3.2.2. 2D 1H-13C NMR Spectra Collection and Processing

The 2D 1H-13C HSQC spectra shown in Figure 3 were collected using a 600 MHz spectrometer. A modified sensitivity enhanced gradient HSQC pulse sequence *hsqcetgpsi2.kc* was applied [44]. The spectral width for the 1H dimension was 11 ppm with the carrier frequency centered at 4.8 ppm. The spectral width for the 13C dimension was 50 ppm with the carrier frequency centered at 23 ppm. The complex points of 1024 and 600 were acquired for the 1H and 13C dimensions, respectively. The resulting acquisition times for 1H and 13C spins were 78 and 40 ms, respectively. The 13C decoupling sequence was GARP with a radio frequency field strength of 1.9 kHz. The coupling constant 1JHC was set to 155 Hz as a compromise between efficient INEPT transfer and T2 signal loss. The recycle delay was 2 s. The number of scans was 16 and the total experimental time was 6 h.

The data processing was performed using NMRPipe [45]. The apodization function of cosine was applied to both dimensions of 1H and 13C. The first point was scaled with a factor of 0.5 before zero-order phase correction. Zero filling of 2048 × 1024 real data points was applied to the 1H and 13C dimensions. The baseline corrections on frequency domains were carried out with a polynomial function under auto mode. The chemical shift reference followed the established procedure [46]. HSQC peaks with *s*/*n* higher than 10 were picked and peak heights were recorded using Sparky (Sparky 3, UCSF).
