Lignin biosynthetic study: Reactivity of quinone methides in the

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Agricultural and Environmental Chemistry

Lignin biosynthetic study: Reactivity of quinone methides in the diastereo-preferential formation of phydroxyphenyl- and guaiacyl-type #-O-4 structures. Xuhai Zhu, Takuya Akiyama, Tomoya Yokoyama, and Yuji Matsumoto J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06465 • Publication Date (Web): 22 Jan 2019 Downloaded from http://pubs.acs.org on January 25, 2019

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Journal of Agricultural and Food Chemistry

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Lignin biosynthetic study: Reactivity of quinone

2

methides in the diastereo-preferential formation of

3

p-hydroxyphenyl- and guaiacyl-type β-O-4

4

structures.

5

Zhu Xuhai,† Takuya Akiyama,*, † Tomoya Yokoyama† and Yuji Matsumoto†

6

†

7

Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan

Wood Chemistry Laboratory, Department of Biomaterial Sciences, Graduate School of

8 9

KEYWORDS: Lignification, para-quinone methide, radical coupling, erythro and threo,

10

gymnosperm

11

ABSTRACT

12

p-Quinone methides are involved in lignin biosynthesis as transient intermediates, and their

13

aromatization step has a great impact on the chemical structure of the resulting lignins. A

14

series of quinone methides (QMs) were synthesized and allowed to react with water in pH 3–

15

7 buffers at 25°C to mimic the formation of p-hydroxyphenyl- and guaiacyl-type (H- and

16

G-type, respectively) β-O-4 structures in gymnosperm plant cell walls. Water addition

17

occurred in 3-methoxy-substituted QMs (G-type QM) with a half-life of 1.4–15 min. In 1 ACS Paragon Plus Environment


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contrast, non-substituted QMs (H-type QM) were very labile; they were aromatized to β-O-4

19

products with a half-life of only 10–40 sec. The rapid aromatization in H-type QMs may

20

provide an advantage over G-type species for efficiently driving the lignin polymerization

21

cycle, which possibly contributes to the development of highly lignified compression wood.

22

In the water addition reaction, the threo isomers of the β-O-4 products were

23

stereo-preferentially formed more than the erythro isomers from both G- and H-type QMs

24

(erythro/threo ratios of 24:76 and 50:50, respectively). The proportion of erythro isomers was

25

higher at lower pH conditions. This pH-dependent trend agrees with findings from the

26

previous study on 3,5-dimethoxy-substituted (syringyl-type (S-type)) QMs; thus, this

27

pH-dependent trend is common in H-, G-, and S-type lignin-related QMs. A higher

28

threo-selectivity was obtained by replacing their β-etherified aromatic rings from G- to

29

H-type. A similar but weaker effect was also observed by replacing the QM moiety from G-

30

to H-type.

31

INTRODUCTION

32

Quinone methides (QMs) are reactive electrophilic species that are involved in the

33

biosynthesis of a variety of natural products.1-5 QMs are formed as transient species in many

34

cases and are stabilized by the addition of a nucleophile, such as a hydroxy group or amino

35

group,3 at the exocyclic methylene or methine group site, which produces benzylic adducts

36

via the aromatization of the benzene ring. On the other hand, relatively stable QMs in the

37

form of diterpenoids and triterpenoids have been isolated from plants.6,7 A generation of

38

para-quinone methide intermediates are also involved in lignin biosynthesis.

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Lignins are major components of vascular plant cell walls. In woody plants, the cell

40

walls contain around 20–35% lignins by weight. They provide mechanical support and 2 ACS Paragon Plus Environment

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chemical reinforcement to the cell walls. This aromatic biopolymer is derived from three

42

monolignols, namely, p-coumaryl, coniferyl, and sinapyl alcohols, which respectively give

43

rise to p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units in the lignin structure.

44

Softwood (gymnosperm) lignins predominately contain G-units with minor levels of H-units,

45

and elevated levels of the latter are in compression wood tissues.8-10 Hardwood (angiosperm)

46

lignins primarily consist of G- and S-units in various proportions, together with minor levels

47

of H-units.

48

Most phenylpropanoid skeletons in lignin contain two asymmetric carbons at the α-

49

and β-positions of the side-chain moiety, with some minor exceptions (e.g., cinnamyl alcohol

50

end-groups). The two chiral centers form in the polymerization process and cause an increase

51

in the structural variety of lignins. One chiral center is generated at the β-position by a β-O-4,

52

β-β, β-5, or β-1 coupling reaction between two radicals arising from monolignol(s) and/or the

53

phenolic end of the growing polymer. QMs are the initial products of these radical-coupling

54

reactions, which are not stereospecific; i.e., the QM bearing a β-asymmetric carbon with

55

either R- or S-configuration occurs in a 1:1 racemic form.11-13

56

The subsequent re-aromatization of QMs gives rise to another chiral center at the

57

α-position (benzylic position). In the formation of arylglycerol-β-aryl ether structures (β-O-4

58

structures), which are the most abundant structures in lignin, β-O-4-aryl ether QMs are

59

aromatized by water addition to produce either the erythro or threo form of β-O-4 structures

60

(Scheme 1). Their stereochemistry is determined by which face of the QM reacts faster with

61

water.4,14,15 The erythro/threo ratio is variable in plants, causing the structural variation in

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lignins.16-18 The erythro/threo ratio is also known to influence the lignin degradation

63

efficiency. For example, the erythro and threo isomers have significantly different behaviors

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in terms of their reactivities under alkaline conditions,17,19-21 as well as in enzymatic and

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nonenzymatic oxidation.22-25 A hydroxy group from a polysaccharide, instead of water, can be

66

added to a β-O-4-linked QM during lignin biosynthesis, which creates a covalent bond

67

between the lignin and polysaccharide components in the cell walls, forming a so-called

68

lignin–carbohydrate complex (LCC) bond.26 Thus, the aromatization process of QM

69

significantly affects the chemical structure and reactivity of lignin and cell components and

70

thereby influences the chemical utilization of plant biomass.

