Article

Finely Designing Dicarboxylic Acid-Based Protic Ionic Liquids

System for Tailoring Lignin Structure via Demethylation
Strategy
Cheng Li 1,2 , Xinyu Xiao 1 , Qizhen Luo 2,3 , Wanting Zhao 2,3 , Wenzhe Xiao 2,3 , Ling-Ping Xiao 1 , Yao Tong 1 ,
Shangru Zhai 1,4, * and Jian Sun 2,3, *

1 Liaoning Key Lab of Lignocellulose Chemistry and BioMaterials, Liaoning Collaborative Innovation Center
for Lignocellulosic Biorefinery, School of Light Industry and Chemical Engineering, Dalian Polytechnic
University, Dalian 116034, China; [email protected] (C.L.); [email protected] (X.X.);
[email protected] (L.-P.X.); [email protected] (Y.T.)
2 Key Laboratory of Molecular Medicine and Biotherapy, Ministry of Industry and Information Technology,
School of Life Sciences, Beijing Institute of Technology, Beijing 100081, China; [email protected] (Q.L.);
[email protected] (W.Z.); [email protected] (W.X.)
3 Beijing Engineering Research Center of Cellulose and Its Derivatives, Advanced Research Institute of
Multidisciplinary Sciences, Beijing Institute of Technology, Beijing 100081, China
4 School of Environmental and Nature Resources, Zhejiang University of Science and Technology,
Hangzhou 310023, China
* Correspondence: [email protected] (S.Z.); [email protected] (J.S.)

Abstract: As one kind of renewable aromatic polymer, lignin is severely underused due
to its chemical recalcitrance. Lignin can endure demethylation modification to improve
its activation by releasing more active functional groups. However, the process suffers
from expensive, corrosive, and toxic issues by employing halogen-containing reagents,
which has become an obstacle to industrial applications. Herein, a series of dicarboxylic
acid-based protic ionic liquids (DAPILs) systems composed of ethanolamine and dibasic
organic acids (e.g., aspartic acid (Asp), glutamic acid (Glu), succinic acid (SA), and glutaric
acid (GA)) with 1~2:1 stoichiometric ratio, have been finely designed for the demethylation
of industrial lignin. With [EOA][GA] treatment, the polyphenol content in lignin was
favorably increased beyond 1.58 times. The structural tailoring and variation were fully
Academic Editor: Rafał M. Łukasik
characterized by 2D HSQC and 1 H NMR. The analysis results indicated that, with the
Received: 10 April 2025 increase of phenolic hydroxyl content in lignin, the β-O-4′ bond was broken and the
Revised: 12 May 2025
content of structural units (S, G) and the S/G ratio of lignin decreased accordingly. After
Accepted: 24 May 2025
the treatment, the used IL and tailored lignin can be recovered over 95%. This novel,
Published: 3 June 2025
halogen-free and environmentally friendly lignin-cutting strategy not only opens avenues
Citation: Li, C.; Xiao, X.; Luo, Q.;
for high-value utilization of lignin but also expands the field of application of dicarboxylic
Zhao, W.; Xiao, W.; Xiao, L.-P.; Tong, Y.;
Zhai, S.; Sun, J. Finely Designing
acid-based protic ionic liquids.
Dicarboxylic Acid-Based Protic Ionic
Liquids System for Tailoring Lignin Keywords: lignin; demethylation strategy; ionic liquids; polyphenol; value-added utilization
Structure via Demethylation Strategy.
Molecules 2025, 30, 2445. https://
doi.org/10.3390/molecules30112445

Copyright: © 2025 by the authors. 1. Introduction

Licensee MDPI, Basel, Switzerland. Lignocellulosic biomass is a renewable, biodegradable, non-toxic, and abundant
This article is an open access article
natural energy source, with the potential to replace fossil fuels in the future [1]. Lignin, a
distributed under the terms and
key component of lignocellulosic biomass, is a major global source of aromatic compounds
conditions of the Creative Commons
Attribution (CC BY) license
and holds significant potential for producing a variety of chemical products [2], including
(https://creativecommons.org/ pharmaceuticals [3], adhesive [4], antioxidants [5], and anti-inflammatory agents [5]. Lignin
licenses/by/4.0/). consists of three structural units: syringyl (S), guaiacyl (G), and p-hydroxyphenyl (H) [6].

