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

Abstract

In the structural analysis of lignins by ¹³C-NMR, signal overlap limits definitive assignment and accurate intensity 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 polymers) 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 between the walls containing ¹³C-enriched and unenriched lignins afforded simplified 2D spectra in which well-separated signals were assigned exclusively to the specifically enriched carbons. This novel NMR technique provides a useful tool for elucidation of entire protolignin in the cell wall of ginkgo xylem.

Keywords

Carbon-13 enrichment, carbon-13-NMR spectroscopy, dehydrogenation polymer (DHP), 2D difference NMR spectroscopy, ginkgo, protolignin, acetylation, dimethyl sulfoxide, xylem, dehydrogenation, spectrum analysis, wood chemistry, nuclear magnetic resonance spectroscopy, carbon isotopes, lignin analysis, plant cell walls, DHP, N-methylimidazole, Ginkgo biloba, polymers, coniferin

Citation

Terashima, Noritsugu; Akiyama, Takuya; Ralph, Sally; Evtuguin, Dmitry; Pascoal, Carlos Neto; Parkas, Jim; Paulsson, Magnus; Westermark, Ulla; Ralph, John. 2009. 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. Holzforschung. Vol. 63, no. 4: pages 379-384.