Conformational analysis relies heavily on the use of scalar coupling constants obtained from NMR spectra. Of particular value are 3-bond 1H-1H coupling constants (3JHCCH) which, given a suitable Karplus equation, reveals the torsion angle between the two coupled protons. Common methods for extracting 3JHCCH from complex spectra include taking slices from a 2D NMR experiment (COSY, HSQC), often with poorly resolved multiplet structure, or 1D- or "selective" versions of 2D experiments (selective TOCSY, NOESY). The latter experiments produce well resolved multiplets, but require a clean "entry point" in the 1H spectrum: an isolated signal for selective excitation that is coupled to the other signals of interest. Our work on the conformation of the furanose rings in the calcium release agent cyclic adenosine diphosphate ribose (cADPR) used selective TOCSY to great effect, but in the corresponding inosine compound (cIDPR) we were confronted with three coupled protons (the 2', 3', and 4' protons of the R-ring) that were not only nearly isochronous (to within a few Hz), non-first order in appearance, and also showed very small couplings to the usual entry point 1' and 5'/5" protons. Lacking a suitable entry point in the 1H spectrum, we turned to the more dispersed 13C spectra as an alternative. We modified the PIP-HSQC (pure in-phase HSQC) sequence published in 2014, converting it from a 2D experiment to a selective 1D version. This selective PIP-HSQC uses a selective 13C pulse (with proton decoupling) for excitation followed by acquisition of a 1H spectrum (without 13C decoupling), producing spectra from which highly accurate 3JHCCH coupling constants may be obtained. These spectra have the advantage appearing much more first order, as the coupling being measured is now not between two nearly isochronous protons each on 12C (H-12C-12C-H) but between one on 12C and one on 13C (H-12C-13C-H), with the latter having been shifted upfield and downfield by 60-70 Hz, one-half the one bond C-H coupling constant.

Carbon-selective methods for extracting accurate 3-bond 1H-1H coupling constants from complex spectra: Selective PIP-HSQC

ACS Fall 2020 Virtual Meeting

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