Lignin-carbohydrate complexes (LCC) can be selectively isolated from lignified secondary walls of woods, for example, bald cypress (Taxodium distichum), birch (Betula platyphylla) (89), spruce (90), beech (Fagus crenata) and pine (Pinus densiflora) (91), and grasses, for ex­ample, sugarcane (Saccharum officinarum) bagasse (92), rice (Oryza sativa) (89), and wheat (Triticum aestivum) straws (93) by fractionation, co-extraction, and co-chromatography of native or derivatized preparations (89, 94-96). Comparative chemical (97) and physico­chemical procedures, including 13C-NMR (97), 2D NMR (98), and FTIR (99) have been used in the characterization of the chemistry of the lignin-polysaccharide linkages.

The carbohydrate portion of LCC from the softwood bald cypress (Taxodium distichum) consists of galactoglucomannan, arabino(4-O-methylglucurono)xylan, and

Figure 4.4 Feruloyl units on neighboring GAX chains in cell walls may be cross-linked by radical coupling into terulate dehydrodimers (structures 1-5) or dehydrotrimers (structure 6). Dotted arrows indicate potential sites tor radical coupling with hydroxycinnamoyl alcohols or lignin oligomers in Iignitying cell walls, resulting in cross-linking of GAXs to lignin. "Ara" is an arabinoturanosyl residue on a GAX. (Reprinted with permission, from Grabber, J. H., Hatfield, R. D., Ralph, J., Zoh, J. & Amrhein, N. (1995) Ferulate cross-linking in cell walls isolated from maize cell suspensions. Phytochemistry, 40, 1077-1082, Fig. 1.)

arabinogalactan. In contrast, the LCCs of the hardwood birch (Betula platyphylla) and rice straw (Oryza sativa) are composed exclusively of 4-O-methylglucuronoxylans and arabino(4-O-methylglucurono)xylan, respectively (89). Laine and coworkers (67) found 4-linked xylan, 3,6-galactan, 4-linked galactan, and 3-linked glucan in the residual LCCs isolated from the kraft pulps of coniferous gymnosperms spruce (P. abies) and pine (Pinus sylvestris). Lawoko and coworkers (90) obtained four major LCC fractions from P abies and characterized a galactoglucomannan LCC containing ~8% of the wood lignin, a glucan LCC containing ~4% of the wood lignin, a xylan-lignin-glucomannan network LCC (xylan > glucomannan) containing ~40% of the wood lignin, and a glucomannan-lignin-xylan net­work LCC (glucomannan > xylan) containing ~48% of the wood lignin. It was concluded that carbohydrate-free lignin, i. e., lignin without covalent bonds to carbohydrates, probably does not occur in spruce wood.

Cellulose is reported to be covalently linked to lignin in both hardwoods and softwoods (100) as judged by the identification of the polysaccharides associated with lignin in the water insoluble fraction after carboxymethyl etherification. In the softwood spruce (Picea jezoensis), more than half the cellulose was linked to lignin, but in the hardwood beech (Fagus crenata), only one-sixth. The major non-cellulosic polysaccharides of spruce wood were also covalently linked to lignin, but in beech wood the extent of their linkage to lignin was low.

43.2.2.1 TYPES OF COVALENT POLYSACCHARIDE-LIGNIN CROSS-LINKS The major types of covalent linkages involved in lignin-carbohydrate associations are shown in Figure 4.5. One of these is indirect, through a bridging hydroxycinnamate molecule, the others are direct covalent linkages. These linkage types are described below.

Ester-ether cross-links. Hydroxycinnamate esters on heteroxylans and dimeric hydroxycin­namate esters bridging heteroxylan (GAX) chains, are etherified through hydroxyl(s) on lignin monomers (Figure 4.3a) and are quantitatively important in secondary walls of grasses (101), wheat (Triticum aestivum) (102-105) and Phalaris acquatica internodes (106), and maize (Zea mays) stover (83). The ether bond maybe at the (З-position of the lignin unit (102, 107-109) or possibly at the a-(benzylic)-position (110). A number of isomeric bridging homo — and hetero-dimers involved in the ester-ether bridging have been characterized (Figure 4.4) (83). The dimers involve mostly FA and dehydrodiferulic acid (DDFA) and to a lesser extent SA (32, 83). pCA does not appear in dimeric form (32, 83). In addition, there is evidence from NMR experiments with 13C-labelled Italian ryegrass (Lolium multiflorum) for other linkages between hydroxycinnamates and lignin monomers that are not easily broken by solvolytic analyses e. g., alkali at high temperatures (4 M NaOH, 170°C, 2 h) (103). Thus C-C, 8- O-4 styryl ether, and biphenyl ether cou­pling of ferulate and diferulates to lignins are not determined and only etherified ferulates are quantified (111-113). These may account for as little as 15% of total cross-linking (83).

