Npr REVIEW NMR Methods for Unravelling the Spectra of Complex Mixtures

View Article Online / Journal Homepage / Table of Contents for this issue NPR Dynamic Article LinksC< Cite this: Nat. Prod. Rep., 2011, 28,78 www.rsc.org/npr REVIEW NMR methods for unravelling the spectra of complex mixtures Ramon Novoa-Carballal,a Eduardo Fernandez-Megia,a Carlos Jimenezb and Ricardo Riguera*a Received 30th July 2010 DOI: 10.1039/c005320c Covering: up to May 2010 The main methods for the simplification of the NMR of complex mixtures by selective attenuation/ suppression of the signals of certain components are presented. The application of relaxation, diffusion and PSR filters and other techniques (J-resolution, TOCSY, etc.), to biological samples, pharmaceuticals, foods, living organisms and natural products are illustrated with examples. 1 Introduction 3.3.3 NMR of pharmaceuticals 2 Relaxation filters 3.3.4 NMR of foods 2.1 Basic concepts 3.3.5 NMR of humic substances 2.1.1 T1, T2 and T1r filters 3.3.6 NMR of biological fluids and tissues 2.1.2 T2 and T1r versus T1 filters 3.3.7 NMR of other complex mixtures 2.1.3 T2 versus T1r filters 4 Other methods 2.1.4 Limitations and practical considerations of relaxa- 4.1 Selective saturation and magnetization transfer tion editing 4.2 TOCSY 2.2 Methodology and state of the art 4.3 J-resolved spectroscopy (JRES) 2.2.1 Pulse sequences: Inversion recovery and spin-echo 4.4 ‘Virtual’ relaxation-edited spectroscopy (RESY) Downloaded on 30/04/2013 15:21:41. 2.2.2 The use of combined techniques 4.5 Paramagnetic spin relaxation (PSR) filtering 2.2.3 Advanced editing of spectra (TOSY and TOPSY) 5 Conclusions Published on 11 October 2010 http://pubs.rsc.org | doi:10.1039/C005320C 2.3 Application of relaxation filters to the NMR analysis 6 Acknowledgements of mixtures 7 References 2.3.1 NMR of biological fluids 2.3.2 NMR of cells and tissues 2.3.3 NMR of whole specimens 1 Introduction 2.3.4 NMR of food 2.3.5 Relaxation vs. diffusion editing For chemists, it is unfortunate that Nature only very rarely 3 Diffusion methods presents its results in the form of pure compounds. Therefore, 3.1 Basic concepts separation and purification of components from complex 3.2 Methodology and the state of the art mixtures has historically occupied a central role in the effort of 3.2.1 Data acquisition researchers, particularly in the field of natural products 3.2.2 Data-processing approaches research. 3.2.3 The use of combined techniques Although very efficient separation methods have been devel- 3.2.4 Enhancement of diffusion coefficient differences oped that provide the pure components ready for structure 3.3 Applications of diffusion NMR spectroscopy to determination, most of the working time is devoted to the NMR analysis of complex mixtures separation/purification steps and not to the structural determi- 3.3.1 NMR of natural product extracts nation. A step further in the optimization of this research has 3.3.2 NMR of natural product mixtures been the implementation of hyphenated techniques such as HPLC–NMR, where the products of the HPLC column enter the NMR spectrometer directly so that no isolation or manip- aDepartment of Organic Chemistry and Centre for Research in Biological ulation of the sample is necessary to obtain the spectra. Still, the Chemistry and Molecular Materials, University of Santiago de investigation of complex mixtures relies on the classical para- Compostela, Jenaro de la Fuente s/n, 15782 Santiago de Compostela, Spain. E-mail: [email protected] digm that has presided over the work of natural products bDepartment of Fundamental Chemistry, Faculty of Sciences, University of chemists for years: first, to separate and purify the components, A Coruna,~ A Coruna,~ Spain and next, to submit the pure compounds to spectroscopic 78 | Nat. Prod. Rep., 2011, 28, 78–98 This journal is ª The Royal Society of Chemistry 2011 View Article Online analysis. This protocol requires the separation of many ‘useless’ its constituents. The mixture is directly submitted to NMR components in order to isolate a single or just a few compounds and the acquisition is implemented in a way that allows the of interest, and stresses the increasing importance of der- data from the components to be obtained with no previous eplication. separation. Nowadays. and thanks to the developments of NMR, it is The most important procedures are based on differences in the not always necessary to separate the components of translational diffusion and NMR relaxation times of the a mixture in order to obtain spectroscopic information from components in the mixture. These differences allow one to obtain Ramon Novoa-Carballal was BS (1984) and PhD (1988) in born in 1979, and received his Organic Chemistry at the BS (2002), MS (2004) and University of Santiago de Com- PhD (2009) in chemistry at the postela (USC), and did post- University of Santiago de Com- doctoral studies at the postela (USC) (supervisors R. University of California at Riguera and E. Fernandez- Santa Cruz (USA) with Dr Phil Megia). During his BS, he Crews (1988–1990) and at the joined the Friedrich Schiller Scripps Research Institute, La University Jena (Germany) for Jolla (USA) with Dr Alfonso two semesters (2000–2001). As Tramontano (1990–1991). He part of his PhD, he had two was assistant professor of short stays at the Imperial Chemistry at USC until 1992 Ramon Novoa-Carballal College London (supervisors A. Carlos Jimenez and then moved to University of Miller and M. Thanou) and the A Coruna,~ where he reached his Stockholm University (super- current position as Full visor G. Widmalm). In 2010 he obtained a Barrie de la Maza Professor of Organic Chemistry in 2009. His research focuses on Fellowship to undertake a postdoc at the University of Bayreuth. the chemistry of bioactive natural products (isolation, structural His research is focused on synthetic modification and character- elucidation, and synthesis), mainly from marine organisms, and the ization (especially by NMR spectroscopy) of polysaccharides and applications of NMR techniques to structural problems, mainly in Downloaded on 30/04/2013 15:21:41. dendrimers of pharmaceutical interest. the determination of the relative configuration of organic mole- cules. He has authored some 80 scientific publications and mono- graphs. Published on 11 October 2010 http://pubs.rsc.org | doi:10.1039/C005320C Eduardo Fernandez-Megia was Ricardo Riguera received his born in 1967 in Vigo, Spain, and PhD in Chemistry from the is currently a Professor Titular University of Santiago de Com- in the Organic Chemistry postela (USC) in 1973. He Department and Centre for carried out postdoctoral studies Research in Biological Chem- at University College London istry and Molecular Materials with Prof. P. Garrat (1974). He at the University of Santiago de was appointed Lecturer in 1978, Compostela (USC). After and in 1990 he became full completing a Ph.D. in 1995 at Professor of Chemistry at USC. USC (supervisor Prof. F. Javier His research is represented by Sardina), he undertook a post- around 200 papers and patents doctoral stay with Prof. Steven covering several topics: bioac- Eduardo Fernandez-Megia V. Ley at the University of Ricardo Riguera tive natural products, medicinal Cambridge (1997–1999). Then, chemistry and NMR methods he returned to the USC as for determination of absolute a Marie Curie Fellow and Prof. Asociado. In 2003, Eduardo configuration. He is now interested on polymeric nanostructures for became a Ramon y Cajal Fellow, followed by a Prof. Contratado biomedical applications and on stimuli responsive dynamic poly- Doctor in 2008. His research has focused on the interface between mers. He has authored three textbooks for students and served the organic and polymer chemistry with emphasis on the preparation of University as Chairman of the Department, Dean of the Faculty well-defined polymeric nanostructures for biomedical applications and Vice-Chancellor. and the development of NMR and SPR tools for their character- ization. This journal is ª The Royal Society of Chemistry 2011 Nat. Prod. Rep., 2011, 28, 78–98 | 79 View Article Online either edited spectra (i.e. spectra where only the components with widely used as a source of information on the dynamics of defined relaxation times or diffusion coefficients are observed), compounds,9 as well as on the study of interactions.10 or to obtain bidimensional spectra (one dimension being the In the case of complex mixtures, the dependence of the chemical shift and the other the diffusion coefficient or relaxation relaxation times on the dynamics and flexibility of each molecule time). Both options have been described for relaxation and opens the possibility to selectively attenuate, or even remove, the diffusion, but bidimensional spectra are only commonly used for signals of the components with shorter spin–lattice (also called diffusion (diffusion-ordered spectroscopy, DOSY). longitudinal) relaxation times, T1 and T1r (longitudinal relaxa- Relaxation filters allow the attenuation/suppression of the tion under spin-lock pulse conditions), or spin–spin (transversal) signals from compounds with high molecular weight, and diffusion relaxation times, T2. filters attenuate the signals from low molecular weight compounds. An illustrative example of relaxation edited spectra is shown in Therefore they can be used to simplify the spectra of a mixture by Fig. 1, where the signals from proteins and lipoproteins (with shorter selectively acting on these two types of components. Other T1, T1r and T2) are almost completely removed from the spectra. procedures, such as the addition of paramagnetic spin relaxation agents (PSR filters), are based on completely different mecha- 2.1.1 T1, T2 and T1r filters. A comprehensive description of nisms, and expand the usefulness of diffusion and relaxation filters the theoretical principles of NMR relaxation is out the scope of to other components independently of their molecular weight.

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