71

Whereas the ratio of erythro to threo forms of β-O-4 structures is close to 50:50 in

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normal softwood lignins, the erythro form dominates in hardwood lignins.16,27,28 For erythro or

73

threo isomers to be produced in excess during lignin biosynthesis, stereo-preferential water

74

addition to the β-O-4-aryl ether QM must occur.15 The syringyl/guaiacyl (S/G) ratio of

75

aromatic nuclei seems to be one of the factors governing the erythro/threo ratio, as has been

76

indicated by structural characterization using NMR and ozonation methods,16,17,29 as well as by

77

an in vitro experiment involving water addition to S-type QM model compounds.15 A positive

78

correlation was clearly found between the S/G ratio and the erythro/threo ratio for the lignins

79

of different hardwood wood species,16,17 and for reaction wood lignins.10,30,31 An NMR

80

INAQUDEATE experiment on 13C-enriched aspen lignin29,32 revealed that the erythro form of

81

β-syringyl ether structures is the most predominant of the four structural types (erythro- and

82

threo-β-syringyl ethers, and erythro- and threo-β-guaiacyl ethers). These findings indicated

83

that the S-unit influences the diastereoselective addition of water to QMs and induces the

84

erythro-preferential formation of β-O-4 structures in hardwood lignins.

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There was a case, however, in which the erythro/threo ratio was not 50:50 in softwood

86

lignin in spite of the absence of the syringyl unit. Previous studies showed that the β-O-4


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structures of lignin in compression wood, which develops eccentric radial growth in both the

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lower side of the leaning woody stem and branch of coniferous gymnosperms in response to

89

longitudinal growth stress,8,9,33,34 were slightly predominant in the erythro form.10,35 The

90

distribution of the erythro/threo ratio in the reaction wood disc was positively correlated with

91

that of the p-hydroxyphenyl/guaiacyl (H/G) ratio, implying that the erythro-preferential

92

formation is related to the H-unit of the aromatic ring type.10

93

In the present study, an experiment involving the addition of water to H-type and

94

G-type QMs was conducted using several β-O-4-aryl ether QM compounds in order to

95

characterize lignin formation in compression wood.

96

OH

lignin 1 5 R''

4 OH

5 R

4 OH

1. Radical coupling reaction Si face (left side)

R R' H, H : p-Coumaryl alcohol (H-type) H, OMe: Coniferyl alcohol (G-type) OMe, OMe: Sinapyl alcohol (S-type)

3 R'

β

H O

α

H

O

4

lignin

R' OH

erythro form

R'''

Re face (right side)

α

4 R'''

R

R'' H

β

OH

β

HO H

2. Water addition to Si face HO

γ

1

lignin

3 R'''

Growing lignin polymers

α

R''

R R' H, H : p-Hydroxyphenyl (H-type) H, OMe: Guaiacyl (G-type) OMe, OMe: Syringyl (S-type)

OH R'' HO

R'

H

β α

H O

R O

lignin

R'''

2. Water addition R to Re face

Quinone methide intermediates

4

R' OH

threo form

-O-4 structures in lignin

Monolignols

97 98

Scheme 1. Formation of erythro and threo forms of β-O-4 structures during lignin

99

biosynthesis.

100 101 102 103

A growing lignin polymer can couple with a monolignol at the phenolic end of the polymer via a radical-coupling reaction to produce a β-O-4-bonded quinone methide (QM). This reactive intermediate is generally re-aromatized by water addition to either an erythro or a threo form of the β-O-4 structure. 5 ACS Paragon Plus Environment


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EXPERIMENTAL

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General. Reagents and solvents were purchased from Fujifilm Wako Pure Chemical Co.

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(Osaka, Japan), Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), or Sigma-Aldrich (Tokyo,

107

Japan), and were used as received. The pH values of the buffers were measured with a Horiba

108

F-52 pH meter with a JF15 electrode (Horiba, Kyoto, Japan). The NMR spectra of the

109

synthesized compounds were recorded with a JEOL JNM-A500 500 MHz spectrometer. The

110

standard JEOL programs of one- and two-dimensional (proton, carbon, DEPT-135, 1H-1H

111

COSY, 1H-13C HSQC, and 1H-13C HMBC) NMR experiments were performed for structural

112

elucidation and the assignment of newly synthesized compounds. The central peak of the

113

residual solvent was used as an internal reference (δH 7.26, δc 77.0 ppm for CDCl3; δH 2.04, δc

114

29.8 ppm for acetone-d6; δH 5.32, δc 53.8 ppm for CD2Cl2). For samples that encountered

115

multiplicity problems with hydroxy group protons, a drop of D2O was added for the OH–OD

116

exchange prior to analysis. The traditional numbering system for lignins18, 36 was used rather

117

than the systematic IUPAC numbering scheme.

118


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HO

R''

γ

B

4

β O

α

3

1

R'

A 4

HO

1

γ

buffered water-dioxane (v/v, 1:1) pH 3.5 – 7 at 25 ºC

3

R

R R' R'' OMe, OMe, H H, OMe, H OMe, H, H H, H, H OMe, OMe, OMe

: : : : :

QM-GG QM-HG QM-GH QM-HH QM-GS

1

B

4

HO

β

α

O

3

1

R'

A 4

O Quinone methides

R''

3

R

OH β-O-4 dimers

β-guaiacyl ethers β-p-hydroxyphenyl ethers β-syringyl ether

R R' R'' OMe, OMe, H H, OMe, H OMe, H, H H, H, H OMe, OMe, OMe

: : : : :

GG HG GH HH GS

119 120

Scheme 2. Water addition to β-O-4-aryl ether quinone methides (QMs) yields β-O-4 dimer

121

products obtained as a mixture of erythro and threo isomers.

122 123 124 125 126 127 128 129

The letters H, G, and S are used to respectively designate the p-hydroxyphenyl, guaiacol, and syringyl nature of the aromatic rings. As shown in the β-O-4 products, we also refer to the A and B rings for the aromatic ring of the phenylpropanoid skeleton and the β-etherified aromatic ring, respectively (e.g., compound GH has a G-type A-ring and an H-type B-ring). QMs also have two rings that are labeled as A for the quinone method moiety and B for the β-etherified aromatic ring. The quinone method moiety was also categorized as H-, G-, and S-type based on the type of A-ring of the corresponding β-O-4 products (e.g., compound QM-GH has a G-type quinone method moiety (A-ring) and an H-type B-ring).

130 131

Preparation of β-O-4 dimer models. Five β-O-4 type lignin model compounds carrying H-,

132

G-, and/or S-type nuclei on the two aromatic rings (Scheme 2) were synthesized according to

133

Adler’s

134

guaiacylglycerol-β-p-hydroxyphenyl ether (GH), guaiacylglycerol-β-syringyl ether (GS),

135

p-hydroxyphenylglycerol-β-guaiacyl

136

p-hydroxyphenylglycerol-β-p-hydroxyphenyl ether (HH). The separation of the erythro and

137

threo isomers of each β-O-4 dimer model was carried out using ion-exchange

method37.

