Profiling of the formation of lignin-derived monomers and dimers from: Eucalyptus alkali lignin

Article abstract of DOI:10.1039/d0gc01658f

Lignin is a renewable and the most abundant aromatic source that can be used for extensive chemicals and materials. Although approximately 50 million tons of lignin are produced annually as a by-product of the pulp and paper industry, it is currently underutilized. It is important to know the structural features of technical lignin when considering its application. In this work, we have demonstrated the formation of low-molecular-weight constituents from hardwood (Eucalyptus) lignin, which produces much more low-molecular-weight constituents than softwood (spruce) lignin, after a chemical pulping process, and analyzed the micromolecular compositions in the alkali lignin after fractionation by dichloromethane (DCM) extraction. By applying analytical methods (gel-permeation chromatography, 2D NMR and GC-MS) with the aid of evidence from authenticated compounds, a great treasure trove of lignin-derived phenolic compounds from Eucalyptus alkali lignin were disclosed. Except for some common monomeric products, as many as 15 new lignin-derived monomers and dimers including syringaglycerol, diarylmethane, 1,2-diarylethanes, 1,2-diarylethenes, (arylvinyl ether)-linked arylglycerol dimers and isomeric syringaresinols were identified in the DCM-soluble fraction. Regarding the formation and evolution of the Cα-condensed β-aryl ether structure, a novel route that is potentially responsible for the high content of β-1 diarylethenes and diarylethanes in the lignin low-molecular-weight fraction, in addition to the β-1 (spirodienone) pathway, was proposed. This work not only provides novel insights into the chemical transformation of S-G lignin during the alkali pulping process, but also discovered lignin-derived phenolic monomers and dimers that can potentially be used as raw materials in the chemical or pharmaceutical industries. This journal is

Full text of DOI:10.1039/d0gc01658f

View Article Online
View Journal
Green
Chemistry
Cutting-edge research for a greener sustainable future
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: C. Zhao, Z. Hu, L.
Shi, C. Wang, F. Yue, S. Li, H. Zhang and F. Lu, Green Chem., 2020, DOI: 10.1039/D0GC01658F.
Volume 20
This is an Accepted Manuscript, which has been through the
Number
May 2018
Pages 1919-2160
9
7
Royal Society of Chemistry peer review process and has been
accepted for publication.
Green
Chemistry
Cutting-edge research for a greener sustainable future
rsc.li/greenchem
Accepted Manuscripts are published online shortly after acceptance,
before technical editing, formatting and proof reading. Using this free
service, authors can make their results available to the community, in
citable form, before we publish the edited article. We will replace this
Accepted Manuscript with the edited and formatted Advance Article as
soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes to the
text and/or graphics, which may alter content. The journal’s standard
Terms & Conditions and the Ethical guidelines still apply. In no event
shall the Royal Society of Chemistry be held responsible for any errors
or omissions in this Accepted Manuscript or any consequences arising
from the use of any information it contains.
ISSN 1463-9262
PAPER
Paul T. Anastas et al.
The Green ChemisTREE: 20 years after taking root with the 12
principles
rsc.li/greenchem

Page 1 of 20
Green Chemistry
View Article Online
DOI: 10.1039/D0GC01658F
Profiling of the Formation of Lignin-derived Monomers and Dimers from Eucalyptus Alkali
Lignin
Chengke Zhao,ab Zhenhua Hu,a Lanlan Shi,a Chen Wang,a Fengxia Yue,*a Suxiang Li,a Han Zhang,a
and Fachuang Lu*a
a
State Key Laboratory of Pulp and Paper Engineering, South China University of Technology,
Guangzhou 510640, China
b Flexible Printed Electronic Technology Center, School of Materials Science and Engineering, Harbin
Institute of Technology (Shenzhen), Shenzhen 518055, China
*Corresponding authors:
Fengxia Yue (yuefx@scut.edu.cn). State Key Laboratory of Pulp and Paper Engineering, South
China University of Technology, Guangzhou 510640, China.
Fachuang Lu (fefclv@scut.edu.cn). State Key Laboratory of Pulp and Paper Engineering, South
China University of Technology, Guangzhou 510640, China.
1/19

Green Chemistry
Page 2 of 20
View Article Online
DOI: 10.1039/D0GC01658F
Abstract
Lignin is renewable and the most abundant aromatic sources that can be used for extensive
chemicals and materials. Although approximately 50 million tons of lignin are produced annually as a
by-product of the pulp and paper industry, it is currently underutilized. It is important to know the
structural features of technical lignin when considering its application. In this work, we have
demonstrated the formation of low-molecular-weight constituents from hardwood (Eucalyptus) lignin,
which produces much more low-molecular constituents than softwood (spruce) lignin, after a chemical
pulping process, and analyzed micromolecular compositions in the alkali lignin after fractionation by
dichloromethane (DCM) extraction. By applying analytical methods (gel-permeation chromatography,
2D NMR and GC-MS) with the aid of evidence from authenticated compounds, a great treasure trove of
lignin-derived phenolic compounds from Eucalyptus alkali lignin were disclosed. Except for some
common monomeric products, as many as 15 new lignin-derived monomers and dimers including
syringaglycerol, diarylmethane, 1,2-diarylethanes, 1,2-diarylethenes, (arylvinyl ether)-linked
arylglycerol dimers and isomeric syringaresinols were identified in the DCM-soluble fraction. Regarding
to the formation and evolution of Cα-condensed β-aryl ether structure, a novel route that is potentially
responsible for the high content of β-1 diarylethenes and diarylethanes in lignin low-molecular fraction,
in addition to the β-1 (spirodienone) pathway, was proposed. This work not only provides novel insights
into the chemical transformation of S-G lignin during alkali pulping process, but also discovered lignin-
derived phenolic monomers and dimers that potentially can be used as raw materials in chemical or
pharmaceutical industries.
Keywords: Structural characterization; Model compound; Depolymerization; Condensation; 2D HSQC
NMR; Phenolic compounds.
2/19

