Oxidative Coupling of Phenols and the Biosynthesis of Lignin - ACS

Chapter 10

Oxidative Coupling of Phenols and the Biosynthesis of Lignin

Downloaded by CORNELL UNIV on May 31, 2012 | http://pubs.acs.org Publication Date: August 13, 1998 | doi: 10.1021/bk-1998-0697.ch010

Gosta Brunow, Ilkka Kilpeläinen, Jussi Sipilä, Kaisa Syrjänen, Pirkko Karhunen, Harri Setälä, and Petteri Rummakko Laboratory of Organic Chemistry, Department of Chemistry, University of Helsinki, P.O. Box 55, FIN-00014 Helsinki, Finland

Our understanding of the structure of lignin is based on our knowledge of its biosynthesis. Lignification in the plant cell wall is initiated by the enzymatic formation of phenoxy radicals from cinnamyl alcohol precursors. The radicals are thought to react without the intervention of enzymes. An essential element in this process is the cross coupling of radicals formed from the monomeric precursors with radicals formed on the polymer chain (end-wise polymerization). The rates of formation of C - C and C-O bonds are governed by the effective concentrations of the different radicals and by the regioselectivity of the coupling reaction. Finally, the stabilization reactions of intermediate quinone methides will, in many cases, determine the outcome of the coupling reaction. Lignin has always baffled researchers in their attempts to determine its chemical structure. All methods of isolation destroy some parts of the biopolymer, making it difficult to find out what structures are artefacts and what structures are missing. A l l analytical methods yield fragmentary, sometimes contradictory, results. For this reason, the study of lignin biosynthesis, the charting of the mechanisms that govern the assembly of the macromolecule in the cell wall, has become such an important approach, more so than for other biopolymers. It is instructive to review briefly the main steps of the development of our current ideas on the structure of lignins and to see how new knowledge about the processes of phenol oxidation has contributed, and still contributes, to this development. The Dehydrogenative Polymerization Theory Although analytical evidence had shown that phenylpropane units form an important part of the structure, the first major breakthrough came when Erdtman in 1933 (7) proposed that the formation of a dimer in the oxidation of isoeugenol could be a model for the biosynthesis of lignin. This example highlights two important features of the dehydrogenative polymerization theory: the formation of carbon-carbon and carbon-oxygen bonds by oxidative coupling in the ortho position on the ring and in the p position of the side chain, and the reaction of nucleophilic oxygen with a

©1998 American Chemical Society

In Lignin and Lignan Biosynthesis; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

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quinone-methide intermediate. This concept opened up the chemistry of lignin and still forms the basis for our understanding of its chemical structure.

isoeugenol dehydrodiisoeugenol The idea was taken up by Freudenberg who, from the late 1930s onwards, developed it using coniferyl alcohol as the lignin precursor. He studied (2) the oxidation of coniferyl alcohol both with enzymes and with inorganic oxidants, and he obtained dehydrogenation polymers (DHPs) that in many respects resembled isolated lignins such as milled wood lignin from spruce, 'Bjorkman lignin'. The bonding patterns that are formed in the oxidation of coniferyl alcohol were revealed by stopping the reaction at a stage when it was possible to isolate dimeric and oligomeric products that lent themselves to exact structural determination. Freudenberg assumed that the bonding pattern in the dimers and oligomers is the same as in the polymer. This view has been modified in later work, as will be shown in later sections, but the basic idea, that lignin is formed by oxidative coupling, soon became established. It became apparent, through hydrolytic and other degradative studies, that most of the important linkages in softwood lignins could have been formed by oxidative coupling of coniferyl alcohol:

atomic centers to which linkages may be formed during oxidative coupling of coniferyl alcohol

It has to be pointed out, however, that the resemblance of a synthetic DHP to a lignin isolated from a plant cell wall rests on the same type of incomplete analytical data as does the structure of the lignin itself. In fact, with more advanced analytical techniques, the structural differences between DHPs and the corresponding lignins have become more and more apparent. The difficulties encountered by those who have attempted to prepare synthetic lignins show that a deeper understanding of the process of lignification is needed before it will be possible to duplicate it in vitro. In Lignin and Lignan Biosynthesis; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

