Biomimetic Oxidation of Monolignol Acetate and p-Coumarate by Silver Oxide in 1,4-Dioxane

Article abstract of DOI:10.1021/acs.jafc.9b07682

Lignin acylated with acetate and/or p-coumarate is common in many herbaceous plants. Herein, the biomimetic oxidation of ?3-acylated monolignols with Ag₂O was studied to understand the effect of ?3-acyl groups on monolignol polymerization. The oxidation of sinapyl acetate gave ?3-acylated and α-acylated β-O-4 dimers in 71 and 9.5% yields, respectively. The oxidation of sinapyl p-coumarate produced ?3-acylated β-O-4 and ?3-acylated tetralin β-β dimers in 53 and 16% yields, respectively. Only the sinapyl alcohol moiety in sinapyl p-coumarate reacted, and the p-coumarate moiety remained unchanged, suggesting that p-coumaric acid is not incorporated into the lignin backbone in the acylated lignins. All of the ?3-acylated monolignols used in this study produced the ?3-acylated β-O-4 dimers, which suggests that the ?3-acylated monolignols act as lignin monomers. The relatively high yields of the β-O-4 dimers indicate that Ag₂O oxidation of the monolignols can be used as an easy method for synthesizing the β-O-4 dimer model compounds.

Full text of DOI:10.1021/acs.jafc.9b07682

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Biofuels and Biobased Materials
Biomimetic Oxidation of Monolignol Acetate
and p-Coumarate by Silver Oxide in 1,4-Dioxane
Ayana Yamashita, Takao Kishimoto, Masahiro Hamada, Daisuke Urabe, and Noriyuki Nakajima
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b07682 • Publication Date (Web): 27 Jan 2020
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Biomimetic Oxidation of Monolignol Acetate and p-Coumarate by
Silver Oxide in 1,4-Dioxane
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Ayana Yamashita, Takao Kishimoto*, Masahiro Hamada, Noriyuki Nakajima, Daisuke
Urabe
Bioorganic Chemistry Laboratory, Department of Biotechnology, Faculty of
Engineering, Toyama Prefectural University, Imizu 939-0398, Japan
*Corresponding author: Takao Kishimoto, Department of Biotechnology, Faculty of
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Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu 939-0398, Japan.
Phone:
+81-766-56-7500
(ext567),
Fax:
+81-766-56-2498,
E-mail:
takao@pu-toyama.ac.jp
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Abstract
Lignin acylated with acetate and/or p-coumarate is common in many herbaceous plants.
Herein, the biomimetic oxidation of -acylated monolignols with Ag2O was studied to
understand the effect of -acyl groups on monolignol polymerization. The oxidation of
sinapyl acetate produced -acylated and -acylated -O-4 dimers in 71% and 9.5%
yields. The oxidation of sinapyl p-coumarate gave -acylated -O-4 dimer and
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-acylated tetralin - dimer in 53% and 16% yields, respectively. Only the sinapyl
alcohol moiety in sinapyl p-coumarate reacted, and the p-coumarate moiety remained
unchanged, suggesting that p-coumaric acid is not incorporated into lignin backbone in
the acylated lignins. All the -acylated monolignols used in this study produced the
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-acylated -O-4 dimers, which suggests that the -acylated monolignols act as lignin
monomers. The relatively high yields of the -O-4 dimers indicate that Ag2O oxidation
of the monolignols can be used as one easy method for synthesizing the -O-4 dimer
model compounds.
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Keywords: coniferyl acetate, sinapyl acetate, sinapyl p-coumarate, -acylated tetralin
- dimer, -acylated -O-4 dimer
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INTRODUCTION
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Lignin is the second most abundant organic compound on earth. Its utilization remains
one of those challenging research topics in the field of biomass conversion. This is
essentially because of its complicated chemical structure. Substructures of lignin
include -O-4, -, -5, 5-O-4, 5-5, and other structures.1 Among them, the -O-4
structure is the most abundant, and its frequency is between 50%–60% in wood lignins.
With a focus on the -O-4 structures, we reported the synthesis of the -O-4 type
artificial lignin polymers.2-4 Also, the existence of both syringyl and guaiacyl units was
effective for producing higher amounts of the β-O-4 structure in natural and
enzymatically synthesized lignin.5
In plant cell walls, natural lignin is synthesized by the dehydrogenative
polymerization of monolignols such as coniferyl, sinapyl, and p-coumaryl alcohol using
laccase and/or peroxidase.1 Lignins from some herbaceous plant like kenaf (Hibiscus
cannabinus), curaua (Ananas erectifolius), sisal (Agave sisalana), and abaca (Musa
textilis) are highly acylated by acetate and p-coumarate groups.6-8 These highly acylated
lignins are characterized by a high frequency of the -O-4 structure and a high S/G
ratio. Miscanthus (Miscanthus × giganteus) lignin is also highly acylated.9 The lignins
from sugarcane (Saccharum spp. hybrids) bagasse and straw are also acylated by acetate
and p-coumarate groups,10 as are all commelinid monocotyledon lignins.11 The highly
acylated lignin has been established to be formed by dehydrogenative polymerization of
-acylated monolignols such as sinapyl acetate and sinapyl p-coumarate.12
In our previous studies, biomimetic oxidation of sinapyl alcohol 1a and sinapyl
acetate 1b were investigated using silver (I) oxide (Ag2O) to better understand the high
frequency of the -O-4 structure in the herbaceous plant lignins.13 The preferential
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formation of the -O-4 dimer was observed by the oxidation of sinapyl acetate 1b. In
this study, oxidative coupling of acylated monolignols was reinvestigated using sinapyl
p-coumarate 1c and coniferyl acetate 1e in addition to sinapyl acetate 1b. The entire
reaction products were analyzed by using 1H-13C correlation heteronuclear
single-quantum coherence (HSQC), nuclear magnetic resonance (NMR) spectra. The
formed dimeric compounds were isolated, and the yields were compared to elucidate the
effects of -acyl groups on the oxidative coupling of monolignols.