Page 3 of 20
Green Chemistry
View Article Online
DOI: 10.1039/D0GC01658F
1. Introduction
Lignin is the largest renewable aromatic source that has great potential to produce extensive
chemicals, biofuels, functional materials and bioactive molecules.1-5 It principally occurs in natural plants
in the form of linear biomacromolecules with the main building blocks of p-hydroxyphenyl (H), guaiacyl
(G) and syringyl (S) units, and the chemical linkages of C-O and C-C bonds.6-10 The block units of lignin
vary from wood species; for example, softwood lignin consists mostly of G unit and very low levels of
H unit, whereas hardwood lignin is primarily composed of S and G units with traces of H unit.6 Among
the interunit linkages, β-aryl ether bond (β-O-4) is most abundant and is relatively labile, providing a
basis for chemical and industrial processes to convert lignin to low-molecular-weight value-added
chemicals.4,11,12
As for pulp and paper production, chemical pulping targets the manufacture of high-quality
cellulosic fiber during chemical pulping processes, in which lignin is abundantly produced as a byproduct
(approximately 50 million tons every year).13 During these processes, the harsh conditions applied result
in a massive and intricate structural alteration of lignin, from depolymerization, condensation, and redox
reactions.14,15 The majority of native ether bonds are destroyed, producing low-molecular-weight
aromatic fragments as well as large polymers with various new unknown moieties. Degradation and
condensation reactions during the pulping processes make the technical lignins even more
inhomogeneous and structure-complicated; consequently, technical lignin valorization becomes more
challenging.16-18
Many efforts, including employing various model compounds aided with advanced analytical
techniques, have been directed toward unraveling the structural characteristics of technical lignins. These
efforts have elucidated some of key pathways associated with the chemical evolution of lignin native
structures (β-O-4 and β-5 and β-β) during pulping process,14,19,20 and provided many significant
perspectives on the structural features of technical lignins, especially on the identification of various new
chemical structures (styryl ethers, stilbenes, diarylmethanes, biaryl, diaresinol, arylglycerol, arylpropanol,
reduced aryl ether, and condensed lactone etc.).15,16,21-24 Such findings are very helpful for lignin post-
processing and valorization strategies. However, efficient valorization of lignin remains the most
challenging part of biomass utilization. Thus, a deeper fundamental understanding of the characteristics
of technical lignins, in particular with regard to the degradation products of lignin during pulping process,
is of crucial importance.
3/19

Green Chemistry
Page 4 of 20
View Article Online
Nowadays, lignin fractionation by using organic solvents prevails at the research stage.10,15,25-28 Such
a strategy can fractionate the complicated lignin mixtures into relatively homogeneous fractions (in terms
of molecular weight and structural functionality),29 and have realized some proof-of-concept applications
in polymer materials, pyrolytic oils and nano particles.30-32 These works have provided some information
on molecular weight, interunit linkages, end-groups, and the content of functional groups of different
fractions. However, very limited research, only one literature reported so far, has touched on the small-
molecular fraction (molecular weight <600 g/mol) from hardwood technical lignin.27 Based on our
knowledge of lignin structural studies, such fraction is presumably consists of lignin-derived monomeric
and dimeric products, although the authors haven’t mentioned that. If so, it will be of great importance
for lignin valorization on account of their potential uses as functional chemicals or valuable precursors
for the production of pharmaceuticals.
DOI: 10.1039/D0GC01658F
In this study, as a continuation of our alkali lignin structural studies, and to address the issues
presented, we fractionated low-molecular-weight (low-Mw) material from Eucalyptus alkali lignin by an
optimized organic solvent (dichloromethane, DCM) extraction, and then analyzed the lignin-derived
monomers and dimers in this fraction via analytical techniques (detailed methods can be seen in
Supplementary Information). In addition, several model compounds were designed and synthesized, and
then used these model compounds as substrates to conduct pulping studies. The degradation products
were then analyzed and identified to reveal the formation of such products during the pulping process.
2. Results and discussion
2.1 Structural fundamental for the formation of low-Mw fraction from hardwood lignin during
chemical pulping processes
Lignin subunit composition and structure in plants varies from species. In general, hardwood lignins
are composed of S and G units, whereas lignin in softwoods are made up of G units and small amounts
of H units. Hardwood and softwood lignins are therefore highly different in their subunit compositions,
thereby the interunit linkages and their consequent reactivities. In consideration of the valorization of
technical lignin, it is of great importance to understand the variation in the nature of such lignins that
originate from their inherent natural variation in lignin subunit composition and structure, as well as the
production process. In this work, four different organic solvents (ethanol, acetone, ethyl acetate, and
DCM) were employed to optimize the extraction process for low-Mw constituents from Eucalyptus alkali
4/19