Molecules 2025, 30, 2445 https://doi.org/10.3390/molecules30112445

Molecules 2025, 30, 2445 2 of 16

The G-type lignin structural unit features a methoxy group at the C3 position. In contrast,
the S-type lignin structural unit possesses two methoxy groups, located at the C3 and C5
positions, respectively. Conversely, the H-type lignin structural unit lacks any methoxy
groups. Lignin is primarily derived from waste pulp in the paper industry, with an
estimated annual global production of approximately 50 million tons [7]. However, only
2% of lignin waste is currently utilized for high-value applications, such as modifications
to enhance utilization, while 98% is used for lower-value purposes, such as combustion
for energy generation [8]. Therefore, research on the modification of lignin to facilitate its
high-value, value-added utilization is of practical importance.
Modifying lignin is also challenging due to its complex aromatic structure, low re-
activity, and poor solubility. The molecular structure of lignin contains several reactive
functional groups, such as aromatic rings, phenolic hydroxyls (Ph-OH), carbonyls, and
methoxyls [9]. These groups enable a variety of chemical reactions, including halogena-
tion, phenolization [10,11], graft copolymerization [12], alkylation and dealkylation [13],
acylation [14], amination [12,15], esterification [16], and hydrolysis [17]. Although these
methods can improve lignin’s reactivity, they are constrained by complex modification
conditions. Therefore, developing novel strategies that can operate under mild conditions
in a short time and are easy to manipulate is an urgent need. Lignin contains numerous
methoxy groups and β-O-4′ bonds in its structure. The depolymerization of the β-O-4′
bond has been reported in the literature, such as the pyrolysis of the β-O-4′ bond to produce
phenolic compounds [18], and electrocatalytic oxidative degradation of the β-O-4′ bond
to yield carboxylic acids, aromatic aldehydes, and other high-value products [19]. During
demethylation, a nucleophilic attack on the methoxy group may lead to the loss of methyl
groups and the release of free Ph-OH [20]. Simultaneously, the cleavage of the β-O-4′ bond
also produces additional Ph-OH, significantly increasing the Ph-OH content in lignin [21].
The complex structure of industrial lignin, coupled with the consumption of various
functional groups during processing, leads to the breaking of ether bonds and a reduction
in reactivity, thereby enhancing lignin’s chemical stability. Traditional demethylation
methods include Lewis acid catalysis [22,23], redox reactions (e.g., bacterial and fungal
processes) [24], thermal cleavage [25], and microwave heating [26]. However, most of these
methods necessitate harsh conditions, such as high pressure, elevated temperatures, metal
catalysts, or halogenated acids, which can lead to equipment corrosion. While microwave
heating has shown effectiveness in lignin modification, its industrialization is hindered by
the high cost of the required equipment. Furthermore, the demethylation process yields
different products under varying catalytic conditions. According to Mei et al., in the
presence of a cocatalyst, methoxyl groups can react with CO and water to produce acetic
acid, achieving a high conversion rate [27]. The demethylation of lignin using sodium
ethyl mercaptan (EtSNa) as a nucleophile produces methyl ethyl sulfide as a by-product,
which has a pungent odor [28]. Under different conditions, lignin also generates various
condensation products. For instance, under acidic conditions, lignin undergoes C-C bond
formation through a possible condensation mechanism, and the acid hydrolysis of β-O-4′
bonds also produces phenylacetaldehyde [29].
Ionic liquids (ILs) are organic molten salts that exist as free ions in the liquid state at
or near room temperature. ILs are frequently used as green solvents for catalysis, organic
synthesis, and material preparation, owing to their unique physicochemical properties,
such as non-flammability, non-volatility, good electrical conductivity, and recyclability [30].
Generally, ILs can be classified into protic, aprotic, zwitterionic, and polymeric types,
which have found wide applications in biomass pretreatment [31], battery electrolytes [32],
additives, membranes [33], and other industrial applications. In biomass pretreatment, ILs
can not only act as solvents to dissolve biomass at appropriate temperatures, but also act as
Molecules 2025, 30, 2445 3 of 16

