Abstract
About four years ago, enantioselective Raman spectroscopy was proposed as a new tool for analyzing chiral media. In recent years, the concept was proven experimentally and different approaches to analyze the data were tested. This article shows how the technique can be implemented and it gives an overview of data evaluation strategies taking the solutions of a chiral pharmaceutical analogue as an example.
Background
There is a high demand for chiral analytical techniques in the life science and pharmaceutical industry. Fast inline monitoring of chiral media for Process Analytical Technology (PAT) is still a challenge. One of the classical approaches in chiral discrimination is breaking the symmetry exhibited by the enantiomers by intermolecular interaction creating a kind of diastereomers. For example, in HPLC chiral stationary phases are used or in NMR spectroscopy chiral agents are added to the chiral analyte. The approach in enantioselective Raman spectroscopy (esR) is different and does not require additives.1 The principle behind esR spectroscopy is an external symmetry breaking of the Raman signal by inserting an achromatic phase shifter, e.g. a double Fresnel rhomb or a super-achromatic half-wave plate, into the collimated beam of the signal collecting unit. This is combined with polarization-resolved detection to gain further qualitative and quantitative information.
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The present article aims at demonstrating the practical application of esR spectroscopy. For this purpose, experimental results of a chiral pharmaceutical analogue 5,6-diphenyl-morpholin-2-one (DPM) diluted in dimethyl sulfoxide (DMSO) are shown. This substance exhibits a relatively high specific optical rotation ([α]D30= -279.6° mL g-1 dm-1 for the levorotary enantiomer)2 and is therefore well-suited as a model for the proof-of-concept. Furthermore, morpholinone derivates are interesting for life sciences as they have potential pharmaceutical applications.3,4
Method
Figure 1 illustrates the esR spectroscopy set-up schematically. It represents a common polarization-resolved set-up with a 90° scat- tering geometry. An additional half-wave retarder, e.g. a super-ach- romatic half-wave plate or a double Fresnel rhomb is implemented into the signal-collecting unit in order to break the symmetry.
Experimental Results
In a first step, the Raman spectrum is recorded as a function of the half-wave retarder orientation angle. The resulting signals are plotted as a 3D surface in Figure 2.
The further analysis focuses on selected spectral regions, which have been found to be particularly useful and effective in the enantiomeric discrimination.5 The maximum intensity occurs at half of the optical rota- tion (α/2). Thus, this approach combines the advantages of conventional polarimetry with the strengths of Raman spectroscopy, as esR spectroscopy provides additional chemical information. Another classical approach is to compare the intensities of peaks associated with vibrational modes exhibiting different symmetries using the racemate as reference. This is illustrated in Figure 3 for the DPM in DMSO case. The intensity differences be- tween the pure enantiomers and the race- mate are obtained with respect to the intensi- ties of the racemate. This analysis shows the regions with the highest intensity differences between the enantiomers and therefore re- veals the retarder orientation angles that provide the most efficient enantioselective discrimination. Due to experimental uncer- tainties, relative intensity differences below about 1% cannot be considered significant. Figure 3 suggests an optimal angle of about +/- 40°.
Multivariate data analysis is another possibility to evaluate esR spectroscopy data. In a recent study, principal component analysis (PCA) was tested as a state-of- the-art chemometric tool. It enables the chiral discrimination in a fully unsupervised manner, which can be the foundation of fully automatic esR approaches.6 Figure 4 highlights the separation of the enantiomers along the second principal component (PC). For media with smaller intensity differences, a discrimination is possible by the analysis of the difference Raman spectra.
Conclusion
Enantioselective Raman spectroscopy is an innovative and non-destructive technique with potential application in PAT. At the cur- rent state, relatively high optical activity is needed to discriminate intensity differences between the enantiomers in the analyzed chiral medium. As the optical rotation is both a function of the concentration and the path length, the problem can be overcome by increasing the path length the Raman sig- nal travels through the sample. The concen- tration cannot be deliberately increased, and also is a datum for inline analytics. Analyte signal path length increase hence could en- able the determination of the enantiomeric ratio of higher diluted samples.
Acknowledgment
The authors gratefully acknowledge financial support from the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG) through grant KI1396/4-1.
References
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- M. Ueda, H. Miyabe, M. Teramachi, O. Miyata, T. Naito, Diastereoselective Intermolecular Radical Addition to Nitrones, Journal of Organic Chemistry 70 (2005) 6653-6660.
- I. W. Ku, S. Cho, M. R. Doddareddy, M. S. Jang, G. Keum, J.-H. Lee, B. Y. Chung, Y. Kim, H. Rhim,S. B. Kang, Morpholin-2-one derivatives as novel selective T-type Ca2+ channel blockers, Bioorganic & Medicinal Chemistry Letters 16 (2006) 5244-5248.
- D. Bardiot, K. Thevissen, K. De Brucker, A. Peeters, P. Cos, C. P. Taborda, M. McNaughton, L. Maes, P. Chaltin, B. P. A. Cammue, A. Marchand, 2-(2-Oxo-morpholin-3-yl)-acetamide Derivatives as Broad-Spectrum Antifungal Agents, Journal of Medicinal Chemistry 58 (2015) 1502-1512.
- C. C. Rullich, J. Kiefer, Enantioselective Raman spectroscopy (esR) for distinguishing between the enantiomers of 2-butanol, Analyst 143 (2018) 3040-3048.
- C. C. Rullich, J. Kiefer, Principal component analysis to enhance enantioselective Raman spectroscopy, Analyst 144 (2019) 2080-2086.
Authors Biographies
Dipl.-Chem. Claudia C. Rullich is a fi nal year Ph.D. student at the division of Engineering Thermodynamics of the University of Bremen, Germany. She holds a degree in Chemistry from the Philipps-University Marburg, Germany. Her research interest focuses on the development of spectroscopic techniques for chiral discrimination.
Prof. Dr. Johannes Kiefer is Chair Professor and Head of the division Technische Thermodynamik at the University of Bremen, Germany. In addition, he is an Honorary Professor at the University of Aberdeen, Scotland, and he holds a guest professorship of the Erlangen Graduate School in Advanced Optical Technologies (SAOT) at the University Erlangen- Nuremberg, Germany. His research interests are the areas of developing and applying spectroscopic techniques for the characterization of advanced materials and processes.