Heteronuclear three-dimensional nmr spectroscopy. A strategy for the simplification of homonuclear two-dimensional NMR spectra

Abstract

Recently, three-dimensional Fourier transform techniques have been suggested (I3) as a possible method to resolve the spectra of complicated systems. Analogous to the comparison of 2D with 1 D NMR, three-dimensional NMR spectroscopy allows an increase in resolution and offers additional correlations for the elucidation of scalarand dipolar-coupled spin networks as compared to 2D NMR. Homonuclear 3D NMR experiments have been reported which emp loy tailored soft pulses (I, 2) to excite only parts of the proton spectral window or which use the narrow frequency band of J couplings for the third dimension (3) in order to reduce the experimental accumulation time. In this communication, we propose heteronuclear 3D NMR spectroscopy to aid in the simplification of two-dimensional proton NMR spectra of isotopically labeled compounds. For proteins, labels can easily be introduced by adding isotopically labeled nutrients to the growth med ium of bacterial expression systems (e.g., Ref. (4)). The experiments described comprise a combination of heteronuclear mu ltiple-quantum correlation (HMQC) (5, 6) with homonuclear COSY and NOESY and may therefore be named HMQC-COSY and HMQC-NOESY. Heteronuclear 3D experiments hold considerable promise for application to larger systems because one of the coherence transfer steps involves scalar couplings much larger than the ‘H linewidths. F igure 1 shows the pulse sequences of the HMQC-COSY and HMQC-NOESY experiments. Analogous to the HMQC experiment, heteronuclear mu ltiple-quantum coherence created by the first two RF pulses evolves during tI effectively with the single-quantum heteronuclear chemical shift. This coherence is converted to proton single-quantum coherence ant iphase with respect to the heteronucleus by the second hetero pulse, which is refocused at the end of the second 7 period. On ly those protons which are coupled to the heteronucleus are frequency labeled during tl . Therefore, the further frequency labeling during t2 under the homonuclear Hamiltonian needs to cover only the heteronuclear coupled protons, thus allowing nonselective RF pulses to be used throughout the sequence. Protons not coupled to the heteronuclei, which may be considered axial peaks in ol, are suppressed by an inversion of the first hetero pulse concomitant with an inversion of the receiver phase (6). Depending on the experiment, the signals arising from heteronuclear coupled protons are converted to COSY (Fg 1A) or NOESY (Fii 1B) type responses. In order to enhance sen-