1. A Chiral Tag Study of the Absolute Configuration of Camphor
June 21st, 2017
University of Virginia
Department of Chemistry
International Symposium on Molecular Spectroscopy
David Pratt, University of Vermont
Luca Evangelisti University of Bologna
Taylor Smart, Martin Holdren, Kevin Mayer, Channing West and Brooks Pate, University of Virginia
Hello, my name is Taylor Smart from the University of Virginia and today I will be speaking about A chiral Tag study of the absolute configuration of camphor

2. 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

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
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).

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(+)
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).

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
While 3 wave mixing works in theory it still has issues that need to be solved before it can be utilized. For instance to determine the absolute configuration of the sample which is determined by the phase of the chiral signal has the issue that we do not know where t0 is. Without knowing where t0 is we can not determine which absolute configuration is which signal. And to our knowledge no one has been able to solve this phase calibration issue. The other issue is that in order to determine the Enantiomeric Excess of the sample you still need a reference sample with a known EE due to the configurations having the same detection window. Which no one is going to use this technique if they have to give you a reference sample with known EE because they can already get the EE of their sample. The other issue with Enantiomeric excess is there is the potential for errors in a sample with high EE

6. Rotational Spectroscopy: The Classical Approach of Chiral Tagging
Enantiomers Diastereomers
Advantages
Enantiomers now have distinct spectra
“Tag” can provide dipole moment
Reference-free EE determination
High enantiopurity limit
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
Due to the unsolved issues in 3 Wave mixing there is an alternative that still uses traditional rotational spectroscopy. This alternative relies on the fact that diastereomers have distinct spectra whereas enantiomers do not, so what is done is a chiral tag is used to complex with your molecule. This will change the enantiomers into diastereomers if the tag is enantiopure. This use of an internal chiral reference is not a new one x-ray crystallography, NMR and even mass spec have used a chiral reference to determine chirality of a molecule. However, these rely on the ability form a single crystal, which is difficult with many compounds, or special solvents that are specific to the molecule you are testing. This chiral tagging system has the advantage that it has broader universality. It can be used on molecules that are difficult for rotational spectroscopy due to being non-polar or low polarity by adding a more polar tag, this would provide a better dipole to the molecule, and increase the sensitivity. And in principal you can determine the EE by looking at the signals of the now diastereomers. This gets around the issues with 3 wave mixing, allowing us to determine absolute configuration and Enantiomeric excess. The great thing about this technique though is that if the computation chemistry is accurate for the complexes it relies on known spectroscopy methods, everything is already in place and the measurement technology is already there.
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

7. Dipole Moment Directionality and Three Wave Mixing Rotational Spectroscopy
Camphor
Analysis Issues for a Small Dipole Moment Component
Potential for quantum chemistry to determine the incorrect sign of the dipole moment product leading to incorrect absolute configuration
Potential to limit measurement sensitivity because pulse durations for optimum signal become too long
Small angle shift (1.5o) changes sign of dipole moment component on c-axis (Z)
Experiment Theory (S-camphor)
A = (72) MHz ma = (23) D (76.2o) A = MHz (0.07%) ma = 3.19 D (75.8o)
B = (47) MHz mb = (6) D (13.7o) B = MHz (0.23%) mb = D (14.1o)
C = (33) MHz mc = (7) D (1.49o) C = MHz (0.26%) mc = 0.10 D (1.94o)
mtot = (22) D mtot = 3.29 D (-6.7%)
In the case of 3 wave mixing there are issues for certain compounds. In the case of this study Camphor was chosen because it has a small dipole component. When there is a small dipole component on the molecule there is the potential for the sign of the dipole moment to change. This raises issues when dealing with 3-wave mixing because that quantum chemistry has to be extremely accurate for these dipole components otherwise the quantum calculations will be wrong due to the wrong sign What we have noticed in other work is that the theory values are still accurate when compared to the experimental values, so far we have not seen an issue with the sign flipping. However, we did utilize the chiral tag method in this case due to those potential issues. If this were not an issue then 3-Wave mixing would be the preferred technique for absolute configuration due to the fact that the chiral tagging method makes clusters whereas 3-Wave mixing does not make clusters of complexes.
B3LYP D3BJ G**