Page 5 of 20
Green Chemistry
View Article Online
DOI: 10.1039/D0GC01658F
lignin. By comparation, DCM showed a better selectivity for the extraction of low-Mw constituents
(shown in Table 1). Thus, DCM was used for fractionation of low-Mw constituents in the following study.
Interestingly, it was found that the Eucalyptus technical lignins contain a much higher content of DCM-
soluble low-Mw fractions than the spruce (softwood) technical lignins (28.2% vs. 1.8% for alkali lignins
and 43.7% vs. 2.4% for kraft lignins) (shown in Table 2). This difference can be interpreted by a different
reactivity caused by the structural difference of hardwood and softwood lignins during chemical pulping
process (Scheme 1).
Table 1 Content (wt.%) and molecular weights of the soluble fractions of Eucalyptus alkali lignin
isolated by using various organic solvents.
Molecular weight
Solvents
Content
Mw
1821
1889
997
Mn
951
927
666
569
PDI
1.91
2.04
1.50
1.37
Ethanol
Acetone
74.8%
66.0%
43.5%
28.2%
Ethyl acetate
Dichloromethane
780
Table 2 Content (wt.%) of DCM-soluble low-Mw fractions from alkali and kraft lignins.
Feedstock
Spruce
Alkali lignin
1.8%
Kraft lignin
2.4%
Eucalyptus
28.2%
43.7%
Despite of the much higher content of β-ether in hardwood lignins, two more reasons could be drawn
forth (Scheme 1). First, the hardwood lignin is more easily depolymerized to produce more lignin-derived
fragments in comparison to softwood lignin, due to the fact that β-aryl ether bonds formed by S units are
easier to be cleaved than those composed of G units;33,34 This is supported by the result from density
functional theory (DFT) calculation that the bond dissociation energy (BDE) of (S-S)-type β-aryl ether
bond is lower than that of (G-G)-type β-aryl ether bond.35,36 On the other hand, S-type fragments are
relatively stable as the C5 positions are substituted by methoxy groups, whereas the G-type fragments
can be consumed by condensation reactions to form recalcitrant C-C linkages (5-5′, 5-α′ and
5/19

Green Chemistry
Page 6 of 20
View Article Online
diarylmethanes) under alkaline pulping conditions.15,16,37 Hence, under the same pulping processes,
technical lignins from hardwood should contain more low-Mw lignin-derived degradation products than
those from softwood lignins.
DOI: 10.1039/D0GC01658F
Scheme 1 Schematic illustration for the different yields of the low-Mw aromatics from softwood and
hardwood lignins after alkaline pulping process. Bond dissociation energies (BDEs) of β-O-4 units
determined by density functional theory (DFT) were reported in the literature.36
2.2 Structural features of low-Mw fractions of Eucalyptus alkali lignin
Molecular weight and molecular weight distribution. The gel-permeation chromatography (GPC)
molecular weight distributions of Eucalyptus alkali and kraft lignins DCM-soluble/insoluble fractions
are shown in Figure 1. Nine reference polystyrene samples with weight-average molecular weight in the
range of 208-49000 g/mol were used for the molecular weight calibration of fractional lignins (Figure
1a). The Mw of DCM-soluble fractions extracted from alkali and kraft lignin were 780 g/mol and 694
g/mol, respectively. In contrast, the Mw of DCM-insoluble fractions were much higher, which were 5331
g/mol for alkali lignin and 1959 g/mol for kraft lignin, respectively. In the chromatogram of the DCM-
soluble fraction from alkali lignin, two individual peaks at the retention time of 16.1 min and 16.6 min
can be observed (Figure 1b) and the corresponding Mw values of these two peaks were 487 g/mol and
315 g/mol, indicating that the DCM-soluble fraction of alkali lignin is rich in monomeric and dimeric
lignin compounds.38 In addition, similar results are observed for the DCM-soluble fraction of kraft lignin,
consistent with the above-mentioned mechanism (Scheme1).
For the DCM-soluble fraction of Eucalyptus kraft lignin, in spite of the higher content, it is normally
is highly sulfurized and its composition is difficult to authenticate by currently available techniques.2,39
Hence, the following work is focused on understanding the lignin small molecules from Eucalyptus alkali
lignin, which is sulfur free and structurally much closer to the degradation products of native lignins.
6/19

Page 7 of 20
Green Chemistry
View Article Online
DOI: 10.1039/D0GC01658F
Figure 1 Molecular weight analysis of DCM-soluble/insoluble fractions from Eucalyptus alkali and
kraft lignins. (a) The standard curve of polystyrenes for molecular weight calibration of lignin
fractions; (b) the molecular weight distribution of DCM soluble/insoluble fractions.
Chemical linkages and end-groups. Cellulolytic enzyme lignin (CEL) was isolated from
Eucalyptus and then analyzed by NMR to represent the native lignin of Eucalypts (Figure S1). The
relative abundance of the substructures were calculated by integrating the contour volumes of the
corresponding correlations in the HSQC spectra, and the percentages of each linkage were β-O-4 (A, β-
aryl ether, 59.6%), β-5 (B, phenylcoumaran, 3.8%), β-β (C, resinol, 10.5%), and β-1 (D, spirodienone,
3.5%) linkages. In addition, low levels of cinnamyl alcohol (CA) and cinnamaldehyde (J) end-groups
were also found in the CEL spectra, at around 1%. The S/G ratio was 2.6. It is obvious that the structure
of the Eucalyptus lignin was significantly modified after the alkaline pulping process. Figure 2 shows
the 2D HSQC NMR spectra of the high-Mw fraction (DCM-insoluble) and low-Mw fraction (DCM-
soluble) from the alkali lignin. Apart from some remaining lignin substructures including β-O-4, resinol
and cinnamyl alcohol, many new structures were observed and identified according to 1H-13C cross peak
assignment (Table S2).16,21,23,24,40-42 These structures include isomeric epiresinols (C′) and diaresinol
(C′′), arylglycerol (Gly), enol ethers (E), β-1/5 stilbenes (SB, or β-1/5 diarylethenes), β-1 diarylethane
(DE), aryl propanol (P), and aldehyde and ketone end-groups. The semi-quantitative analysis of
characteristic correlations was performed based on volume integration to indicate the relative abundance
of structural units. Aromatic S2,6 and G2 peaks were chosen as the reference.
7/19

