1. A CHIRAL TAGGING STRATEGY FOR DETERMINING ABSOLUTE CONFIGURATION AND ENANTIOMERIC EXCESS BY MOLECULAR ROTATIONAL SPECTROSCOPY
Luca Evangelisti and Walther Caminati
Dipartmento di Chimica “Giacomo Ciamician”, Universita Di Bologna
David Patterson
Department of Physics, Harvard University
Yunjie Xu and Javix Thomas
Department of Chemistry, University of Alberta
Channing West and Brooks H. Pate
Department of Chemistry, University of Virginia
ISMS 2017 RG03

2. Acknowledgements
This work supported by the National Science Foundation (CHE ) and
The Virginia Biosciences Health Research Corporation (Frank Gupton: VCU, Justin Neill: BrightSpec)
Special thanks for work on chiral tag rotational spectroscopy:
Luca Evangelisti
Nathan Seifert, Lorenzo Spada
Dave Patterson, Walther Caminati, Yunjie Xu, Javix Thomas, David Pratt, Smitty Grubbs, Galen Sedo, Mark Marshall, Helen Leung, Kevin Lehmann, Justin Neill
Frank Marshall, Marty Holdren, Kevin Mayer, Taylor Smart, Reilly Sonstrom, Channing West
Ellie Coles, Elizabeth Franck, John Gordon, Julia Kuno, Pierce Eggan, Victoria Kim, Ethan Wood, Megan Yu

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: The Search For a Universal Tool
For “N” chiral centers
2N isomers
2N-1 unique diastereomers
2 enantiomers per diastereomer
Molecules with multiple chiral centers pose an issue for current techniques
Image Credit:
Enantiomers: Mirror images of each other that are not superimposable and have opposite configurations at their stereocenters
Diastereomers: Distinct compounds that have different configurations at one or more, but not all of the stereocenters
The idea of chiral analysis is not a new one in chemistry, and it is still a prevalent problem today. The issue comes from the ability to detect all stereoisomers of a chiral molecule. Diastereomers are molecules that have different configurations at one or more, but not all of their stereocenters, while enantiomers are molecules that are mirror images of each other and have the opposite configurations at their stereocenters. A chiral molecule that has N chiral centers has 2 to the N isomers with 2 to the N-1 being unique diastereomers, each of those diastereomers has an enantiomer. As the number of chiral centers increases the number of isomers increases exponentially increasing the difficulty to detect them all. For example the cyclization of Citronellal which has a total of 3 stereocenters has 8 isomers, 4 unique diastereomers. Which means you have to also be able to detect the 4 other enantiomers. The main issue is being able to detect these enantiomers from each other as there are already current methods for detecting diastereomers. The ultimate goal for universal chiral analysis would be the ability to use a single technique on multiple chiral centers for full analysis with high throughput screening, that gives the ratio of enantiomers and diastereomers and can determine the absolute configuration of a high enantiopurity sample.
Cyclization of citronellal has 3 final stereocenters
8 total forms with 4 distinct structures
Fractional abundance of all 8 forms
A single diasteriomer is wanted for the desired flavor
Isopulegol is an intermediate to menthol which is produced at 3,000 tons per year
Absolute configuration
Fractional abundance
Universally applicable
Rapid analysis
Handle multiple chiral centers
Minimal consumption of sample
Identify all enantiomers, diastereomers, conformers, and isotopologues
Analysis from reaction mixture
High speeds, optimally flow chemistry
EE of reagents show up in products
Quantitative ratio for each of the isomers
-Synthesis of isopulegol is an intermediate step to making menthol (3000 tons per year in production).
-There are 3 chiral centers making 8 isomers with 4 distinct diastereomers with each having its own enantiomer
-In this case, 1 chiral center is locked in making 4 distinct structures with 2 pairs of enantiomers
-In the synthesis, only one diastereomer is sought after for the desired menthol flavor/effects
-”Ryōji Noyori shared the 2001 Nobel Prize in Chemistry for the stereoselective synthesis of menthol (94% ee)”.
Distinct geometries/diastereomers yield distinct rotational spectra, but enantiomers/mirror images yield same rotational spectra similar to seeing the shadow of a hand but not knowing if its right or left
Other examples of good uses: Flow chemistry control, asymmetric catalyst screening
Need for universally applicable chiral analysis methods
Quantitative ratios of all stereoisomers
Complex mixture analysis
Rapid monitoring

