Lignin and Lignan Biosynthesis - American Chemical Society

Chapter 20

Downloaded by UCSF LIB CKM RSCS MGMT on November 26, 2014 | http://pubs.acs.org Publication Date: August 13, 1998 | doi: 10.1021/bk-1998-0697.ch020

Efficient Ether Cleavage in Lignins: The Derivatization Followed by Reductive Cleavage Procedure as a Basis for New Analytical Methods Fachuang Lu and John Ralph

1

U.S. Dairy Forage Research Center, U.S. Department of Agriculture-Agricultural Research Service, 1925 Linden Drive West, Madison, WI 53706

A new method is introduced for cleaving α- and β-alkyl ethers in lignins. Acetyl bromide treatment dissolves the lignin or whole cell wall material, cleaving benzyl aryl ethers, acetylating primary hydroxyl groups, and brominating benzylic positions. The key step is reductive cleavage of the resulting α-bromo-β-ethers to give cinnamyl acetates. Finally, the product is acetylated to minimize the number of components and to facilitate G C separation. The method has been named the 'DFRC' procedure, an acronym for the methodology involved (Derivatization Followed by Reductive Cleavage). Low molecular mass products can be extracted from the crude mixture; G C analysis gives data similar to those from analytical thioacidolysis. The basic method itself can be adapted for numerous other analytical determinations of value to lignin researchers. Lignin is a complex polymer derived from free-radical coupling reactions of hydroxycinnamyl alcohols (7, 2). Although lignification is not random, it is still widely held that there are no regularly repeating structures of any significant length. Certainly there is enormous stereochemical heterogeneity (3). The β-ether interunit linkage predominates in most natural lignins and there is significant evidence for sequences of β-ether linked units of three or more along the polymer chain (4, 5). It is well recognized that cleaving ether linkages in lignin effects a dramatic degree of depolymerization. Cleaving ethers is the basis of chemical pulping and various lignin analytical methods. If lignin were a truly random infinite linear polymer with randomly incorporated β-ether linkages (it is not!), Figure 1 shows the 'mer' proportions, from simple probability theory, resulting from cleavage of all β-ethers. For example, with 40% randomly distributed β-ethers, some 16% monomers would be expected, and 84% of the product would be hexamers or smaller. Similarly, if 50% of the interunit linkages are β-ethers, 25% monomers and 94% hexamers or smaller would result from cleaving all β-ethers. Although natural lignins are not correctly modeled by such an infinite, linear, random polymer, these numbers give crude estimates of the expected amounts and distributions of products. Conversely, if the values obtained in practice vary widely from these numbers, the actual distribution might give some clues to the grouping of lignin interunit linkages. Corresponding author.

