NASCA‐HMBC, a New NMR Methodology for the Resolution of Severely Overlapping Signals: Application to the Study of Agathisflavone Gaétan Bayiha Ba Njock, a,b,c Trixie Ann Bartholomeusz, a Mohammadali Foroozandeh, b Dieudonné Emmanuel Pegnyemb, c Philippe Christen a * and Damien Jeannerat b ABSTRACT: Introduction – Standard NMR 2D heteronuclear HMBC spectra have a low resolution in the indirect carbon dimension, making it very difficult to assign signals to individual carbons when their chemical shifts are < 0.3 ppm apart. Objective – To establish spectral aliasing for HMBC experiments to improve the resolution in the carbon dimension without increasing the total experimental time and avoiding ambiguities in the observed chemical shifts. Methodology – The NASCA‐HMBC (Non‐ambiguous Assignment by Superposition of Coupled Aliased HMBC) methodology combines a pair of HMBC spectra recorded with slightly different carbon windows to provide typically one order of magnitude increase in the resolution and unambiguous chemical shifts. Results – The application of this methodology to a biflavonoid found in Ouratea gilgiana resulted in spectra with a sufficiently high resolution to make the assignment straightforward and report, for the first time, the full assignment of agathisflavone. Conclusion – The methodology should find many applications in dimeric and oligomeric compounds such as peptides, carbohydrates, polyketides and other cases where signal clustering is expected. Copyright © 2011 John Wiley & Sons, Ltd. Keywords: spectral aliasing; high‐spectral resolution; NMR; biflavonoids; Ouratea gilgiana Introduction Usually, spectral information present in one‐ and two‐dimensional NMR spectra, including 1 H, 13 C, 2D COSY, HSQC and HMBC, are combined to determine the connectivity of atoms and assign the structure of a compound. Two‐dimensional spectra are very powerful because they directly identify atoms that are one or a few bonds apart through the scalar coupling of their nuclei. When recorded with the classical acquisition parameters, two‐ dimensional heteronuclear 1 H– 13 C experiments (HSQC, HMBC, HMQC) resolve the signals of protons provided that the carbons differ by more than about 0.3 ppm. In most cases, this limitation is not problematic and accidental overlap of a couple of carbons does not jeopardise the determination of the coupling network. But when studying dimeric structures such as the biflavonoid agathisflavone (Fig. 1), signals of the different units almost systematically resonate as pairs of signals only a few hertz apart and the assignment of signals is seriously hampered, as illustrated in Fig. 2. When determining structures using long‐range HMBC correla- tions, the intrinsic difficulty of distinguishing between two‐ and three‐bond correlations gets dramatically worse when signals cannot be unambiguously assigned to individual carbons. The number of candidate structures therefore increases dramatically (Bayiha Ba Njock et al., 2010), which also increases the risk of misassignment, resulting in a wrong structure. Some aspects about the origin of the problem are presented below, together with a very practical solution to resolve clusters of signals. The low resolution in the 13 C dimension of 2D NMR spectra is due to the indirect detection of the carbon chemical shifts. In classical set‐up, typically only 128–512 points are recorded, while thousands of points are acquired in 1D experiments. Increasing this number of points (usually called “time increments”) by a factor of 20 would increase the resolution towards the limit imposed by relaxation and homonuclear couplings, but it would come with a 20‐fold increase in experimental time, making this solution unrealistic. Spectral aliasing (Eggenberger et al., 1988; Schmieder et al., 1991; Jeannerat, 2007) is a very powerful method to improve spectral resolution in the carbon dimension without increasing the experimental time. The most practical implementation * Correspondence to: Dr. P. Christen, Section of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Quai E. Ansermet 30, CH‐1211 Geneva 4, Switzerland. E-mail: philippe.christen@unige.ch a Section of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Quai E. Ansermet 30, CH‐1211 Geneva 4, Switzerland b Department of Organic Chemistry, University of Geneva, Quai E. Ansermet 30, CH‐1211 Geneva 4, Switzerland c Department of Organic Chemistry, Faculty of Sciences, University of Yaounde I, P.O. Box 812, Yaoundé, Cameroon Phytochem. Anal. 2012, 23, 126–130 Copyright © 2011 John Wiley & Sons, Ltd. Research Article Received: 22 February 2011; Revised: 30 March 2011; Accepted: 30 March 2011 Published online in Wiley Online Library: 19 May 2011 (wileyonlinelibrary.com) DOI 10.1002/pca.1333 126