Direct ester linkages. Carboxylic acid groups of uronic acids on matrix polysaccharides e. g., GAX and HGA may esterify alcoholic hydroxyls on lignin monomers (Figure 4.6). These alkali labile linkages have been reported in LCC from the woods of spruce (Picea sitchenis), pine (Pinus resinosa) and aspen (Populus tremuloides) and fibers from the

(^): p-coumaroyl feruloyl

Figure 4.5 Schematic diagram showing possible covalent crosslinks between polysaccharides and lignins in secondarily thickened cell walls of commelinid monocotyledons, including grasses (o)p-coumaryl, (•) feruloyl, (•-•) dehydrodiferuloyl residues. (a) direct ester linkage;(b) direct ether linkage;(c) ferulic acid esterified to polysaccharide;(d) p-coumaric acid esterified to lignin; (e) hydroxycinnamic acid etherified to lignin; (f) ferulic acid ester-ether bridge;(g) dehydrodiferulic acid ester-ether bridge. (Reprinted with permission, from liyama, K., Lam, T. B-T. & Stone, B. A. (1994b) Covalent cross-links in the cell wall. Plant Physiology, 104, 315-320, Fig. 2.)

eudicotyledons jute (Corchorus capsularis) and mesta (Hibiscus cannabinus), and the commelinid monocotyledon pineapple (Ananas comosus) (2). Borohydride reduction of aspen LCC leads to the loss of lignin and the formation of 4- O-methyl glucose from 4-O-methylglucuronic acid residues presumably from a glucuronoxylan (114). In one case, the LCC from beech (Fagus crenata) wood, the entity on the lignin involved in the ester linkage has been identified as a benzyl unit using 2,3-dichloro-5,6-dicyano — 1,4-benzoquinone (DDQ) and conjugate acid oxidation (91, 115). There is also direct evidence from 2D heteronuclear 3H-13C NMR for the presence of direct ester linkages in an acetylated LCC preparation from Eucalyptus globulus wood (98). In this LCC no benzyl ester was detected but the у -position of a lignin unit is esterified by a carboxyl group

of a uronic acid which was either GalA or GlcA or both. The extent of esterification was estimated as 9 per 100 lignin monomeric units. Pectin lyase treatment of birch (Betula maximowiczii) wood suggested that lignin maybe esterified to HGA (116).

Benzyl ether linkages. Linkages between hydroxyls on matrix polysaccharides and lignin monomers (Figure 4.7) have been described on the basis ofstudies with model compounds and observations on LCC fractions (2, 83, 85). These ether linkages have been character­ized by DDQ oxidation. In normal and compression woods of Japanese red pine (Pinus densiflora) (117), galactoglucomannan and (1^4)-p-galactan were bound to the lignin

preferentially through the C(O)6 position of the hexoses and the 4- O-methylglucurono — arabinoxylan was bound to the lignin through C(O)2 and C(O)3 positions of xylose units (Figure 4.7). Benzyl ether linkages in P. densiflora LCC have also been detected by ozonol — ysis (118). DDQ oxidation of beech (Fagus crenata) wood LCC showed that xylan was ether-linked through C(O)2 and C(O)3 (117). Phenolic benzyl ether linkages, in contrast to their non-phenolic counterparts, are susceptible to alkaline hydrolysis (1 M NaOH, 100°C). They are also sensitive to acid hydrolysis (0.3 M H2S04, 120°C). The lignins in the beechwood LCC are 100 times larger than those in the pinewood LCC but are less frequent (119).

Phenyl glycoside linkages. Glycosylation of lignin phenolic monomer hydroxyls or sidechain hydroxyls on lignins by monosaccharides, oligosaccharides, or polysaccharides (Figure 4.8) is a form of carbohydrate-lignin association that has experimental sup­port (95, 120, 121). The presence of a phenyl glycoside linkage was reported in the galactoglucomannan-rich LCC from spruce (P. abies) wood (90). There is direct evidence from 2D heteronuclear :H-13C NMR for the presence of phenyl glycosides in an acetylated LCC preparation from Eucalyptus globulus wood (98). The content of phenyl glycoside moieties in the acetylated LCC was ~0.08 per monomeric lignin unit. Phenyl glycosidic linkages are acid labile but stable to alkalis.