294

U.S. government work. Published 1998 American Chemical Society

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

295


Analytical thioacidolysis (6, 7) is today one of the most effective methods for efficiently cleaving ether bonds in lignin to produce identifiable monomeric and dimeric degradation products which can be quantitated. In this chapter we describe an even more efficient method for cleaving lignin a- and β-ethers. The similar release of fragments obtained by this approach also lends itself to development of an analytical method or, as we suggest below, a series of methods. It has already become so important in our own work that a description of the basis of the methodologies is presented here with the aim of hastening their further development. Analytical methodologies such as those described here that identify aspects of composition, structure, and regiochemistry are valuable aids in understanding lignin biosynthesis as well as degradation pathways. The Reaction of Lignins with Acetyl Bromide Acetyl bromide (AcBr) is well-known for its ability to facilitate dissolution of lignocellulosic materials in acetic acid. A rapid and simple method for lignin determination using AcBr (8) has been widely applied (9) and modified for various purposes (11-13). Iiyama and Wallis (14), after observing the changes in UV-spectra and UV-specific absorption coefficients of lignin model compounds after AcBr treatment, proposed that AcBr acted as an acylating reagent similar to acetyl chloride, acylating hydroxyls (both aliphatic and phenolic) present in lignin to produce acetylated derivatives. Nimz (75) had previously demonstrated aryl ether cleavage, bromination and acetylation of a phenylcoumaran model compound using AcBr. It was also suspected that AcBr might acylate aromatic rings of lignin structural units in some cases in the presence of perchloric acid (14). Later, they examined the reactions of AcBr (with and without perchloric acid) with lignin model compounds and saccharides (16). They found that the rates of the various reactions were in the order O-acetylation > bromine substitution » β-ether cleavage « C-acetylation > demethylation. Their reactions of veratrylglycerol-β-guaiacyl ether GG-lb (Figure 2), a 4-O-etherified β-ether model compound, gave rather complex arrays of products as determined by HPLC (16). The reactions included full acetylation, acetylation/ccbromination, and β-ether cleavage. We have discovered (77) that remarkable efficiency and selectivity can be obtained under milder conditions than those described previously ( 16). Acetyl bromide can effect three types of reactions of value in the study of lignins: acetylation, benzylic bromination, and α-ether cleavage (Figure 3) (77). With the correct choice of solvents and conditions, these reactions are more selective and higher yielding than those that result under the conditions used in lignin determination methods based on AcBr ( 16). Acetylation of Hydroxyl Groups. Acylation of alcohols and phenols with acyl halides using basic or acidic catalysts is well known, although AcBr is only rarely used for preparative purposes. Results (77) from a series of model compounds 1 (Figure 2) showed that primary γ-OHs were readily acetylated by AcBr treatment. Although secondary benzylic hydroxyls, when treated with AcBr in A c O H or 1,4-dioxane, were also acetylated, they subsequently formed oc-bromo derivatives 7 in almost quantitative yield (Figure 3) as discussed below. Phenolic OHs were acetylated more slowly than primary (γ-) or secondary benzylic (a-) OHs. These reactions are probably acid-catalyzed since small amounts of water in the samples will produce HBr. Bromination of Benzyl Alcohols. Zawadowski (18) observed the bromination of benzylic hydroxyl groups with AcBr, and Iiyama and Wallis (16) proposed that bromides were formed among the products of their reactions. Bromotrimethylsilane (TMSBr) and HBr are known to effectively form benzyl bromides from benzyl alcohols, and these bromides have been utilized in schemes to generate lignin model


296

0.5

0.7 beta 0.6 beta 0.5 beta 0.4 beta 0.3 beta 0.2 beta 0.1 beta


ω 0.2-

0.0-

~l 2

1—Γ" 3 4

9

I I 10 11 12 13 14 15 16 17 18 19 20

Mer

Figure 1. Proportion of monomers, dimers and higher mers expected from β-ether cleavage of an infinite linear polymer with randomly disposed linkages having proportions of β-ether linkages ranging from 10% to 70%. R'O 2

R 0, R'O a b c d

R=H R = Me R = Ac R = Pr

Ar .Ar'

2

1R'=R =H 2

2R'=Ac, R = H 3R

1

Ar

Η ι Ar

2

= R = Ac 1

2

4 R = H , R = Ar' 1

6R'=H 1

7 R = Ac

2

5 R = Ac, R = Ar*

Figure 2. Shorthand notation for structures. Compounds are named using P, G and S (to identify p-coumaryl, guaiacyl and syringyl moieties) using the Ar ring followed by the A r ' ring (if there is one), then the compound number and the phenolic hydroxyl derivatization indicator a-d. For example, GG-la denotes free phenolic guaiacylglycerol-β-guaiacyl ether. Later in the manuscript, trimers follow the same format specifying Ar, Ar', and the third A r " ring (not shown), e.g. GSS-la.



Figure 3. Reaction of β-aryl ether model compounds with AcBr acetylated a-bromo-compounds 7; a-aryl ethers are cleanly cleaved.