reactants to react with biomass, thereby achieving the effect of biomass modification [34–36].
Sun et al. employed a protic ionic liquid, ethanolamine acetate ([EOA][OAc]), for the one-
pot production of cellulosic ethanol, achieving ethanol yields of up to 70% of the theoretical
maximum. The IL, [EOA][OAc], synthesized through a simple acid-based reaction, is
biocompatible and cost-effective for bioethanol production under optimal pH and high
loading conditions [37]. More recently, the same group demonstrated the use of prepared
lignin polyphenols as tanning agents for chrome-free eco-leather production, aiming to
address issues in the leather industry, such as the need for harsh conditions and toxic
reagents [38,39]. Despite the successes of [EOA][OAc], the volatile nature of acetic acid
can lead to changes in the composition of the IL system, affecting the demethylation
performance during recycling. Furthermore, the IL contains only one active carboxylic
acid site, so the potential of protic ionic liquid in lignin demethylation requires further
exploration. Therefore, in pursuit of a more sustainable treatment process, there is a
growing demand to design IL systems with multiple carboxylic acid active sites to efficiently
tailor lignin structures.
Inspired by the above viewpoints, a continuous endeavor is undertaken to develop
and explore new strategies toward lignin demethylation for the high-value-added utiliza-
tion. Herein, eight dicarboxylic acid-based protic ionic liquids (DAPILs) were prepared at
different stoichiometric ratios using ethanolamine (EOA) as the positive ion and aspartic
acid (Asp), glutamic acid (Glu), succinic acid (SA), and glutaric acid (GA) as the anions.
Lignin was demethylated using these different DAPILs (Scheme 1), respectively. As shown
in Scheme 1, this scheme contains two different approaches, namely the demethylation
of lignin (⃝),
1 and the demethylation accompanied by the breaking of ether bonds simul-
taneously (⃝, 2 ⃝).
3 The lignin samples obtained from different DAPILs treatments were
systematically characterized by Fourier Transform Infrared Spectroscopy (FTIR), Ultraviolet
Spectroscopy (UV), Gel Permeation Chromatography (GPC), two-dimensional Heteronu-
clear Single Quantum Coherence (2D HSQC), and Nuclear Magnetic Resonance (NMR) to
reveal the structural changes of lignin.

Scheme 1. Demethylation processing of lignin by DAPILs.

2. Results and Discussions

2.1. Preparation and Characterization of DAPILs
To screen the most effective DAPILs from the eight prepared candidates of ILs, the
purified alkali lignin (AL) was selected as the substrate. The weight ratio of AL to DAPIL
Molecules 2025, 30, 2445 4 of 16

was 1/4. To lower the viscosity of the system, 0.5 mL of water was added, and the mixture
was pretreated for 2 h. At the end of the experiment, the contents of Ph-OH in the pretreated
lignin (PAL) were determined using UV spectrophotometry. To some extent, the newly
formed Ph-OH group in PAL represented the impact on the demethylation of AL. Figure 1
shows DAPILs and the basic tests performed on their performance, where each of the
eight DAPILs is shown in Figures S1 and S2, which show the structural formula for the
eight DAPILs. For example, a DAPIL with a 2:1 stoichiometric ratio of EOA to Asp is
referred to as [EOA]2 [Asp], and a DAPIL with a 1:1 stoichiometric ratio is referred to as
[EOA][Asp]. Figure 1b illustrates the pH and conductivity of the DAPILs, indicating that
the pH is higher when the molar ratio of ethanolamine to dibasic acid is 2:1. Given that
Ph-OH tends to be weakly acidic, alkaline conditions are not conducive to the generation
of PhOH. As shown in Figure 1a, the PHC produced by DAPIL-pretreated lignin under
identical conditions at a molar ratio of 1:1 was slightly higher than that at a molar ratio of
2:1. The conductivity, which influenced the transfer of active protons, reached its maximum
at [EOA][GA] (Figure 1b). In summary, when the pH is weakly acidic, the conductivity of
[EOA][GA] and [EOA][SA] are at large values.

