Measurement of small one-bond proton-carbon residual dipolar coupling constants in partially oriented (13)C natural abundance oligosaccharide samples: analysis of heteronuclear (1)J(CH)-modulated spectra with the BIRD inversion pulse

Two 2D J-modulated HSQC-based experiments were designed for precise determination of small residual dipolar one-bond carbon-proton coupling constants in (13)C natural abundance carbohydrates. Crucial to the precision of a few hundredths of Hz achieved by these methods was the use of long modulation intervals and BIRD pulses, which acted as semiselective inversion pulses. The BIRD pulses eliminated effective evolution of all but (1)J(CH) couplings, resulting in signal modulation that can be described by simple modulation functions. A thorough analysis of such modulation functions for a typical four-spin carbohydrate spin system was performed for both experiments. The results showed that the evolution of the (1)H-(1)H and long-range (1)H-(13)C couplings during the BIRD pulses did not necessitate the introduction of more complicated modulation functions. The effects of pulse imperfections were also inspected. While weakly coupled spin systems can be analyzed by simple fitting of cross peak intensities, in strongly coupled spin systems the evolution of the density matrix needs to be considered in order to analyse data accurately. However, if strong coupling effects are modest the errors in coupling constants determined by the "weak coupling" analysis are of similar magnitudes in oriented and isotropic samples and are partially cancelled during dipolar coupling calculation. Simple criteria have been established as to when the strong coupling treatment needs to be invoked.