Deciphering Complex Chemical Structures with COSY NMR
Nuclear magnetic resonance (NMR) spectroscopy is now a workhorse technique for many scientists. With the right technical knowledge, NMR can be used for qualitative and quantitative analysis of sample composition and chemical structures. Advances in software support and automated analysis codes
have made NMR now a sufficiently accessible tool that it is widely used in many areas of science, including in the analysis of the purity of complex reaction products.1
To enhance the chemical information that can be recovered from NMR spectra, NMR measurements often use different radio frequencies to look at various types of nuclei, such as carbon-13, phosphorous-31 and hydrogen-1. If a single NMR measurement cannot provide sufficient information to solve a chemical structure, and there are insufficient NMR active nuclei present to solve the structure by looking at different elements, multidimensional NMR methods can be an incredibly useful suite of methods.2 Multidimensional methods use more complex radiofrequency pulse sequences to examine different physical phenomena through the generation of multidimensional datasets.
One of the most widely used two-dimensional (2D) NMR methods is correlated spectroscopy (COSY) NMR. COSY NMR reveals information on correlations between NMR active nuclei in the form of cross-peaks in a 2D map. The appearance of these cross-peaks says which particular, in the case of 1H-COSY NMR, protons are coupled – or separated by up to four chemical bonds.3
How does COSY NMR work?
The 2D spectrum from a COSY NMR experiment is plotted by comparing the frequencies of a single isotope. The diagonal peaks have the same frequency on each axis and so form the diagonal of the map, and the cross-peaks appear displaced from this diagonal. The diagonal peaks are the same information that would be contained in the equivalent 1D NMR experiment.
The off-diagonal information in COSY NMR is the key to the technique's usefulness. It is the off-diagonal elements that describe the spin-spin coupling between isotopes and, therefore, can be used to interpret spectra that have significant spectral congestion in the 1D spectrum. The spin-spin coupling can provide information on the distance and orientation of relative isotopes and be used to recover structural information. Some care has to be taken to avoid misinterpretation of artefacts in the COSY NMR spectrum, but these are rarely in phase with the desired signals.
Variations of COSY NMR include COSY NMR-90/COSY NMR-45, where the first pulse suppresses or enhances the diagonal peaks. Another is double quantum filtered COSY NMR (DQF COSY NMR), which changes the relative phase of the diagonal and cross peaks in the 2D spectrum.
Applications of COSY NMR
The biological and chemical sciences have extensively used COSY NMR as a structure elucidation tool. For some applications, the focus may just be product identification or quality control of samples
, including for therapeutics, but COSY NMR can be used for advanced structural identification of even large biological systems such as proteins.4
The wealth of alternative pulse sequences for COSY NMR and the ability to perform complementary measurements to fully understand the chemical origin of contributions to a sample have also helped make it invaluable for analysing complex structures.
Tackling Complex Structures
Other techniques can be combined with COSY NMR-based approaches to help with the identification of complex structures. For amino acid sequences that make up protein backbones, there is always an NH-C-CO feature, and the N can be substituted for the NMR active 15N isotope. Using polarization transfer from the protein and a specific pulse sequence, the peaks corresponding to the 15N-1H structures can be isolated and identified, a key step in assembling a full macromolecule structure.
Labelling with other NMR active isotopes is another common way of helping identify particular regions in a complex biomolecule though there are many smaller proteins (< 10 kd) that have been fully identified without the need for labelling.5
Some proteins may not be amenable to crystallization, and NMR allows the measurement of proteins in their natural solution-phase environment. Developing highly sensitive probes and instrumentation is also helping reduce COSY NMR acquisition times
for more rapid forensic analysis of samples.
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- Pauli, G. F., Gödecke, T., Jaki, B. U., & Lankin, D. C. (2012). Quantitative 1H NMR. Development and potential of an analytical method: An update. Journal of Natural Products, 75(4), 834–851. https://doi.org/10.1021/np200993k
- Kanelis, V., Forman-Kay, J. D., & Kay, L. E. (2001). Multidimensional NMR methods for protein structure determination. IUBMB Life, 52(6), 291–302. https://doi.org/10.1080/152165401317291147
- Martineau, E., Dumez, J. N., & Giraudeau, P. (2020). Fast quantitative 2D NMR for metabolomics and lipidomics: A tutorial. Magnetic Resonance in Chemistry, 58(5), 390–403. https://doi.org/10.1002/mrc.4899
- Brinson, R. G., & Marino, J. P. (2019). 2D J-correlated proton NMR experiments for structural fingerprinting of biotherapeutics. Journal of Magnetic Resonance, 307, 106581. https://doi.org/10.1016/j.jmr.2019.106581
- Bax, A. (1989). Two-dimensional NMR and protein structure. Annual Review of Biochemistry, 58, 223–256. https://doi.org/10.1146/annurev.bi.58.070189.001255