133 Regioselectivity and Stereoselectivity of Radical Coupling


The coniferyl alcohol radicals form C - C bonds and C - 0 bonds between one another, but the factors governing the distribution of these bond types are unknown. What we know is that the P -position is the most reactive, which means that in the most frequent coupling modes the (3-carbon is involved in one of the moieties participating in the coupling reaction. The substitution pattern on the aromatic rings undoubtedly influence the selectivity of the coupling (3). As an example, the work of Sarkanen and Wallis (4) demonstrated that steric effects favor 'racemoid' coupling in the P~p dimerization of isoeugenol:

steric effects favor

"racemoid" coupling

The steric requirements of a 'sandwich' transition state can be visualized, as shown above, as leading to a particular stereochemical outcome from coupling, but the factors determining the actual ratios of P~P', p-5' and p-0-4' coupling are not known. One factor that may influence the final distribution of bonding types is the occurrence of irreversible hydrolytic reactions. For instance, in §-0-X coupling, the formation of the final product involves the addition of water to an intermediate quinone methide, and this reaction requires an acid catalyst. The P-5' and the P~P' dimer formation, on the other hand, requires no water. Thus the availability of water and an acid catalyst may influence the formation of P-0-4' dimers at the expense of other structures (5).



134

OH

nucleophile

Cross Coupling In the mixture of dimers and polymers that are formed on the oxidation of coniferyl alcohol in vitro, the proportion of p-0-4' structures is always lower than in natural lignins. When the coniferyl alcohol is added slowly to the oxidizing medium ('Zutropf'), the proportion of Q-O-4' structures rises, approaching that in natural lignins. This finding points to the importance of cross coupling between a coniferyl alcohol radical and a phenoxy radical generated on the growing polymer chain. This process, called 'end wise' polymerization by Sarkanen (6), is favored at low concentrations of coniferyl alcohol. At high concentrations of coniferyl alcohol, dimerization is favored, but when the concentration of coniferyl alcohol approaches zero, coupling between end-groups occurs, yielding 5-5' biphenyl (and 5-0-4') structures:

5-5' We have thus identified another important factor controlling the process by which the structure of lignin is formed: the concentration of coniferyl alcohol in the zone where the growth of the lignin chain occurs. But why does cross coupling lead to higher amounts of (3-0-4' structures? In this case consideration of the transition state may provide a reasonable explanation. Perhaps the 'sandwich' transition state In Lignin and Lignan Biosynthesis; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

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leading to the $-0^4' structure may be more sterically favored than, for instance, the transition state leading to a 0-5' coupling:

Redox Potentials Phenoxy radicals tend to dimerize and the dimerization is controlled by the reactivities and the concentrations of the radicals. For coupling between two different phenoxy radicals to occur (cross coupling), their respective reactivities and concentrations must, in combination, be such as to favor the process. One physical parameter which is important in this connection is the redox potential of the phenol. In the case of phenols important in lignin biosynthesis, very few measurements have been published. Figure 1 shows a rough estimate of the influence of substituents on the redox potentials of phenols. It has been constructed by extrapolation of data from two references (7, 8). The scale is not proportional; the data are intended to show the effects of 0-methoxyl and /?-(3-hydroxy)propenyl substituents on the oxidation potential of the phenol, and one 5-5' biphenyl is included. [The numbers on the left are taken from ref. (8) and refer to the oxidation potential of the phenolate; the numbers on the right are approximations of substituent effects taken from ref (7).] When a mixture of phenols is oxidized, the phenols will react in the order shown, starting with the lowest on the scale. Phenols on the same level of the scale should form cross coupling products easily when equimolar mixtures are oxidized. Under other circumstances it is to be expected that the phenols react without significant cross coupling. Only if the phenol with the higher redox potential is present in large excess is cross coupling to be expected. Assuming that cross coupling at equimolar concentrations occurs only between phenols of equal oxidation potential, it is possible to draw some important conclusions: in the case of softwood lignin, coniferyl alcohol has a lower oxidation potential than the phenols on the polymer that do not have a conjugated double bond. For cross coupling to occur, the concentration of the more reactive phenol must be kept low. The phenol with the higher oxidation potential has to be in large excess. The different redox potentials of the structural units in lignins can also lead to inhomogeneities. It has, for instance, been found that reaction wood and cell culture lignins are rich in p-hydroxyphenyl units, and that more than 90% of these units are In Lignin and Lignan Biosynthesis; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.


