Practical advances in conversion of lignocellulosic biomass to bioethanol require improvement of current knowledge about the composition and chemical diversity of lignin. This research aimed to perform mass spectrometry-based metabolomic profiling of grasses to fill in these knowledge gaps. Six grasses, and the hardwood poplar, were investigated including: corn stover, wheat straw, rice straw, switchgrass, Miscanthus, and sorghum. Methanolic extracts of untreated biomass contained intact lignin constituents that were profiled using ultrahigh performance liquid chromatography-time-of-flight mass spectrometry (UHPLC-TOF MS). Most extractives exhibited molecular masses consistent with phenolic compounds. Derivatives of the flavonoid tricin were prominent in extracts of all grasses but were not detected in poplar. Multiplexing of non-selective collision-induced dissociation (CID) in UHPLC-MS analyses provided evidence that more than 90% of tricin is incorporated in compounds other than the small number of abundant tricin derivatives. Wide-mass window tandem mass spectrometry (MS/MS) analyses provided further evidence for incorporation of tricin, p-coumarates, and other monolignols up to 4 kDa molecular mass. Mass spectrometric profiling of grass extractives also revealed abundant phenolic acid esters of glycerol, with 1-p-coumaroyl-3-feruloylglycerol the most abundant in corn stover. Profiles yielded evidence that these esters undergo dehydrodimerization via reactions of to form diferulate esters. The presence of these novel compounds demonstrates that glycerol esters undergo oligomerization similar to classical monolignols. In addition, extracts contained conjugates of tricin with 1-p-coumaroyl-3-feruloylglycerol, and provide further evidence of.Extracts of corn stover also contained numerous oligomers of the ester sinapyl p-coumarate. Using UHPLC-HRMS, MS/MS, and 1D and 2D NMR spectra, the most abundant isomer is formed by 8-8 coupling of two sinapyl p-coumarate groups followed by formation of a tetrahydrofuran (THF) core with oxygen bridging between position-7 carbons of each sinapyl group. Larger phenolic constructs containing this compound in their core were also annotated using UHPLC-MS/MS that were formed by addition of either more oxidized sinapyl p-coumarate groups or oxidized coniferyl alcohols. Compounds of this category were detected up to m/z 2000 in agreement with wide-mass window MS/MS analyses.High molecular weight lignins may have low solubility, and to explore the range of lignin composition, γ-valerolactone (GVL) was evaluated as a solvent for electrospray ionization (ESI) –MS and as a mobile phase component for reversed phase liquid chromatography. GVL yielded simpler ESI spectra of phenolic substances due to reduced adduct multiplicity for each molecule. GVL in the mobile phase resulted in faster elution relative to methanol, while chromatographic resolution for major extractives of corn stover was retained. GVL also exhibited differential retention selectivity for lignin compounds compared to methanol which offers benefits for separation and analysis of polymers and large natural products. Taken together, these findings suggest diversity of lignin components in grasses is extensive despite a limited range of lignin precursors. Recognition of the nature of tricin and p-coumarate ester derivatives provides a foundation for novel strategies for deconstructing lignins and converting biomass to renewable fuels and chemical feedstocks.
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