Basic Lignin Chemistry David Wang’s Wood Chemistry Class Lignin n Lignin is the second abundant and important organic substance in the plant world. n The incorporation of lignin into the cell walls of plants gave them the chance to conquer the Earth’s land surface. n Lignin increased the mechanical strength properties to such an extent that huge plants such as trees with heights of even more than 100 m can remain upright. Lignin n Lignin is derived from the Latin word for wood (lignum). de Candolle firstly introduced this term in 1819. (Cellulose was named by Anselme Payer in 1838) n In 1897, Peter Klason studied the composition of lignosulfonates and put forward the idea that lignin was chemically related to coniferyl alcohol. n In 1907, Klason proposed that lignin is a macromolecular substance. After ten year (1917) Klason further purposed that coniferyl alcohol units are joined together by ether linkages. Lignin n Definition q A very irregular, randomly cross-linked polymer of phenylpropane units joined by many different linkages. q A polymer derived from the phenylpropanoid compound, coniferyl alcohol and related alcohols. q Amorphous, cross-linked polymers that occur uniquely in vascular plants. q Lignin may be defined as an amorphous, polyphenolic material arising from an enzyme-mediated dehydrogenative polymerization of three phenylpropanoid monomers, conniferyl, synapyl and p- coumaryl alcohols. Lignin n Lignin precursors (three cinnamyl alcohol) q p-Coumaryl alcohol q Coniferyl alcohol q Sinapyl alcohol Lignin Precursors and Aromatic Constituents Lignin o Lignification n Formation lignin in wood o Lignin function n Supportive agent → mechanical strength n Antioxidant → protection n Sealant and reinforcing agents → bonding cellulose and hemicellulose together n Cross linker → cross link carbohydrates Lignin Isolation oGeneral n Lignin can be isolated from extractives-free wood as an insoluble after hydrolytic removal of polysaccharides. n Lignin can be hydrolyzed and extracted from wood or converted to a soluble derivative. Lignin Isolation — Removing the polysaccharides n Klason lignin q Klason lignin is obtained after removing the polysaccharides from extractives-free wood by hydrolysis with 72% sulfuric acid. n Cellulolytic enzyme lignin (CEL) q The polysaccharide may be removed by enzymes from finely divided wood meal. This method is tedious, but the resulting CEL retains its original structure essentially unchanged. n Dioxane containing water and HCl q Considerable changes in its structure occur. Lignin Isolation — Removing the polysaccharides n Milled wood lignin (MWL) q Björkman lignin q It is the best preparation known as far and it has been widely used for structure studies. q Wood meal is ground in a ball mill either dry or in the presence of non- swelling solvents (ex. toluene), the cell structure of the wood is destroyed and a portion of lignin (usually less than 50%) can be obtained from the suspension by extraction with a dioxane-water mixture. q MWL preparation always contain some carbohydrate materials. Lignin Isolation — Removing the polysaccharides n Lignin preparation by artificial q Dehydrogenation polymer (DHP) Coniferyl alcohol was treated with H2O2 in the presence of peroxidase enzyme yield synthetic lignin named DHP. q Released suspension culture lignin (RSCL) RSCL was isolated suspension cultures of spruce wood cells as a secretion products (RSCL represents a carbohydrate-free coniferous lignin) Lignin Isolation — Removing the lignin o Lignosulfonates n Soluble lignin derivates, lignosulfonates, are formed by treating wood at elevated temperatures with solutions containing sulfur dioxide and hydrogen sulfite ions. o Sulfate lignin or Kraft lignin n Lignin is dissolved as alkali lignin when wood is treated at elevated temperature (170 °C) with NaOH, or better, with a mixture of NaOH and Na2SO4. n Lignin is further converted to an alkali soluble derivative a solution of hydrochloric acid and thioglycolic acid at 100 °C. Measurement on Lignin Content n Softwood ¨Softwood lignin can be determined gravimetrically by klason method. ¨Normal softwood contains 26-32% lignin while the lignin content of compression wood is 35-40%. n Hardwood ¨The lignin present in hardwood is partly dissolved during the acid hydrolysis and hence the gravimetric values must be corrected for the “acid-soluble lignin” using UV spectrophotometer. ¨Normal hardwood contains 20-25% lignin. Tropical hardwood can have a lignin content exceeding 30%. Tension wood contains only 20-25% lignin. Biosynthesis and structure of Lignin Lignin are polymers of phenylpropane units. Many aspects in the chemistry of lignin still remain unclear. The ability to synthesize lignin has been essential in the evolutionary adaptation of