Figure 1. (a) Effect of eight DAPILs on phenolic hydroxyl content in al-modified lignin at different
temperatures (conditions: 2 h, DAPILs to lignin AL mass ratio is 4:1). (b) Conductivity and pH
of DAPILs.

2.2. Demethylation and Structural Characterization of Lignin

Experiments on the demethylation effects of lignin at different temperatures (i.e., 70,
90, and 110 ◦ C) were carried out on the eight DAPILs for 2 h. As depicted in Figure 1a,
the demethylation effects of all eight ILs were better at 90 ◦ C, which was attributed to
the nucleophilic attack of the anions of the ILs on the methoxyl group in the lignin to
demethylate the lignin and then transformed to free Ph-OH. In contrast, demethylation was
less effective at 70 ◦ C, which was probably since lower temperatures were not favorable
for nucleophilic reactions. But at higher temperatures, the simultaneous condensation
reaction of lignin demethylation increases, resulting in a decrease in Ph-OH. Since Ph-OH
was weakly acidic, more acidic conditions favored demethylation. Whereas the reaction
system was more acidic when the EOA content was low, DAPILs with a stoichiometric
ratio of 1:1 were therefore more favorable for lignin demethylation.
By summarizing the demethylation effects of eight DAPILs reacted under different
reaction conditions for 2 h, [EOA][GA] reached a higher demethylation effect at 90 ◦ C for
2 h, and the PHC was determined to be 3.91. Therefore, [EOA][GA] was chosen as the
Molecules 2025, 30, 2445 5 of 16

main DAPIL to continue the experiment, and [EOA]2 [GA] DAPIL was used as a control
experiment. The two DAPILs were subjected to different reaction times (e.g., 1, 2, 3, 4, 6,
and 8 h) on AL at 90 ◦ C. As depicted in Figure 2a, after treatment with the two DAPILs, the
content of PHCs in AL increased. Specifically, when AL was modified by [EOA][GA], its
PHC reached the maximum value of 4.29 at 3 h and then began to decrease, while that of
AL modified by [EOA]2 [GA] could reach a maximum value of 4.36 at 6 h.

Figure 2. (a) Effect of DAPILs with different stoichiometric ratios on AL modification at the same
temperature (90 ◦ C) for different times. (b) Infrared spectra of AL before and after modification
(PAL1: [EOA][GA], 90 ◦ C, 3 h; PAL2: [EOA]2 [GA], 90 ◦ C, 6 h). (c) UV spectra before and after lignin
modification (AL, PAL1, PAL2). (d) 1 H NMR after acetylation of AL, PAL1 and PAL2.

To verify whether there was a change in the functional groups of lignin before and after
modification, FT-IR characterizations were conducted (Figure 2b). The peaks at 1026 cm−1
and 825 cm−1 indicated that the AL used contains H, G, and S structural units, whereas
the peaks at 1601 cm−1 , 1501 cm−1 and 1442 cm−1 represent the vibration of the aromatic
backbone in the lignin [40]. The slight change of the peak at 1126 cm−1 indicates that the
aromatic ring of lignin is modified. Moreover, the breakage of the ester bonds may also
generate Ph-OH. The peak at 1705 cm−1 represents the stretching vibration of the lignin
carbonyl group [41]. The peaks at 2939 and 2846 cm−1 are the C-H stretching vibrations of
methyl (-CH3 ) and methylene (-CH2 ) [42], respectively. It was observed that both those
peaks were slightly reduced after AL modification, which also indicated the demethylating
effect of [EOA][GA] on AL. The larger peak at 3400 cm−1 is a phenol/aliphatic hydroxyl
(-OH) stretching vibration [43]. From the results of the FTIR spectra, we can see that
different ratios of [EOA][GA] have a role in the demethylation process of AL, and the basic
skeleton of lignin remains unchanged after the modification.
The demethylation effect of different ratios of DAPILs on AL was reflected to some
extent by the calculation of PHC using UV differential absorption method. As shown
in Figure 2c, the UV absorption spectra of Ph-OH of lignin were shown at 250~400 nm,
where the two characteristic absorption peaks of Ph-OH were displayed at 300 and 360 nm,
respectively. The peak intensity was proportional to the concentration of lignin dissolved
in dioxane-aqueous solution at 360 nm (i.e., AL: 10.5 mg, PAL1: 7.5 mg, and PAL2: 5.9 mg,
respectively). Although the weighing of AL was greater than that of PAL1 and PAL2, the
Molecules 2025, 30, 2445 6 of 16

