Accurate measurement of homonuclear coupling constants using JHH-TOCSY

Abstract

The homonuclear JHH coupling constant is one of the most important NMR parameters. In complex molecules with quite long relaxation times, E.COSY (1) in combination with the DISCO procedure (2) seems to be the best homonuclear method for quantifying J couplings. Other homonuclear methods are z-filtered COSY (3)) J-6 spectroscopy (4)) and selective excitation (5). These methods fail, however, in molecules with short spin-spin relaxation times where the linewidth is in the range of coupling constants. For these situations, some triple-resonance methods have been proposed recently (6-8), which can be used for 15Nand “Ccuenriched peptides and proteins. Their pulse sequences include a heteronuclear coherence transfer from “N to 13C. Additionally the magnetization transfer using a 13C spin lock works well for completely 13C-enriched molecules (9). In these spectra, the signal is split in Fi by the ‘JCH coupling constant, whereas the 3J(HN-H~) coupling constant can be extracted from the displacement in F2. From these methods, precisely estimated coupling constants are obtained, and they are, in distinction to E.COSY, independent of the linewidth. Until now no similar method has been proposed that could be used for molecules at natural abundance. For this purpose, we have developed two new pulse sequences shown in Figs. la and 1 b, called JHH-TOCSY. Sequence 1 a is a H,H correlation and sequence 1 b is a H,X correlation. As for the methods mentioned above, these sequences provide the JHH splitting for XH groups only, whereas XH2 groups show no splitting. For overlapping XH and XH2 groups, and for spectral simplification, XH selection may be used as shown in Fig. 1 c. Figure 1 d shows the 30 extension of sequence 1 a with and without XH selection. JHH-TOCSY is well suited to molecules at natural abundance or for 13Cor “N-monolabeled compounds. In comparison with the triple-resonance methods, the sequences are much shorter, because there is no need for the long 1 /( 2 JCN) delay. The evolution of the magnetization after 2, is as follows (only the relevant operators are shown); cf. Fig. la: