1. Applications of Molecular Rotational Spectroscopy for Chiral Analysis Rotational Spectrum and Carbon Atom Structure of Dihydroartemisinic Acid
Luca Evangelistsi, Lorenzo Spada, Nathan Seifert, and Brooks H. Pate
Department of Chemistry, University of Virginia
Justin Neill and Matt Muckle
BrightSpec, Inc.
Frank Gupton
Virginia Commonwealth University
Hughes-Wilson Stark Modulation Spectrometer
Phys. Rev. 71, 562 (1947)
Commercialized by Hewlett-Packard in 1971

2. 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)

3. Unmet Needs in Analytical Chemistry: Routine Analysis of Molecules with Multiple Chiral Centers
Many traditional pharmaceutical compounds exhibit multiple chiral centers, requiring methods that can at least separate the potential enantiomers and diastereomers from the API. Even more desirable is a method that can separate each of the potential isomeric impurities for accurate quantitation; however, this is rarely accomplished.

4. Chiral Analysis of Isopulegol:
Three chiral centers: 23 isomers
Four diastereomers each with enantiomers
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
Each asymmetric carbon: R or S
Distinct Enantiomer
Geometry Pair
RRR SSS
RRS SSR
RSR SRS
RSS SRR
M06-2X/ g(d,p)
* B2PLYP D G**

5. Comparison with GC Analysis for the Alfa Aesar Sample
Diastereomer Analysis (GC/MS): Sample consumption 1 mg
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)

6. Unmet Needs in Pharmaceutical Process Analytical Chemistry:
Real-time Chiral Analysis for Molecules with Multiple Chiral Centers
Current methods rely on chromatography (HPLC):
Long development times for analytical procedure
Analysis can take min
Prevents the use of flow chemistry production
Estimate cost is $50B/yr

7. Simulated Rotational Spectra as a Function of Molecular Size
Solketal C6H12O3
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, (2015).
Ambroxide C16H28O
Target size for applications
in pharmaceuticals
Cedrol Dimer
A MHz
B MHz
C MHz
A-type rotational spectra; equal dipole moment
SPCAT Simulations at T = 1.5 K

8. 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: ( % of range)
RMS Frequency Error in Fit: 6-10 kHz (~10% of FWHM)
Hamiltonian: Watson Distortable Rotor
(8 parameters)
“Simple rules, not simple patterns” – Automated Spectral
Fitting
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, (2015).

9. Low Cost Production of the Antimalarial Drug Artemisinin (Clinton Foundation)
DHAA
Artemisinic
Acid
Enantiopurity of final product set by reagent by a non-epimerizing asymmetric carbon.
Process Needs:
Determination of diastereomer excess for DHAA.
Real-time reaction product monitoring (< 1min)

10. Chiral Analysis Required is for Diastereomer Ratio Measurements
Measurement Needs: (Frank Gupton, Justin Neill, Matt Muckle)
1) Diastereomer ratio to show better than 90:10 purity
2) Measurement times less than 1 minute
3) Monitoring of unreacted artemisinic acid and over-reduced tetrahydroartemisic acid
4) Detection from a sample matrix that uses ethanol as the solvent
5) Sample concentration is approximately 15 mM (15 nmol/mL)

11. The Diastereomers from Epimerization Are Easily Distinguishable by Rotational Spectroscopy
Dihydroartemisinic Acid
C15H24O2
MW 236
A MHz
B MHz
C MHz
A MHz
B MHz
C MHz
Two conformers observed (approximately a H-atom shift so similar constants)
Theory: HF/ g(d,p)

12. Broadband Molecular Rotational Spectrum of DHAA
(Reduced bandwidth for 13C analysis)

13. Acknowledgements
National Science Foundation (Chemistry, CCI, MRI, I-Corps)
National Radio Astronomy Observatory
VA NC Alliance LSAMP
University of Virginia
Virginia Biosciences Health Research Corporation
BrightSpec, Inc.
David Pratt, Steve Shipman, Bob Field, David Perry, Tom Gallagher
Mike McCarthy, Tony Remijan, Joanna Corby, Phil Jewel, Susanna Widicus-Weaver
Rick Suenram, Frank Lovas, David Plusquellic
Zbyszek Kisiel, George Shields, Berhane Temelso, Jeremy Richardson, Stuart Althorpe, David Wales, Alberto Lesarri, Sean Peebles, Rebecca Peebles, Gamil Guirgis, Jim Durig, Isabelle Kleiner, Bob McKellar, Kevin Lehmann
Frank Gupton
Pate Broadband Rotational Spectroscopy Group
Gordon Brown, Kevin Douglass, Brian Dian, Steve Shipman
Matt Muckle, Justin Neill, Dan Zaleski, Brent Harris, Amanda Steber, Nathan Seifert,
Cristobal Perez, Simon Lobsiger, Luca Evangelisti, Lorenzo Spada, Jonathan Warren

14. Is Rotational Spectroscopy up for the Challenge
Is Rotational Spectroscopy up for the Challenge? Quantitative chiral analysis of molecules with multiple chiral centers performed directly on reaction samples.
Can we identify molecules unambiguously at 500 Da?
Can we develop efficient conformational analysis methods
Distinguishability of conformers and diastereomers
Accuracy of quantum chemistry methods
Ability to resolve isotopologues
Can we achieve sensitivity for high purity measurements (>99.5%)?
Sampling methods for large molecules
Spectral clutter at 1000:1 especially in complex sample matrices
Instruments for low-frequency rotational spectroscopy
Potential for cavity-enhanced instrument for rapid monitoring
Can we meet the challenges of a single instrument for complete chiral analysis?
Diastereomer and enantiomer analysis
Enantiomer analysis in the high enantiopurity limit (ee > 99.5)
Three wave mixing or chiral tagging