These

were

guaiacylglycerol-β-guaiacyl

ether

(HG),

ether

(GG),

and



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138

chromatography with borate solution as the eluent.38–39,40 The configurations of the separated

139

β-O-4 dimers (erythro or threo) were determined by the ozonation method, which yields

140

erythronic and threonic acids from the erythro and threo isomers, respectively.41 NMR data

141

can be found in the Electronic Supplementary Information (ESI, page S1–S3).

142

Preparation of QMs. Five β-O-4-aryl ether QM compounds (QM-HH, QM-HG, QM-GH,

143

QM-GG, and QM-GS in Scheme 2) were synthesized from the corresponding five β-O-4

144

model compounds (HH, HG, GH, GG, and GS, respectively) by mild alkaline treatment of

145

the benzyl bromides,14 which were prepared using trimethylsilyl bromide (TMSiBr) according

146

to Ralph’s method.42,43 The general procedure for the preparation of QMs is outlined below.

147

β-O-4 dimer (0.1 mmol, as a mixture of erythro and threo isomers) was transferred

148

into a small vial with a screw cap and dissolved in chloroform (1 mL). TMSiBr (0.2 mmol)

149

was added to this solution, and it was shaken for 90 sec at room temperature to generate

150

benzyl bromide. The reaction mixture was shaken with saturated aqueous NaHCO3 (1 mL) for

151

10 sec. The organic layer was collected, washed with saturated NaCl, and dried over Na2SO4.

152

The resulting pale-yellow solution of QM was diluted 1000-fold with dioxane for the

153

subsequent water addition experiment and used without further purification. The deuterated

154

chloroform solution of the QM was prepared in a separate experiment for NMR structural

155

determination in the same way using deuterated chloroform as a reaction solvent instead of

156

chloroform. The chloroform-d solution of the crude QM was transferred into an NMR tube

157

and kept in liquid N2 before NMR measurement.

158

Most of the β-O-4-aryl ether QM compounds (QM-HG, -GH, -GG, and -GS) were

159

prepared as described above. For the preparation of QM-HH, dichloromethane (or

160

dichloromethane-d2) was used as a reaction solvent instead of chloroform because of the 8 ACS Paragon Plus Environment

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limited solubility of the β-O-4 dimer HH in chloroform. For all QM solutions, any residual

162

NMR peak resulting from the β-O-4 dimer was not found on their 1H NMR spectrum (see

163

Figure S3–S7 in ESI).

164

Compound QM-HH: 1H NMR (CD2Cl2, 500 MHz), δH: 3.85 (1H, dd, J = 12.1, 4.0 Hz, γ1),

165

3.95 (1H, dd, J = 12.1, 6.6 Hz, γ2), 5.34 (1H, m, β), 6.33 (1H, broad-dd, J = 10, 2 Hz, A3),

166

6.42 (1H, br-dd, J = 10, 2 Hz, A5), 6.45 (1H, d, J = 8.9 Hz, α), 6.90 (2H, br-dd, J = 7, 1 Hz,

167

B3 and B5), 6.98 (1H, br-td, J = 7, 1 Hz, B1), 7.10 (1H, br-dd, J = 10, 3 Hz, A2), 7.27 (2H,

168

br-t, J = 7 Hz, B2 and B6), 7.61 (1H, br-dd, J = 10, 3 Hz, A6). 13C NMR (CD2Cl2, 125 MHz)

169

δC: 65.4 (γ), 76.8 (β), 116.5 (B3 and B5), 122.5 (B1), 129.5 (A3), 130.3 (B2 and B6), 130.9

170

(A5), 133.9 (A6), 134.0 (A1), 142.0 (A2), 146.1 (α), 158.0 (B4), 187.3 (A4).

171

Compound QM-HG: 1H NMR (CDCl3, 500 MHz), δH: 3.82 (1H, dd, J = 12.1, 4.0 Hz, γ1),

172

3.87 (3H, s, B3-OMe), 3.91 (1H, dd, J = 12.1, 6.9 Hz, γ2), 5.19 (1H, m, β), 6.38 (1H, broad-d,

173

J = 10 Hz, A3), 6.40 (1H, br-d, J = 12 Hz, A5), 6.53 (1H, d, J = 8.6 Hz, α), 6.85–6.87 (2H, m,

174

B5 and B6), 6.92 (1H, d, J = 8.0 Hz, B2), 7.03 (1H, m, B1), 7.10 (1H, br-dd, J = 10, 2 Hz,

175

A2), 7.47 (1H, br-dd, J = 10, 2 Hz, A6). 13C NMR (CDCl3, 125 MHz) δC: 55.8 (B3-OMe),

176

64.8 (γ), 79.5 (β), 112.2 (B2), 119.1 (B5), 121.2 (B6), 124.1 (B1), 129.3 (A3), 130.4 (A5),

177

133.2 (A1), 133.2 (A6), 141.6 (A2), 144.6 (α), 146.5 (B4), 150.8 (B3), 187.0 (A4).

178

Compound QM-GG (mixture of syn- and anti-isomers (7:3)): 1H NMR (CDCl3, 500 MHz),

179

δH: syn-isomer, 3.72 (3H, s, A3-OMe), 3.83 (1H, broad-dd, J = 12, 3 Hz, γ1), 3.87 (3H, s,

180

B3-OMe), 3.94 (1H, br-dd, J = 12, 7 Hz, γ2), 5.16 (1H, m, β), 6.31 (1H, d, J = 8.3 Hz, α),

181

6.42 (1H, d, J = 9.5 Hz, A5), 6.49 (1H, d, J = 2.0 Hz, A2), 6.85–6.87 (2H, m, B5 and B6),

182

6.92 (1H, d, J = 8.0 Hz, B2), 7.02 (1H, m, B1), 7.02 (1H, m, A6); anti-isomer, 3.75 (3H, s,

183

A3-OMe), 3.78 (1H, m, γ1), 3.87 (3H, s, B3-OMe), 3.88 (1H, m, γ2), 5.19 (1H, m, β), 6.24 9 ACS Paragon Plus Environment


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(1H, br-d, J = 2 Hz, A2), 6.42 (1H, m, α), 6.44 (1H, br-d, J = 10 Hz, A5), 6.85–6.87 (2H, m,

185

B5 and B6), 6.92 (1H, d, J = 8.0 Hz, B2), 7.02 (1H, m, B1), 7.42 (1H, br-dd, J = 10, 2 Hz,