8. Determination of Absolute Configuration by
Chiral Tag Rotational Spectroscopy
Enantiomers of molecules have identical rotational spectra
Complexes of enantiomers with an enantiopure “chiral tag” form diastereomers that have different rotational spectra
To determine the absolute configuration of camphor we added a tagging molecule to it. This was so that any enantiomers in the sample would become diastereomers due to the addition of the chiral tagging molecule. This can be done with either a racemic camphor sample and an enantiopure tag or with an enantiopure camphor sample and racemic tag. Either way this will result in homo or hetero chiral samples which is why we look at the spectra for the lowest energy conformations of both hetero and homochiral simulations.
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

9. The Problem R,R-Camphor S,S-Camphor Given an unknown sample of Camphor
Which Camphor is it?
R Camphor or S Camphor?
Add enantiopure tagging molecule
Results in either homo or hetero chiral complexes
Compare values to theoretical values
R,R-Camphor
S,S-Camphor
The problem analytically is if you were to receive an unknown sample of camphor. And you know that it is enantiopure. How do you know which camphor it is, is it R camphor or is it S camphor. Well what you can do with this technique is attach an enantiopure tagging molecule to it. Then you just have to solve whether the spectra you are getting are in the homochiral or heterochiral families, based on your quantum calculations.

10. Chiral Tag Rotational Spectroscopy and Isomers
Heterochiral
Complexes
Homochiral
Complexes
Expect a few isomers to be formed in the pulsed jet expansion.
Spectroscopy “match” is to the set of high abundance isomers
Requires accurate structures, thorough isomer searches, and accurate energies from computational chemistry … …
0.92
0.94
Relative Electronic Energy (kcal/mol)
Say you put R Butynol on your Camphor sample. You expect isomers to form of the Butynol Camphor complex during the injection into the chamber. As such there will be different complexes based on how the Butynol attached to the Camphor. Now when we look at the energetics of the different forms we see that heterochiral and homochiral families have similar energetics. There are two lower level conformations and then a jump in energy up to the next lowest conformation. So, what we expect to see is two dominant spectra whether the camphor is R or S. We can compare these constants solved for by quantum chemistry to the experimental spectra and see which family of conformations the unknown camphor and R-Butynol match up with. Which will tell you what Camphor you are working with. To predict the spectra and the energies of these different complexes we used the computational chemistry method B3LYP D3BJ def2TZVP. This method does not take into account the vibrational corrections. However, to do so would increase the computation time by a large factor and the accuracy of including the vibrational corrections is unreliable due to low frequency nodes in the clusters, and the vibrational corrections may be very similar due to the similarity of the structures. If you are interested in learning more about the use of this computational method the talk on Thursday RG03 on Solketal will focus more on that.
Lowest Energy
Homochiral
0.16
0.16
Second Lowest
Energy Heterochiral
0.00
0.05
Lowest Energy
Heterochiral
B3LYP D3BJ def2TZVP

11. Methodology Monomer spectra: Cut out monomer signals
Enantiopure samples: Identify the homochiral or heterochiral spectra
Based on quantum calculations
S and R Camphor result in different spectra
What we first do is get spectra of the monomer, just your unknown camphor. What you do with that is cut it out of your future experimental spectra. Otherwise your data would be dominated by the signals of camphor. You then can look at your experimental spectra and compare it to the spectra generated by the quantum chemistry calculations. If the Camphor is R camphor then you expect to match up the homochiral spectra with it, if its S Camphor then the heterochiral spectra should match up.
Lowest energy isomers found by the quantum chemistry method B3LYP D3BJ def2TZVP
Validated on Solketal talk RG03 on Thursday
Low Frequency measurements in the 2-8 GHz range as molecules peak intensity decreases in frequency as the number of heavy atoms increases
Spectral Properties:
Measurement Bandwidth: 6000 MHz
FWHM Resolution: 60 kHz
RMS Frequency Error in Fit: 6-10 kHz (~10% FWHM)
Hamiltonian: Watson Distortable Rotor
Analogous to NMR and XRAY crystallography.
NMR uses special solvents to detect chirality of a molecule by the anisotropy effect of an aromatic group contained in a chiral supplement with a known absolute configuration
XRAY crystallography uses an internal chiral reference of another chemical, the difficulty comes from making a crystal, due to the necessity of a single crystal makes this method far from universal

