FT-MRR Analysis for Continuous Manufacturing Diastereomer Analysis Isopulegol Experiment Enantiomeric Excess Analysis

Application of Fourier Transform Molecular Rotational Resonance Spectroscopy for Rapid Chiral Purity Characterization

Matt Muckle, Justin Neill, BrightSpec, Inc., Charlottesville, VA Brooks H. Pate, Department of Chemistry, University of Virginia Introduction Chiral analysis of molecules with multiple chiral centers requires identifying the diastereomers and measuring their enantiomeric excess (ee). Molecular rotational spectroscopy can perform both of these analyses, without chromatographic separation.* This work examines the ability of rotational spectroscopy to identify diastereomers, conformational isomers, and enantiomers for measurements with a dynamic range of 1000 or more using quantum chemistry estimates of the molecular structure and dipole moment. *David Patterson, Melanie Schnell, John M. Doyle, “Enantiomer-specific detection of chiral molecules via microwave spectroscopy”, Nature 497, 475 (2013). Isopulegol Experiment

• Spectra measured using broadband chirped pulse excitation to measure • Chiral intermediate in the synthesis of menthol 6 GHz of instantaneous bandwidth • 3000 tons of menthol are produced per year • A single diastereomer is responsible for flavor • Broad composition analysis – for • Three Chiral centers (8 total isomers) characterizing new species, unknown identification

• Typical spectrum at >1000:1 S/N (99.9% diastereomer purity) in ~12 C. Perez, S. Lobsiger, N. A. Seifert, D. P. Zaleski, B. hours Diastereomer Analysis Temelso, G.C. Shields, Z. Kisiel, B. H. Pate, Chem. Phys. Enantiomeric Excess Analysis Lett. 571, 1 (2013).

Spectra from two different sources Enantiomer Analysis Results The Alfa isopulegol sample was Three Wave Mixing Level (Alfa Aesar & Sigma Aldrich) were Diagram 414 measured using FT-MRR analyzed by three wave mixing FT- 844.3 MHz Chiral GC: ~50:50 (not fully resolved) spectroscopy. The resulting MRR. The measurement produced a 404 spectra were matched to spectra signal that was proportional to the FT-MRR Three Wave Mixing: 57(-):43(+) sum of the abundance of both chiral 5101.1 MHz predicted by theory to confirm Three Wave Mixing Measurements on Several Isopulegol Samples isomers and a signal that was their identity. Relative line GC/MS FT-MRR* intensities (experimental) are used proportional to the difference in the 5945.4 MHz abundance of the isomers. The ratio to determine the abundance of 303 Isopulegol 66% 60% of the two signals yielded the % EE in each diastereomer. Results for Alfa Neoisopulegol 23% 33% the sample (right). (푁 −푁 ) sample were compared to GC/MS Isoisopulegol 6% 4% 푒푒 = 푅 푆 x 100% (푁푅+푁푆) results. Neoisoisopulegol 4% 3%

* Different lot number; similar CoA FT-MRR Analysis for Continuous Manufacturing

Current methods rely on chromatography (HPLC): Artemisinin: A Model for Rapid Analysis 1) Long method development times A continuous flow synthesis for artemisinin (a vital anti- 2) Analysis can take 10-30 min malarial) starting from biologically synthesized artemesinic 3) Prevents the use in flow chemistry production acid has been developed by the Frank Gupton group at VCU. 4) Estimated cost is $50B/yr FT-MRR will be used to monitor, in real time, the isomeric FT-MRR Method: purity of a stable intermediate, dihydroartemisinic acid, and ultimately of artemisinin. 1) Shorter method development times • Consistent sampling methods • No columns to develop • Requires a single “master” broadband experiment • Requires simple quantum chemistry calculations 2) Measurements < 5 min 3) Allows constant rapid sampling for continuous FTMRR measurements (right) have been made to determine manufacturing candidate lines for DHAA analysis and characterize detection limits.

Roberts, Leslie. “Malaria wars” Science 22 Apr 2016: Vol. 352, Issue 6284, pp. 398-405 A new cavity-enhanced FT-MRR instrument is currently under Acknowledgements: DOI: 10.1126/science.352.6284.398 development to directly interface with the continuous flow This work supported by NSF MRI CHE-0960074, University of Bologna (LS), and ERS/EU Marie Curie process at VCU. This instrument will reduce analysis times to Postdoctoral Research Fellowship Program (LE), Virginia Biosciences Health Research Cooperation minutes in order to give live feedback on reaction progress and selectivity.