International Symposium on Molecular Spectroscopy, 71st Meeting Champaign-Urbana, June 20-24, 2016 Chiral analysis of isopulegol by Fourier Transform Molecular Rotational Spectroscopy Luca Evangelistsi1, Nathan Seifert2, Lorenzo Spada1, Brooks H. Pate Department of Chemistry University of Virginia 1 Dipartmento di Chimica “Giacomo Ciamician” Bologna 2Department of Chemistry, University of Alberta http://faculty.virginia.edu/bpate-lab/

The Problem from the Physical Chemistry Perspective: Rotational Spectroscopy of Single Molecules The Problem from the Physical Chemistry Perspective: Modern computational chemistry produces structures and conformational potential energy surfaces that provide accurate estimates of the spectroscopic constants and relative energies. Is there a reason to perform the measurement? The Dream of Analytical Chemistry: A rapid spectroscopy technique where molecules can be unambiguously identified (especially in a mixture) by direct comparison of experimental spectroscopic constants to theoretical estimates. Library-free detection (the ability to perform analysis without a previously prepared, high purity reference sample)

Rotational Spectroscopy in Analytical Chemistry Can’t compete with “gold standard” methods that use mass spectrometry/ion detection: GC/MS (HPLC methods) Reduction in the cost of consumables and technical expertise required Samples that can’t use chromatography (reactive) – direct mixture analysis Monitoring applications requiring “real-time” quantification (~1 min) Analysis of isomers Isotopologues: Site-specific stable isotope analysis Chiral Analysis: Diastereomers and Enantiomers Isomer ratios are required Major Strength: Potential for Library-Free Analysis (Molecule identification by theory alone)

Chiral Analysis for Molecules with Multiple Chiral Centers 1) Quantitative Analysis of Diastereomers Resolution for molecules with multiple chiral centers 2) Absolute Configuration Determination of left-handed or right-handed form 3) Enantiomeric Excess Measurements Internal calibration for accurate, linear ee measurement Dihydroartemisinic Acid Image Credit: http://doktori.bme.hu/bme_palyazat/2013/honlap/Bagi_Peter_en.htm R S

Chiral Analysis of Isopulegol: Three chiral centers: 23 isomers Four diastereomers each with enantiomers Goal: 99.5% purity levels (regulatory) Isopulegol Diasteromers: OH Iso CH3 E(cm-1)* Isopulegol: Eq Eq Eq 0 Neoisopulegol: Ax Eq Eq 197 Isoisopulegol: Eq Eq Ax 550 Neoisoisopulegol: Ax Eq Ax 971 * B2PLYP D3 6-311++G** M06-2X/6-311++g(d,p)

Low Frequency (2-8 GHz) Chirped-Pulse Fourier Transform Microwave Spectrometer General Spectral Properties: Measurement Bandwidth: 6000 MHz FWHM Resolution: 60 kHz (105 data channels) Transitions in a Spectrum: 20-200 (0.02-0.2% of range) RMS Frequency Error in Fit: 6-10 kHz (~10% of FWHM) AUTOFIT C. Perez, S. Lobsiger, N. A. Seifert, D. P. Zaleski, B. Temelso, G.C. Shields, Z. Kisiel, B. H. Pate, Chem. Phys. Lett. 571, 1 (2013). N.A. Seifert et al., “Autofit, an Automated Fitting Tool for Broadband Rotational Spectra, and Applications to 1-Hexanal”, J. Mol. Spectrosc. 312, 13-21 (2015).

Isopulegol 5% of bandwidth Isopulegol has three chiral centers with four diastereomers. Isopulegol is an intermediate in the synthesis of menthol which is produced at 3,000 tons per year. A single diastereomer is needed because only a single diastereomer of menthol has the desired flavor. Ryōji Noyori shared the 2001 Nobel Prize in Chemistry for the stereoselective synthesis of menthol (94% ee). 5% of bandwidth Carlos Kleber Z. Andrade*, Otilie E. Vercillo, Juliana P. Rodrigues and Denise P. Silveira, J. Braz. Chem. Soc., 15, 813-817, 2004.