186

A6). 13C NMR (CDCl3, 125 MHz) δC: syn-isomer, 55.3 (A3-OMe), 55.8 (B3-OMe), 64.6 (γ),

187

79.6 (β), 103.9 (A2), 112.2 (B2), 118.6 (B5), 121.2 (B6), 123.7 (B1), 128.3 (A5), 133.9 (A1),

188

141.1 (α), 141.3 (A6), 146.6 (B4), 150.5 (B3) 153.3 (A3), 181.0 (A4); anti-isomer, 55.2

189

(A3-OMe), 55.8 (B3-OMe), 65.0 (γ), 79.3 (β), 111.6 (A2), 112.2 (B2), 119.0 (B5), 121.2

190

(B6), 123.9 (B1), 129.6 (A5), 132.5 (A6), 133.7 (A1), 141.1 (α), 146.5 (B4), 150.5 (B3),

191

152.5 (A3), 181.3 (A4). Stereochemical assignments (syn- or anti-isomer) were made by

192

comparison with the reported data of the same compounds,44 typically by using the chemical

193

shift data of A2, A6, and A3-OMe protons.

194

Compound QM-GH (mixture of syn- and anti-isomers, major and minor isomers ratio =

195

6:4): 1H NMR (CDCl3, 500 MHz), δH: major isomer, 3.77 (3H, s, A3-OMe), 3.88 (1H,

196

broad-dd, J = 12, 4 Hz, γ1), 3.98 (1H, br-dd, J = 12, 7 Hz, γ2), 5.31 (1H, m, β), 6.22 (1H, d, J

197

= 8.6 Hz, α), 6.40 (1H, d, J = 9.5 Hz, A5), 6.59 (1H, s, A2), 6.88 (2H, br-d, J = 8 Hz, B3 and

198

B5), 6.98 (1H, m, B1), 6.99 (1H, m, A6), 7.26 (2H, br-t, J = 8 Hz, B2 and B6); minor isomer,

199

3.73 (3H, s, A3-OMe), 3.85 (1H, br-dd, J = 12, 4 Hz, γ1), 3.95 (1H, br-dd, J = 12, 7 Hz, γ2),

200

5.35 (1H, m, β), 6.21 (1H, s, A2), 6.31 (1H, d, J = 8.6 Hz, α), 6.50 (1H, br-d, J = 10 Hz, A5),

201

6.88 (2H, br-d, J = 9 Hz, B3 and B5), 6.98 (1H, m, B1), 7.26 (2H, br-t, J = 9 Hz, B2 and B6),

202

7.54 (1H, br-d, J = 10 Hz, A6).

203

(A3-OCH3), 64.7 (γ), 76.5 (β), 103.7 (A2), 115.8 (B3 and B5), 122.1 (B1), 128.2 (A5), 129.7

204

(B2 and B6), 134.0 (A1), 141.2 (A6), 141.7 (α), 153.4 (A3), 157.3 (B4), 181.0 (A4); minor

205

isomer, 55.2 (A3-OMe), 65.2 (γ), 76.1 (β), 111.5 (A2), 115.9 (B3 and B5), 122.0 (B1), 129.7

206

(B2 and B6), 129.8 (A5), 132.4 (A6), 133.9 (A1), 141.8 (α), 152.4 (A3), 157.2 (B4), 181.2

207

(A4).

13

C NMR (CDCl3, 125 MHz) δC: major isomer, 55.4


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208

Compound QM-GS (mixture of syn- and anti-isomers, major and minor isomer ratio = 7:3):

209

1

210

= 4 Hz, γ), 3.82 (6H, s, B3-OMe and B5-OMe), 5.07 (1H, m, β), 6.43 (1H, br-d, J = 10 Hz,

211

A5), 6.56 (1H, d, J = 8.6 Hz, α), 6.58 (1H, br-d, J = 2 Hz, A2), 6.59 (1H, br-d, J = 8 Hz, B2

212

and B6), 7.04 (1H, br-t, J = 8 Hz, B1), 7.09 (1H, br-dd, J = 10, 2 Hz, A6); minor isomer,

213

3.77 (3H, s, A3-OMe), 3.75 (2H, br-d, J = 4 Hz, γ), 3.82 (6H, s, B3-OMe and B5-OMe), 5.09

214

(1H, m, β), 6.30 (1H, br-d, J = 2 Hz, A2), 6.43 (1H, br-d, J = 10 Hz, A5), 6.58 (1H, br-d, J =

215

8 Hz, B2 and B6), 6.65 (1H, d, J = 8.9 Hz, α), 7.03 (1H, br-t, J = 8 Hz, B1), 7.51 (1H, br-dd,

216

J = 2, 10 Hz, A6). 13C NMR (CDCl3, 125 MHz) δC: major isomer, 55.1 (A3-OMe), 56.0

217

(B3-OMe and B5-OMe), 64.1 (γ), 80.4 (β), 104.6 (A2), 105.2 (B2 and B6), 124.8 (B1), 128.2

218

(A5), 133.2 (A1), 134.9 (B4), 141.7 (α), 141.9 (A6), 153.0 (A3), 153.3 (B3 and B5), 181.2

219

(A4); minor isomer, 55.2 (A3-OMe), 56.1 (B3-OMe and B5-OMe), 64.3 (γ), 79.8 (β), 105.2

220

(B2 and B6), 112.0 (A2), 124.7 (B1), 129.1 (A5), 133.3 (A6), 133.4 (A1), 134.8 (B4), 141.3

221

(α), 152.5 (A3), 153.3 (B3 and B5), 181.4 (A4).

222

General procedure for water addition to QMs.15 A dioxane solution of QM (3 mL)

223

prepared as described above (originating from 0.3 μmol of β-O-4 dimer) was transferred into

224

a 14 mL screw vial. To the QM solution, citrate–phosphate buffer at a pH of 2.4, 3.2, 3.9, 4.5,

225

or 5.3 (3 mL) was added to start the reaction with water. This marked the reaction time of 0

226

sec. After immediate shaking of the reaction mixture for a few seconds to make the mixture

227

homogeneous, an aliquot of the mixture (approx. 3 mL) was transferred into a quartz UV cell

228

(1 cm square, TOS-UV-10; Toshin Riko Co., Ltd., Japan), which was placed in a UV–visible

229

spectrometer (Jasco V-660, Jasco International Co. Ltd., Tokyo, Japan) equipped with a

230

circulating water bath (cooling-circulator CB-15, Iuchi Seieido Co. Ltd., Japan). The reaction

231

mixture was maintained at 25°C, and monitoring of the reaction progress was started at the

H NMR (CDCl3, 500 MHz), δH: major isomer, 3.72 (3H, s, A3-OMe), 3.75 (2H, broad-d, J



Page 12 of 32

232

reaction time of 20 sec. The citrate–phosphate buffers mentioned above were prepared by

233

mixing 0.01 M citric acid and 0.02 M disodium phosphate in different proportions so that

234

after mixing with an equal volume of dioxane, the resulting buffered dioxane–water mixture

235

(1:1, v/v) indicated the desired pH value (3.5, 4.5, 5.5, 6.0, and 7.0).

