Measurement of J CH NMR Spin-Couplings in Saccharides: Non-First-Order Behavior in ¹ H NMR Spectra Introduced by Selective ¹³ C Labeling

The incorporation of ¹³C isotopes into saccharides introduces additional signal multiplicity into the ¹H NMR spectra caused by ¹³C-¹H spin-coupling to the highly abundant ¹³C spins. Large ¹JCH values have been exploited to relieve non-first-order (strong coupling) effects in these spectra by spitting the signal of a ¹H spin that overlaps the signal arising from a mutually coupled ¹H spin. Using NMR spin simulation and complementary experimental studies, we show that this approach is valid only under specific spectral conditions. When the homonuclear JHH value between the strongly coupled hydrogens exceeds ∼6.5 Hz, the underlying multiplet exposed by ¹JCH splitting will experience a chemical shift offset due to strong coupling that complicates the measurement of JCH values if this hydrogen is also spin-coupled to the ¹³C-labeled carbon. Since the magnitude of the offset scales with the magnitude of the ³JHH between the strongly coupled hydrogens after a threshold of ∼6.5 Hz is reached, antiperiplanar hydrogens in saccharide pyranosyl rings are particularly vulnerable to this complication. The offset is not eliminated when spectral data are collected in two (and presumably higher) dimensions. This heretofore under-appreciated effect causes significant errors in the measurement of JCH values, especially those having dynamic ranges of <4-5 Hz. This behavior will be more difficult to recognize and treat in larger oligosaccharides that contain ¹³C enrichment at one or more carbons when multidimensional spectra are collected to improve the spectral resolution.