298 quinone methides (79, 20). Model compounds la and lb, which are often used as representatives of β - 0 - 4 ' linked structural elements predominant in softwood lignins, were completely converted to acetylated α-bromo derivatives 7b and 7c after AcBr treatment in acetic acid or 1,4-dioxane (Figure 3) (77). This observation confirms the nature of the products proposed by Iiyama and Wallis (16). The cleanliness of AcBr reactions is illustrated in Figure 4, which depicts H N M R spectra of crude total products from AcBr treatment of various model compound of the type 1 (see Figure 2). AcBr is therefore synthetically useful for the selective bromination of benzylic hydroxyl groups in the presence of other primary or secondary hydroxyls. Additionally, if stereochemical integrity is not an issue, AcBr may also be used to selectively acetylate primary and/or phenolic hydroxyls in the presence of benzylic hydroxyls because the resulting benzylic bromide can be readily hydrolyzed (acetone/water) back to the benzylic hydroxyl groups without affecting the acetates. It was previously found (77) that acetylation of primary γ-hydroxyl groups proceeded faster than that of benzylic hydroxyls during the AcBr treatment in acetic acid. The α-bromo product 7 is formed via the α-acetate intermediate 3 (e.g. 3b from lb). Therefore, Iiyama and Wallis' observation (16) of diacetates is an indication of either too short a reaction time or that the bromides 7 were partially hydrolyzed in subsequent work-up. Treatment of model compound threo-GG-la with AcBr in A c O H or 1,4-dioxane resulted in a mixture of 2 diastereomers (about 1:1) of the α-bromo derivatives GG-7c, suggesting an S I reaction via the benzyl carbocation ion. Conversion of the benzyl acetate to the bromide will occur in free-phenolic or 4-etherified compounds but is very slow with 4-O-acetates. No bromide formation was observed when the fully acetylated compound GG-3c was used. This could be because little HBr is produced when compounds with no hydroxyls react; much of the reactivity is presumably attributable to HBr. The completeness of reactions in dioxane or acetic acid implies that phenolic acetylation by AcBr is much slower than benzylic alcohol acetylation.


1

Cleavage of Non-cyclic Benzyl Aryl Ethers. The presence and quantity of noncyclic benzyl aryl ether (a-O-4') substructures in lignin have been controversial (2723). Methods for distinguishing a- from β-ethers are becoming more important because of the fundamentally different mechanisms for their formation (nucleophilic addition to quinone methides vs. radical coupling) and the consequent biochemical implications (24). There are several methods used to cleave and quantitate benzyl aryl ether substructures. Perhaps the simplest is a mild acid hydrolysis (25). The increase in phenolic content is measured by employing reaction conditions which cleave β-Ο^Φ' ethers more slowly than α - Ο ^ ' ethers. Recently, we developed a bromotrimethylsilane (TMSBr) method which works effectively on lignin model compounds (26) but was not suitable for quantitative analysis of α-aryl ether linkages in lignin. This is because the reagent rapidly reacts with water while lignin has very low solubility in totally anhydrous solvents that are compatible with TMSBr (e.g. dioxane). We assumed that AcBr would efficiently cleave non-cyclic benzyl aryl ethers although this was not specifically mentioned by Iiyama and Wallis (16). Cyclic benzyl aryl ether lignin model compounds are efficiently cleaved (75). AcBr has been used for the cleavage of cyclic ethers under more drastic conditions. For example, refhixing simple cyclic ethers with AcBr and Lewis acid catalysts yielded acetoxy-bromoalkanes (27). We found that the benzyl aryl ether model compound GG-4b was cleaved rapidly and cleanly, without affecting the β-ether after >12 h (77). N M R spectra of the products snowed that the α-bromo acetylated product GG-7b and 2-methoxyphenyl acetate G-8c were formed essentially quantitatively in A c O H or 1,4-dioxane. The mechanism of α-ether cleavage by AcBr presumably follows an S 1 process resulting in a mixture of α-bromo diastereomers GG-7b. The possibility that the cleavage reaction follows an S 2-like mechanism with fast isomerization cannot be ruled out; anchimerically assisted reactions followed by bromide isomerization have recently been observed in this laboratory (26). N

N


299

GG-7C (from GG-1a)


jlki_J

7.0

6.5

6'.0

5'.5

5Τθ

GS-7b (from GS-1b)

JUL_ l L i u J 7\δ

6\S

6'.0

5'.5

5.0

SS-7C (from SS-1a)

7.0

6'.5

6'0

ι

5'.5

n 5'.0

a

11 Ujl J

4'.5

4.0

SS-7b (from SS-1b)

. i l l _jl ppm

7.0

6.5

6.0

5.5

5.0

4.5

4.0

Figure 4. Partial *H N M R spectra of crude AcBr reaction products from a variety of dimeric β-aryl ether model compounds.


