1. Absolute Configuration of 3-methylcyclohexanone by Chiral Tag Rotational Spectroscopy and Vibrational Circular Dichroism
Luca Evangelisti, Dipartimento di Chimica G. Ciamician, Università di Bologna; Martin S. Holdren, Kevin J. Mayer, Taylor Smart, Channing West, Brooks Pate, Department of Chemistry, University of Virginia

2. Chiral Analysis: The Search For a Universal Tool
For “N” chiral centers
2N isomers
2N-1 unique diastereomers
2 enantiomers per diastereomer
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
Need for universally applicable chiral analysis methods
Quantitative ratios of all stereoisomers
Complex mixture analysis
Rapid monitoring
Provides unique problem to analytical chemistry
Issue in ability to determine all stereoisomers for a chiral molecule and molecules with multiple chiral centers
Stereoisomers consist of enantiomers and diastereomers
Enantiomers have a chiral center giving them a mirror image compound that is non super imposable
As # of chiral centers increase, goes as…
Diastereomers have no mirror image, different geometry
Example
To a synthetic chemist…
MY TALK will be about if you were given one enantiomer and asked what it was, how would you do it with the highest confidence

3. 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
First we look at diastereomers
Rotational spectroscopy is a great technique for diastereomer analysis
Subtle changes in mass distribution
Use CP-fourier transform microwave specstroscopy utilizing 4-5 nozzles which increases sensitivity and decreases sample consumption
High agreement between theory and experiment making it library free without need for reference
Freq range good for complexes and larger molecules
High res technique making it good for mixture analysis
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).

4. 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(+)
Problem is enantiomers – same mass distribution or moments of inertia.
Different approach taken by … with measurement principle …
Pictures
Use cycle of transition to create coherence, transfer of coherence, and detection of the final coherence through
mutually orthogonal polarizations incorporating mu a,b,c dipole vectors to produce a chiral signal fid that would have the two handednesses out of phase 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).

5. Challenges of Three Wave Mixing
Absolute Configuration (AC):
Enantiomeric Excess (EE):
Since AC is determined by the phase of the chiral signal, t0 must be known
Phase Calibration is currently unsolved
Needs a reference sample with known EE due to single detection window for enantiomers
Potential for errors in high EE limit
Problem in AC is t0
Other problems in EE but discussed previously

6. Classical Approach of Chiral Tag Rotational Spectroscopy
Enantiomers Diastereomers
Advantages
Universal Van der Waals attraction
“Tag” can provide dipole moment
Reference-free EE determination
High enantiopurity limit
Distinct spectra
Disadvantages
Spectral complexity from complexes
Fraction of molecules complexed can be low (<10%) limiting sensitivity
Accuracy of quantum chemistry for complexes needs to be determined
S-Butynol
S-3MCH
R-Butynol
S-3MCH
Want to get around those issues
Use classical approach
Know that diastereomers are great for rotational spectroscopy
Uses simple universal van der waals attractions and hydrogen bonds to form complexes making homo chiral (SS) or heterochiral (RS) making diastereomers
If molecule didn’t have a dipole, the tag can bring it,
Its reference free,
Has a high enantiopurity limit, and of course produces distinct spectra as seen below
Add some complexity in making complexes
Fraction for ONE isomer as low as 10%
Need good quantum computational chemistry
FIND AC BY KNOWING TAG AC AND SPECTRA PRODUCED

7. Chiral molecules may absorb left or right circularly polarized light
Vibrational Circular Dichroism
Image credit:
Chiral molecules may absorb left or right circularly polarized light
Another good way to find AC is by VCD.
Differential ability for molecules to absorb right or left circularly polarized light in their vibrational modes.
Normal ir
VCD with + and – peaks.
Computational work to match spectra to compound which works well and is a benchmark for 3MCH
S-(-)-pinene
ΔAx105
Frequency
He, Y.; Wang, B.; Dukor, R. K.; Nafie, L. A. ChemInform 2011, 42 (50).
BioTools - VCD (Vibrational Circular Dichroism) Info (accessed Jun 1, 2017).