PHC content increased from 2.75% for AL to 4.29% and 4.36%, an increase of 56% and 58%,
respectively. All these data also indicated that the two DAPILs underwent different degrees
of demethylation of AL.
The 1 H NMR spectra of acetylated Al, PAL1 and PAL2 are shown in Figure 2d. The
proton signal peak at a peak represents the hydrogen proton displacement of aldehyde in
the internal standard p-nitrobenzaldehyde (NBA) molecule, a and peaks b and c represent
the chemical displacement of hydrogen protons on the benzene ring of the internal standard
NBA, respectively. The hydrogen proton signal at 3.82 ppm is the lignin methoxyl signal
peak (d peak), and the hydrogen proton signal peaks at 2.24 ppm (e peak) and 1.90 ppm
(f peak) represent aromatic and aliphatic protons in the acetyl group [44]. The results of
calculating the methoxy content and PHC based on the internal standard NBA content
and signal peaks are summarized in Table 1. After demethylation, the methoxy content
decreased from 1.11 mmol/g for AL to 0.45 mmol/g for PAL1 and 0.61 mmol/g for PAL2,
respectively. However, the PHC increased from 0.50 mmol/g for AL to 0.58 mmol/g for
PAL1 and 0.61 mmol/g for PAL2, respectively. Compared with PAL2, PAL1 has a higher
methoxy removal rate and a lower PHC, which may be due to the acidic environment of
PAL1 leading to a small part of Ph-OH condensation [45].

Table 1. Relative signal intensities of methoxy and hydroxy groups in 1 H NMR.

Chemical Shift
FAr-OH FMeO
Sample M/mg Mi /mg Ii Iar-OH IMeO (mmol/g) (mmol/g)
δ10.3–10.1 δ2.46–2.11 δ4.0–3.71
AL 40 5 1 4.05 8.86 0.50 1.11
PAL1 40 5 1 4.68 3.64 0.58 0.45
PAL2 40 5 1 4.93 5.93 0.61 0.61
F: relative content of specific functional groups (mmol/g). Mi : weight of the inner label NBA (mg). Ii : peak area
of internal standard NBA. M: weight of lignin samples.

Elemental analysis was performed on AL, PAL1, and PAL2, as shown in Table S1. The
fact that there was some reduction in carbon content after lignin demethylation treatment
indicated the demethylation effect [46]. On the contrary, the small amount of elemental
N in the unreacted lignin was due to the fact that corn stover belongs to the gramineous
family of plants, which consisted of carbohydrates and a small number of proteins. The
increase in N content in modified lignin may be related to the trace residue of DAPIL. This is
because we observed extremely tiny peaks at 1651 cm−1 and 864 cm−1 in Figure 2b, which
respectively represent the vibration peaks of N-H and -NH2 [47]. Furthermore, in Figure 2b,
we observed the characteristic peak of C-N at 1365 cm−1 [48], which indicates that at
90 ◦ C, with the increase of reaction time, the increase of elements N and H after DAPILs
treatment for different times might be due to the combination of ethanolamine and lignin in
DAPILs. Many Ph-OH exist in modified lignin (PAL1), while ethanolamine contains amino
(-NH2 ) and -OH groups. Under high temperature conditions, ethanolamine undergoes
an amination reaction with Ph-OH in lignin to produce a small amount of aminated
lignin [49,50]. This has been reported in other literature and seems to be a common feature
of pretreatment of lignin with amines [51,52]. Moreover, PAL2 was observed to have
more ethanolamine in comparison to that of PAL1, which could be due to the fact PAL2
was more basic with a reaction time of 6 h. Therefore, the increase in content was more
significant [53]. And the degree of demethylation was calculated through the change of
methoxy content. The demethylation degrees of AL by [EOA][GA] and [EOA]2 [GA] were
59.3% and 45%, respectively.
Molecules 2025, 30, 2445 7 of 16

