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
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 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. n Thioacidolysis
q Thioacidolysis is solvolysis in dioxane-ethanethiol with boron trifluoride etherate. 乙醚合三氟化硼 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 (Et2O-BF3, 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. 硫代醋酸
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. 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 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 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. 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 C9-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
only low amount in softwood
Spruce lignin 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). 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-O- methyl glucuronic acid as a bridge group.
An ether linkage to An ether linkage to galactoglucomannan xylan through an through a galactopyranose arabinofuranose unit. unit. 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. 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)
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 Polymer Properties of Lignin n The Mw 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 2.0 -3.0 for softwood MWL.