236

The disappearance of QMs and their half-life (t1/2) in the buffered dioxane–water

237

mixture was determined from the decrease in UV absorbance.45,46 The absorbance data at 304

238

nm was collected every 1 sec from the reaction time of 20 sec to 120 min for QM-GG, -GH,

239

and -GS, and every 0.2 sec from the reaction time from 20 sec to 10 min for QM-HG and

240

-HH. The collected data were fitted with an exponential function to estimate the UV

241

absorbance at a reaction time of 0 sec (Absinitial). The gram extinction coefficient of the QM at

242

304 nm was calculated using the Absinitial (see Table S1 for the Absinitial values and

243

pseudo-first-order reaction rate constants (kobs) for the disappearance of the QMs).

244

The precise concentrations of the prepared QM were not determined since the QMs

245

were not stable enough to be purified and weighed. However, their 1H NMR spectra indicated

246

that each crude QM solution was essentially pure and contained only small amounts of

247

unknown products, as indicated by the peaks differing from those of QM. Additionally, any

248

residual starting material (β-O-4 dimer) was not found in the 1H NMR spectra for all crude

249

QM solutions (Figure S3–S7). Thus, the β-O-4 yield was expressed as the yield of the

250

successive two reaction steps consisting of the QM formation (from β-O-4 dimers to QM) and

251

water addition (from QM to the β-O-4 isomer products).

252

The amounts of erythro and threo β-O-4 dimers in the crude products of the water addition

253

experiments were determined by HPLC analyses. After QM completely disappeared, an

254

aliquot of the reaction mixture (1 mL) was mixed with an internal standard (IS) of 12 ACS Paragon Plus Environment

Page 13 of 32


255

3,4-dimethoxyacetophenone (0.05 μmol) in MeOH and passed through a hydrophilic PTFE

256

membrane filter (Millex-LG, 0.2 μm, Millipore). The reaction mixture was analyzed by a

257

high-performance liquid chromatograph (HPLC, LC-10A, Shimadzu Co., Kyoto, Japan)

258

equipped with an SPD-M10A detector (280 nm, Shimadzu Co.).

259

The conditions for the HPLC analysis were as follows: column, Luna 5u C18 (2) 100

260

A (150 mm × 4.6 mm; Phenomenex, Inc., Torrance, CA, USA); oven temperature, 40 °C;

261

flow rate, 1.0 mL min−1. The β-O-4 dimers GG, GH, and HH in the reaction products were

262

separated using a binary gradient system consisting of CH3OH and H2O initially at 15:85 ratio

263

(v/v), and then increased linearly over 7.5 min to 25:75, and finally increased linearly over

264

42.5 min to 35:65 (55 min total). The retention times of the erythro and threo isomers were

265

35.8 and 38.8 min, respectively for GG, 30.0 and 27.5 min for GH, and 27.0 and 24.6 min for

266

HH. The retention time of 3,4-dimethoxyacetophenone (IS) was 32.4 min. The β-O-4 dimer

267

HG in the products was analyzed using a binary gradient system: the initial CH3OH/H2O (v/v)

268

15/85 was maintained isocratically for 7.5 min and then linearly increased over 27.5 min to

269

25:75, followed by a final linear increase over 15 min to 35:65 (55 min total). The retention

270

times of the products were 46.0 and 46.8 min for the erythro and threo isomers of HG,

271

respectively, and 44.8 min for IS. The β-O-4 dimer GS in the products was analyzed using a

272

binary gradient system: Initially, CH3OH/H2O (v/v) 15:85 was increased linearly over 7.5 min

273

to 25:75 and then increased linearly over 27.5 min to 35:65, and finally linearly increased

274

over 15 min to 75:25 (55 min total). The retention times of products were 39.2 and 42.1 min,

275

respectively, for the erythro and threo isomers of GS, and 27.1 min for IS.

276

The calibration curves obtained for the erythro and threo isomers of the β-O-4 dimers

277

were Y = 2.55X + 0.058 and Y = 2.38X + 0.048, respectively, for GG; Y = 3.62X + 0.022



Page 14 of 32

278

and Y = 3.31X + 0.046, respectively, for GH; Y = 6.51X + 0.065 and Y = 6.77X + 0.060,

279

respectively, for HH; Y = 3.86X − 0.012 and Y = 3.61X + 0.027, respectively, for HG; and Y

280

= 3.56X + 0.024 and Y = 3.42X + 0.013, respectively, for GS. Y is the molar ratio of each

281

β-O-4 diastereomer to IS (e.g., erythro-GG/IS molar ratio), and X is the peak area of each

282

β-O-4 diastereomer to IS (e.g., erythro-GG/IS area ratio).

283 284 285 286 287


Page 15 of 32


288

Table 1. UV absorbance and specific absorption coefficient of lignin model compounds in a

289

buffered dioxane–water solution (1:1, v/v, pH = 7) at 25°C. Compound λ max.a

ε 304 nmb

(nm)

(g-1Lcm-1)

QM-HH

306

92.8

QM-HG

304

81.5

QM-GH

307

68.4

QM-GG

306

61.2

QM-GS

304

49.9

β-O-4 dimer

λ max.

ε maxc

(nm)

(g-1Lcm-1)

Quinone methide

290 291 292 293

HHa

273

HGa

276

12.1

GHa

279

12.2

GGa

278

15.7

GSa

278

8.8

9.6

a

Wavelength of maximum absorbance. bGram extinction coefficient of quinone methides (QMs) at 304 nm are estimated from the UV absorbance data, which were collected starting at the reaction time of 20 sec (see experimental section and Table S1 in the ESI for details). c Gram extinction coefficient at λmax.

294



Page 16 of 32

295

(a)

1

QM-GG λmax

Absorbance

0.8

0.6

QM-GG 1 min 5 min 10 min 15 min 30 min 60 min 90 min 120 min GG

0.4

0.2 GG λmax

0

250

300

350

400

Wavelength (nm) 1

UV absorbance at 304 nm

(b) 0.9

QM-GG QM-GH QM-HG QM-HH

0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

0

10

20

30

40

50

60

70

Reaction time (min)

296 297

Figure 1. Disappearance of quinone methides (QMs) in a buffered dioxane–water solution

298

(1:1, v/v, pH = 7 at 25°C. a) UV absorbance spectra of QM-GG and a β-O-4 dimer GG. b)

299

Changes in the absorbance at 304 nm for four QMs (QM-GG, -GH, -HG, and -HH). See also

300

Figure S1 for the UV spectra of the other QMs, and Figure S2 for the disappearance of QMs

301

under different pH conditions.

302 303 304 16 ACS Paragon Plus Environment

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305

RESULTS AND DISCUSSION

306

A lignin model study using a β-O-4-aryl ether QM compound QM-GG (shown in Scheme

307

2) has been developed by Nakatsubo et al.14 Since then, subsequent studies have been

308

conducted for many purposes, such as studies on the reactivity of QMs toward different

309

nucleophiles14,47-49 and the characterization of the syn- and anti-geometric isomers of

310

3-methoxy substituted (G-type) QMs.42,50,51 This β-O-4-aryl ether QM has been used to

311

synthesize novel lignin model compounds for LCC linkages,52-55 dibenzodioxocin,56 and

312

anthrahydroquinone adducts.57,58

313

The reaction conditions in the water addition experiments on the QM were modified

314

by Brunow et al.15 to make them more suitable for mimicking the rearomatization step in

315

lignin biosynthesis. This was then applied to G-type and 3,5-dimethoxy-substituted (S-type)

316

QMs in a biosynthetic study of hardwood lignin. We conducted similar in vitro experiments

317

using non-substituted (H-type) QMs according to the Brunow’s method15 to mimic the

318

formation of β-O-4 structures in softwoods, especially in compression wood.

319

Brunow et al.15 prepared three types of β-O-4-aryl ether QMs, QM-GG, QM-GS, and

320

QM-SS (shown in Scheme 2), from corresponding β-O-4 dimer models: GG, GS, and

321

syringylglycerol-β-syringyl ether (SS), respectively. The water addition experiments were

322

conducted in a buffered 1:1 dioxane–water solution within a wide range of pH values (3–7).

323

The erythro/threo ratios of the resultant β-O-4 dimers in terms of the greatest ratio were in the

324

order SS > GS > GG at pH 3 (the ratio was 3 for SS, 2 for GS, and 1 for GG). In contrast,

325

under conditions at pH 4–7, threo-preferential formation was observed for the GG- and

326

GS-types, and no water adducts were found in the SS-type products. The stereo-structural



Page 18 of 32

327

feature of the β-O-4-structures in hardwood lignin was in line with the experimentally

328

observed erythro-preferential formation at a pH of 3.

329

We newly prepared three β-O-4-aryl ether QMs, QM-HH, -HG, and -GH (Scheme 2)

330

from their corresponding p-hydroxyphenyl-type β-O-4-dimeric compounds: HH, HG, and

331

GH, respectively. The QMs QM-GG and QM-GS were also prepared and subjected to the

332

water addition experiments, and the results were compared with those previously obtained by

333

Brunow et al.15

334

The β-O-4-aryl ether QMs (QM-HH, -HG, -GH, -GG, and -GS) were allowed to

335

react with water in the buffered dioxane–water (1:1, v/v) solution at 25°C under neutral and

336

mildly acidic conditions at pH values ranging from 3.5 to 7 (Scheme 2). The reaction progress

337

was monitored by the UV absorbance at 304 nm. Figure 1a shows the spectral changes of

338

QM-GG as the reaction time progressed. The disappearance of the QM was determined on

339

the basis of a decrease in the absorbance at 304 nm. The β-O-4 dimer products GG appeared,

340

which can be recognized by the absorbance peak at 278 nm but could not be quantified using

341

the UV absorbance because of the overlapping of the absorbance peak of GG with the

342

shoulder of the intensive peak of QM-GG at this wavelength. Thus, the β-O-4 product yield

343

was determined separately by HPLC analysis. The β-O-4 product yield was expressed as the

344

yield of the successive two-step reaction consisting of QM formation (from β-O-4 dimers to

345

QM) and water addition (from QM to the water adduct, i.e., the β-O-4 dimer products). This

346

is due to the difficulty in determining the precise concentrations of the prepared QMs that

347

were not stable enough to be purified and weighed. Their 1H NMR spectra suggest that each

348

crude QM solution was essentially pure and contained only a small amount of unknown

349

products as indicated by the peaks other than those of QM. Any residual β-O-4 dimer, i.e., the


Page 19 of 32


350

starting material, was not found in the 1H NMR spectra of all crude QM solutions (see

351

Experimental section for the details of the preparation of QMs). These data for the β-O-4

352

product yields were used only for determining whether the water adducts (erythro and threo

353

β-O-4 dimers) were obtained as the major products in this reaction and were not used to draw

354

any other conclusions.

355 356

Reactivity of QMs with water

357

The disappearance of the QM, which was monitored by the absorption at 304 nm

358

(Table 1, Figure 1, and Figure S1), was well-fitted with an exponential function in most cases

359

of the reactions (see Table S1 and Figure S2 for the correlation coefficient and kobs). A

360

comparison of the reaction rates based on the t1/2 of the QMs was made (Table 2). The QM, as

361

expected, disappeared faster under more acidic conditions in all cases. Noticeable differences

362

in t1/2 were found between the G- and H-type QMs. Water addition occurs with G-type QMs

363

(QM-GG and -GH), to form guaiacylglycerol-β-aryl ethers (GG and GH), with a t1/2 of 1.4–

364

15 min. In contrast, the H-type QMs (QM-HH and -HG) were very labile and transformed

365

into p-hydroxyphenylglycerol-β-aryl ethers (HH and HG), with a t1/2 of 10–40 sec. t1/2 tended

366

to be shorter in the order QM-HH < QM-HG < QM-GH < QM-GG, regardless of the pH

367

conditions. The β-O-4 dimer products showed satisfactory yields in the cases of these four

368

QMs (70–94% yield), indicating that water addition was the main reaction in these

369

experiments although minor unknown side-reactions must have also occurred. The relatively

370

clear presence of the isosbestic point in the spectral change during the progress of the water

371

addition to QM-GG (Figure 1a; see also Figure S1 for the other QMs) suggests that the

372

conversion of QM-GG to the corresponding β-O-4 dimer GG proceeded rather quantitatively. 19 ACS Paragon Plus Environment


Page 20 of 32

373

Thus, it is possible to compare the reaction rate of the water addition based on the

374

disappearance of the QMs.