8. Challenges with VCD Reduced signals (103 – 104 below IR)
R-3-methylcyclohexanone
89:11 E:A
Axial *
Axial *
Equatorial *
Equatorial
Gaussian - Studying Chirality with Vibrational Circular Dichroism
(accessed Jun 1, 2017)
Some challenges, though
Small signals
Need high concentrations which might mean solvent interactions will interfere or mess up computational work
Conformationally flexible molecules provide a challenge. These pop up in VCD shown here where stephens and devlin
show a spectra
Want a quantitative way to measure absolute configuration
Reduced signals (103 – 104 below IR)
Need for high concentrations
Conformers and complex mixtures produce challenges due to spectral overlap
Experimental *
Devlin, F. J.; Stephens, P. J. Journal of the American Chemical Society 1999, 121 (32), 7413–7414.
DFT/B3PW91/TZ2P 

9. The Problem: Is it S or R? Measurement: Challenges: Given unknown 3MCH
Introduce racemic tag
Measure with one enantiopure tag (R)
Subtract to get other set
Heterochiral
Homochiral
Experimental
Lowest
% Error
2nd Lowest
A (MHz)
(7)
1096.1
0.1598
1351.0
1186.8
7.790
1238.2
B (MHz)
(5)
470.4
2.041
395.6
430.1
7.138
425.0
C (MHz)
(4)
386.3
1.588
373.8
357.7
6.280
412.0
m a,b,c (D)
3.7,0.8,1.4
3.3, 1.4, 0.7
3.6,1.4,0.8
3.6, 1.1,0.07
So in our approach of chiral tagging
First given pure unknown 3MCH
Measure with racemic tag to get both heterochiral and homochiral complexes
Measure with pure tag, lets say R
Challenges:
How much difference does the tag make in larger molecules?
Isomer searches may get difficult and time consuming

10. The Problem: Is it S or R? Measurement: Challenges: Given unknown 3MCH
Introduce racemic tag
Measure with one enantiopure tag (R)
Subtract to get other set
Heterochiral
Homochiral
Experimental
Lowest
% Error
2nd Lowest
A (MHz)
(7)
1096.1
0.1598
1351.0
1186.8
7.790
1238.2
B (MHz)
(5)
470.4
2.041
395.6
430.1
7.138
425.0
C (MHz)
(4)
386.3
1.588
373.8
357.7
6.280
412.0
m a,b,c (D)
3.7,0.8,1.4
3.3, 1.4, 0.7
3.6,1.4,0.8
3.6, 1.1,0.07
Measure with pure tag, lets say R
Could subtract to get other set of complexes if we wanted to from racemic measurement
Compare rotational constants
2 lowest energy isomers of heterochiral and homochiral complexes
Experiment lines up with low uncertainty to heterochiral
Either SR or RS, known its an R tag, must be S-3MCH, proven true
Challenges:
How much difference does the tag make in larger molecules?
Isomer searches may get difficult and time consuming

11. S-3MCH is found and matches true AC
The Problem: Is it S or R? R S
Measurement: S
Given unknown 3MCH
Introduce racemic tag
Measure with one enantiopure tag (R)
Subtract to get other set
S-3MCH is found and matches true AC
Heterochiral
Homochiral
Experimental
Lowest
% Error
2nd Lowest
A (MHz)
(7)
1096.1
0.1598
1351.0
1186.8
7.790
1238.2
B (MHz)
(5)
470.4
2.041
395.6
430.1
7.138
425.0
C (MHz)
(4)
386.3
1.588
373.8
357.7
6.280
412.0
m a,b,c (D)
3.7,0.8,1.4
3.3, 1.4, 0.7
3.6,1.4,0.8
3.6, 1.1,0.07
Some challenges:
Larger compound means tag might not make as noticeable of a difference,
More isomers formed as compound size increases and number of docking sites increase
Want to improve confidence in absolute configuration determination
Challenges:
How much difference does the tag make in larger molecules?
Isomer searches may get difficult and time consuming

