Boosting the NMR Assignment of Carbohydrates with Clean In‐Phase Correlation Experiments

Abstract

Novel CLIP‐COSY based homo‐ and heteronuclear correlation experiments are reported for the rapid, semi‐automated NMR assignment of small to medium‐sized molecules. The homonuclear CLIP‐COSY and corresponding relayed experiments employ the perfect‐echo based mixing sequence for in‐phase coherence transfer between directly and/or indirectly coupled proton spins. The combined analysis of the resulting CLIP‐COSY and relayed spectra made it possible to easily track down, layer by layer, the proton–proton connectivity network. In larger molecules the narrow chemical shift range of protons may, however, compromise the efficacy of the homonuclear correlation based assignment strategy. To overcome this limitation, an HSQC variant of the CLIP‐COSY experiment has now been devised. Combined treatment of HSQC‐CLIP‐COSY (relayed) and standard HSQC spectra facilitates simultaneous and semi‐automatic assignment of 1H and 13C resonances of medium‐sized molecules, such as pentasaccharides. The recently introduced PSYCHE broadband homonuclear decoupling scheme has been also implemented into the devised homo‐ and heteronuclear CLIP‐COSY based experiments, resulting in fully decoupled high‐resolution pure‐shift correlation spectra.

perfectBASH: Band‐selective homonuclear decoupling in peptides and peptidomimetics

“…In this section, we want to illustrate the utility of F 1 perfect BASH decoupled 2D experiments to the spectral analysis of α‐peptides. Following the generalized experiment design outlined in Figure c, we implemented F 1 perfect BASH homonuclear decoupling in the indirect dimensions of various homonuclear correlation techniques, namely TOCSY, NOESY, EASY‐ROESY, CLIP‐COSY, and relayed CLIP‐COSY experiments, to demonstrate the broad applicability of this approach and to provide a valuable experimental toolbox to potential users of these techniques. The full pulse sequence codes for all five 2D experiments implemented can be found in the Supporting Information.…”

“…For sequential signal assignment, it is useful to combine TOCSY spectra, which map out the full spin system, together with COSY techniques, which provide the assignment of next neighbors within the spin system. To provide the user with the complementary COSY techniques to the F 1 perfect BASH TOCSY just presented, we implemented the same F 1 decoupling approach for the recently presented CLIP‐COSY and relayed CLIP‐COSY experiments. These experiments are amenable to pure shift acquisition approaches in both of their spectral dimensions, due to the fully absorptive in‐phase signal character they provide for both cross and diagonal peaks.…”

“…Scalar coupling is refocused at time point IV before mixing, whereas chemical shifts have experienced a net evolution for t 1 at the same time point. Homonuclear mixing can be enforced using elements for isotropic mixing along the coupling network (TOCSY), for cross‐relaxation via the nuclear Overhauser effect (NOESY or EASY‐ROESY), or for in‐phase and relayed in‐phase COSY transfer (CLIP‐COSY and relayed CLIP‐COSY). The delays δ 1 – δ 4 in (c) are set to δ 1 = δ 3 = 2 p 16 + 3 d 16 , δ 2 = p 16 + d 16 , and δ 4 = p 16 + 2 d 16 .…”

“…This situation is shown in the generalized pulse sequence in Figure c. Homonuclear mixing may be performed via isotropic mixing along the scalar coupling network with TOCSY, via the nuclear Overhauser effect using NOESY or EASY‐ROESY, and via in‐phase or relayed in‐phase COSY transfer (CLIP‐COSY and relayed CLIP‐COSY). The timing slightly differs from the interferogram‐based 1D perfect BASH sequence, to achieve chemical shift evolution for t 1 and scalar coupling removal just before the beginning of the mixing element.…”

Towards perfect NMR: Spin‐echo versus perfect‐echo building blocks

“…The unquestionable utility of the ZQF element is demonstrated by its successful incorporation in most of the classical NMR experiments, such as J ‐resolved, COSY/RELAYED‐COSY, TOCSY, NOESY, ROESY, nemoDIAG, G‐SERF, DOSY, HSQC, HSQC‐TOCSY, HSQMBC or HSQMBC‐TOCSY, among others. In general, the ZQF introduces some loss of sensitivity because it removes detectable magnetization and lengthens the duration of the sequence, but this is more than compensated for with the improvement in spectral quality.…”