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-) a¹³C-ω_I-half-filtered TOCSY spectrum yields the³J(H^N-C^β) coupling constant, which can be used to remove ambiguity in the π{variant} angle determination from³J(H^N-H^α). In cyclo(-d-Pro-Phe-Phe-Lys(Z)-Trp-Phe-) a¹⁵N-ω_I-half-filtered TOCSY was applied to individually assign the diastereotopic β-methylene protons via the³J(H^∇-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.