Structure Elucidation Challenges: How Can Advanced Spectroscopy Overcome Them?

When new compounds are made, structure elucidation is an important step in confirming the novelty of the new compound.


Structure Elucidation Challenges: How Can Advanced Spectroscopy Overcome Them?

Structure elucidation is an essential part of chemical synthesis and development. When new compounds are made, structure elucidation is an important step in confirming the novelty of the new compound. The information from structure elucidation methods is also particularly important for understanding whether new compounds have potential as therapeutics.1 Many biological interactions have some ‘shape dependence’ – where the therapeutic can only interact with the biological target if it has the right structural features, which is why structural elucidation is so prized in the pharmaceutical industry.2

Working out the chemical formula or the elemental composition of a sample can be done with techniques such as elemental analysis and high mass resolution mass spectrometry.3 Structural elucidation though can be more difficult. Particularly for biological and organic samples where there may be a large number of carbon atoms but in different spatial locations and with different relative orientations, whatever method is used for structural elucidation needs to be capable of differentiating between atomic sites that have the same element.

Nuclear Magnetic Resonance (NMR)

One of the most successful structural elucidation techniques has been nuclear magnetic resonant (NMR) spectroscopy. NMR is a very widely adopted technique in the chemical and biological sciences and consider a definitive verification method for new compounds for patents and publications.4 Many organic chemistry journals require an NMR spectrum of the compound in order to consider publication of a paper proposing a new molecule.

In order for a nucleus to be ‘NMR’ active it needs to have non-zero nuclear spin, normally meaning an odd number of protons and/or neutrons. What this means is a number of specific isotopes give rise to NMR signals, with the most popular being 13C and 1H. One isotope that can be a very powerful tool in structural elucidation studies is 19F.

Fluorine is not an element typically found in biological molecules and that is what has made it such a useful tool for structural elucidation in even the most complex proteins.5 The proliferation of multidimensional NMR techniques, such as COSY and NOSY, has simplified structural elucidation for many compounds by providing direct access to coupling and spatial information. However, for many more complex systems, running a single NMR experiment of a single isotope is insufficient to perform a unique structural elucidation.

By using elements that are not naturally found in a structure and specific biolabeling techniques that can insert fluorine isotopes into targeted areas of the molecular structure. The fluorine can then be used as a probe of the local environment for structural elucidation and give more straightforward and uncongested NMR spectra as there may only be a few fluorine isotopes present.

Solid States

NMR methods for structural elucidation can be used on solid state samples as well, whether that is full materials or crystalline samples. The richness of the structural elucidation information provided is why solid state (SS) NMR and NMR crystallography is now being used as a routine method to evaluate the stability and provide quality assurance for pharmaceuticals.

SS-NMR uses many of the same approaches in terms of pulse sequencies and multidimensional techniques for structural elucidation as solution phase NMR but the sample quality can be somewhat more critical. The advantage of being able to measure solids as this can cover samples such as crystalline pharmaceuticals as well as biomaterials like bone and hair.

Structural Elucidation

Other highly successful methods for structural elucidation in biological samples include electron diffraction (ED). ED is a popular method as it can be performed with laboratory sources and provides direct structural information, though there can be specific sample requirements in terms of the sample thickness to be measured.

The structural elucidation is performed through a reconstruction of the diffraction pattern that is often performed in combination of quantum chemical modelling, that can evaluate the relative energetic stability of different structural arrangements of the sample. The diffraction patterns can then be simulated and compared to the experimental data to evaluate the best fits.
Electron diffraction is often combined with microscopy methods or imaging approaches such as electron energy loss imaging. Electron energy loss imaging is a spatially resolved method where a sample is exposed to an electron beam of a known, narrow range of energies and the energy loss of those electrons following interaction with the sample is measured.

Electron energy loss is an ideal method for looking at many materials and examples of its use include mapping the sp2 and sp3 hybridized carbon sites in diamond-like carbon films. Especially compared to ED, energy loss methods have generally low dispersion but by using signal processing methods on the images obtained, this restriction can be overcome.

The right choice of advanced structural elucidation method can provide invaluable characterization information and physical insight into the behavior of therapeutics or screening for new therapeutic candidates.

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  1.  Nicolaou, K. C., & Snyder, S. A. (2005). Chasing Molecules That Were Never There : Misassigned Natural Products and the Role of Chemical Synthesis in Modern Structure Elucidation Angewandte. Natural Products Synthesis, 44, 1012–1044.
  2. Morozov, D., Mironov, V., Moryachkov, R. V, Shchugoreva, I. A., Artyushenko, P. V, Zamay, G. S., Kolovskaya, O. S., Zamay, T. N., Krat, A. V, Molodenskiy, D. S., Zabluda, V. N., Veprintsev, D. V, Sokolov, A. E., Zukov, R. A., Berezovski, M. V, Tomilin, F. N., Fedorov, D. G., Alexeev, Y., & Kichkailo, A. S. (2021). The role of SAXS and molecular simulations in 3D structure elucidation of a DNA aptamer against lung cancer. Molecular Therapy: Nucleic Acid, 25, 316–327.
  3. Lin, Y., Yu, Q., Hang, W., & Huang, B. (2010). Progress of laser ionization mass spectrometry for elemental analysis — A review of the past decade. Spectrochimica Acta Part B: Atomic Spectroscopy, 65(11), 871–883.
  4. Elyashberg, M. (2015). Trends in Analytical Chemistry Identification and structure elucidation by NMR spectroscopy. Trends in Analytical Chemistry, 69, 88–97.
  5.  Arntson, K. E., & Pomerantz, W. C. K. (2016). Protein-Observed Fluorine NMR: A Bioorthogonal Approach for Small Molecule Discovery. Journal of Medicinal Chemistry, 59, 5158–5171.




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