MATERIALS AND METHODS
General.
All the chemicals were purchased from one of the following companies: Tokyo
Chemical Industry Co., Ltd. (Tokyo, Japan), Fujifilm Wako Pure Chemical Corporation
(Osaka, Japan) or Sigma-Aldrich Japan (Tokyo, Japan), and were used without any
purification. The NMR spectra were recorded with a Bruker AVANCE II 500 FT-NMR
(500 MHz) spectrometer, Bruker AVANCE II 400 FT-NMR (400 MHz) in acetone-d6
or chloroform-d. The central peaks of the residual solvents were used as internal
references. We assigned both the erythro and threo forms of the -O-4 dimers based on
the comparison with other similar lignin model compounds.4,14 High-resolution
electrospray-ionization time-of-flight mass spectrometry (ESI-ToF-MS) was conducted
with a Bruker micrOTOF.
Synthesis of Monolignols.
Sinapyl alcohol 1a and coniferyl alcohol 1d were synthesized from the
corresponding ethyl cinnamate.15 Sinapyl acetate 1b, sinapyl p-coumarate 1c, and
coniferyl acetate 1e were synthesized by reported procedures.16 NMR data for the
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acylated monolignols are found in the Supporting information (SI, page s2).
Oxidative Coupling of Monolignols.
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To a stirred solution of AgNO3 (0.44 g, 2.59 mmol) in H2O (2.6 mL), NaOH
(0.114 g, 2.85 mmol) in H2O (1.0 mL) was added dropwise under dark at room
temperature. The reaction mixture was kept for 5 min. The precipitate was filtered off,
washed with H2O, and completely dried under vacuum over P2O5 to afford Ag2O. The
freshly prepared Ag2O (212 mg, 0.91 mmol) was added portion-wise to a stirred
solution of 1c (100 mg, 0.28 mmol) in 1,4-dioxane (3 mL) at room temperature. The
reaction mixture was kept for 4.5 h, filtered using Celite, and dropped into a flask
containing concentrated HCl (0.35 mL)/H2O (20 mL)/ tetrahydrofuran (THF, 20 mL).
The resulting mixture was kept for another 30 min, diluted with ethyl acetate, and then
washed with saturated NaHCO3 and brine. The mixture was then dried over Na2SO4,
and concentrated to dryness under vacuum. The entire reaction products were analyzed
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by H–13C HSQC NMR spectroscopy. The preparative thin-layer chromatography
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(TLC) of the reaction products using ethyl acetate/ hexane/ benzene (9:4:1, v/v/v) gave
the -O-4 dimer 2c (60 mg, erythro:threo = 73:27) and the - dimer 4c (18 mg). The
erytho-2c and threo-2c were further separated by TLC (ethyl acetate/ hexane/ benzene,
9:4:1, v/v/v) for NMR analysis.
By using a similar procedure, sinapyl alcohol 1a (100 mg, 0.48 mmol) gave
syringyl glycerol -sinapyl alcohol ether 2a (73 mg), syringyl glycerol -sinap aldehyde
ether 3a (4.2 mg), syringaresinol 4a (5.5 mg), and sinap aldehyde 6a (1.1 mg). From
sinayl acetate 1b (100 mg, 0.40 mmol), the -acylated -O-4 dimer 2b (77 mg), and the
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-acylated -O-4 dimer 3b (10 mg) were obtained. Coniferyl alcohol 1d (100 mg, 0.56
mmol) produced guaiacyl glycerol -coniferyl alcohol ether 2d (79 mg) and the -5
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dimer, dehydrodiconiferyl alcohol 5d (12 mg). From coniferyl acetate 1e (100 mg, 0.45
mmol), the -acylated -O-4 dimer 2e (54 mg), the -acylated -O-4 dimer 3e (24 mg)
and the -acylated -5 dimer 5e (5.1 mg) were obtained. These isolated products were
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1
analyzed by H, 13C, HSQC, heteronuclear multiple-bond coherence (HMBC) and
nuclear Overhauser effect spectroscopy (NOESY) NMR spectra. NMR data for all
coupling products are given in Tables 1 and 2, and are also found in the Supporting
information (SI, pages S3-S11). The oxidative coupling of monolignols was carried out
in duplicate. Table 3 summarizes the isolated dimer yields. These were calculated on the
basis of the average yields of each reaction product.
2a: erythro: high resolution ESI-TOF-MS, calculated for C22H28O9Na+ 459.1626,
found 459.1643.
2a: threo: high resolution ESI-TOF-MS, calculated for C22H28O9Na+ 459.1626,
found 459.1634.
2b: erythro: high resolution ESI-TOF-MS, calculated for C26H32O11Na+
543.1837, found 543.1821.
2b: threo: high resolution ESI-TOF-MS, calculated for C26H32O11Na+ 543.1837,
found 543.1820.
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2c: erythro: H NMR (400 MHz, acetone-d6 and 1 drop of D2O),  7.65 (d, J =
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15.9 Hz, 1H, B′), 7.55 (d, J = 8.6 Hz, 2H, B2′/6′), 7.44 (d, J = 8.6 Hz, 2H, A2′/6′), 7.31
(d, J = 16.0 Hz, 1H, A′), 6.89 (d, J = 8.6 Hz, 2H, A3′/5′, or B3′/5′), 6.87 (d, J = 8.6 Hz,
2H, A′/′ or B3′/ ′), 6.86 (s, 2H, B2/6), 6.75 (s, 2H, A2/6), 6.69 (br d, J = 15.9 Hz,
1H, B,), 6.40 (dt, J = 6.3, 15.9 Hz, 1H, B), 6.40 (d, J = 15.9 Hz, 1H, B′), 6.17 (d, J
= 16.0 Hz, 1H, A′), 4.99 (d, J = 3.5 Hz, 1H, A), 4.82 (dd, J = 1.1, 6.3 Hz, 2H, B),
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4.61 (m, 1H, A), 4.47 (dd, J = 7.6, 11.8 Hz, 1H, A), 4.25 (dd, J = 3.2, 11.8 Hz, 1H,
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A), 3.88 (s, 6H, BOMe), 3.81 (s, 6H, AOMe); C NMR (500 MHz, acetone-d6 and 1
drop of D2O),  167.4 (BCO), 167.3 (ACO), 160.8 (B4′), 160.6 (A4′), 154.4 (B3/5),
148.6 (A3/5), 145.8 (B′), 145.2 (A′), 136.9 (B4), 136.0 (A4), 134.5 (B), 133.4 (B1),
131.9 (A1), 131.1 (B2′/6′), 131.0 (A2′/6′), 126.9 (A1′, B1′), 124.5 (B), 116.8 (A3′/5′,
B3′/5′), 115.6 (A′), 115.4 (B′), 104.9 (B2/6), 104.7 (A2/6), 84.8 (A), 73.5 (A),
65.3 (B), 64.0 (A), 56.7 (OMe), 56.6 (OMe); high resolution ESI-TOF-MS, calculated
for C40H40O13Na+ 751.2361, found 751.2365.
2c: threo: 1H NMR (400 MHz, acetone-d6 and 1 drop of D2O),  7.65 (d, J = 16.0
Hz, 1H, B′), 7.55 (d, J = 8.6 Hz, 2H, B2′/6′), 7.49 (d, J = 8.6 Hz, 2H, A2′/6′), 7.40 (d,
J = 16.0 Hz, 1H, A′), 6.89 (d, J = 8.6 Hz, 4H, A3′/5′, B3’/5’), 6.86 (s, 2H, B2/6), 6.77
(s, 2H, A2/6), 6.68 (br d, J = 15.8 Hz, 1H, B), 6.40 (dt, J = 6.2, 15.8 Hz, 1H, B), 6.40
(d, J = 16.0 Hz, 1H, B′), 6.28 (d, J = 16.0 Hz, 1H, A′), 4.99 (d, J = 6.9 Hz, 1H, A),
4.81 (dd, J = 1.1, 6.2 Hz, 1H, B), 4.41 (dd, J = 3.4, 11.7 Hz, 1H, A), 4.33 (m, 1H,
A), 4.06 (dd, J = 4.8, 11.7 Hz, 1H, A), 3.87 (s, 6H, BOMe), 3.77 (s, 6H, AOMe); 13C
NMR (500 MHz, acetone-d6 and 1 drop of D2O),  167.5 (BCO), 167.3 (ACO), 160.9
(B4′), 160.8 (A4′), 154.0 (B3/5), 148.5 (A3/5), 145.9 (B′), 145.5 (A′), 137.8 (B4),
136.3 (A4), 134.3 (B), 133.4 (B1), 132.0 (A1), 131.1 (A2′/6′, B2′/6′), 126.8 (A1′, B1′),
124.5 (B), 116.7 (A3′/5′, B3′/5′), 115.4 (A′), 115.3 (B′), 105.5 (A2/6), 104.9 (B2/6),
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86.8 (A), 74.7 (A), 65.3 (B), 65.1 (A), 56.6 (OMe × 2); high resolution
ESI-TOF-MS, calculated for C40H40O13Na+ 751.2361, found 751.2362.
2d: erythro: high resolution ESI-TOF-MS, calculated for C20H24O7Na+ 399.1414,
found 399.1410.
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2d: threo: high resolution ESI -TOF-MS, calculated for C20H24O7Na+ 399.1414,
found: 399.1426.
2e: erythro: high resolution ESI-TOF-MS, calculated for C24H28O9Na+ 483.1626,
found 483.1616.
2e: threo: high resolution ESI-TOF-MS, calculated for C24H28O9Na+ 483.1626,
found 483.1614.
3a: erythro: high resolution ESI-TOF-MS, calculated for C22H26O9Na+ 457.1469,
found 457.1466.
3b: erythro: high resolution ESI-TOF-MS, calculated for C26H32O11Na+
543.1837, found 543.1835.
3b: threo: high resolution ESI-TOF-MS, calculated for C26H32O11Na+ 543.1837,
found 543.1837.
3e: erythro: high resolution ESI-TOF-MS, calculated for C24H28O9Na+ 483.1626,
found 483.1609.
3e: threo: high resolution ESI-TOF-MS, calculated for C24H28O9Na+ 483.1626,
found 483.1613.
4a: high resolution ESI-TOF-MS, calculated for C22H26O8Na+ 441.1520, found
441.1522.
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4c: H NMR (500 MHz, acetone-d6 and 1 drop of D2O),  7.63 (d, J = 16.0 Hz,
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1H, A′ or B′), 7.59 (d, J = 16.0 Hz, 1H, A′ or B′), 7.50 (d, J = 8.0 Hz, 2H, A2′/6′
or B2′/6′), 7.48 (d, J = 8.1, 2H, A2′/6′ or B2′/6′), 6.85 (d, J = 8.0 Hz, 2H, A3′/5′ or
B3′/5′), 6.84 (d, J = 8.1 Hz, 2H, A3′/5′ or B3′/5′), 6.74 (s, 1H, B2), 6.67 (s, 2H, A2/6),
6.40 (d, J = 16.0 Hz, 1H, A′ or B′), 6.33 (d, J = 16.0 Hz, 1H, A′ or B′), 4.89 (br s,
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1H, B), 4.52 (m, 1H, B), 4.50 (m, 1H, A), 4.42 (dd, J = 5.9, 11.2 Hz, 1H, B), 4.29
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(dd, J = 2.6, 11.8 Hz, 1H, A), 4.00 (d, J = 9.5 Hz, 1H, A), 3.85 (s, 3H, B3OMe), 3.74
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(s, 6H, AOMe), 3.15 (s, 3H, B5OMe), 2.55 (m, 1H, A), 2.23 (m, 1H, B); C NMR
(500 MHz, acetone-d6 and 1 drop of D2O),  167.8 (CO), 167.7 (CO), 160.7 (A4′, B4′),
148.4 (A3/5), 147.9 (B3), 146.8 (B5), 145.6 (A′ or B′), 145.4 (A′ or B′), 140.5
(B4), 139.5 (A1), 135.6 (A4), 131.1 (A2′/6′ or B2′/6′), 131.0 (A2′/6′ or B2′/6′), 131.0
(B1), 127.1 (B6), 127.0 (A1′, B1′), 116.7 (A3′/5′, B3′/5′), 115.8 (A′ or B′), 115.7 (A′
or B′), 108.1 (A2/6), 108.0 (B2), 69.3 (B), 65.9 (B), 63.4 (A), 58.8 (B5OMe), 56.9
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(AOMe), 56.5 (B3OMe), 44.8 (A), 41.3 (B), 41.2 (A); high resolution
ESI-TOF-MS, calculated for C40H40O13Na+ 751.2361, found 751.2354.
5d: high resolution ESI-TOF-MS, calculated for C20H22O6Na+ 381.1309, found
381.1281.
5e: high resolution ESI-TOF-MS, calculated for C24H26O8Na+ 465.1520, found
465.1495.
RESULTS AND DISCUSSION
Identification of Reaction Products from Oxidative Coupling of Acylated
Monolignols by Silver Oxide.
Oxidative coupling of acylated and non-acylated monolignols, including sinapyl
alcohol 1a, sinapyl acetate 1b, sinapyl p-coumarate 1c, coniferyl alcohol 1d, and
coniferyl acetate 1e were investigated using Ag2O as shown in Figure 1. Ag2O was
freshly prepared from AgNO3 and NaOH, completely dried under vacuum over P2O5,
and subsequently used. This is because water content of Ag2O affected the yield and
proportions of the reaction products in our preliminary experiments. The water
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containing reaction system was also reported to affect the coupling products from
coniferyl alcohol 1d.17 The acylated monolignols were reacted in 1,4-dioxane to form
quinone methide intermediates. These quinone methides were quenched in aqueous
HCl/THF solution. A summary of the reaction products from the oxidative coupling of
acylated monolignols are shown in Figure 2. Compounds 4b and 4d were detected only
1
by H-13C HSQC NMR spectroscopic analysis of the reaction mixtures, whereas other
products were isolated by TLC. The isolated products were identified by the use of
NMR spectroscopy and high resolution mass spectrometry.
Oxidation of sinapyl alcohol 1a and sinapyl acetate 1b by Ag2O was
reinvestigated using completely dried Ag2O. The oxidative coupling of sinapyl alcohol,
1a gave erythro and threo isomers of the -O-4 dimer, syringyl glycerol -sinapyl
alcohol ether, 2a in a yield of 70% as shown in Table 3. The yield of 2a was higher than
that in our previous study. This is most likely because the water content of Ag2O in this
study was lower than in the previous study.13 Small amounts of syringyl glycerol
-sinap aldehyde ether 3a, syringaresinol 4a, and sinap aldehyde 6a were also obtained
from 1a. In contrast, sinapyl acetate 1b gave erythro and threo isomers of the
-O-acetylated -O-4 dimer 2b in a 71% yield. In addition to 2b, the -O-acetylated
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-O-4 dimer 3b was also obtained in a 9.5% yield. This was formed from 2b by the
acetyl migration after the oxidative coupling of 1b as shown in Figure 3. The acetyl
migration product 3b was not obtained in our previous study.13 The non-traditional
tetralin type - dimer 4b rather than syringaresinol 4a was detected by HSQC NMR
analysis of the entire reaction products from the oxidation of 1b as shown in Figure 4A.
The tetralin type - dimer 4b was not identified in our previous study.13 The HSQC
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NMR signals of 4b were tentatively assigned because the isolation of 4b failed. The
tetralin - structures seemed to be formed by the hydration step of the quinone methide
intermediate under the acidic reaction conditions used in this investigation as shown in
