Lignin

Objective: a general understanding of how lignin is formed and its "overall structure."

Important Concepts
Monolignols
Free Radical Polymerization
Predominant Interunit Linkages
 
As far as chemists and biochemists are concerned, lignin is a nasty substance to deal with. While polysaccharides are composed of specific carbohydrates linked by glycosidic bonds, lignin structure appears random and unorganized. Whether this lack of organization is actually the case is somewhat controversial, and the structure of lignin and how it is formed is an active area of research, with scientific groups worldwide arguing about what the true structure is and how it is formed. Why all the interest? Simply put, lignin is what the paper manufacturers want out of wood...manipulating trees to make less lignin or a different type of lignin which is more easily pulped is a tremendous economic issue.
Monolignols
There are three monmers that make up almost all lignin found in nature. They are biosynthesized in plants via the shikimic acid pathway and their structures and names are shown below:

p-Coumaryl alcohol is a minor component of grass and forage type lignins. Coniferyl alcohol is the predominant lignin monomer found in softwoods (hence the name). Both coniferyl and sinapyl alcohols are the building blocks of hardwood lignin.
Biosynthesis
Lignin is biosynthesized by a free radical coupling reaction. Enzymes termed peroxidases and laccases initiate the process by breaking the covalent bond between the phenolic oxygen and its hydrogen. The bond is broken in such a way that one electron stays with the oxygen and the other goes with the hydrogen. This generates a free radical. Just like free radicals in society, these molecules tend to be somewhat reactive. The initial free radical attempts to stabilize itself by moving the electron throughout the molecule by a process known as resonance stabilization. In doing this, a series of resonance-stabilized free radicals are formed which are in constant equilibrium with one another. When one free radical meets another, they combine to share their electrons making a new covalent bond. Two monomers would come together to make a dimer. The dimer still has a free phenolic hydroxyl and an enzyme can remove the hydrogen and electron to make another free radical. The dimer can combine with a monomer or another dimer making an even larger molecule (trimer or tetramer). This process continues until a polymeric structure results.
 
How this process is initiated and controlled is poorly understood. Understanding the process of lignification is the first step required in order to rationally manipulate it to provide cell wall material more amenable to standard pulping processes. Some scientists feel that there is no real control over the polymerization process, whereas others feel that there is a high level of control. In fact a recent paper in Science [275 (1997) 362-366] goes so far as to state that "...oxidative coupling with monolignols then gives rise to the macromolecular lignins. It is inconceivable, however, that lignin formation would be left to the vagaries of such a wide range of enzymes, or be realized in a haphazard fashion." This statement has been challenged by another paper in Science [277 (1997) 235-239] where a Loblolly pine with abnormal lignin was studied. This investigation concluded that there is considerable "plasticity" in the polymerization process---in essence a haphazard process. Anyway......the major resonance stabilized free radicals for coniferyl alcohol are shown below:

Of the five free radicals shown above, the most important are structures a, b and c. The new bonds that are formed are either carbon-carbon (b+b, b+c) or carbon-oxygen (a+b, a+c). If the monomer is sinapyl alcohol (2 methoxyl groups), the major radicals are a and b. Free radical coupling is much more difficult if there are several groups attached at the site of the free radical (ie., structures d and e), so little, if any coupling occurs with these free radicals.
Lignin Isolation
One of the problems with investigating lignin biosynthesis and structure is that you can't really get it out of the cell wall without modifying its structure. If one uses chemical means, how does one know what reactions occurred during processing so that you can separate them from the original structure? It's very difficult, and usually some assumptions are taken. The most realistic lignin one can obtain is by ball milling extractive-free wood. In this process wood is pulverized by shaking wood meal in amongst ball bearings for long periods of time. A portion of the lignin can then be extracted with certain solvents. This does not remove all of the lignin, only a portion of it. One can, however, determine lignin contents in wood samples and/or wood pulps by chemical and gravimetric means. Softwood lignin contents are on the order of 26-32% and hardwood lignin contents range from 20-28%.
Lignin Structure
The free radical polymerization process generates a series of inter-unit linkages. The predominant linkage is the so-called beta-O-4 llinkage which results from the condensation of free radicals a and b. The intermediate of this coupling is called a quinone methide, which usually rearranges (with the addition of water) to form the stable beta-O-4 ether bond. About 40-60% of all interunit linkages in lignin is via this ether bond, and it is this bond that is usually broken during delignification (pulping) processes. The pdf file below shows the major interunit linkages in wood.
Lignin Substructures.
 
Scientitsts have been often concerned about generating a picture of lignin, placing different linkages together in different proportions to provide a visual representation. Since the overall "ordered/random" nature of lignin is not known, it is probably best just to assume that the major interunit linkages are present in different percentages. The proportion of major interunit linkages for spruce and birch are shown below:
Linkage Type Dimeric Structure Spruce (%) Birch (%)
Beta-O-4 (a + b) Arylglycerol-beta-aryl ether  50 60
Beta-5 (b+ c) Phenylcoumaran 10 6
5-5 (c+ c) Biphenyl 10  5
 Beta-Beta (b + b) Pinoresinol 5 5
 5-O-4 (c + a) Diaryl ether  5 5
 Others ---  10 10
The a + b coupling providing the beta-O-4 linkage is the predominant interunit linkage. This is the interunit bond which is broken in pulping operations.
 
As can be seen, getting a handle on lignin structure can be difficult. To look at differences in lignin structure between species or during pulping operations, chemists have devised chemical techniques to measure certain functional groups in wood. These functional groups are often presented in the literature as functional groups per 100 monomer units. In other words, if I have a lignin polymer that is linked together with 100 monomers (lignin molecule with a DP of 100), how many of these specific functional groups will be present. The major functional groups determined are methoxyl content, phenolic hydroxyl content and benzyl alcohol (hydroxyl group on the alpha carbon) content. Representative values are shown below:
Functional Group Softwood Lignin Hardwood Lignin
Methoxyl 95 150
Phenolic Hydroxyl 23 12
Benzyl Alcohol 35 45
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