Identification of Conformers and Diastereomers: Computational Chemistry and Structure Determination Species Theory Experiment Percent Error Isopulegol 1948.4 1949.96485(299) 0.08   700.5 700.16883(113) -0.04 591.1 591.63903(111) -0.08 Neo isopulegol 2136.0 2138.08622(207) 0.10 675.2 676.60098(76) 0.21 626.8 625.97792(73) -0.13 Neo isoisopulegol 1706.2 1713.8188 0.44 783.3 779.69047 -0.46 674.2 672.63379 -0.23 Iso isopulegol 1941.5 1942.46933 0.05 746.1 747.16774 0.14 706.7 704.69217 -0.28 Theory: B2PLYP D3 6-311++G** Structure Determination using 13C and 18O Isotopologue Spectra Directly Confirms Conformer and Diastereomer Geometry Isopropenyl Dihedral Angle Hydroxyl Dihedral Angle

Diastereomer Ratio in Aldrich Sample Molecule identification is made through high accuracy matching of the rotational transition frequencies – relative intensity information is quantitative but less accurate. A rich rotational spectrum is produced through a small set of spectroscopic parameters giving many detection windows. Because the measurement is made under ideal gas conditions, the spectroscopic signature is independent of the sample matrix. 0.33% of bandwidth Spectrum Simulation for Neoisopulegol at 0.1% Diastereomer Abundance

Physical Principles for FT-MRR Analysis of Enantiomers D. Patterson, M. Schnell, and J.M Doyle, Nature 497, 475- 478 (2013). D. Patterson and J.M. Doyle, Phys. Rev. Lett. 111, 023008 (2013). The Sign of the Product of the Dipole Moment Vector Components is Unique to an Enantiomer J.U. Grabow, Angew. Chem. 52, 11698 (2013). V.A Shubert, D. Schmitz, D. Patterson, J.M Doyle, and M. Schnell, Angew. Chem. 52, (2013). The Excitation Pulses and Chiral Signal have Mutual Orthogonal Polarization Energy 000 110 101 µa : 2307.52 MHz µb : 1673.842 MHz µc : 3981.36 MHz Simon Lobsiger, Cristobal Perez, Luca Evangelisti, Kevin K. Lehmann, Brooks H. Pate, “Molecular Structure and Chirality Detection by Fourier Transform Microwave Spectroscopy”, J. Phys. Chem. Lett. 6, 196-200 (2015).

Three Wave Mixing Measurements 303 404 414 5101.1 MHz 5945.4 MHz 844.3 MHz μ a μ b μc Optimal Pulse Sequence: π/2 - π   Simon Lobsiger, Cristobal Perez, Luca Evangelisti, Kevin K. Lehmann, Brooks H. Pate, “Molecular Structure and Chirality Detection by Fourier Transform Microwave Spectroscopy”, J. Phys. Chem. Lett. 6, 196-200 (2015).

Features of Microwave Three Wave Mixing Spectroscopy The chiral signal has an amplitude comparable to regular rotational transitions. The chiral signal is background free. The chiral signal is triply resonant and compatible with complex sample mixtures. Actual digitized data points are shown along with sine wave fit. Simon Lobsiger, Cristobal Perez, Luca Evangelisti, Kevin K. Lehmann, Brooks H. Pate, “Molecular Structure and Chirality Detection by Fourier Transform Microwave Spectroscopy”, J. Phys. Chem. Lett. 6, 196-200 (2015).

Normalized Enantiomeric Excess Measurements Three Wave Mixing Measurements on Several Isopulegol Samples Optimal Pulse Sequence: π/2 - π 𝑒𝑒= ( 𝑁 𝑅 − 𝑁 𝑆 ) ( 𝑁 𝑅 + 𝑁 𝑆 ) 303 404 414 5101.1 MHz 5945.4 MHz 844.3 MHz Normalized ee Measurement Results Aldrich Analytical Reference Sample: 1.4437 (Certificate of Analysis: 99.9: 0.1) Aldrich Sample: 1.4393 99.6 : 0.4 Alfa Aesar Sample: 0.1937 56.7 : 43.3

Comparison with GC Analysis for the Alfa Aesar Sample Diastereomer Analysis (GC/MS): Sample consumption 1µg GC/MS CP-FTMW* Isopulegol 66% 60% Neoisopulegol 23% 33% Isoisopulegol 6% 4% Neoisoisopulegol 4% 3% enantiomer Analysis Chiral GC: ~50:50 (not fully resolved) Three Wave Mixing: 57(-):43(+) (Isopulegol only) Retention Time (min) * Different lot number; similar CoA Retention Time (min)

Conclusions BROOKS PATE  WK04 Spectroscopy expected to allow confident structural analysis for molecules up to 500 Da even for direct analysis of samples that are complex mixtures Instruments for broadband FT-MRR spectroscopy can approach 10,000:1 dynamic range in a measurement (12 hours, ~200 mg) but require advances in source technology for volatilizing solids Analytical chemistry is still in early stage of development with technique advances and measurement validation needed in phase calibration (absolute configuration), quantitative ee measurements, pulsed jet characterization (clusters), and spectroscopic library development BROOKS PATE  WK04 DHAA

Acknowledgements Pate Group BrightSpec NSF MC-IOF 328405 RSC