5. Rotational Spectroscopy for Chiral Analysis: Three Wave Mixing for Enantiomers
The sign of the product of dipole vector components are opposite for enantiomers
D. Patterson, M. Schnell, and J.M Doyle, Nature 497, (2013).
D. Patterson and J.M. Doyle, Phys. Rev. Lett. 111, (2013).
J.U. Grabow, Angew. Chem. 52, (2013).
V.A Shubert, D. Schmitz, D. Patterson, J.M Doyle, and M. Schnell, Angew. Chem. 52, (2013).
mambmc(-)
mambmc(+)
Identification of enantiomers by traditional rotational spectroscopy is impossible due to the enantiomers having the mass distribution and overall dipole. However with the recent findings of Hirota, Patterson, Schnell and Doyle the ability to detect the different enantiomers. This relies on the fact that the sign product of the dipole vector components are opposite for enantiomers. As you can see mew a and b are equal and in the same direction however mew c is in the opposite direction. This provides researchers with a physical means of measuring left or right handedness. This is performed using mutually orthogonal polarized excitation corresponding to mew a, mew b and mew c. We detect the FID that is perpendicular to both excitation pulses, which is the chiral signal. The phase of that signal corresponds to the absolute configuration while the amplitude corresponds to the Enantiomeric excess. mb
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).

6. Determination of Absolute Configuration by Chiral Tag Rotational Spectroscopy: Enantiomers-to-Diastereomers
Enantiomers of molecules have identical rotational spectra
Complexes of enantiomers with an enantiopure “chiral tag” form diastereomers that have different rotational spectra
Heterochiral Complex
A = MHz ma = 3.3 D
B = MHz mb = 1.6 D
C = MHz mc = 0.9 D
Homochiral Complex
A = MHz ma = 3.1 D
B = MHz mb = -1.9 D
C = MHz mc = D
Lowest Energy Isomers: B3LYP D3BJ def2TZVP

7. Determination of Enantiomeric Excess using Chiral Tag
Enantiopure Chiral Tag ((S)-3-butyn-2-ol)
Analogy to Chromatography
Heterochiral Spectrum
Homochiral Spectrum
Different enantiomers gives signals in distinct detection windows
Background free detection permits determination of high EE when peaks are highly resolved
Rotational spectroscopy can be used to identify which enantiomer gives each signal
Rotational spectroscopy has the potential for significant decreases in analysis time
(Chiral GC Example: 30 min)
Enantiomer populations converted to different
diastereomers with distinct spectra

8. Rotational Spectroscopy for Chiral Analysis: Diastereomers
Chirped-Pulse FTMW Spectroscopy
Extreme sensitivity to changes in mass distribution
Agreement with Theory: “Library-Free” Diastereomer Identification
Low Frequency (2-8 GHz): Peak Transition Intensity of Large Molecules
High Resolution + Broadband Coverage: Mixture Analysis
Rotational spectroscopy is great for diastereomers because it is able to detect subtle changes in mass distribution with high accuracy added to the fact that diastereomers have unique rotational spectra. In order to detect the diastereomers we utilize a chirped pulse Fourier transform spectrometer. This system uses multiple nozzles, up to 5, which increases the sensitivity of the broadband signals while reducing sample consumption and measurement time. We measure the diastereomers at a lower frequency, 2-8 GHZ, this is good for larger molecules due to the peak intensity for larger molecules and complexes shifting to lower frequencies. It provides high resolution which is good for mixture analysis, the quantum chemistry calculations are highly accurate which makes it a library free identification system as you can match up the experimental spectra with the quantum calculations. An added improvement over other techniques is that it has higher sensitivity to the mass distribution of the molecules.
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).

9. Isopulegol Nomenclature for Stereoisomers RSS: Methyl: R Hydroxyl: S
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).
RSS: Methyl: R
Hydroxyl: S
Isopropenyl: S
(-)-isopulegol: RRS
(+)-isopulegol: SSR
(+)-neoisopulegol: RSS
(-)-neoisopulegol: SRR
Cyclohexyl Ring Conformation:
Isopropenyl in the equatorial position is the most stable chair isomer
Isopulegol and neoisopulegol allow isopropenyl and methyl groups to be equatorial.
RSS
RRS
RSR
RRR
Carlos Kleber Z. Andrade*, Otilie E. Vercillo, Juliana P. Rodrigues and Denise P. Silveira, J. Braz. Chem. Soc., 15, , 2004.