136

Figure 1. Estimates of substituent effects on the oxidation potentials of phenols relevant to the biosynthesis of lignin.


137 unetherified (9). This can be explained by the high oxidation potential of such units that makes them unreactive in phenolic coupling when they have become incorporated into the lignin. Experiments on Cross Coupling


We have carried out a number of cross coupling experiments to demonstrate the constraints on cross coupling between phenols of unequal redox potential. Coniferyl alcohol and syringyl units without a conjugated double bond in the side chain can be assumed to have about equal oxidation potentials. Accordingly, oxidation of a mixture of coniferyl alcohol and the syringyl p-0-4' model compound 1 yielded the cross coupling product 2 in good yield:

OH

OH

1 H 0 /horseradish peroxidase 2

2

The oxidation of p-coumaryl alcohol together with a guaiacyl model compound yielded, among other cross coupling products, a similar p-ether but in lower yields. When this experiment is carried out with coniferyl alcohol and a guaiacyl model compound or sinapyl alcohol with a syringyl model compound, no cross coupling products were isolated. A rule of thumb can be formulated on basis of these results: when the monolignol and the dilignol have the same number of methoxyls, no cross coupling is obtained with equimolar mixtures; to obtain cross coupling, the monolignol must have fewer methoxyls than the dilignol. To sum up, the concentration of coniferyl alcohol (or other cinnamyl alcohol) in the reaction zone is a major factor determining the structure of the growing polymer. It is possible that this concentration varies with time and in different regions of the cell wall, giving rise to inhomogeneity in the lignins formed in these regions. The


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following scheme shows how the competition between dimerization and cross coupling reactions can lead to different structures in the lignin.

Cross-coupling occurs between phenolic cinnamyl alcohols at particular relative concentrations

cross-coupling

fJ-5' P-l' Knowledge of the mechanisms involved in the oxidative coupling reactions and the role of cross coupling versus dimerization allows us to predict under what conditions a given structure is formed. This gives us deeper insight into the structure of lignin and is an invaluable aid for interpreting analytical data. In the following sections we are going to demonstrate the value of this perspective by considering some structural units that have been studied in our laboratory. The Non-cyclic Benzyl Aryl Ethers The existence of non-cyclic benzyl aryl ethers or a - 0 - 4 ' bonds in lignins has been debated for some time (10). The evidence for their existence has been based on hydrolytic results from lignin preparations and on the fact that such structures are prominent among the oligomers isolated by Freudenberg (77). In our experiments we were able to repeat the results of Freudenberg: a quinone methide with a (i-aryl ether in the side chain did indeed react with vanillyl alcohol forming a non-cyclic benzyl aryl ether: OH


139 The problem is that attempts to find spectroscopic evidence for the existence of such structures in lignin have yielded mostly negative results (72). Minute amounts have been detected in a C-13 enriched sample of a poplar lignin (13) but no evidence for non-cyclic benzyl aryl ethers has been found in softwood lignins. A possible explanation for this apparent contradiction is that the biosynthesis of lignin occurs at a pH lower than that typically used in the dehydrogenation experiments. We have found that quinone methides in aqueous solution react with water in preference to a phenol when the pH is lower than 4 (Table I). The vanishingly small amounts that have been detected in N M R spectroscopic studies may thus be explained by a low pH during lignin biosynthesis. The reaction of the quinone-methide intermediate with water seems to be rate determining in the formation of P-0-4' structures in lignins.


Table I. The Relative Rates of Addition of Nucleophiles to Quinone Methides pH range

addition reaction

stereochemistry

5-7

carboxylic acids > phenols > water

erythro for acids and phenols, threo for water

3-5

water » carboxylic acids

Oxidative Coupling of Phenols and the Biosynthesis of Lignin - ACS

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