Figure 5. However, the -acylated tetrahydrofuran type - structure was not detected in
this investigation.18, 19
Oxidative coupling of sinapyl p-coumarate 1c attracted our interest, because 1c
has two phenolic hydroxy groups in sinapyl alcohol and p-coumarate moieties. Both
sinapyl alcohol and p-coumarate moieties may in principal participate in the radical
coupling reactions, as observed with coniferyl ferulate.20 However, in the presence of
excess Ag2O, only the sinapyl alcohol portion of 1c coupled with each other and the
-acylated -O-4 dimer 2c was obtained with a 53% yield. HMBC NMR analysis of 2c
237
showed a clear correlation between A-H and B-C4. This confirmed the coupling
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between A-C and B-O4 in the sinapyl alcohol moiety of 1c as shown in Figure 6. Only
the sinapyl alcohol moiety of 1c participated in the coupling reactions, and the
p-coumarate moiety remained unchanged even in the presence of excess Ag2O. These
results obtained are reasonably explained by the presence of electron-donating methoxy
groups in the sinapyl alcohol moiety, which promotes the oxidation reaction of phenols.
Also it is reported that in enzymatic oxidation of a mixture of methyl p-coumarate and
sinapyl alcohol by using isolated corn wall peroxidase, methyl p-coumarate was readily
oxidized by the peroxidase, and the produced p-coumarate radical transferred to sinapyl
alcohol. Therefore, only sinapyl alcohol underwent a radical coupling reaction.21 These
results suggest that p-coumaric acid is esterified in the acylated lignins, but is not
incorporated into lignin backbone by radical coupling mechanism as has been
established previously.12
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The -p-coumaroylated tetralin type - dimer 4c was also isolated from the
reaction mixtures from sinapyl p-coumarate 1c, which also indicates that the
p-coumarate moiety in 1c remained unchanged. The yield of 4c from sinapyl
1
p-coumarate 1c was 16%. The relative configuration of 4c was assigned by H and
NOESY NMR spectra. The coupling constant between AH and AH is 9.5 Hz, which
indicates that AH and AH is in the trans configuration. The correlation between
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AH and BH was observed by NOESY spectra, but not between AH and BH This
indicates the trans-trans-trans configuration of 4c.
The oxidative coupling of coniferyl alcohol 1d by Ag2O was reported in the
literature.16 The main reaction product was the -5 dimer 5d with 50% yield in CH2Cl2,
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whereas in CH2Cl2/H2O (1:1, v/v), the - dimer 4d was formed preferentially with
25% yield. In contrast, in 1,4-dioxane, we obtained the -O-4 dimer 2d (70% yield) and
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the -5 dimer 5d (14% yield) on the occasion of using freshly prepared and completely
dried Ag2O. These results play major roles in suggesting that the reaction solvent and
water content in the reaction system affect the reaction products obtained from the
oxidation of coniferyl alcohol 1d by Ag2O.
Oxidative coupling of coniferyl acetate 1e by Ag2O has not previously been
reported. Oxidation of 1e in 1,4-dioxane produced the -acetylated -O-4 dimer 2e
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(46% yield), the -acetylated -O-4 dimer 3e (21% yield), and the -acetylated -
dimer 5e (7.6% yield). The -acetylated dimer 3e was produced by the acetyl migration
of the -acetylated -O-4 dimer 2e as shown in Figure 3. The - coupling products
were not obtained at all. The formation of the -acetylated - dimer 5e from 1e
suggests the presence of such structures in the acylated native lignin from herbaceous
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plants.
Effect of Acyl Groups on the Oxidative Coupling of Monolignols.
The effect of the -acyl groups on the oxidative coupling of monolignols was
evaluated by two methods. One such method is the isolated dimer yields from each
monolignol as shown in Table 3. Another is the relative signal intensities of the interunit
linkages in the entire reaction products identified on each monolignol by using H–13C
1
HSQC NMR spectra as shown in Figure 4 and Table 4.
The total -O-4 dimer yields from the monolignols were calculated as the sum of
the yields of compounds 2 and 3, which includes syringyl glycerol -sinap aldehyde
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ether 3a and the -acetylated -O-4 dimers 3b and 3e. The total -O-4 dimer yield from
sinapyl acetate 1b was 80%. This was higher than what was obtained from sinapyl
alcohol (74% yield). The 1H–13C HSQC NMR spectra showed that the relative intensity
of the -O-4 structure in the entire reaction products from sinapyl acetate 1b was higher
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than that from sinapyl alcohol 1a. The frequency of the - structure from 1b was lower
than that from 1a. The current results are in good agreement with our previous results in
which the -O-4 structures were preferentially formed from sinapyl acetate 1b.
Therefore, it can be stated that the -acetyl group affected the oxidative coupling of
sinapyl alcohol 1a. Furthermore, the acetyl group increased the frequency of the -O-4
structures and decreased the - structures.13
In contrast to sinapyl acetate 1b, the total -O-4 dimer yield from sinapyl
p-coumarate 1c was lower than that from sinapyl alcohol 1a. The tetralin - dimer 4c
was obtained in significant yield. HSQC NMR analysis also indicated that the relative
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intensity of the -O-4 structure in the entire reaction products from 1c was significantly
lower than that from 1a. Whereas the relative intensity of the - structure obtained
from 1c was much higher than that from sinapyl acetate 1b. These results showed that
the -p-coumarate group decreased the frequency of the -O-4 structures and increased
the frequency of the - structures.
We note that the total -O-4 dimer yield from coniferyl alcohol 1d (70 % yield)
was similar to that from coniferyl acetate 1e (67% yield), even though the isolated -5
