2D-NMR (HSQC) difference spectra between specifically 13C-enriched and unenriched protolignin of Ginkgo biloba obtained in the solution state of whole cell wall material

In the structural analysis of lignins by 13C-NMR, signal overlap limits definitive assignment and accurate inten­ sity measurement. Selective labeling by 13C-enrichment of a specific carbon in lignin enhances its signal intensity in the spectrum. Further enhancement of the specifically labeled carbons can be realized via difference spectra created from the enriched and unenriched samples. Difference 2D 13C-1H correlation (HSQC) NMR spectra, derived from the spectra of specifically 13C-enriched lignin model polymers (so-called dehydrogenation poly­ mers) and their unenriched counterparts, take advantage of the enhanced dispersion afforded by both 13C and 1H chemical shifts, diminishing the difficulties arising from the signal-overlap problem and aiding in definitive signal assignments. In this research, protolignin in xylem cell walls was specifically 13C-enriched at all of the individual phenylpropanoid side-chain carbons by feeding 13C­ enriched coniferins to growing stems of Ginkgo biloba. The whole xylem fractions containing 13C-enriched and unenriched protolignins were dissolved in a mixture of N-methylimidazole and DMSO, and then acetylated. Solution state 2D-NMR (HSQC) spectra of the acetylated whole cell wall were acquired. Difference spectra be­ tween the walls containing 13C-enriched and unenriched lignins afforded simplified 2D spectra in which well-sep­ arated signals were assigned exclusively to the specif­ ically enriched carbons. This novel NMR technique provides a useful tool for elucidation of entire protolignin in the cell wall of ginkgo xylem.