1. Complexes of Small Chiral Molecules: Propylene Oxide and 3-Butyn-2-ol
Luca Evangelisti
Dipartimento di Chimica G. Ciamician
Università di Bologna
Channing West, Ellie Coles, Brooks Pate
Department of Chemistry
University of Virginia

2. Chiral Analysis For “N” chiral centers 2N stereoisomers
2(N-1) unique geometries
2 enantiomers per diastereomer
Image Credit:
Need for universally applicable enantiomeric excess methods
Quantitative ratios of all stereoisomers
Complex mixture analysis
Rapid monitoring
Enantiomers – mirror images but not superimposable
Diastereomers – molecules with multiple chiral centers that are not mirror images (distinct geometries)
Carlos Kleber Z. Andrade*, Otilie E. Vercillo, Juliana P. Rodrigues and Denise P. Silveira, J. Braz. Chem. Soc., 15, , 2004.

3. Three Wave Mixing: Enantiomer Analysis
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).
mambmc(-)
mambmc(+)
J.U. Grabow, Angew. Chem. 52, (2013).
V.A Shubert, D. Schmitz, D. Patterson, J.M Doyle, and M. Schnell, Angew. Chem. 52, (2013). 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).

4. Challenges of Three Wave Mixing: Enantiomeric Excess
Absolute Configuration (AC):
Phase dependence
Small dipole component can produce erroneous results
Enantiomeric Excess (EE):
Needs a reference sample with known EE due to single detection window for enantiomers
Potential for errors in high EE limit

5. Rotational Spectroscopy: Chiral Tagging
By complexing with a separate chiral molecule
Enantiomers Diastereomers
S-Butynol
R-Butynol
Advantages
Enantiomers now have distinct spectra
“Tag” can provide dipole moment
Reference-free EE determination
High enantiopurity limit
S-3MCH
S-3MCH
The basic idea, since rot spec yields different spectra for distinct geometries/mass distributions, let’s turn enantiomers into diastereomers by ‘tagging’ the molecule with a small molecule like Butynol that interacts via a hydrogen bond and van der Waals attractions in the pulsed jet.
Structures are clearly different when looking at the mirror image of the butynol and then attaching the 3MCH below
Show difference In two structures. Include spectra to show difference?
Advantages
No reference sample needed for EE measurement
Preferred for high EE measurements
Good chemical selectivity
Can find absolute configuration from theoretical spectra
Dipole moment can be provided by tag
Disadvantages
Need for accurate quantum mechanical structures
Fraction of molecule complexed may be low (<10%)
Spectral complexity through isomers of complexes

6. Rotational Spectroscopy for Chiral Analysis: Diastereomers
Chirped-Pulse FTMW Spectroscopy
Low Frequency (2-8 GHz): Peak Transition Intensity of Large Molecules
High Resolution + Broadband Coverage: Mixture Analysis
Extreme sensitivity to mass distribution
Agreement with Theory: “Library-Free” Diastereomer Identification
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).

7. Ideal Case: Enantiopure 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)
(1R)-(+)-verbenone +
(S)-(-)-3-butyn-2-ol
(1S)-(-)-verbenone +
(S)-(-)-3-butyn-2-ol
Enantiomer populations converted to different
diastereomers with distinct spectra

8. Calibration and Analysis for Enantiomer Excess Determination
53.6% EE
Include Kevin’s histogram and previous value for EE  Chiral tagging works
Chiral tagging as analytical technique (pharma  4 decimal places)
What do we need to know?
High tag enantiopurity

9. Actual Measurement Conditions: Effects Due to the Enantiomeric Excess of the Chiral Tag
Chiral Tag Has High Enantiopurity: Alfa Aesar Sample
(S)-(-)-butynol: 99.3%
(R)-(+)-butynol: 0.7%
EE: 98.6%
Sample mole fractions:
Chiral Tag: (-): 1- Analyte: (-): (1-f+)
(+):  (<<1) (+): f+
ee = (1-2) ee = (1-2f+)
Note:
EE = ee x 100
Linear Assumption: Number Density of Complex Proportional to the
Product of Number Densities for the Enantiomers
More general nomenclature
Based on optical rotational properties
When delta = 0 -> simple
When delta=/= 0 -> apparently not simple
Homochiral Species:
Tag(-) + Analyte(-): (1-) (1-f+)
Tag(+) + Analyte(+):  f+
Heterochiral Species:
Tag(-) + Analyte(+): (1-) f+
Tag(+) + Analyte(-):  (1- f+)