dimer yield from 1d was higher than that from 1e. The HSQC NMR analysis indicated
that the relative frequency of the -O-4 structure in the reaction products from coniferyl
alcohol 1d was similar to that from coniferyl acetate 1e. The relative frequency of the
- structure in the reaction products from coniferyl alcohol 1d was also similar to that
from coniferyl acetate 1e. Thus, the -acetyl group did not have a significant effect on
the oxidative coupling of coniferyl alcohol 1d.
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In conclusion, all the -acylated monolignols used in this study coupled with each
other to produce the -acylated -O-4 dimers, which suggests that the -acylated
monolignols act as lignin monomers and polymerize to produce the acylated lignin, as
has been established previously.12 The relatively high yields of the -O-4 dimers
indicate that the oxidation of the monolignols with Ag2O can be used as one easy
method used for synthesizing the -O-4 dimer lignin model compounds. The oxidative
coupling of sinapyl p-coumarate 1c yielded the -acylated -O-4 dimer. Only the
sinapyl alcohol moiety in sinapyl p-coumarate coupled with each other and the
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p-coumarate moiety remained unchanged even in the presence of excess Ag2O, which
suggests that p-coumaric acid is not incorporated into lignin backbone in the acylated
lignins by the radical coupling mechanism. Also, the tetralin - structures were formed
by the oxidative coupling of the -acylated sinapyl alcohols, including sinapyl acetate
1b, and sinapyl p-coumarate 1c under the acidic reaction conditions. The acyl migration
products, including the -acetylated -O-4 dimers 3b and 3e, were formed only from
the -acetylated monolignols. The -acetylated -5 dimer was also produced from
coniferyl acetate 1e. The -acyl groups in monolignols affect the oxidative coupling of
monolignol, but the effects vary depending on the type of the acyl groups and the
monolignols. These results provided some meaningful new insight into the effects of the
-acyl groups on the monolignol polymerization and the acylated lignin structures.
ABBREVIATIONS USED
HSQC, heteronuclear single-quantum coherence; HMBC, heteronuclear multiple-bond
coherence; NMR, nuclear magnetic resonance; NOESY, nuclear Overhauser effect
spectroscopy; THF, tetrahydrofuran.
AUTHOR INFORMATION
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Corresponding Author
*Phone:
+81-766-56-7500
(ext.
567).
Fax:
+81-766-56-2498.
E-mail:
takao@pu-toyama.ac.jp
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Funding
A part of this work was supported by JSPS KAKENHI Grant Number 15K14777.
Supporting information
1H NMR and C NMR data for acylated monolignols and the reaction products from
13
oxidative coupling of monolignols and acylated monolignols.
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REFERENCES
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polymers. Org. Biomol. Chem. 2005, 3, 1067−1073.
(3) Kishimoto, T.; Uraki, Y.; Ubukata, M. Chemical synthesis of β-O-4 type artificial
lignin. Org. Biomol. Chem. 2006, 4, 1343−1347.
(4) Kishimoto, T.; Uraki, Y.; Ubukata, M. Synthesis of β-O-4 type artificial lignin
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Influence of syringyl to guaiacyl ratio on the structure of natural and synthetic lignins.
J. Agric. Food Chem. 2010, 58, 895−901.
(6) Ralph, J. An unusual lignin from Kenaf. J. Nat. Prod. 1996, 59, 341−342.
(7) del Río, J. C.; Rencoret, J.; Marques, G.; Gutiérrez, A.; Ibarra, D.;Santos, J. I.;
Jiménez-Barbero, J.; Zhang, L.; Martínez, Á. T. Highly acylated (acetylated and/or
p-coumaroylated) native lignins from diverse herbaceous plants. J. Agric. Food Chem.
2008, 56, 9525−9534.
(8) Martínez, Á. T.; Rencoret, J.; Marques, G.; Gutiérrez, A.; Ibarra, D.;
Jiménez-Barbero, J.; del Río, J. C. Monolignol acylation and lignin structure in some
nonwoody plants: A 2D NMR study. Phytochemistry 2008, 69, 2831–2843.
(9) Villaverde, J. J.; Li, J.; Ek, M.; Ligero, P.; de Vega, A. Native lignin structure of
miscanthus × giganteus and its changes during acetic and formic acid fractionation. J.
Agric. Food Chem. 2009, 57, 6262−6270.
(10) del Rio, J. C.; Lino, A. G.; Colodette, J. L.; Lima, C. F.; Gutierrez, A.; Martinez, A.
T.; Lu, F.; Ralph, J.; Rencoret, J. Differences in the chemical structure of the lignins
from sugarcane bagasse and straw. Biomass and Bioenergy 2015, 81, 322–338.
(11) Karlen, S. D.; Free, H. C. A.; Padmakshan, D.; Smith, B. G.; Ralph, J.; Harrisc, P.
J. Commelinid monocotyledon lignins are acylated by p-coumarate. Plant Physiol.
2018, 177, 513–521.
(12) Ralph, J. Hydroxycinnamates in lignification, Phytochem. Rev. 2010, 9(1), 65–83.
(13) Kishimoto, T.; Takahashi, N.; Hamada, M.; Nakajima, N. Biomimetic oxidative
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coupling of sinapyl acetate by silver oxide: Preferential formation of β-O-4 type
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Model
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(15) Quideau, S.; Ralph, J. Facile large-scale synthesis of coniferyl, sinapyl, and
p-coumaryl alcohol. J. Agric. Food Chem. 1992, 40, 1108–1110.
(16) Zhu, Y.; Regner, M.; Lu, F.; Kim, H.; Mohammadi, A.; Pearson, T. J.; Ralph, J.
Preparation of monolignol γ-acetate, γ-p-hydroxycinnamate, and γ-p-hydroxybenzoate
conjugates: Selective deacylation of phenolic acetates with hydrazine acetate, RSC Adv.
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(18) Lu, F.; Ralph, J.; Morreel, K.; Messensc, E; Boerjanc W. Preparation and relevance
of a cross-coupling product between sinapyl alcohol and sinapyl p-hydroxybenzoate.
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reactions involving sinapyl acetate in kenaf lignins. Org. Biomol. Chem. 2008, 6,
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into lignin enhances the alkaline delignification and enzymatic degradation of cell walls.
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as a radical transfer mechanism in grass lignin formation. Planta 2008, 228, 919–928.
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Table 1. 1H NMR data for the oxidative coupling products in acetone-d6.
Cmpd A/B



