Incorporation of C-13-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

A suite of four samples of xylem tissue from Ginkgo (Ginkgo bilobo L.) shoo ts grown in a medium containing coniferin C-13-labeled at differing side-ch ain carbon atoms were studied using thermal and chemical degradation method s in combination with molecular-level isotopic analyses. The aims of the st udy were threefold: (1) to verify conclusions drawn from Nuclear Magnetic R esonance experiments previously performed on the same tissue samples, (2) t o 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 t he label is retained in its original position on the sidechain. These two r esults 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 inv estigations of the lignin macromolecule. Indeed, the isotopic enrichment of specific degradation products provides new evidence for certain types of l inkages 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 confidenc e in the selective labeling approach for elucidation of the structure and b iosynthesis of lignin. Isotopic mass balance calculations reveal that certa in 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.