Abstract
Ferulate-mediated cross-linking of plant cell wall polymers has various implications on the quality of plant based food products, forage digestibility, and biomass utilization. Besides dehydrodiferulic acids (DFA), dehydrotriferulic acids (TriFA) gained increasing interest over the past two decades, because they potentially cross-link up to three polymers. Here, we describe a separation strategy to obtain several TriFA as analytical standard compounds from a reaction mixture after radical coupling of ethyl ferulate. By using silica flash chromatography, Sephadex LH-20 chromatography, and reversed phase HPLC, six known TriFA as well as three previously unidentified ferulic acid trimers were obtained, and their structures were characterized by mass spectrometry and NMR spectroscopy (1H, HSQC, COSY, HMBC, and NOESY). The novel trimers were identified as 5-5/8-8(cyclic)-, 8-8(noncyclic)/8-O-4-, and, tentatively, 5-5/8-8(noncyclic)-TriFA. Natural occurrence of these TriFA in plant cell walls was demonstrated by LC-MS/MS analyses of alkaline cell wall hydrolyzates.
Highlights
Ferulic acid is widely distributed in plant cell walls
We described the generation of TriFA by radical coupling of ethyl ferulate using the TMEDA complex previously described by Lu et al (2012) to produce dehydrodiferulic acids (DFA) (Waterstraat et al, 2016)
Eluting fractions from the initial fractionation by silica flash chromatography can be purified after saponification by reversed phase RP-HPLC to obtain 8-O-4/8-5(noncyclic)-TriFA, 8-O-4/8-O-4-TriFA, and 8O-4/8-5(cyclic)-TriFA (Waterstraat et al, 2016)
Summary
Ferulic acid is widely distributed in plant cell walls. Esterified to arabinoxylans, larger amounts of ferulic acid can be found in monocotyledonous plants, within the Poaceae family (Smith and Hartley, 1983; Harris and Trethewey, 2010). After washing the combined ethyl acetate fractions with acidic NH4Cl solution (2 mL of 1 M HCl plus 20 mL of saturated NH4Cl), the organic solvent was removed by rotary evaporation. The fraction obtained by rinsing the silica flash chromatography column with ethyl acetate and subsequent saponification, contained a large variety of TriFA, dehydrotetraferulic acids, partially saponified oligomers, and additional unknown reaction products.
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