analysis of regioisomers, which has applications in pharmaceutical synthesis. Rotational spectroscopy offers important advantages over other chemical analysis techniques resulting from extreme sensitivity to molecular mass distribution, high accuracy molecular structure (and, therefore, rotational constant) predictions from quantum calculations, and the combination of high-spectral resolution and dynamic range. The sensitivity and range of the technique allow low abundance regioisomer impurities to be quantitatively measured without chemical separation. The challenge for rotational spectroscopy is that actual measurement challenges in pharmaceutical chemistry involve molecules in the 200-500 Da mass range. Unique features of the rotational kinetic energy levels lead to decreasing peak transition strength as the molecular size increases as well as a decrease in the frequency where the strongest transitions occur. However, special features of the high-J limit (semiclassical limit) of the rigid rotor Hamiltonian potentially mitigate these experimental difficulties and suggest that rotational spectroscopy can be applied to much larger molecules than previously expected. The rotational spectra of a series of high vapor pressure large molecules in both the limiting prolate and oblate limits are analyzed. For the prospect of analyzing larger molecules, the prolate limit appears to offer major advantages. However, experiments using pulsed-jet sources show rapid relaxation of these high-energy rotational levels and, at present, appear to reduce the major advantages suggested by the rotational spectroscopy in the high-J limit.

Homochirality is omnipresent in nature on Earth in which life predominately utilizes one handedness of a chiral molecule over another. It is considered a biomarker that can aid in the search for life elsewhere in the solar system on places like Mars, Titan, Europa, and Enceladus. The 2013 planetary science Decadal Survey recommends “a detailed characterization of organics to search for signatures of biological origin, such as molecules with preferred chirality or unusual patterns of molecular weights” as a key future investigation for determining the possibility of life beyond Earth. Mass spectrometers are the primary choice for chemical detection and identification of simple organics for planetary and astrobiology investigations. However, mass spectrometry alone cannot address the challenge of successfully deconvolving mixtures of structurally complex organic molecules of approximately the same molecular weight; this includes lacking the chirality detection capability required for analysis of chiral molecules. ChiralSpec can provide synergetic measurements to mass spectrometers for planetary science and is funded by the NASA Planetary Instrument Concept for the Advancement of Solar System Observations (PICASSO) program. ChiralSpec is a millimeter-wave spectrometer operated in two modes: (1) chirality detection mode, based on a novel three-wave mixing; and (2) survey mode, with the instrument acting as a traditional millimeter-wave spectrometer to characterize chemical composition and quantify abundance of planetary samples. ChiralSpec extends the work done on microwave three-wave mixing into higher frequencies of light where size, weight, and power of many components of the instrument can be reduced. We will report on the current state of this instrument and its future developments. G-band (180-200GHz) and W-band (70-90GHz) excitation channels have been designed, tested, and optimized to show that power requirements are met and that molecular emission can be detected for many two level systems for a test case molecule, propylene oxide. Three-wave mixing experiments are on-going, and we will report our findings.

Over half of pharmaceuticals, both among the top 100 drugs by prescription totals and new U.S. Food and Drug Administration (FDA) approvals, contain at least one chiral center. Moreover, most new chiral pharmaceuticals are synthesized as a single isomer. Therefore, it is important to be able to determine the primary isomer generated by a synthetic process as well as the presence of any other isomers - preferably directly on the intermediate compounds where each chiral center has been introduced. Molecular rotational spectroscopy, with its sensitivity to small changes in structure and ability to identify compounds directly from electronic structure theory, can be a powerful tool in this application. The present study concerns dolutegravir, an HIV integrase inhibitor developed by GlaxoSmithKline and approved by the FDA in 2013. Efforts are ongoing at the Medicines for All institute in Richmond, Virginia to develop a stereoselective flow synthesis for dolutegravir to reduce its cost and increase availability.R.E. Ziegler, B.K. Desai, J.-A. Jee, B.F. Gupton, T.D. Roper, and T.F. Jamison, Angew. Chem. Int. Ed., 2018, 130, 7299-7303.s part of a new route development, an intermediate with two chiral centers was assessed by rotational spectroscopy to determine which diastereomer was the predominant one formed by the process. Notably, NMR was unable to conclusively determine this, but rotational spectroscopy unambiguously determined that the synthetic route produced the correct stereochemistry. This result suggests that rotational spectroscopy can be a useful complement to other analytical characterization methods in organic process development.

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Footnotes:

R.E. Ziegler, B.K. Desai, J.-A. Jee, B.F. Gupton, T.D. Roper, and T.F. Jamison, Angew. Chem. Int. Ed., 2018, 130, 7299-7303.A

We report on our recent research on applying Terahertz molecular sensing to quantitative analysis of human breath. A recently developed tabletop THz gas sensor has been demonstrated to detect a range of breath volatiles at a part per billion/trillion level of dilution. In a recent project we developed several statistical models of fatigue based on THz analyses of expired human breath. Breath of ten subjects was sampled over the course of a 40-hour sleep deprivation study performed by Navy Medical Research Unit – Dayton (NAMRU-D) at Wright Patterson Air Force Base. The breath-fatigue models presented here predict the reaction times measured by Psychomotor Vigilance Task test along the timeline of sleep deprivation study. This promising application of THz gas sensing hold a lot of potential for a range of civilian and military applications.

Quantitative analytical gas sampling is of great importance in a range of environmental, safety, and scientific application. In this article we present the design, operation, and performance of a recently developed table top Terahertz spectroscopic molecular sensor capable of rapid (minutes) and sensitive (part per trillion level of dilution) detection of a wide range of gaseous analytes with ‘absolute’ specificity. The technique presented in this paper excels at detecting light polar volatile compounds which often challenge the capabilities of competing gas sensing techniques.