Green Chemistry
Page 8 of 20
View Article Online
DOI: 10.1039/D0GC01658F
Figure 2 2D HSQC NMR spectra of high-Mw (DCM-insoluble) and low-Mw (DCM-soluble) fractions
from Eucalyptus alkali lignin.
As shown in Figure 2, a very low level of native β-O-4 linkage was detected in high-Mw fraction
of alkali lignin that accounts for 4.7% of all aromatic groups, whereas it was absent in the low-Mw
fraction. Resinol structures are relatively stable in the alkaline media that can be observed in both high-
and low-Mw fractions, although some of them were transformed into epiresinols and diaresinols. Such
alkali-catalyzed epimerization occurs only when the associated phenolic groups are free.16,22 The epimers
are easily identified in the spectrum of low-Mw fraction, but hardly observed in the high-Mw fraction.
The total content of β-β-linked structures (resinol, epiresinol, and diaresinol) was 11.4% in low-Mw
8/19

Page 9 of 20
Green Chemistry
View Article Online
DOI: 10.1039/D0GC01658F
fraction, which is much higher than that of the high-Mw fraction (7.7%). The possible reason is that these
resinol-related units are easy to be released with the base-promoted depolymerization of hardwood lignin.
Gly and vinyl ether (or enol ether, E) structures come from the derivation of β-O-4 linkages through
different reaction pathways based on the etherified or free nature of associated phenolic groups.20,39 These
structures are significant, and prominently higher in alkali lignins than that of normal kraft lignins.23 In
line with the previous report,16 much more Gly presented in high-Mw fraction (14.8%) than that in low-
Mw fraction (5.4%). This is because the stability of this end-group highly depends on the etherified
phenolic nature whereas the high-Mw fraction contains more aryl ether bonds.24 Except for cis-trans
isomerism, vinyl ether linkages in hardwood alkali lignins have four different structural forms based on
different combinations of G and S units. Unfortunately, it is difficult to distinguish these correlations
from each other, the correlations of E(G-G) overlaps with the signals of E(S-G), and the correlations of
E(G-S) overlaps with the signals of E(S-S).24 Based on the analysis of the spectra, the content of vinyl
ether linkage was in a comparable value (~9%) in two fractions, while the content of (G/S-S)-linked vinyl
ethers was two times higher than that of (G/S-G)-linked vinyl ethers.
As condensed structures, β-1/5 diarylethenes (SB1 and SB5) and β-1 diarylethane (DE) were
identified in the low-Mw fraction, but are totally absent in the high-Mw fraction. SB5 is formed by the
degradation of phenylcoumaran,25 and its content is very low due to the low levels of phenylcoumaran
in hardwood native lignin. SB1 accounts for 3.6% of all aromatics in the low-Mw fraction, which can be
produced by β-1 unit (spirodienone) (Pathway 1 in Scheme 2).25 Normally, it is found to be more in the
highly-depolymerized lignin fraction, and is considered to be quite easily released from lignin polymer
during chemical processes, potentially due to the quinoid end-group in spirodienone that restricts the
chain branching.7,16 It is worth noting that a novel pathway differing from Pathway 1 might occur that
accounts for the formation of SB1 and DE structures. In this pathway, the (β-1)-condensed C-C linkages
can be generated from β-O-4 linkages after formation of Cα-condensed structure (T1) in alkali media
(Pathway 2 in Scheme 2). A part of this pathway was proved previously.19 This pathway is supported by
the reaction products of synthetized model compounds (simplified β-O-4 and Cα-condensed model
compounds) (Figure S2, S3 and S4). Although it is still difficult to distinguish the dominant pathway
guiding the formation of SB1 in hardwood alkali lignin, the pathway 2 can be an explanation for the high
content of this structure in some softwood kraft lignins (4-7%),25 because the content of spirodienone in
softwood lignin is quite low.43 β-1 diarylethanes (DEs), to the best of our knowledge, is first assigned in
9/19

