Basic Lignin Chemistry

Transcription

1 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.

2 Lignin n Lignin is derived from the Latin word for wood (lignum). de Candolle firstly introduced this term in (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.

3 Lignin n Lignin precursors (three cinnamyl alcohol) q p-coumaryl alcohol q Coniferyl alcohol q Sinapyl alcohol Lignin Precursors and Aromatic Constituents

4 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.

5 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 nonswelling 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.

6 Lignin Isolation Removing the polysaccharides n Lignin preparation by artificial q Dehydrogenation polymer (DHP) Coniferyl alcohol was treated with H 2 O 2 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 Na 2 SO 4. n Lignin is further converted to an alkali soluble derivative a solution of hydrochloric acid and thioglycolic acid at 100 C.

7 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.

8 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.

9 Outline of the Biosynthetic Pathway of Phenylpropanoids 1. Shikimate pathway phosphoenol pyruvic acid D-erythrose 4-phosphate 3-deoxy-D-arabino-heptulosonic acid 7-phosphate 3-dehydroquinic acid NADP-linked shikimate dehydrogenase phosphochorismic acid (1) shikimic acid 3-dehydroshikimic acid phosphorylated phenylpyruvic acid chorismic acid prephenic acid arogenic acid p-hydroxyphenylpyruvic acid

10 2. Cinnamate pathway PAL phenylalanine ammonia-lyase cinnamic acid TAL tyrosine ammonia-lyase p-hydroxy cinnamic acid or 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 feruloyl adenylate feruloyl CoA thio ester hydroxycinnamoyl-coa reductase oxidoreductase coniferyl aldehyde 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 alcohol oxidoreductase is obviously one of the key enzymes controlling the specificity of the lignin precursors.

11 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

12 A Metabolic Channel Model for Independent Pathways to G and S Monolignols A Representation of the Random Model for Lignin Formation

13 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 C 6 C 3 precursors of the coniferyl alcohol type via enzymatic dehydrogenation. n Freudenberg and co-worker ( ) 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.

14 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.

15 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) Lignin 1. KOH, Methylation 3. KMnO 4 10% veratric acid minor isohemipinic acid The formation of isohemipinic acid support the occurrence of condensed structure (β-5 or γ-5) dehyrodiveratric acid

16 Nitrobenzene oxidation Softwood Hardwood Vanillin (25% of lignin) Lignin Nitrobenzene oxidation Grass syringaldehyde p-hydroxybenzaldehyde Hydrolysis Lignin H 2 /CAT Ethnolysis Hibert ketons Lignin 2% HCl in EtOH

17 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 is a better solvent for lignins than is ethanol). q The majority of the acidolysis monomers originate from arylglycerol β-aryl ether structure. q Formation of monomeric phenols from β-o-4 structures in lignin during acidolysis.

18 n Thioacidolysis q Thioacidolysis is solvolysis in dioxane-ethanethiol with boron trifluoride etherate. q It is an acid-catalyzed reaction with results in the depolymerization of lignins. q As in acidolysis, thioacidolysis proceeds mainly by cleavage of arylglycerol-β-aryl ether linkages. q In contrast to acidolysis, thioacidolysis is preformed in anhydrous media, ether-cleaving reagent combines a hard Lewis acid (Et 2 O-BF 3, boron trifluoride etherate), and a soft nucleophile (EtSH, ethanethiol) n Thioacetolysis q Thioacetolysis-lignin samples are subjected to a treatment with thioacetic acid and boron trifluoride. q The cleavage of the ether bonds by using thioacidolysis is equally specific, but more complete than by thioacetolysis. q The reaction products are separated as TMS (trimethylsilyl ethers by GC.

19 n Permanganate oxidation Much of our current knowledge about structure of lignin in wood and pulp is based on results obtained from permanganate oxidation. This method involved the selective degradation of all aliphatic side chains attached to aromatic moieties structures henceforth referred to as acids. The original permanganate oxidation method of methylated lignin has also been considerably improved when it is performed at alkaline instead of neutral conditions. n Permanganate oxidation The methylated fragments are seperated by GC and identified MS Reaction sequence for the oxidative degradation of lignin with potassium permanganate

20 l Major carboxylic acid methyl esters formed in the oxidation of lignin with potassium permanganate Type of Linkages and Dimeric Structure n It is clear that phenylpropane units are joined together both with C-O-C (ether) and C-C linkages. C-O-C linkages is dominant; approximately 2/3 or more. The rest are the C-C type. n Proportions of different type of linkages connecting the phenylpropane units in lignin