2
5
-
-
-
-
-
-
-
-
-
-
-
-
6
OMe
3.79
3.87
3.78
3.89
3.79
3.88
3.77
3.86
3.81
3.88
3.77
3.87
OAc
2a e
2a t
2b e
2b t
2c e
2c t
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
4.98 (4.3)
6.54 (15.9)
4.96 (7.0)
6.51 (15.9)
4.92 (3.3)
6.64 (15.8)
4.90 (7.1)
6.63 (15.8)
4.99 (3.5)
6.69 (15.9)
4.99 (6.9)
6.68 (15.8)
4.88 (5.3)
6.49 (15.9)
4.88 (5.6)
6.50 (15.9)
4.90 (5.1)
6.60 (15.8)
4.89 (5.7)
6.61 (15.9)
5.00 (4.5)
7.62 (15.9)
6.11 (5.1)
6.64 (15.8)
6.16 (8.0)
6.65 (15.8)
5.96 (4.9)
6.61 (15.8)
6.02 (7.2)
6.61 (15.9)
4.66 (3.3)
4.00 (9.5)
4.89
4.18
3.84-3.87, 3.45 (3.0, 11.9)
6.71
6.79
6.71
6.79
6.74
6.79
6.68
6.83
6.71
6.82
6.75
6.86
6.77
6.86
-
6.37 (5.2, 15.9) 4.21 (5.2)
3.97
6.36 (5.0, 15.9) 4.19 (5.0)
4.52 4.37 (7.4, 11.8), 4.10 (2.8, 11.8) 6.68
6.34 (6.3, 15.8) 4.68 (6.3) 6.83
4.24 4.30 (2.9, 11.9), 3.90 (4.3, 11.9) 6.71
6.33 (6.3, 15.8) 4.67 (6.3) 6.82
4.61 4.47 (7.6, 11.8), 4.25 (3.2, 11.8) 6.75
6.40 (6.3, 15.9) 4.82 (1.1, 6.3) 6.86
4.33 4.41 (3.4, 11.7), 4.06 (4.8, 11.7) 6.77
6.40 (6.2, 15.8) 4.81 (1.1, 6.2)
-
3.67 (3.6, 12.1), 3.31 (3.2, 12.1) 6.74
6.79
-
-
1.85
2.03
1.94
2.03
-
-
-
6.86
7.09
7.04
-
2d e
2d t
2e e
2e t
4.32
3.80-3.82, 3.70 (3.7, 11.8)
6.74 (8.1) 6.87 (8.1) 3.80*1
6.91 (8.3) 6.86 (8.3) 3.84*1
6.74 (8.3) 6.88 (8.3) 3.78*1
-
6.26 (5.3, 15.9) 4.17 (5.3)
-
4.27
6.27 (5.4, 15.9) 4.17 (5.4)
4.60-4.62 4.35 (6.7, 11.9), 4.29 (2.9, 11.9) 7.08
6.23 (6.4, 15.8) 4.65 (6.4) 7.09
4.57 4.23 (3.4, 11.9), 3.99 (6.5, 11.9) 7.08
6.25 (6.5, 15.9) 4.66 (6.5) 7.13
4.30 3.88 (6.8, 12.5), 3.49 (2.8, 12.5) 6.73
6.79 (7.7, 15.9) 9.68 (7.7)
3.70 (4.0, 11.9), 3.47 (5.6, 11.9) 7.07
-
7.07
7.06
6.87 (8.3) 3.87*1
-
6.75 (8.1) 6.87 (8.1) 3.79
6.95 (8.3) 6.91 (8.3) 3.83
6.76 (8.1) 6.89 (8.1) 3.79
7.04 (8.3) 6.94 (8.3) 3.87
1.88
2.01
1.92*1
2.02*1
-
3a e
3b e*2
3b t
3e e
3e t
-
-
-
-
-
-
6.73
7.15
3.81*1
3.95*1
3.82*1
3.89*1
3.82
7.15
-
4.42
3.81, 3.54
6.31-6.38
4.32
3.58 (12.2), 3.23 (12.2)
6.73
6.85
6.73
6.85
1.94
2.04
1.98*1
2.02*1
1.91*1
2.02*1
-
6.35 (6.3, 15.8) 4.69 (6.3)
4.65-4.66
6.24 (6.3, 15.8) 4.65 (6.3)
4.56
3.64 (2.9, 12.2), 3.46 (5.2, 12.2) 7.03*1 6.78 (8.1) 6.88 (8.1) 3.80*1
6.24 (6.4, 15.9) 4.65 (6.4)
7.11*1 7.06 (8.3) 6.94 (8.3) 3.85*1
3.89
3.71 (5.7, 11.6), 3.61 (4.5, 11.6) 7.10*1 6.76 (8.1) 6.90 (8,1) 3.82*1
7.13*1 6.99 (8.2) 6.92 (8.2) 3.83*1
4a
4c
3.09
2.55
2.23
4.21, 3.82
6.66
6.67
6.74
-
-
-
6.66
6.67
-
3.80
A
B
4.50, 4.29 (2.6, 11.8)
4.52, 4.42 (5.9, 11.2)
3.74
-
3.85 (C3)
3.15 (C5)
-
5d
5e
A
B
A
B
5.44 (6.2)
6.50 (15.8)
5.49 (7.2)
6.63 (15.8)
3.51
3.83-3.86, 3.74-3.78
6.99
6.93
6.78 (8.1) 6.84 (8.1) 3.78
6.96 3.83
6.82 (8.1) 6.85 (8.1) 3.80
7.03 3.85
-
6.22 (5.6, 15.8) 4.16 (5.6)
3.76
6.23 (6.5, 15.8) 4.65 (6.5)
-
-
4.41 (5.5, 11.0), 4.32, 7.5, 11.0) 7.02
7.03
2.02
1.98
-
410
411
412
413
414
415
*1 A/B was not determined.
*2
All signals were from the mixture of erythro and threo isomers. Some signals were
from HSQC NMR spectra.
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Table 2. 13C NMR data for the oxidative coupling products in acetone-d6.
Cmpd
A/B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B