The molecular weight of AL before and after modification was examined by GPC.
As shown in Table S2, the molecular weight (Mw ) of lignin samples all increased after
modification, and the increase in the Mw of PAL3 was particularly significant. This was
possibly because PAL3 was subjected to an acidic condition, which was favorable to the
condensation of lignin and led to an obvious increase in the Mw of the modified lignin.
Moreover, it was observed that the Mw and Mn of PAL2 increased, which was due to partial
condensation of lignin under a longer reaction time at 90 ◦ C [54]. The increase in Mw
during the modification process caused the lignin to form a highly cross-linked structure,
resulting in an increase in storage modulus and greater rigidity of the lignin. Therefore, the
modified lignin with higher Mw was commonly used to prepare resins [55].
To further investigate the mechanism of lignin demethylation and the changes of
functional groups, the 2D HSQC NMR analysis was performed and the results are displayed
in Figure 3 and Table 2. Table S3 is the 2D HSQC belonging table of various units in
AL [56]. It is worth noting that the results of the lignin side chain region in Figure 3a–c
show that the β-O-4′ content decreased after AL demethylation modification. In the
samples of PAL1- and PAL2-modified lignin, the content of S unit and the value of S/G
showed a decreasing trend (Figure 3d–f). The β-O-4′ content decreased from 36.14% to
23.52% and 28.91%, respectively. Moreover, the contents of Aα (δC /δH 72.31/4.84 ppm),
Aγ (δC /δH 60.18/3.53 ppm), Aβ (G/S-G) (δC /δH 84.08/4.37 ppm), Aβ (G/S-S) (δC /δH
86.23/4.09 ppm), A’β (δC /δH 82.29/4.85 ppm), Bγ (δC /δH 69.81/3.50 ppm), Cγ (δC /δH
66.69/4.01 ppm), and Eα (δC /δH 79.24/5.57 ppm) were found to be decreased to different
degrees, and the Cγ peak even disappeared (Figure 3a–c). A group of representative
signal strength measurement results are presented in Table S4. Those results indicated that
the breaking of β-O-4′ structure also contributed to a fraction of low molecular weight
polyphenols after the demethylation of AL (as shown in Scheme 1).

Table 2. 2D HSQC NMR analysis of different lignin samples.

Inter-Unit Linkages (/100 Ara ) Lignin Aromatic Units (%) S/G

Lignin Sample
β-O-4′ β-β′ β-5′ S G H
AL 36.14 12.04 7.22 66.46 17.3 19.23 3.66
PAL1 23.52 9.41 4.7 44.77 26.86 28.35 1.66
PAL2 28.91 9.6 4.81 51.51 25.75 22.72 2

In the aromatic region of lignin, the peaks of the S, G, and H units could be observed
before and after lignin modification. Notably, the peak intensities of the S and G units were
found to be reduced after the modification, and the value of S/G decreased from 3.66 to
1.66. The significantly reduced peak strength of the S unit as compared to that of the G unit
was due to the higher methoxyl content of the S unit, which was more prone to fracture
than the G unit. In addition to the basic unit structure, the correlation signals of p-coumaric
acid (pCA) and ferulic acid (FA) were observed in the aromatic region. The decrease in
the intensity of pCA and FA in PAL1 and PAL2 in comparison with that of AL may be
due to the preferential breaking of the ester bonds of pCA and FA during the modification
process [57].
Molecules 2025, 30, 2445 8 of 16

Figure 3. HSQC NMR spectra of lignin samples, denoted as AL, PAL1, and PAL2. (a–c) The side chain
regions of AL, PAL1, and PAL2. (d–f) The aroma regions of AL, PAL1, and PAL2. (g) Detection of the
group key structures of the lignin fractions, including changes in the units of the side chain bonds and
the aromatic regions: (A) β-O-4′ bonds, (B) resin structure formed by β-β′ bonds, (C) phenyl formed
by β-x-5′ and α-O-4 bonds coumarin structure, (E) α, β-diaryl ether substructures, (G) guaiacyl unit,
(S) syringyl unit, (H) p-hydroxyphenyl unit, (pCA) p-coumarate, and (FA) ferulate.