Green Chemistry
Page 10 of 20
View Article Online
DOI: 10.1039/D0GC01658F
the alkali lignin fraction (the dimeric diarylethane products also are identified by GC-MS in follow-up
analysis). The methylene groups “-CH2-” in DEs with different aromatic substituent groups have inverse
cross signals (versus “=CH-” and “-CH3”) at the 13C-1H correlation of 39.8/2.74-2.29 ppm according to
the synthetized reference compounds in literatures (Figure 2).40,41 Similar with SB1, this structure mainly
distributes into the low-Mw fraction, accounting for about 2% of all aromatics. The presence of DEs
implies that the formal reduction of quinone methide or SB1 structure is possible in the complicated
alkaline pulping system.
Scheme 2 Two possible pathways for the formation of diarylethenes during alkali pulping process.
Pathway 1 was previously proposed;25 Pathway 2 is proposed based on the analysis of the products
from related model compounds. 1,1-diarylethene was identified in the products of simplified Cα-
condensed model compound (Figure S3 and S4), whereas it was not observed neither in the 2D HSQC
NMR spectra (according to the NMR data of its structural analogs44) of all lignin fractions nor by the
GC-MS analysis of the low-Mw lignin fraction (Section 2.4).
Aldehyde and ketone end-groups in alkali lignin are derived from alkali-catalyzed oxidation of
aromatic side-chains.2,25 These groups are primarily present in the low-Mw fraction that are related to the
vanillin (VA), syringaldehyde (SA), acetovanillone (AV) and acetosyringone (AS) identified in the
HSQC spectrum of low-Mw fraction.
In comparison to the Eucalyptus CEL, the low-Mw fraction has a very high S/G ratio (4.8), whereas
10/19

Page 11 of 20
Green Chemistry
View Article Online
DOI: 10.1039/D0GC01658F
the S/G ratio of high-Mw fraction (2.4) is slightly lower than that of CEL (2.6). This supports the point
that the S-incorporated lignin fragments are more facile to depolymerize compared to G-incorporated
lignin fragments during the chemical pulping process, as discussed above.
2.4 Identification of lignin-derived monomers and dimers in low-Mw fraction
The low-Mw fraction was subjected to acetylation to analyze the lignin monomers and dimers by
GC-MS. Figure 3 shows the primary aromatic monomers identified in the low-Mw fraction. These
monomers include guaiacol (G1), vanillin (G2), and acetovanillone (G3) constituted by G units, and
syringol (S1), syringaldehyde (S2), acetosyringone (S3), and syringylglycerol (S4) constituted by S units.
The S-type monomers had a significantly higher content than the G-type monomers. The total content of
identified S-type monomers was 19.85 mg/g DCM-soluble fraction, whereas the total content of
identified G-type monomers was 5.13 mg/g (Table S3). G1 and S1 can be produced by retro-aldol
reaction of hardwood lignin in alkali media.15 G2, G3, S2 and S3 are the common products from the
oxidative depolymerization of hardwood lignin.45-49 Vanillin has been reported to be isolated from kraft
lignin.50 S4, as a monomer with glycerol side-chain, was first identified in the hardwood alkali lignin
with the aid of a reference compound. However, in consideration of its instability at high temperature
under base conditions due to the presence of free-phenolic group, this compound was potentially
generated after the pulping process (see the discussion below).
Figure 3 Aromatic monomers identified in the low-Mw fraction from Eucalyptus alkali lignin by GC-
MS.
11/19

Green Chemistry
Page 12 of 20
View Article Online
DOI: 10.1039/D0GC01658F
Figure 4 Lignin dimers identified in low-Mw fraction of the Eucalyptus alkali lignin by GC-MS.
Scheme 3 Possible routes for the formation of lignin-derived dimers during alkaline pulping process.
12/19

Page 13 of 20
Green Chemistry
View Article Online
DOI: 10.1039/D0GC01658F
As shown in Figure 4, a total of fourteen lignin-derived dimers were identified in the low-Mw
fraction of Eucalyptus alkali lignin (MS information seen in Section 2.2 in supplementary material).
These compounds including biaryl compound (D1), 1,2-diarylethanes (D2 and D3), diarylmethane (D4),
diarylethenes (D5, D6 and D7), (arylvinyl ether)-linked arylglycerols (D8, D9, D10 and D11), and
isomeric syringaresinols (D12, D13 and D14). D1 is produced by the radical coupling reaction of
guaiacols produced by retro-aldol reaction of lignin under alkaline condition, in which the C1 and C5
positions are preferred for coupling reaction (Route 1, in Scheme 3).15,51 Diarylmethane is formed by the
phenol-formaldehyde reaction,52 of which the formaldehyde can be produced by elimination of γ-CH2OH
in phenolic β-O-4 or β-5 structure. trans-(β-1)-Diarylethene and (β-1) diaryethane structures have been
proved to be present in low-Mw fraction lignin by 2D HSQC NMR. The formation of these structures is
related to two distinct pathways that have been discussed above (Scheme 2). Four (arylvinyl ether)-linked
arylglycerols have a similar structure that contains vinyl ether linkage and glycerol side-chain, with
different aromatic units. Apparently, the content of S-S enol ether linked dimer (D11) is the dominant
component in four types of dimers, which is much higher than that of the dimers incorporating G units
(D8, D9 and D10), in agreement with the results from 2D HSQC NMR. Each dimer consists of cis and
trans isomers that can be clearly identified in the chromatogram according to the MS information
(Section 2.2, in supplementary material). These structural dimers can be produced by phenolic tandem
β-aryl ether fragments (β-O-4/β-O-4), in which one of the β-O-4 units undergoes the phenolic reaction
pathway to form arylvinyl ether linkage, whereas the other β-O-4 unit suffers degradation reaction to
produce a glycerol terminal unit following the non-phenolic pathway (Route 3). Although these dimers
are notably identified, they are labile and tend to be degraded under acidic conditions or in air owing to
the presence vinyl ether bonds,24 which also explains the presence of phenolic arylglycerol monomer
(S4) in the low-Mw fraction lignin. The original β-β units can undergo base-catalyzed epimerization to
generate epiresinol and diaresinol once the associated phenolic group is generated from lignin
depolymerization.16 From the GC-MS analysis, only isomeric syringaresinols were substantially
identified, which can be interpreted by that (1) S monomers are more prone to form β-β linkage rather
than G monomers during lignification;7 (2) The β-aryl ether bonds incorporating with S units are more
sensitive to chemical processing.53 In three resinol products, the syringaresinol (D14) is most abundant,
followed by episyringaresinol (D13) and diasyringaresinol (D12), in accordance with the result from
semiquantitative analysis by 2D NMR. Quantitative analysis by GC-MS shows that the content of
13/19

