Sensitivity enhancement by sequential data acquisition for 13C-direct detection NMR

Abstract

13C-direct detection NMR has several advantages compared to proton detection, including a tendency to relax slower and wider chemical shift range. However, the sensitivity of 13C-direct detection is much lower than that of proton detection because of its lower gyromagnetic ratio. In addition, a virtual decoupling procedure is often performed to remove peak splitting in the 13C-direct detection axis, which further reduces the sensitivity to 1/√2. In this study, to enhance the sensitivity of 13C-direct detection experiments, we developed a HCACO-type new pulse sequence in which anti-phase (AP) and in-phase (IP) signals are acquired sequentially in a single scan. The developed experiment was tested on an amino acid (valine) and two proteins (streptococcal protein G B1 domain (GB1) and α-synuclein). The AP and IP spectra were successfully obtained in all cases. Using these spectra, IPAP virtual decoupling was performed, and peak splitting was successfully removed. The sensitivity of the experiment was increased by 1.43, 1.26 and 1.26 times for valine, GB1 and α-synuclein, respectively, compared to the conventional HCACO experiment. In addition, we developed another HCACO-type pulse sequence, where AP and IP signals are simultaneously acquired in a single FID. The sensitivity of the experiment was increased by 1.40 and 1.35 times for valine and GB1, respectively. These methods are potentially applicable to other 13C-direct detection experiments that measure one-bond correlations and will further extend the utility of the 13C-direct detection method, especially for structural analyses of intrinsically disordered proteins.