Determination of heteronuclear long-range couplings to heteronuclei in natural abundance by two- and three-dimensional NMR spectroscopy.

A method to determine heteronuclear long-range couplings to carbon and nitrogen at natural abundance is presented and applied to two cyclic hexapeptides and the peptidomacrolide FK506. The method is applicable for proton-bearing heteronuclei. By introduction of heteronuclear half-filters in two- or three-dimensional experiments the spectra exhibit an E.COSY pattern when executed without heteronuclear decoupling. The extraction of the heteronuclear coupling constants is therefore independent of linewidth. In cyclo(-Ala-Ala-Ala-Pro-Ala-Pro-) a 13C-omega 1-half-filtered TOCSY spectrum yields the 3J(HN-C beta) coupling constant, which can be used to remove ambiguity in the phi angle determination from 3J(HN-H alpha). In cyclo(-D-Pro-Phe-Phe-Lys(Z)-Trp-Phe-) a 15N-omega 1-half-filtered TOCSY was applied to individually assign the diastereotopic beta-methylene protons via the 3J(H beta-N). In FK506 a 3D-HMQC-TOCSY without heteronuclear decoupling is used to obtain a number of heteronuclear coupling constants to carbons. These values have been applied for the assignment of diastereotopic methylene protons and determination of dihedral angles in the cyclic portion of the molecule.