73.6

88.1

1
2
3
4
5
6
OMe
56.7
OAc
CO
2a e
61.0
132.7
134.5
132.8
134.6
131.6
133.5
132.0
133.4
131.9
133.4
132.0
133.4
134.4
133.0
133.9
133.1
133.6
132.1
133.2
132.1
104.9
104.6
105.6
104.6
104.5
104.9
105.4
104.9
104.7
104.9
104.9
105.5
111.5
111.1
111.5
110.9
111.3
111.3
111.5
111.2
148.5 135.9
154.3 136.1
148.4 136.3
154.1 136.6
148.6 136.0
154.5 136.5
148.5 136.4
153.9 137.8
148.6 136.0
154.4 136.9
148.5 136.3
154.0 137.8
148.1 146.8
152.0 148.7
148.2 147.4
151.8 149.3
148.1 146.9
152.0 149.0
148.2 147.1
151.8 149.5
148.5 104.9
154.3 104.6
148.4 105.6
154.1 104.6
148.6 104.5
154.5 104.9
148.5 105.4
153.9 104.9
148.6 104.7
154.4 104.9
148.5 104.9
154.0 105.5
115.2 120.5
119.4 120.3
115.3 120.7
119.7 120.4
115.3 120.4
119.1 120.7
115.4 120.6
118.7 120.8
-
-
-
-
-
-
-
-
129.9 130.9 63.2
74.3 89.9 61.6
129.9 131.1 63.2
73.1 84.4 63.5
134.4 124.3 65.3
74.7 86.7 64.8
134.3 124.3 65.3
73.5 84.8 64.0
134.5 124.5 65.3
74.7 86.8 65.1
134.3 124.5 65.3
73.9 86.8 61.9
130.0 129.7 63.4
73.9 88.6 62.0
129.9 129.8 63.4
73.2 83.3 64.0
134.4 123.1 65.5
73.8 84.1 64.2
134.3 123.2 65.5
73.9 88.3 61.2
153.3 129.3 194.0 131.2*3 105.0*1 154.7 139.5*3 154.7 105.0*1 56.7*1
2a t
56.7
56.7
2b e
2b t
2c e
2c t
20.9*1 170.9
20.8*1
20.9*1 170.9*1
20.8*1 170.8*1
-
-
-
-
-
-
-
-
20.9*1 171.0
20.8*1 170.9
20.9*1 170.9
20.7*1 170.9
-
-
56.7*1
56.6*1
56.7*1
56.6*1
56.6
56.6
56.4
56.3
56.3
167.3
167.4
167.3
167.5
-
-
-
-
2d e
2d t
2e e
2e t
56.4
56.4*1
56.3*1
56.4*1
56.3*1
3a e
3b e*2
3b t
3e e*4
3e t
132.8*3 107.2*1 148.5 136.0*3 148.5 107.2*1 56.8*1
-
-
75.2
85.3
60.7
56.5*1
56.4*1
56.7*1
56.6*1
56.4
76.9
85.2
61.4
133.1*3 105.8
128.9*3 104.9
148.6 136.7*3 148.6 105.8
154.0 137.2*3 154.0 104.9
121.9
21.1*1 171.4*1
20.9*1 170.7*1
21.1*1 170.0
20.9*1
21.0*1 171.4*1
20.9*1 170.6*1
-
-
-
134.3 124.0 65.4
75.2 83.8 61.4
134.4 123.1 65.5
75.9 84.1 61.4
134.3 122.8 65.6
86.9
44.8
69.3
131.9
129.4
131.5
129.5
133.3
139.5
131.0
112.5*1
111.3*1
112.1*1 148.4 147.5
111.1*1 151.3 149.6
104.5
108.1
108.0
118.6 120.7
115.7 121.1
117.9 120.7
148.8 104.5
148.4 108.1
146.8 127.1
56.3
56.3
56.7
56.9
56.5(C3)
58.8(C5)
56.5
56.4
56.5*1
56.4*1
4a
4c
55.4
41.2
41.3
72.5
63.4
65.9
148.8 136.3
148.4 135.6
147.9 140.5
-
A
B
167.8*1
167.7*1
5d
5e
A
B
A
B
88.6
54.9
64.7
134.5
132.0
133.4
131.5
110.6
111.8
110.6
112.2
148.5 147.4
145.3 149.0
148.5 147.7
145.5 149.5
115.8 119.7
130.5 116.2
115.8 120.0
129.3 116.4
-
-
-
-
130.6 128.4 63.5
89.1 51.2 66.0
134.9 122.2 65.6
20.9*1 170.9
20.8*1 171.0
418
419
420
421
422
423
424
*1 A/B was not determined.
*2
All signals were from the mixture of erythro and threo isomers. Some signals were
from HSQC NMR spectra.
*3 Tentatively assigned.
*4 Only the main signals were determined because of low signal intensity.
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Page 22 of 31
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Table 3. Isolated yields of reaction products from the oxidative coupling of monolignols
and acylated monolignols by Ag2O.
-

