Accurate measurements of multiple-bond 13C-1H coupling constants from phase-sensitive 2D INEPT spectra.

Measurements of multiple-bond 13C-)H coupling constants are of great interest for the assignment of nonprotonated 13C resonances and the elucidation of molecular conformation in solution. Usually, the heteronuclear multiple-bond coupling constants were measured either by the J(CH) splittings mostly in selective 2D spectra or in 3D spectra, which are time consuming, or by the cross peak intensity analysis in 2D quantitative heteronuclear J correlation spectra (1994, G. Zhu, A. Renwick, and A. Bax, J. Magn. Reson. A 110, 257; 1994, A. Bax, G. W. Vuister, S. Grzesiek, F. Delaglio, A. C. Wang, R. Tschudin, and G. Zhu, Methods Enzymol. 239, 79.), which suffer from the accuracy problem caused by the signal-to-noise ratio and the nonpure absorptive peak patterns. Concerted incrementation of the duration for developing proton antiphase magnetization with respect to carbon-13 and the evolution time for proton chemical shift in different steps in a modified INEPT pulse sequence provides a new method for accurate measurements of heteronuclear multiple-bond coupling constants in a single 2D experiment.