375

Apart from the structural difference in the QM moiety (A-ring), the type of

376

β-etherified ring (B-ring) also influenced the reaction rates. Upon replacement of the B-ring

377

from the p-hydroxyphenyl with the guaiacyl ring, t1/2 became longer regardless of the type of

378

A-ring (t1/2: QM-XH < QM-XG, where X is either H or G). More specifically, in the case of

379

the 3-methoxy-substituted QMs (G-type), t1/2 of the β-O-4-guaiacyl ether QM was up to 1.2–

380

1.4 times longer than that of the β-O-4-p-hydroxyphenyl ether QM (t1/2: QM-GH < QM-GG)

381

at a pH of 5.5–7, although they have similar half-lives at pH 3.5 and 4.5. A similar trend was

382

observed for non-substituted QMs, in which t1/2 was 1.1–2 times longer for the

383

β-O-4-guaiacyl ether QM in the range of pH 5.5–7.0 (t1/2: QM-HH < QM-HG).

384

The reaction rate, however, was more influenced by the type of QM moiety (A-ring)

385

(t1/2: QM-HX < QM-GX, where X is either H or G). More specifically, the β-O-4-guaiacyl

386

ether QM became less reactive when the non-substituted QM moiety was replaced with the

387

3-methoxy-substituted moiety under all pH conditions, and t1/2 was up to 27 times longer (t1/2:

388

QM-HG < QM-GG).

389

β-O-4-p-hydroxyphenyl ether QMs reacted much slower at all pH conditions, with t1/2 of up to

390

39 times longer at a pH of 5 (t1/2: QM-HH < QM-GH). The water addition reaction was

391

slowed because of the methoxy group at the 3-position of the QM moiety.

In

addition,

the

3-methoxy-substituted

QM

in

the

392


Page 21 of 32


393

Table 2. Half-life of quinone methides (QMs) in buffered dioxane–water solution (1:1, pH =

394

3.5, 4.5, 5.5, 6, and 7) at 25°C and the yields of β-O-4 dimer products. Entry

395 396

pH

Half-life Products Yield erythro:thr (t1/2) (mol%) eo QM-HH 3.5 11 sec HH 94 30:70 4.5 15 sec HH 85 29:71 5.5 17 sec HH 80 24:76 6.0 17 sec HH 89 24:76 7.0 18 sec HH 83 24:76 QM-HG 3.5 GG. That is, a higher

473

threo-selectivity was obtained by replacing the β-etherified aromatic nucleus of the QMs

474

(B-ring) from guaiacyl-type to p-hydroxyphenyl-type. This phenomenon was observed with

475

both non-substituted and 3-methoxy-substituted QMs under all pH conditions (as observed by

476

comparing QM-GG with QM-GH and QM-HG with QM-HH).

477

The structural type of the QM moiety (A-ring), which governs the reactivity of the

478

QMs, also influenced the stereo-selectivity to a smaller extent. A higher threo-selectivity was

479

obtained by replacing the QM moiety from methoxy-substituted type with a non-substituted

480

one under every pH condition. The effect of this replacement, however, was moderate (as

481

shown by the comparison of QM-GG with QM-HG and QM-GH with QM-HH). The

482

structural type of β-etherified ring influenced the erythro/threo ratio more than the QM

483

moiety did (B-ring effect > A-ring effect).

484

Implications from the erythro/threo ratio of the water adducts



Page 26 of 32

485

Yeh et al.35 and Nawawi et al.10 reported that the erythro/threo ratio of β-O-4

486

structures in compression wood is slightly but clearly higher than 1.0, whereas the ratio in

487

opposite wood is exactly 1.0. Although the erythro/threo ratios obtained in the present model

488

experiments were lower than 1.0, the ratios obtained under lower pH conditions were closer to

489

the values reported for compression wood lignins in comparison with the ratios obtained at

490

higher pH conditions. Hence, the results obtained in the present study are in line with the

491

proposal by Brunow et al.15 that, “the pH of the medium in which lignin biosynthesis occurs is

492

lower than has been assumed until now”. This statement was made in relation to guaiacyl

493

and/or syringyl lignins. However, even the erythro/threo ratios obtained at lower pH values

494

by the present experiments still differed from the ratios for compression wood lignins

495

determined by Yeh et al.35 and Nawawi et al.10 This model experiment may not perfectly

496

mimic the re-aromatization step in lignin biosynthesis. An explanation of the gap between the

497

model experiment and lignin structure made by exploring a reaction condition that leads to

498

erythro-preferential formation in H-type QM would be of use. Such experimentation may

499

contribute to our further understanding of the lignin polymerization conditions in cell walls.

500

The effects of the solvent conditions14 and a more detailed understanding of the effects of

501

β-etherified structures and their size may deserve more attention.

502

ASSOCIATED CONTENT

503

Supporting Information. The Supporting information is available free of charge on the ACS

504

Publication website. The UV absorbance spectra of QMs (Figure S1), Pseudo-first-order

505

reaction rate constants (kobs) for the disappearance of β-O-4-aryl ether QMs (Table S1, Figure

506

S2), NMR data of β-O-4 model compounds and the 1H NMR spectra of β-O-4 model

507

compounds and their corresponding QMs (Figure S3-S7) (PDF).


Page 27 of 32


508

AUTHOR INFORMATION

509

Corresponding Author

510

* Takuya Akiyama. * Wood Chemistry Laboratory, Department of Biomaterial Sciences,

511

Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku,

512

Tokyo

513

+81-3-5841-5262; fax: +81-3-5841-2678.

514

ACKNOWLEDGMENT

515

This work was supported by the Japan Science and Technology Agency (JST), PRESTO

516

(JPMJPR12B1), and the Grant-in-Aid for Challenging Exploratory Research (15K14767)

517

from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT).

518

REFERENCES

519 520

1. Peter, M. G., Chemical modifications of biopolymers by quinones and quinone methides. Angewandte Chemie International Edition in English 1989, 28, 555-570.

