Dynamic nuclear polarization-enhanced, double-quantum filtered 13C-13C dipolar correlation spectroscopy of natural 13C abundant bone-tissue biomaterial.

Here, we describe a method for obtaining a dynamic nuclear polarization (DNP)-enhanced double-quantum filtered (DQF) two-dimensional (2D) dipolar 13C-13C correlation spectra of bone-tissue material at natural 13C abundance. DNP-enhanced DQF 2D dipolar 13C-13C spectra were obtained using a few different mixing times of the dipolar-assisted rotational resonance (DARR) scheme and these spectra were compared to a conventional 2D through-space double-quantum (DQ)-single-quantum (SQ) correlation spectrum. While this scheme can only be used for an assignment purpose to reveal the carbon-carbon connectivity within a residue, the DQF 13C-13C dipolar correlation scheme introduced here can be used to obtain longer distance carbon-carbon constraints. A DQF pulse block is placed before the DARR mixing scheme for removing dominant 13C single-quantum (SQ) signals because these SQ 13C signals are overwhelmingly large compared to those 13C-13C dipolar cross-peaks generated and therefore saturate the dynamic range of the NMR detection. This approach exhibits strong enough 2D cross-peaks in a dipolar 13C-13C correlation spectrum and potentially provides pairwise 13C-13C dipolar constraints because the dipolar truncation effect as well as multi-step signal propagations involving a spin cluster that contains more than two spins can be ignored probabilistically. To obtain fast signal averaging, AsymPolPOK was used to provide a short 1H DNP signal build-up time (1.3 s) and to expedite our MAS DNP NMR acquisitions while still maintaining a satisfactory DNP enhancement factor (ε = 50). Under long DARR mixing, a t1-noise-like artifact was observed at a site that possesses a large chemical shift anisotropy (CSA) and a few different strategies to address this problem were discussed.