12. Improving Confidence: The “Gold Standard”
Obtain structure through 13C substituted isotopologue in natural abundance * *
Gold standard of rotational spectroscopy : obtain 3-D structure through C-13 substituted isotopolougues in natural abundance
Subtle change in mass by substituting one carbon produces a signature spectra.
Need increase in time and sample consumption but higher confidence in AC S
Complete 3-D structure tells us absolute configuration
105 increase in time and sample consumption R

13. Properties of the Butynol Tag
Potential Energy Surface
Heterochiral
Homochiral
Homochiral and heterochiral complexes are extremely similar with a close gap in energy levels
Dug deeper into butynol tag
Docking uses hydrogen bond and acetylene piece electron density towards ring exposing methyl and hydrogen with noticeable difference
Looks like conformation change which looking at PES plot shows hardly an energy cost between the two. See both hetero and homo
Look at most important atom in complex
Plot shows carbon atom position parameters between homo and heterochiral
Largest change in methyl position

14. Properties of the Butynol Tag
Potential Energy Surface
Heterochiral
Homochiral
Homochiral and heterochiral complexes are extremely similar with a close gap in energy levels
What if we could isotopically label that carbon
High confidence in AC without long runs or lots of sample consumption

15. Homochiral Methyl Substituted Heterochiral Methyl Substituted
Isotopic Labeling of the Tag
Potential Energy Surface
Heterochiral
Homochiral
Homochiral Methyl Substituted
Heterochiral Methyl Substituted
Theoretical
Fitted
% Error
A (MHz)
(19)
(25)
B (MHz)
(3)
(6)
0.0351
C (MHz)
(3)
(5)
0.0200
Distance(Å)
4.43
4.48
1.12
3.78
3.77
0.264
Look at ABC rotational constants and distance from center of mass of that substituted carbon position
Scaled theoretical value predicted from normal species
% errors much less than before in just looking at complex as a whole
Get gold standard AC deterimination by just looking at the most important atom position
Raise confidence level of absolute configuration determination with less sample consumption and less time

16. Butynol Methyl Carbon Atom Position Analysis
3-methylcyclohexanone
Homochiral: Theory (4.43, 0.07, 0.12) R = 4.43
Experiment (4.48, 0.11, 0.14) R = 4.48
Heterochiral: Theory (3.57, -0.85, 0.91) R = 3.78
Experiment (3.55, 0.86, 0.89) R = 3.77
Camphor
Homochiral: Theory (-5.04, -0.18, -0.43) R = 5.06
Experiment (5.07, , ) R = 5.10
Heterochiral: Theory (-4.10, 0.88, 0.94) R = 4.30
Experiment ( 4.13, 0.90, 0.92) R = 4.32
See this exact same thing through out the lab
Strong change in atom position for that methyl carbon due to the docking of the tag
Could instead label hydrogens for a similar effect if that was more cost effective,
So again, we can get the gold standard absolute configuration quantitatively through looking at just the most important atom position
Verbenone
Homochiral: Theory (3.92, 1.38,-0.57) R = 4.20
Experiment (4.02, 1.37, 0.44) R = 4.27
Heterochiral: Theory (5.03, 0.00, -0.01) R = 5.03
Experiment (5.08, 0, ) R = 5.07

17. 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, Smitty Grubbs, Galen Sedo, Dave Pratt
Mark Marshall, Helen Leung, Kevin Lehmann, Justin Neill
Frank Marshall, Kevin Mayer, Taylor Smart, Reilly Sonstrom, Channing West
Ellie Coles, Elizabeth Franck, John Gordon, Julia Kuno, Pierce Eggan, Victoria Kim, Ethan Wood, Megan Yu

18. Conclusions
Rotational Spectroscopy has similar features to VCD but has better:
Molecule sensitivity
Conformer specificity
Mixture compatibility
High confidence AC determination via theoretical spectra
EE determination (previous talks)
Limitations:
Size limitations needs further evaluation in Rotational Spectroscopy whereas VCD may have an advantage
Significant improvements in AC determination may be possible through singly-substituted chiral tags
Pinpoint positions of atoms that can differentiate homochiral and heterochiral complexes may have reliability close to that of the ‘Gold Standard’ level without the added time and sample consumption of natural abundance of 13C along with the possibility of spectral complexity due to digging out 13C from lower population isomers and low intensity peaks