521 522

2. Hemingway, R. W.; Foo, L. Y., Condensed tannins - quinone methide intermediates in procyanidin synthesis. J Chem Soc Chem Comm 1983, 1035-1036.

523 524

3. Andersen, S. O., Insect cuticular sclerotization: A review. Insect Biochem Molec 2010, 40, 166-178.

525 526 527

4. Ralph, J.; Schatz, P. F.; Lu, F.; Kim, H.; Akiyama, T.; Nelsen., S. F., Quinone methides in lignification. In Quinone methides, Rokita, S., Ed. John Wiley & Sons: Hoboken, NJ, 2009; pp 385-420.

528 529

5. Chen, F.; Tobimatsu, Y.; Havkin-Frenkel, D.; Dixon, R. A.; Ralph, J., A polymer of caffeyl alcohol in plant seeds. P Natl Acad Sci USA 2012, 109, 1772-1777.

530 531 532

6. Kupchan, S. M.; Karim, A.; Marcks, C., Taxodione and taxodone, two novel diterpenoid quinone methide tumor inhibitors from Taxodium distichum. J. Am. Chem. Soc. 1968, 90, 5923-5924.

533 534

7. Gunatilaka, A. A. L.; Fernando, H. C.; Kikuchi, T.; Tezuka, Y., 1H and 13C NMR analysis of three quinone-methide triterpenoids. Magn. Reson. Chem. 1989, 27, 803-807.

535 536

8. Timell, T. E., Chemical properties of compression wood. In Compression wood in gymnosperms, Springer-Verlag: Berlin, 1986; Vol. 1, pp 289-408.

113-8657,

Japan.

E-mail:

[email protected];

tel:



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9. Nanayakkara, B.; Manley-Harris, M.; Suckling, I. D.; Donaldson, L. A., Quantitative chemical indicators to assess the gradation of compression wood. Holzforschung 2009, 63, 431-439.

540 541 542

10. Nawawi, D. S.; Akiyama, T.; Syafii, W.; Matsumoto, Y., Characteristic of β-O-4 structures in different reaction wood lignins of Eusideroxylon zwageri T. et B. and four other woody species. Holzforschung 2017, 71, 11-20.

543 544 545

11. Akiyama, T.; Magara, K.; Meshitsuka, G.; Lundquist, K.; Matsumoto, Y., Absolute configuration of β- and α-asymmetric carbons within β-O-4-structures in hardwood lignin. J. Wood Chem. Technol. 2015, 35, 8-16.

546 547

12. Ralph, J.; Peng, J. P.; Lu, F. C.; Hatfield, R. D.; Helm, R. F., Are lignins optically active? J. Agric. Food. Chem. 1999, 47, 2991-2996.

548 549

13. Ogiyama, K.; Kondo, T., On pinoresinol type of structural units in lignin. Tetrahedron Lett. 1966, 2083-2088.

550 551

14. Nakatsubo, F.; Sato, K.; Higuchi, T., Enzymic dehydrogenation of p-coumaryl Alcohol. IV. Reactivity of quinonemethide. Mokuzai Gakkaishi 1976, 22, 29-33.

552 553 554

15. Brunow, G.; Karlsson, O.; Lundquist, K.; Sipila, J., On the distribution of the diastereomers of the structural elements in lignins: the steric course of reactions mimicking lignin biosynthesis. Wood Sci. Technol. 1993, 27, 281-286.

555 556 557 558

16. Akiyama, T.; Goto, H.; Nawawi, D. S.; Syafii, W.; Matsumoto, Y.; Meshitsuka, G., Erythro/threo ratio of β-O-4-structures as an important structural characteristic of lignin. Part 4: Variation in the erythro/threo ratio in softwood and hardwood lignins and its relation to syringyl/guaiacyl ratio. Holzforschung 2005, 59, 276-281.

559 560 561

17. Nawawi, D. S.; Syafii, W.; Tomoda, I.; Uchida, Y.; Akiyama, T.; Yokoyama, T.; Matsumoto, Y., Characteristics and reactivity of lignin in Acacia and Eucalyptus woods. J. Wood Chem. Technol. 2017, 37, 273-282.

562 563 564

18. Ralph, J.; Lundquist, K.; Brunow, G.; Lu, F.; Kim, H.; Schatz, P. F.; Marita, J. M.; Hatfield, R. D.; Ralph, S. A.; Christensen, J. H.; Boerjan, W., Lignins: Natural polymers from oxidative coupling of 4-hydroxyphenyl- propanoids. Phytochemistry Reviews 2004, 3, 29-60.

565 566 567

19. Miksche, G. E., Zum alkalischen abbau p-alkoxy-arylglycerin-β-arylätherstrukturen des lignins: Versuche erythro-veratrylglycerin-β-guajacyläther. Acta Chem. Scand. 1972, 26, 3275-3281.

568 569 570

20. Criss, D. L.; Collier, W. E.; Fisher, T. H.; Schultz, T. P., Alkaline hydrolysis of nonphenolic β-O-4 model diastereomers: Substituent effect on the leaving phenoxide. Holzforschung 1998, 52, 171-174.

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21. Shimizu, S.; Yokoyama, T.; Akiyama, T.; Matsumoto, Y., Reactivity of lignin with different composition of aromatic syringyl/guaiacyl structures and erythro/threo side chain structures in β-O-4 type during alkaline delignification: As a basis for the different degradability of hardwood and softwood lignin. J. Agric. Food. Chem. 2012, 60, 6471-6476.

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22. Jonsson, L.; Karlsson, O.; Lundquist, K.; Nyman, P. O., Stereospecificity in enzymatic and nonenzymatic oxidation of β-O-4 lignin model compounds. FEBS Lett. 1990, 276, 45-48.

578 579

23. Bohlin, C.; Andersson, P. A.; Lundquist, K.; Jonsson, L. J., Differences in stereo-preference in the oxidative degradation of diastereomers of the lignin model compound

der mit


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708 709

TABLE OF CONTENTS

710 HO

OH H

Water addtion

H

β

O

HO H

R'

Water addtion

α

R O

711

4

R R' OMe, OMe : H, OMe : OMe, H : H, H :

Water-dioxane (v/v, 1:1)

β α

H O

HO

4

H

β α

H O

R'

pH 3.5 - 7 at 25 ºC QM-GG QM-HG QM-GH QM-HH

OH

R'

R

R

OH

OH

erythro isomer

β-O-4-aryl ether quinone methides

4

threo isomer

β-O-4 structures

712 713


Lignin biosynthetic study: Reactivity of quinone methides in the

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