300


Summary of Lignin Reactions with AcBr Lignin dissolution by AcBr is an extremely effective process (8, 10, 12, 14,17). The N M R spectra shown in Figure 4 are for crude products from AcBr reactions with various syringyl/guaiacyl lignin model β-ethers. N M R spectra of milled tissue lignins following AcBr treatment (Figure 5) show that the bromides are also produced as major products from polymeric lignins. Solubilization with AcBr provides a promising means for characterizing whole lignins by N M R spectroscopy. The relative rates of reactions observed between AcBr and β-aryl ether lignin model compounds under mild conditions are: primary (γ-) O H acetylation > benzyl aryl ether cleavage/ bromination > secondary benzylic (a-) O H acetylation > benzyl acetate to benzyl bromide substitution » phenolic O H acetylation. Under these conditions, no β-aryl ether cleavage or demethylation (reported previously under harsher conditions (16)) were detectable. The cleanliness and reproducibility of these reactions suggest that lower reaction temperatures should be examined for AcBr lignin determination methods. The selective bromination of benzyl alcohols by AcBr is synthetically useful. Selective cleavage of benzyl aryl ethers by AcBr may provide a new technique for the analysis of a-aryl ether linkages and for quantitating hydroxycinnamic acids that are passively incorporated into lignin (24, 28). Such applications are being evaluated in our laboratory. Reductive Cleavage of β-Bromo Ethers The major contribution here (29, 30) is the recognition that reaction with AcBr has produced compounds that are almost ideally susceptible to reductive cleavage. The β-bromo ethers 7 that are produced are amenable to formal two-electron transformations (Figure 6). Related ethers have been cleaved using Zn dust (31) or Cr(II)en (32, 33), for example. Reaction of the lignin or lignin model products with Zn° in dioxane/acetic acid/water at room temperature produces the cleaved products within minutes. N M R spectra of the crude products resulting from AcBr treatment followed by reaction with Zn° are shown in Figure 7. It should be noted that the 'adduct' mechanism (34, 35) for the cleavage of free phenolic β-ethers by reduced anthraquinone species is a formal two-electron process with considerable similarity when viewed in general terms from the perspective of Figure 6b. The adduct 11 between AHQ " and a lignin quinone methide undergoes Grob fragmentation to cleave the β-ether bond. The reaction with lignin α-ketones 12 also occurs readily (Figure 6b) but is not as convenient for lignin reactions since lignins oxidized at the benzylic position are particularly insoluble and intractable (29). The actual mechanism presumably involves single electron transfer steps rather than a concerted two-electron reduction (Figure 6c) although precedence for the latter is known (36). Similar reactions with 1,2-dibromides where the bromines can be oriented in anti (180°) or syn (or 0°) periplanar positions show complete stereo selectivity, implicating concerted two-electron reduction (36-38). In our case, a stereoselective reaction (Figure 6d) should lead to -50:50 trans.cis products since the starting bromides equilibrate to a 50:50 syn:anti (threo:erythro) mixture. However, since the products are -95% trans, the mechanism cannot be concerted and must rather involve carbanion intermediates (Figure 6c). It is possible to run the Zn° reaction under anhydrous conditions to produce aryl propenyl derivatives 10 (e.g. eugenol G-10 in Figure 6a). However, we were not able to find conditions that would cleanly give such products without some of the cinnamyl acetates 9 also being formed in larger amount. That limitation, the increased sensitivity of the reaction to the exact conditions, and the realization that the hydroxycinnamyl acetates 9 were more diagnostic of the lignin structure, led to our exclusive development of the method based on the production of hydroxycinnamyl acetates. Thus the two steps involving AcBr treatment followed by reductive cleavage cleanly produces cis- and trans -hydroxycinnamyl acetates 9 (Figure 8). These could 2


301

'

GG-7c

(from GG-1a)


GG-7b (from GG-1b)

Jl β

ppm

Bromides from Pine ML

80

75

70

β

il Ύ

65

60

55

60

55

y Bromides from Kenaf ML

ppm

80

75

70

65

13

Figure 5. Partial C N M R spectra (sidechain region) of AcBr reaction products from guaiacyl model compounds and pine milled wood lignin, and syringyl models and a kenaf milled lignin. Analogous bromides are clearly formed in lignins and model compounds.