10. Enantiomeric Excess Measurements
Linear Assumption: Signal Level for an Individual Rotational Transition
SignalHOMO = CHOMO * ( [Tag(-)][Analyte(-)] + [Tag(+)][Analyte(+)] )
SignalHETERO = CHETERO * ( [Tag(-)][Analyte(+)] + [Tag(+)][Analyte(-)] )
Signal Normalization:
For a racemic tag sample ( = 0.5):
SignalHOMO = CHOMO * 0.5
SignalHETERO = CHETERO * 0.5
Effect: For any PAIR of heterochiral and homochiral transitions in the spectrum
Normalization
What do you do when you don’t know the enantiopurity of the tag?
Measure racemic -> normalization
Transitions have unequal intensity due to intrinsic transition strength, isomer populations, and instrument intensity calibration
N = (SignalHOMO/SignalHETERO ) The ratio of the transition intensities using racemic tag

11. Enantiomeric Excess Measurements
Linear Assumption: Signal Level for an Individual Rotational Transition
SignalHOMO = CHOMO * ( [Tag(-)][Analyte(-)] + [Tag(+)][Analyte(+)] )
SignalHETERO = CHETERO * ( [Tag(-)][Analyte(+)] + [Tag(+)][Analyte(-)] )
EE Determination:
Using the high enantiopurity tag ( << 1):
SignalHOMO = CHOMO * [(1-) (1-f+) +  f+]
SignalHETERO = CHETERO * [(1-) f+ +  (1- f+) ]
Effect: For any PAIR of heterochiral and homochiral transitions in the spectrum
Intensity Changes with use of High Enantiopurity Tag
Highe enant purity tag
Product of the tag ee and analyte ee
How do we get tag ee
Calculate the Normalized Signal Ratio (R):
Normalized Signal
With Racemic Tag
Normalized Signal
Ratio (R)
R = N * (SignalHETERO/SignalHOMO )
Result:

12. Calibration of Butynol and Propylene Oxide Chiral Tags
Butynol: AUTOTAG (Walther Caminati)
Measure the spectrum of racemic butynol and high enantiopurity butynol and use the rotational transitions of the homochiral and heterochiral dimer spectra
EE calibration does not require assignment of the dimer spectra
(for the high enantiopurity sample, homochiral complexes approximately double in intensity and heterochiral complexes nearly disappear when compared to the intensities in the racemic spectrum)
Sample Composition:
(S)-butynol: 99.05(15)%
(R)-butynol: 0.95(15)%
Certificate of Analysis:
(S)-butynol: 99.3%
(R)-butynol: 0.7%

13. Calibration of Butynol and Propylene Oxide Chiral Tags
Propylene Oxide: Calibration with Butynol
The lowest energy isomer of the propylene oxide dimer has a small dipole moment limiting the measurement sensitivity.
The transition intensities for the propylene oxide – butynol complex are a factor of 10 higher than the strongest transitions observed for the propylene oxide dimer (RR2).
The homochiral and heterochiral complexes of propylene oxide – butynol have been assigned and verified by Kraitchman substitution structures and can be used for chiral tagging
Homochiral
Heterochiral

14. Calibration of Butynol and Propylene Oxide Chiral Tags
Butynol Dimer Present in this Measurement: Can Calibrate both Butynol and Propylene Oxide

15. Enantiomeric Excess of Verbenone Revisited
Using Auto Tag Calibration of Butynol:
EE = 98.1(3)
The verbenone EE determinations are:
Four Nozzle: EE = 53.0(5.9)
Single Nozzle: EE = 52.7(4.8)
Certificate of Analysis: EE = 53.6%

16. Acknowledgements
This work supported by the National Science Foundation (CHE ) and
The Virginia Biosciences Health Research Corporation
Special thanks for work on chiral tag rotational spectroscopy:
Luca Evangelisti
Dave Patterson, Yunjie Xu, Walther Caminati, 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, Ellie Coles, Elizabeth Franck, John Gordon, Julia Kuno, Pierce Eggan, Victoria Kim, Ethan Wood, Megan Yu

17. Conclusion
Rotational spectroscopy has the potential to be a powerful analytical tool for determining enantiomeric excess in the high EE limit
Correction for tag enantiopurity is necessary for accurate analytical work
Results are reproducible and narrowly distributed in the high EE limit

18. Effect of Intensity Fluctuations Between Racemic and Enantiopure Measurements
Modeling of EE Determination using 5% intensity fluctuation on transitions
Distribution width is linear in (100-EE) – amount of enantio impurity
Distribution width is linear in the intensity fluctuation