21 Functional Groups n Lignin polymer contains characteristic methoxyl groups, phenolic hydroxyl groups, and some terminal aldehyde groups in the side chain. n Only relatively few of the phenolic hydroxyls are free; most of them are occupied through linkages to the neighboring phenylpropane units. n The syringly units in hardwood lignin are extensively etherified. n Alcoholic hydroxyl groups and carbonyl groups are introduced into the final lignin polymer during the dehydrogenative polymerization process. Functional Groups n In some wood species substantial amounts of the alcoholic hydroxyl groups are esterified with p- hydroxybenzoic acid or p-hydroxycinnamic acid. Ester of p-hydroxybenzoic acid are typical in aspen lignin. p-hydroxycinnamic acid are abundant in bamboo and grass lignin.

22 Lignin Formula o The formula of lignin presented in its final shape in 1968 by Freudenburg for softwood lignin (spruce) has attained general acceptance. n This scheme for spruce lignin represents 18 phenylpropane units as a section of the total molecule which was assumed to consist of more than 100 units in the native state. o Adler (1977) gave a structural scheme for spruce lignin comprising 16 prominent C 9 -units, mainly derived from the results of oxidative degradation experiment. A Structure Segment of Softwood lignin Proposed by Adler β-6 glyceraldehyde-2-aryl ether pinoresinol only low amount in softwood Spruce lignin

23 Softwood Lignin Model designed by Computerized Evaluation Loblolly pine lignin (Glasser, 1981) Lignin Formula n In addition, formulas for hardwood lignins have been suggested as well. n The structure concept of beech lignin (Nimz, 1974).

24 Lignin-Carbohydrate Bonds n The possible existence of covalent bonds between lignin and polysaccharides has been a subject of much debate and intensive studies. n It is obviously and now generally accepted that such chemical bonds must exist, and the term lignin-carbohydrate complex (LCC) is used for the covalently bonded aggregates of this type. n Chemical bonds have been reported between lignin and practically all the hemicellulose constituents (even between lignin and cellulose). These linkages can be either of ester or ether type and even glycosidic bonds are possible. Examples of suggested LCC bonds An ester linkage to xylan through 4-Omethyl glucuronic acid as a bridge group. An ether linkage to xylan through an arabinofuranose unit. An ether linkage to galactoglucomannan through a galactopyranose unit.

25 Lignin-Carbohydrate Bonds o Ether linkages are more stable and common between lignin and carbohydrates. n The α-position is even in this case the most possible connection point between lignin and hemicellulose. o LCC bridge groups n Arabinose unit (HO-2 or HO-3) n Galactoglucomannans, the galactose unit (HO-3) n In middle lamella and primary wall (pectic polysaccharides) galactan HO-6 in galactose arabinan HO-5 in arabinose Lignin-Carbohydrate Bonds o Glycosidic linkages for LCC n Benzylic alcohol group, which is the most probable connection point, the phenolic group may also be partly occupied through glycosidation. n The glycosidic linkages are easily cleaved with acid.

26 Classification and Distribution of Lignin l Lignin can be divided into several classes according to their structure elements l Guaiacyl lignin It is a largely polymerization product of coniferyl alcohol. Guaiacyl lignin occurs in almost all softwoods. l Guaiacyl-syringyl lignin The typical lignin of hardwood, this type of lignin is a copolymer of coniferyl and sinapyl alcohol. The ratio varing from 4:1 to 1:2 for the two monomeric units. l p-hydroxyphenyl lignin Compression wood, which has a high proportion of phenylpropane units of the p-hydroxyphenyl type in addition to the normal quaiacyl units. Transverse Section of a Spruce Tracheid Photographed in UV Light (240 nm)

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28 Classification and Distribution of Lignin n In hardwood, there are still many uncertainties involved, because of the more heterogeneous nature of the wood and the presence of both quaiacyl and syringyl units in the lignin. q The lignin located in the secondary wall of hardwood fibers has a high content of syringly units whereas larger amounts of guaiacyl units are present in the middle lamella lignin. q The vessel in birch seem to contain only guaiacyl lignin, whereas syringyl lignin predominates in parenchyma cell. Polymer Properties of Lignin n The limitation for studying the macromolecular properties for lignin : q Low solubility in most solvents q It will cause degradation during the isolation process. q The polymer properties of lignin is depend on its location in the cell wall. n The method for characterizing the polymer properties of lignin: q Vapor pressure osmometry q Light scattering q Ultracentrifugation

29 Polymer Properties of Lignin n The M w of softwood lignin around 20000, the lower values have been reported for hardwood lignin. n Compared with cellulose, the polydiversity of lignin is relatively high, roughly for softwood MWL.