-O-4
-5
Recovered
Monomer monomer
(%)
Oligomer
(wt%)
(%)
(%)
2
3
Total
4
5
1a (S)
5.6 ± 2.6
3.5
±
±
70 ± 0.4
74 ± 0.2 5.3 ± 0.3
80 ± 3.3
10 ± 5.4
0.6
9.5
1.7
1b (Sac)
5.2 ± 0.4
71 ± 3.3
12 ± 2.6
2.4 ± 4.5
1c (Spc)
1d (G)
8.0 ± 0.7
0.6 ± 0.9
3.0 ± 1.0
53 ± 4.8
70 ± 7.7
53 ± 4.8 16 ± 3.6
70 ± 7.7
14 ± 4.5 17 ± 2.5
7.6± 5.3 28 ± 2.7
1e (Gac)
46 ± 4.5 21 ± 2.4 67 ± 6.9
429
430
431
432
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Journal of Agricultural and Food Chemistry
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Table. 4 Relative intensities of interunit linkages from HSQC NMR spectra of the entire
reaction products obtained by the oxidative coupling of monolignols by Ag2O. The
reaction products contain dimers, oligomers, and unreacted monomers.
Monomer
1a (S)
-O-4 (%)
93 ± 0.6
98 ± 0.3
80 ± 3.4
75 ± 4.3
75 ± 3.7
- (%)
6.8 ± 0.6
1.7 ± 0.3
20 ± 3.4
1.6 ± 0.1
-5 (%)
1b (Sac)
1c (Spc)
1d (G)
23 ± 4.4
26 ± 3.7
1e (Gac)
439
440
441
442
443
444
445
446
447
448
449
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Page 24 of 31
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469
OR2



1) Ag2O/1,4-dioxane, rt
Oxidative coupling
products
1
6
2) conc. HCl/THF/H2O, rt
2
5
R1
OMe
3
4
OH
1
2
1a
1b
1c
1d
1e
: R = OMe, R = H (S)
1
2
: R = OMe, R = COCH3 (Sac)
1
2
: R = OMe, R = COCHCHC6H4OH (Spc)
1
2
: R = H, R = H (G)
1
2
: R = H, R = COCH3 (Gac)
Figure 1. Oxidative coupling of acylated monolignols by Ag2O
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Journal of Agricultural and Food Chemistry
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477
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483
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504






R2O
R1
O
HO MeO
HO
R1
O
OR2
OAc


O




B
B
B
HO
HO
AcO



O
4
4

4


OMe
OMe
OMe
A
A
A
R1
OMe
MeO
OMe
R1
OMe
OH
OH
OH
1
2
1
3a
2a
2b
2c
2d
2e
3b
3e
: R = OMe, R = H
: R = OMe
1
2
1
: R = OMe, R = COCH3
: R = H
1
2
: R = OMe, R = COCHCHC6H4OH
1
2
: R = H, R = H
1
2
: R = H, R = COCH3

OR2
OH

OMe
OH





OR2
MeO
HO
O
OR2
OR2
B
O




5
B



R1
R1
H
H
4
OMe

OMe
O



A
A
O
HO
MeO
OMe
MeO
OMe
HO
MeO
OH
OMe
OH
1
2
2
6a
4a
4d
4b
4c
5d
: R = OMe
: R = COCH3
: R = H
1
2
2
5e
: R = H
: R = COCHCHC6H4OH
: R = COCH3
Figure 2. Oxidative coupling products from acylated monolignols using Ag2O.
Stereochemistry is a relative configuration.
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O
O
A
R1
O
OAc
B
HO
OMe
3b, 3e
R1
OMe
OH
1
2b
2e
: R = OMe
1
: R = H
Figure 3. Acetyl migration of -acetylated -O-4 dimers 2b and 2e.
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Journal of Agricultural and Food Chemistry
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525
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528
529
530
531
532
533
534
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539
540
541
542
543
544
545
546
547
548
549
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551
552
553
554
555
556
557
558
Figure 4. HSQC NMR spectra of the entire reaction products from oxidative coupling of
A) sinapyl acetate 1b, B) sinapyl p-coumarate 1c, and C) coniferyl acetate 1d by Ag2O
in acetone-d6.
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Page 28 of 31
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H
H
O
OH
OR
MeO
O
MeO
HO
OR
OR
OR
OR
1. Ag2O
2. aq HCl
OMe
MeO
Quinone methide
MeO
MeO
MeO
OMe
OMe
OMe
OH
O
O
H
1b, 1c
OH
OR
RO
MeO
HO
OR
OR
OMe
OH
MeO
HO
O
OMe
MeO
OMe
OMe
OMe
OH
Tetrahydrofuran -
4b, 4c
581
582
583
584
585
586
587
588
589
590
591
592
593
Figure 5. Formation of tetralin type - dimers 4b and 4c from oxidative coupling of
the -acylated sinapyl alcohols by Ag2O. Stereochemistry is a relative configuration.
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Journal of Agricultural and Food Chemistry
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HO
O
O
'
O MeO

O
'
'
B
HO

O
OH
4'
4

OMe
A
MeO
OMe
OH
2c
612
613
614
615
616
617
618
619
620
621
622
623
624
625
626
627
628
629
Figure 6. The -p-coumaroylated -O-4 dimer 2c from sinapyl p-coumarate 1c
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