302

a)

AcO.

AcO ,Ar'

2e'

Ar

Ar

HO HO


b)

(i)

c)

(ii)

10

HO

Ar 11

(ii)

AcO

X

OAr

Br—-

N

Ar 12

(i) Concerted

Br

Ar

9

7

2Θ

—OAr

+

Η

β

θ γ

+

Α

γ

ο

θ

Sequential

Br—J—I—OAr -

Bp

+

τ

-

-OAr

+

Η

ArO

0

OAr'

d)

AcO. Br/,,

AcO.

"Ο Ar

.Ar*

=

OAc

Hv^Ar

2c" concerted

Br

Η Ar

7 5v/i

9 trans

(threo) AcO.

Η

(E) OAr"

Bp,,,

OAc Ar

7

Figure 6 (a). Reductive cleavage of β -bromoethers 7 to give hydroxycinnamyl acetates 9; compounds 10 can be formed in stronger acid, (b) Three examples of conceptually similar formally two-electron processes: (i) cleavage of ethers by anthraquinone via adduct mechanism (see text); (ii) reduction of a-keto-β-ethers with Zn/H efficiently cleaves the ether; (iii) bromides proced by AcBr treatment are cleavable through formally twoelectron processes, (c) Two alternative reaction mechanisms for reductive cleavage: (i) concerted two-electron reduction; (ii) two sequential singleelectron steps via an intermediate carbanion. (d) Stereochemistries expected from concerted two-electron reduction of the two bromide isomers of 7; since products from a 50:50 syn:anti-l favor trans-9 highly over the cisisomer, the mechanism must be sequential. +


303

S-9C (from SS-1a)


x i 7.0

6'.5

.

Ote-

7'.0

6'.0 '

' 5'.5

' 5'.0 '

' 4'.5 '

' 4'.0 '

S-9c (from SG-1a)

β«

6'.5

6'.0

'

'

5'.5

'

JUL

5'.0

'

'

'

4'.5

G-9C (from GG-1a)

J i L j II e'.o

' 5'.5

Cinnamyl Acetates G-9c from Pine ML

6'.5

6'.0

Cinnamyl Acetates S-9c (and G-9c) from Kenaf ML

JJJ' ppm

U J l

7.0

6.5

6.0

5.5

5.0

4.5

4.0

l

Figure 7. Partial H N M R spectra of crude products from full D F R C treatments of model dimers 1 and milled tissue lignins from pine and kenaf. The production of cinnamyl acetates 9 (Figure 6a) is clearly evident, even in the lignins.


304


be separated by G C and quantitated directly at this stage. Indeed there are some advantages to doing so as we shall see later. However, such an approach necessitates separately quantitating free phenolic and phenol-acetylated compounds for each monomer aromatic ring (S, G, or P); free phenolic components arise in the reduction step from originally etherified structures, whereas phenolic acetates are produced from initially free-phenolic or α-etherified units in the AcBr step (Figure 8). In order to obtain the simplest and most easily quantifiable mixture, the obvious solution was to simply acetylate the product before G C quantitation. Thus the third step, acetylation, produces fully acetylated hydroxycinnamyl acetates 9c, which are stable and ideal for GC quantitation (and GC-MS identification). Attempts to Convert cis/trans Isomers to Single Products Although the trans.cis ratio is high, it would be advantageous to convert the isomers to a single product thereby allowing even easier quantitation. The simplest way to remove isomers from the products 9 would seem to be hydrogénation (Figure 9). Regrettably this apparently simple step is capricious and difficult to realize in practice in our hands. The cinnamyl esters are readily hydrogenated to arylpropane derivatives 14 in competition with the desired double-bond saturation reaction to give propyl acetates 13. Hydrogenolysis is normally considered to be less significant with Pt than Pd catalysts, but this was not borne out on these substrates. Hydrogénation without first acetylating any free phenols is a particular problem. Hydrogénation with Rh/C in EtOAc was the best condition we found; the hydrogenolysis products from acetylated phenolic models comprised

Lignin and Lignan Biosynthesis - American Chemical Society

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