10. Analysis of TCI America Sample
Optical Rotation of Enantiopure (-)-isopulegol: -22o (neat)
(Reference Optical Rotation from Aldrich CoA: Chiral GC + Optical Rotation)
At 94% pure isopulegol, but with a -6.1o specific rotation, the expected enantiomeric excess is ~30% with assumption of achiral impurities.
Pierce Eggan, Victoria Kim, Ethan Wood, Megan Yu, Luca Evangelisti

11. Measurement Methodology
Broadband Rotational Spectrum of the Monomer
Used for diastereomer analysis
Broadband Rotational Spectrum with Racemic Tag: Propylene Oxide
Forms both homochiral and heterochiral complexes
Isolate complexes by cutting the monomer spectrum
Broadband Rotational Spectrum with Enantiopure Tag
Forms either homochiral or heterochiral complex
Isolate one set of complexes by cutting the monomer spectrum
Isolate the other set by cutting this spectrum from the racemic tag

12. Is (-)-isopulegol (RRS) the predominant enantiomer?
Spectroscopic Analysis of Complex with (S)-propylene oxide:
Assessment of Absolute Configuration of Isopulegol from Spectroscopy:
Experiment: A = MHz Theory RRS: A = MHz (-2.4%) Theory SSR: A = MHz
B = MHz B = MHz (-0.6%) B = MHz
C = MHz C = MHz (-1.1%) C = MHz
(-0.1%)
(-1.4%)
(-1.1%)
B3LYP D3BJ def2TZVP

13. Is (-)-isopulegol (RRS) the predominant enantiomer?
High Confidence Determination of Absolute Configuration from Substitution Structure
(Analogous to Internal Chiral Reference X-ray Crystallography)
Experiment is known to use
(S)-propylene oxide as the tag
Structure of RRS Isopulegol with Correct Stereochemistry

14. What is the EE for (-)-isopulegol?
The expectation from the Certificate of Analysis is an EE of 35% (assumes pure isopulegol)
Uses 25 transitions for each complex
EE=74.7%
75% EE Reference Sample from Enantiopure
(-)-isopulegol and Racemic isopulegol mixture
EE=74.6%
(S)-Propylene Oxide: (eeTag) = 0.998

15. Is there an impurity that could be affecting the optical rotation measurement?
(+) – Neoisopulegol is formed in this reaction with a specific rotation of +36o
Identification of Neoisopulegol by Comparison to Quantum Chemistry Rotational Constants
Species
Theory
Experiment
Percent Error
Isopulegol
1948.4
(299)
-0.39
700.5
(113)
-0.81
591.1
(111)
-0.23
Neo isopulegol
2151.4
(207)
-0.62
678.9
(76)
-0.34
630.0
(73)
-0.64

16. Are the chiral properties of the stereoisomer impurity consistent with the (presumed) reaction chemistry?
RRS
RSS
RRS
EE=74.7%
EE of reaction products set by the EE of the citronellal reagent.
RSS
EE=74.1%
Assessment of Absolute Configuration of Neoisopulegol from Spectroscopy:
Experiment: A = MHz Theory RSS: A = MHz (-1.5%) Theory SRR: A = MHz
B = MHz B = MHz (-0.6%) B = MHz
C = MHz C = MHz (-1.1%) C = MHz
B3LYP D3BJ def2TZVP

17. Is the analysis by rotational spectroscopy consistent with the optical rotation characterization?
Chiral Tag Rotational Spectroscopy Analysis Results
Stereoisomer Percent Optical Rotation (Pure)
(-)-isopulegol RRS o
(+)-isopulegol SSR o
(+)-neoisopulegol RSS o
(-)-neoisopulegol SRR o
Net Optical Rotation: -6.5o TCI Certificate of Analysis: -6.1o

18. Conclusions
Chiral tag rotational spectroscopy has the potential to be a quantitative analytical tool for routine analysis of molecules with multiple chiral centers
Coupled with complementary three-wave mixing capabilities, rotational spectroscopy offers the full range of chiral analysis capabilities
Potential for high speed monitoring of stereoisomers using cavity-enhanced spectrometers (Balle-Flygare)
There is much to validate in the methodology
Development of sampling methods for large molecules is a high priority

19. Chiral Analysis by Molecular Rotational Spectroscopy
Unmet Needs in Analytical Chemistry: Routine Analysis of Molecules with Multiple Chiral Centers
Validation of Accurate Methods in Quantum Chemistry
Automated
Spectral Assignments
(Autofit, PGOPHER, JB95, AABS)
Hamiltonians for Molecular Rotation
(Watson Hamiltonian, SPCAT,BELGI)
Molecular Structures from Isotopologue Analysis (Kraitchman,R0,rm)
Pulse Sequences for FTMW Spectroscopy
(Three Wave Mixing)
Chiral Analysis by Molecular Rotational Spectroscopy
Conformational Properties of Molecules
Design of Nozzle Sources and Properties of Pulsed Jets (Cooling)
Structures of Weakly Bound Complexes
High-Sensitivity
Broadband Spectrometers
(CP-FTMW)
High-Speed
Cavity-Enhanced Spectrometers
(Balle-Flygare)