plants from an aquatic environment to land. Although researchers have studied lignin for more than a century, many aspects of its biosynthesis remain unresolved. The monolignol biosynthetic pathway has been redrawn many times and remains a matter of debate. n Ethnolysis ¨The hydrolytic treatment of wood or lignin with dilute alcoholic hydrochloric acid under pressure was the original method for obtaining defined phenylpropenoid ketones (Hibbert ketones) by splitting β-aryl ether linkages. n Lange (1954) who applied UV microscopy at various wavelength directly on thin wood sections, obtaining spectrum typical of aromatic compounds. Biosynthesis of Lignin Precursor o Precursors of lignin n Gymnosperms: coniferyl alcohol (guaiacyl unit) n Angiosperms: coniferyl alcohol + sinapyl alcohol (guaiacyl unit + syringyl unit) n Grasses: p-coumaryl alcohol (p-hydroxyphenyl unit) o Lignin precursors are generated from D-glucose through reaction of enzyme. Outline of the Biosynthetic Pathway of Phenylpropanoids 1. Shikimate pathway phosphoenol pyruvic acid 3-dehydroquinic acid 3-deoxy-D-arabino-heptulosonic D-erythrose 4-phosphate acid 7-phosphate NADP-linked shikimate dehydrogenase 3-dehydroshikimic acid phosphochorismic acid shikimic acid (1) phosphorylated phenylpyruvic acid 苯丙胺酸 arogenic acid prephenic acid chorismic acid p-hydroxyphenylpyruvic acid 酪氨酸 2. Cinnamate pathway PAL cinnamic acid phenylalanine ammonia-lyase TAL p-hydroxy cinnamic acid or tyrosine ammonia-lyase p-coumaric acid hydroylase caffiec acid O-methyl transferase (OMT) ferulic acid 5-hydroxyferulic acid sinapic acid — Reduction of Ferulic Acid to Coniferyl Alcohol hydroxycinnamate-CoA ligase hydroxycinnamoyl-CoA reductase feruloyl adenylate feruloyl CoA thio ester • These enzyme in angiosperms reduce both coniferyl and sinapyl aldehyde almost equally, but the gymnosperm enzymes are remarkably specific for coniferyl aldehyde. • O-methyltransferase, p-hydroxycinnamyl oxidoreductase alcohol oxidoreductase is obviously one of the key enzymes controlling the specificity of coniferyl aldehyde the lignin precursors. Pathways to monolignols. The complete metabolic grid of reactions is shown. All enzyme reactions shown with a solid arrow have been demonstrated to occur in vitro. Reactions shown in smaller type may not occur in vivo. The reactions shown in green seem the most likely route to G lignin in vivo. The reactions in red represent those reactions consistent with both in vivo and in vitro evidence for being involved speci.cally in S lignin biosynthesis. The intermediate in orange is common to both G and S lignin pathways. Major Segments of Phenylpropanoid Metabolism in Vascular Plants as Currently Understood A Metabolic Channel Model for Independent Pathways to G and S Monolignols A Representation of the Random Model for Lignin Formation Polymerization of Lignin n Erdman (1930) studied the oxidative dimerization of various phenols in the biogenesis of natural products and reached the conclusion that lignin must be formed α,β-unsaturated C6C3 precursors of the coniferyl alcohol type via enzymatic dehydrogenation. n Freudenberg and co-worker (1940-1970) had demonstrated the polymerization of precursors to lignin in nature does indeed occur as Erdman described. n One-electron transfer from coniferyl alcohol by enzymatic dehydrogenation yield resonance-stabilized phenoxy radicals. n Oligomeric products formed through coupling of coniferyl alcohol radicals n Endwise β-O-4 coupling of a coniferyl alcohol radical with a growing lignin group radical to an intermediate quinone methide (3), which is stabilized to a quaiacylglycerol-β-aryl ether (4) structure through addition of water. Type of Linkages and Dimeric Structure l Common linkages between the phenylpropane units Lignin Structure n Methods based on classical organic chemistry led to the conclusion, already by 1940, that lignin is build up phenylpropane units. ¨Permanganate oxidation (methylation softwood lignin) 10% veratric acid 1. KOH, 170℃ 2. Methylation 藜蘆酸 3. KMnO4 Lignin minor isohemipinic acid 異半派酸 The formation of isohemipinic acid support the occurrence of condensed structure (β-5 or γ-5) dehyrodiveratric acid — Nitrobenzene oxidation Softwood Vanillin (25% of lignin) Hardwood Nitrobenzene oxidation Lignin syringaldehyde Grass p-hydroxybenzaldehyde — Hydrolysis H /CAT Lignin 2 — Ethnolysis Hibert ketons 2% HCl in EtOH Lignin n Acid hydrolysis (Acidolysis) q The term acidolysis refers specifically to the refluxing of lignin or lignocellulose with 0.2 M HCl in dioxane-water (9:1, v/v). q Acidolysis has a close relationship to ethanolysis (heating of lignin in HCl in ethanol). Both treatment result in the degradation of lignin with formation of substantial amounts of arylpropanes (but, dioxane-water