Green Chemistry
Page 14 of 20
View Article Online
DOI: 10.1039/D0GC01658F
syringaresinol, episyringaresinol, and diasyringaresinol is 36.85 mg, 23.3 mg, and 5.69 mg of per gram
DCM-soluble fraction, respectively (Table S3).
Overall, we have identified a series of lignin-derived monomers and dimers in low-Mw fraction
isolated from Eucalyptus alkali lignin. It is meaningful not only for the fundamental understanding of
chemical transformation of hardwood lignin during alkaline processing, but also for the potential
industrial and medical applications for these identified aromatic small molecules. For instance,
syringaresinols, which are normally obtained by chemical/biological synthetic strategies or by isolation
from some special natural plants, are promising for preparation of epoxy-amine resins, and have anti-
inflammatory and anti-microbial activities;54,58 Guaiacol, acetovanillone, vanillin, syringol,
syringaldehyde, and acetosyringone are basic chemical raw materials with antioxidative/antibacterial
properties;59 Arylglycerols, diarylethanes, and diarylethenes with featured structures potentially can be
used as pharmaceutical precursors.
Although this work hints a sustainable and low-cost supplementary source for these valuable
chemicals and raw materials, the isolation and separation of these compounds are still challenging. DCM
has shown a high selectivity for extraction of low-Mw fraction from technical lignins, whereas it is
normally associated with acute and/or chronic toxicity in humans, and long-time half-life in
environment.60 These issues make it important to reduce the use of this undesirable solvent, and look for
some greener alternative solvents, in consideration of the large-scale implementation in industry. In
addition, the low-Mw fraction of alkali lignin is a quite complex product mixture (Figure 4), due to the
structural complexity of lignin and the non-specificity of reactions involved in pulping process. The
separation and purification of individual compound, to some extent, in a relatively pure form, is high-
cost in consideration of diversiform constituents. Nevertheless, further fractionation protocols to screen
the lignin-derived small molecules into a series of mixtures with various functionalities would be cost-
efficiency to pave the way for more efficient valorization of this by-product.
3. Conclusion
Hardwood lignin, in comparison with softwood lignin, with abundant S units is more facile to be
depolymerized during alkaline pulping, and also undergoes relatively low levels of condensation reaction
due to the presence of the C5 substituent on S units. In this work, Eucalyptus alkali lignin was fractionated
by dichloromethane extraction to afford a low-Mw fraction and a high-Mw fraction, and these two
14/19

Page 15 of 20
Green Chemistry
View Article Online
DOI: 10.1039/D0GC01658F
fractions were analyzed. It was found that the substructures: epiresinol and diaresinol (with β-β linkages),
β-1 condensed diarylethane and diarylethene units, as well as aldehyde and ketone end-group-derived
units, were primarily present in the low-Mw fraction, whereas the content of arylglycerol was much
higher in high-Mw fraction and the content of enol ether units were comparable in two fractions. A new
pathway related to the formation and chemical production of Cα-condensed β-aryl ether structures was
proposed that potentially explained the high content of β-1 type of diarylethane and diarylethene
structures in the low-Mw fraction, in addition to the traditional reaction pathway from native
spirodienone structures. By further analysis of the low-Mw fraction, a series of novel lignin-derived
phenolic monomers (syringaglycerol) and dimers (including a biaryl dimer, a diarylmethane dimer, 1,2-
diarylethanes, 1,2-diarylethenes, (arylvinyl ether)-linked arylglycerol dimers, and isomeric
syringaresinols) were identified. The disclosure and identification of these lignin-derived phenolic
products in quite substantial amounts in low-Mw fraction isolated from hardwood alkali lignin provides
new insights of technical lignins, and thereby aids the development of efficient valorization strategies for
the production of valuable chemicals or materials from industrial byproducts.
Acknowledgement
This work was supported by the National Natural Science Foundation of China (31870560,
31770621), Fundamental Research Funds for the Central Universities (2018MS52), Nature Science
Foundation of Guangdong Province (2018A030313840), State Key Laboratory of Pulp and Paper
Engineering (2018TS07), and Guangdong Province Science Foundation for Cultivating National
Engineering Research Center for Efficient Utilization of Plant Fibers (2017B090903003).
Conflict of interest statement
There are no conflicts to declare.
References
1. A. J. Ragauskas, G. T. Beckham, M. J. Biddy, R. Chandra, F. Chen, M. F. Davis, B. H. Davison, R.
A. Dixon, P. Gilna, M. Keller, P. Langan, A. K. Naskar, J. N. Saddler, T. J. Tschaplinski, G. A.
Tuskan and C. E. Wyman, Science, 2014, 344, 1246843-1246843.
2. W. Schutyser, T. Renders, S. Van den Bosch, S. F. Koelewijn, G. T. Beckham and B. F. Sels,
15/19

