Incorporation of 13C-Labeled Coniferyl Alcohol into Developing Ginkgo biloba L. Lignin Revealed by Analytical Pyrolysis and CuO Oxidation in Combination with Isotope Ratio Monitoring-Gas Chromatography-Mass Spectrometry

Summary A suite of four samples of xylem tissue from Ginkgo (Ginkgo biloba L.) shoots grown in a medium containing coniferin 13C-labeled at differing side-chain carbon atoms were studied using thermal and chemical degradation methods in combination with molecular-level isotopic analyses. The aims of the study were threefold: (1) to verify conclusions drawn from Nuclear Magnetic Resonance experiments previously performed on the same tissue samples, (2) to investigate degradation mechanisms and (3) to quantify the proportion of labeled material in each sample. Isotopic analysis of specific degradation products revealed the presence of the label exclusively within lignin-derived (phenolic) products and that the label is retained in its original position on the side-chain. These two results clearly indicate that there is no “scrambling” of carbon atoms as a result of thermal or chemical degradation, and thus lend strong support to analytical pyrolysis and chemolysis as viable approaches for structural investigations of the lignin macromolecule. Indeed, the isotopic enrichment of specific degradation products provides new evidence for certain types of linkages within the lignin polymer. The distribution and isotopic composition of the degradation products also strongly suggest an origin from newly-formed lignin as opposed to DHP-type products or unreacted substrate. As such, the data provides added confidence in the selective labeling approach for elucidation of the structure and biosynthesis of lignin. Isotopic mass balance calculations reveal that certain pyrolysis and CuO oxidation products show enhanced labeling which may be indicative of preferential incorporation of their specific precursors into the growing lignin macromolecule or heterogeneous lignin deposition.