Highly Acylated (Acetylated and/or p-Coumaroylated) Native Lignins from Diverse Herbaceous Plants JOS ´ E C. DEL R´ IO,* ,† JORGE RENCORET, † GISELA MARQUES, † ANA GUTI ´ ERREZ, † DAVID IBARRA, ‡ J. IGNACIO SANTOS, ‡ JES ´ US JIM ´ ENEZ-BARBERO, ‡ LIMING ZHANG, § AND ´ ANGEL T. MART ´ INEZ ‡ Instituto de Recursos Naturales y Agrobiologı ´a de Sevilla, CSIC, P.O. Box 1052, 41080 Seville, Spain; Centro de Investigaciones Biolo ´gicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain; and Fiber and Polymer Technology, Royal Institute of Technology (KTH), SE-100 44 Stockholm, Sweden The structure of lignins isolated from the herbaceous plants sisal (Agave sisalana), kenaf (Hibiscus cannabinus), abaca (Musa textilis) and curaua (Ananas erectifolius) has been studied upon spectroscopic (2D-NMR) and chemical degradative (derivatization followed by reductive cleavage) methods. The analyses demonstrate that the structure of the lignins from these plants is highly remarkable, being extensively acylated at the γ-carbon of the lignin side chain (up to 80% acylation) with acetate and/or p-coumarate groups and preferentially over syringyl units. Whereas the lignins from sisal and kenaf are γ-acylated exclusively with acetate groups, the lignins from abaca and curaua are esterified with acetate and p-coumarate groups. The structures of all these highly acylated lignins are characterized by a very high syringyl/guaiacyl ratio, a large predominance of -O-4′ linkages (up to 94% of all linkages), and a strikingly low proportion of traditional -′ linkages, which indeed are completely absent in the lignins from abaca and curaua. The occurrence of -′ homocoupling and cross-coupling products of sinapyl acetate in the lignins from sisal and kenaf indicates that sinapyl alcohol is acetylated at the monomer stage and that, therefore, sinapyl acetate should be considered as a real monolignol involved in the lignification reactions. KEYWORDS: Lignin; herbaceous plants; sinapyl acetate; sinapyl p-coumarate; 2D-NMR; HSQC; DFRC; sisal; kenaf; abaca; curaua INTRODUCTION Lignins are complex natural biomacromolecules characteristic of vascular plants, where they provide mechanical support. In addition, lignin waterproofs the cell wall, enabling transport of water and solutes through the vascular system, and plays a role in protecting plants against pathogens (1). The lignin polymer results from the random oxidative coupling of p-hydroxycin- namyl monolignols mediated by laccases and/or peroxidases (2, 3). The three primary monolignols are p-coumaryl, coniferyl, and sinapyl alcohols, which produce, respectively, p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) phenylpropanoid units when incorporated into the lignin polymer. However, it is now widely accepted that other monomers also participate in coupling reactions giving rise to the lignin macromolecule. This is the case of γ-acylated (with acetate, p-hydroxybenzoate and/or p-coumarate groups) lignins, which have been discovered in many plants. Different grass lignins are partially p-coumaroylated, and some hardwood lignins such as poplar, aspen, or willow lignins are p-hydroxybenzoy- lated (3-11). Acetylated lignin units have also been reported to occur in many plants (12-16). Characteristic products from sinapyl and coniferyl acetate coupling have been detected upon degradative techniques (Py-GC/MS and derivatization followed by reductive cleavage, DFRC) in the lignin of several plants characterized by having a high S/G ratio such as sisal, kenaf, abaca, and jute (15, 16). Previous studies have shown that lignin from these plants is acetylated exclusively at the γ-position of the side chain and that this acetylation occurred predominantly on S units (12-16). Moreover, these studies have provided strong evidence that sinapyl acetate is implicated as a monomer in lignification in several plants and that the naturally acetylated lignin derives not from acetylation of the lignin polymer but from polymerization of preacetylated monolignols (14, 16, 17). The same seems also to occur with sinapyl p-coumarate and sinapyl p-hydroxybenzoate (17-19). We have recently shown, using a previously developed modification of the DFRC method to allow detection of natural acetate groups (13) (the so-called DFCR′ methodology) that lignin γ-acetylation is widespread, and probably ubiquitous, * Author to whom correspondence should be addressed (e-mail delrio@irnase.csic.es; telephone +34 95 462 4711; fax +34 95 462 4002). † Instituto de Recursos Naturales y Agrobiologı ´a de Sevilla, CSIC. ‡ Centro de Investigaciones Biolo ´gicas, CSIC. § Royal Institute of Technology (KTH). J. Agric. Food Chem. 2008, 56, 9525–9534 9525 10.1021/jf800806h CCC: $40.75  2008 American Chemical Society Published on Web 09/30/2008