Green Chemistry
Page 16 of 20
View Article Online
DOI: 10.1039/D0GC01658F
Chemical Society Reviews, 2018, 47, 852-908.
3. C. Li, X. Zhao, A. Wang, G. W. Huber and T. Zhang, Chemical Reviews, 2015, 115, 11559-11624.
4. Y. Cao, S. S. Chen, S. Zhang, Y. S. Ok, B. M. Matsagar, K. C. W. Wu and D. C. W. Tsang,
Bioresource Technology, 2019, 291, 121878.
5. W. Lan and J. S. Luterbacher, ACS Central Science, 2019, 5, 1642-1644.
6. W. Boerjan, J. Ralph and M. Baucher, Annual Review of Plant Biology, 2003, 54, 519-546.
7. J. Ralph, G. Brunow, P. J. Harris, R. A. Dixon, P. F. Schatz and W. Boerjan, Lignification: are
Lignins Biosynthesized via simple Combinatorial Chemistry or via Proteinaceous Control and
Template Replication?, Wiley-Blackwell, 2008.
8. J. Ralph and L. L. Landucci, NMR of Lignins, CRC Press, Taylor & Francis Group, Boca Raton,
2010.
9. C. Crestini, F. Melone, M. Sette and R. Saladino, Biomacromolecules, 2011, 12, 3928-3935.
10. F. Yue, F. Lu, S. Ralph and J. Ralph, Biomacromolecules, 2016, 17, 1909-1920.
11. A. Beste and A. C. Buchanan, Chemical Physics Letters, 2012, 550, 19-24.
12. M. Lei, S. Wu, J. Liang and C. Liu, Journal of Analytical and Applied Pyrolysis, 2019, 138, 249-
260.
13. A. Grossman and W. Vermerris, Current Opinion in Biotechnology, 2019, 56, 112-120.
14. G. Gellerstedt, A. Majtnerova and L. Zhang, C R Biol, 2004, 327, 817-826.
15. N. Giummarella, P. A. Lindén, D. Areskogh and M. Lawoko, ACS Sustainable Chemistry &
Engineering, 2020, 8, 1112-1120.
16. C. S. Lancefield, H. L. J. Wienk, R. Boelens, B. M. Weckhuysen and P. C. A. Bruijnincx, Chemical
Science, 2018, 9, 6348-6360.
17. L. Dessbesell, M. Paleologou, M. Leitch, R. Pulkki and C. Xu, Renewable and Sustainable Energy
Reviews, 2020, 123, 109768.
18. B. Pang, S. S. Lam, X. J. Shen, X. F. Cao, S. J. Liu, T. Q. Yuan and R. C. Sun, ChemSusChem,
2020, 13, 1-10.
19. G. Josef and P. Ingegerd, Canadian Journal of Chemistry 1979, 33, 580-622.
20. G. Josef, Wood Science and Technology, 1980, 14, 241-266.
21. S. Constant, H. L. J. Wienk, A. E. Frissen, P. d. Peinder, R. Boelens, D. S. van Es, R. J. H. Grisel,
B. M. Weckhuysen, W. J. J. Huijgen, R. J. A. Gosselink and P. C. A. Bruijnincx, Green Chemistry,
16/19

Page 17 of 20
Green Chemistry
View Article Online
DOI: 10.1039/D0GC01658F
2016, 18, 2651-2665.
22. F. Tran, C. S. Lancefield, P. C. J. Kamer, T. Lebl and N. J. Westwood, Green Chemistry, 2015, 17,
244-249.
23. C. Zhao, J. Huang, L. Yang, F. Yue and F. Lu, Industrial & Engineering Chemistry Research, 2019,
58, 5707-5714.
24. C. Zhao, S. Li, H. Zhang, F. Yue and F. Lu, International Journal of Biological Macromolecules,
2020, 152, 411-417.
25. C. Crestini, H. Lange, M. Sette and D. S. Argyropoulos, Green Chemistry, 2017, 19, 4104-4121.
26. S. N. Sun, M. F. Li, T. Q. Yuan, F. Xu and R. C. Sun, Industrial Crops and Products, 2012, 37,
51-60.
27. X. Jiang, D. Savithri, X. Du, S. Pawar, H. Jameel, H.-m. Chang and X. Zhou, ACS Sustainable
Chemistry & Engineering, 2016, 5, 835-842.
28. C. L. Chambon, V. Fitriyanti, P. Verdía, S. M. Yang, S. Hérou, M.-M. Titirici, A. Brandt-Talbot, P.
S. Fennell and J. P. Hallett, ACS Sustainable Chemistry & Engineering, 2020, 8, 3751-3761.
29. M. Gigli and C. Crestini, Green Chemistry, 2020, DOI: 10.1039/d0gc01606c.
30. C. Gioia, M. Colonna, A. Tagami, L. Medina, O. Sevastyanova, L. A. Berglund and M. Lawoko,
Biomacromolecules, 2020, 21, 1920-1928.
31. S. Chen, H. Cheng and S. Wu, Fuel, 2020, 270, 117520.
32. M. Ma, L. Dai, J. Xu, Z. Liu and Y. Ni, Green Chemistry, 2020, 22, 2011-2017.
33. S. Shimizu, T. Yokoyama, T. Akiyama and Y. Matsumoto, Journal of Agricultural and Food
Chemistry, 2012, 60, 6471-6476.
34. S. Shimizu, T. Yokoyama and Y. Matsumoto, Journal of Wood Science, 2015, 61, 529-536.
35. J. M. Younker, A. Beste and A. C. Buchanan, Ⅲ, Chemphyschem, 2011, 12, 3556-3565.
36. R. Parthasarathi, R. A. Romero, A. Redondo and S. Gnanakaran, The Journal of Physical Chemistry
Letters, 2011, 2, 2660-2666.
37. D. Kanungo, R. C. Francis and N.-H. Shin, Journal of Wood Chemistry and Technology, 2009, 29,
227-240.
38. F. Yue, F. Lu, M. Regner, R. Sun and J. Ralph, ChemSusChem, 2017, 10, 1-7.
39. R. Rinaldi, R. Jastrzebski, M. T. Clough, J. Ralph, M. Kennema, P. C. A. Bruijnincx and B. M.
Weckhuysen, Angewandte Chemie International Edition, 2016, 55, 8164-8215.
17/19