12. Absolute Configuration: Spectral Comparison
10% Camphor complexed in the lowest Homochiral conformation
3% complexed in the second lowest Homochiral conformation
When we took the experimental data of the unknown camphor we compared it to the two lowest conformations for both the hetero chiral and homochiral families. What you see on the left is our experimental data compared to the heterochiral families, so what we would expect to see if our camphor was S Camphor. What you can tell is that it doesn’t look like it aligns well. Then on the right when we compared it to the homochiral conformations what you can see is that it matches up pretty much spot on. So, you would infer that the Camphor is most likely R camphor. If this is the case what we see is that there is about 10 % Camphor complexed in the lowest energy conformation and 3% in the second lowest conformation. There’s still a lot of camphor not accounted for, most likely in other conformations or still being detected as the monomer, which is more likely.

13. Absolute Configuration by Rotational Constant Comparison
Compare experimental rotational constants to the theoretical
Experimental spectra compared to simulated spectra of 2 lowest energy forms
First Camphor R-Butynol Complex
Experimental
Theoretical Lowest Energy Homochiral
Theoretical Lowest Energy Heterochiral
(23)
(-0.21%)
975.3
(63)
(-0.93%)
320.1
(65)
(-0.97%)
301.6
Dipole component values all very similar for the isomers: Can’t be used to confirm analysis
Second Camphor R-Butynol Complex
Experimental
Theoretical Second Lowest Energy Homochiral
Theoretical Second Lowest Energy Heterochiral
(22)
(-0.06%)
(74)
(-1.61%)
290.8
(79)
(-1.47%)
274.2
Besides just comparing the spectral families, we can look at the rotational constants. We can compare our experimental constants of the unknown camphor to the theoretical values for both homo and hetero chiral families. Then you just need to see which 2 lowest energy conformations match up to the two you are seeing experimentally. In our case when you look at the lowest energy conformations, the most abundant isomer. What you can see here is that our experimental data is very close to the homochiral simulated constants and can’t be confused with the heterochiral as the constants are a match better match for the homochiral. This is the same case for the second lowest energy conformations, the second most abundant isomer we detected. The values were once again closer to the homochiral constants then those of the hetero chiral constants. This tells us that the Camphor we were using was R camphor, and we can confirm this because we bought it.

14. Determination of Absolute Structure from Isotopologue Analysis
Enantiomers for the lowest energy homochiral complex from quantum chemistry
Experimental carbon atom positions from isotopologue analysis (Kraitchman)
DON’T TALK ABOUT XRAY INTERNAL REFERENCE
Measurement uses (R)-butynol as the chiral tag so the absolute structure of the sample is known:
(R)-camphor

15. 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:
David Pratt
Luca Evangelisti
Dave Patterson, Yunjie Xu, Walther Caminati, Javix Thomas, Smitty Grubbs, Galen Sedo
Mark Marshall, Helen Leung, Kevin Lehmann, Justin Neill
Frank Marshall, Marty Holdren, Kevin Mayer, Reilly Sonstrom, Channing West
Ellie Coles, Elizabeth Franck, John Gordon, Julia Kuno, Pierce Eggan, Victoria Kim, Ethan Wood, Megan Yu

16. Conclusion and Final refinements
However
Absolute configuration of a chiral molecule can be determined by rotational spectroscopy
Unambiguous result with X-ray quality
Camphor is a simple starting case with only one effective chiral center
Stable molecule with no conformational changes
More complex chiral molecule with conformational changes
Future work
Accuracy of quantum chemistry needs to be explored for larger complexes