Green Chemistry
Page 18 of 20
View Article Online
DOI: 10.1039/D0GC01658F
40. F. Alonso, P. Riente and M. Yus, Tetrahedron, 2009, 65, 10637-10643.
41. S. Thangaraj, W. S. Tsao, Y. W. Luo, Y. J. Lee, C. F. Chang, C. C. Lin, B. J. Uang, C. C. Yu, J. H.
Guh and C. M. Teng, Tetrahedron, 2011, 67, 6166-6172.
42. F. R. Chang, Y. C. Chao, C. M. Teng and Y. C. Wu, Journal of Natural Products, 1998, 61, 863-
866.
43. J. Ralph, C. Lapierre and W. Boerjan, Current Opinion in Biotechnology, 2019, 56, 240-249.
44. D. P. Ojha and K. R. Prabhu, The Journal of Organic Chemistry, 2012, 77, 11027-11033.
45. J. Huang, C. Zhao and F. Lu, Polymers, 2018, 10, 1077.
46. H. Guo, D. M. Miles-Barrett, B. Zhang, A. Wang, T. Zhang, N. J. Westwood and C. Li, Green
Chemistry, 2019, 21, 803-811.
47. R. Ma, M. Guo and X. Zhang, Catalysis Today, 2018, 302, 50-60.
48. Y. Liu, C. Li, W. Miao, W. Tang, D. Xue, C. Li, B. Zhang, J. Xiao, A. Wang, T. Zhang and C.
Wang, ACS Catalysis, 2019, 9, 4441-4447.
49. S. Liu, L. Das, D. N. Blauch, C. Veronee, C. Dou, J. Gladden, N. Sun and A. M. Socha, Green
Chemistry, 2020, 22, 3917-3926.
50. E. D. Gomes and A. E. Rodrigues, Separation and Purification Technology, 2020, 239, 116551.
51. N. Giummarella, I. V. Pylypchuk, O. Sevastyanova and M. Lawoko, ACS Sustainable Chemistry &
Engineering, 2020, DOI: 10.1021/acssuschemeng.0c03776.
52. D. J. Yelle and J. Ralph, International Journal of Adhesion & Adhesives, 2016, 70 26–36.
53. E. M. Anderson, M. L. Stone, R. Katahira, M. Reed, W. Muchero, K. J. Ramirez, G. T. Beckham
and Y. Roman-Leshkov, Nature Communications, 2019, 10, 2033.
54. M. Habib, M. Trajkovic and M. W. Fraaije, ACS Catalysis, 2018, 8, 5549-5552.
55. M. Janvier, L. Hollande, A. S. Jaufurally, M. Pernes, R. Ménard, M. Grimaldi, J. Beaugrand, P.
Balaguer, P. H. Ducrot and F. Allais, ChemSusChem, 2017, 10, 738-746.
56. F. Bonzanini, R. Bruni, G. Palla, N. Serlataite and A. Caligiani, Food Chemistry, 2009, 117, 745-
749.
57. A. S. Jaufurally, A. R. S. Teixeira, L. Hollande, F. Allais and P.-H. Ducrot, ChemistrySelect, 2016,
1, 5165-5171.
58. V. K. Bajpai, M. B. Alam, K. T. Quan, M.-K. Ju, R. Majumder, S. Shukla, Y. S. Huh, M. Na, S. H.
Lee and Y. K. Han, Scientific Reports, 2018, 8, 9216.
18/19

Page 19 of 20
Green Chemistry
View Article Online
DOI: 10.1039/D0GC01658F
59. M. P. Vinardell and M. Mitjans, International Journal of Molecular Sciences, 2017, 18, 1219.
60. J. P. Taygerly, L. M. Miller, A. Yee and E. A. Peterson, Green Chemistry, 2012, 14, 3020-3025.
19/19

Green Chemistry
Page 20 of 20
View Article Online
DOI: 10.1039/D0GC01658F
Graphical abstract:
Identification and mechanism study of lignin-derived monomers and dimers from
fractionation of eucalyptus alkali lignin provide new insights into lignin valorization.