2.3. Molecular Computation

The reactions of the lignin model compound with two different types of DAPILs
at the bond level were analyzed by molecular simulations. As depicted Figure 4, the
carboxyl groups (-COOH) of both ILs could interact with the lignin model compounds in
the system of two bidentate ILs. According to simulations performed with the Multiwfn
software (gaussian 16 A03) [58], the bond orders for the C31-O30 bond were 0.880 and 0.897,
and for the C14-O19 bond were 0.848 and 0.886, depending on whether [EOA][GA] or
[EOA]2 [GA] was used. This positive correlation existed between bonding level and bonding
stability. Accordingly, a positive correlation between bonding level and bonding stability
Molecules 2025, 30, 2445 9 of 16

was observed. The higher the bonding energy level, the more stable the bonding [59]. A
high bonding level also indicated that the proton-coupled electrons were involved in the
bond formation, and the density of electrons increased the gravitational attraction between
each other, thus improving the bond stability. Based on the calculation results, it can be
concluded that the bonding level of the lignin model compounds is lower in the [EOA][GA]
simulation system, in which the carboxyl group of DAPIL has a higher density of electron
cloud, which is more likely to undergo nucleophilic reaction with C31-O30, allowing the
-CH3 to be shed and free Ph-OH to be formed. Moreover, in the [EOA][GA] system, the
acidic environment catalyzed the breaking of the lignin β-O-4′ bond, which also increased
the PHC [60]. Combined with the previously reported literature [61], we speculate that
DAPIL acts as a catalytic cleavage of β-O-4′ bonds in addition to acting as a solvent during
demethylation. This suggests that in addition to the demethylation effect of the increase in
Ph-OH, the breaking of the β-O-4′ bond is also an important cause (Figure S4).

Figure 4. Computational simulations of the interaction between DAPIL and lignin model
compounds. (a) [EOA][GA]-2-(2-methoxyphenoxy)-1-phenylethan-1-ol and (b) [EOA]2 [GA]-2-(2-
methoxyphenoxy)-1-phenylethan-1-ol.

2.4. DAPIL Cycle Performance

Finally, in addition to the circular performance of [EOA][GA], we also compared the
demethylation efficiency of [EOA][GA] with other traditional demethylation reagents, as
shown in Figure 5 and Table S5 [20,27,62–65]. As shown in Figure 5a, after the third cycle,
the PCH of lignin decreased from 5.24% in the first cycle to 5.08%, a reduction of 0.16%. It
was only 0.26% lower than AL after demethylation of fresh DAPIL. It also shows that it has
good recyclability. When compared with the content of phenolic hydroxyl groups depicted
in Figure 2a, an increase is observed, likely attributable to the inherent heterogeneity
of lignin. The challenge of homogenizing lignin to enhance its high-value utilization
remains a significant hurdle that demands our attention. In addition, 1 H NMR analysis was
performed on the DAPIL before and after the reaction, as shown in Figure S3. The DAPIL
is recovered according to the method described in Section 2.4. The peak at 6.90 ppm is the
peak of DAPIL active H, and the peaks at 3.05 ppm and 3.35 ppm are the peaks of amide.
The formation of amide is the reaction of the ethanolamine and carboxyl group under the
condition of heating in this experiment [66]. Most of the peaks of RDAPIL did not shift
after cycling. However, due to the consumption of a small amount of ethanolamine in the
Molecules 2025, 30, 2445 10 of 16

generation of amide, the electron cloud density around the proton decreases, causing the
peak of active hydrogen to shift to a lower area (from 6.9 ppm to 7.7 ppm). The spectra
did not reveal any characteristic peaks of lignin, which also indicated that lignin was
essentially separated from the DAPIL and did not require further purification. As shown
in Figure 5b and Table S5, chemical methods such as pyrolysis, the biological method, and
Lewis acid can demethylate lignin, split aryl ether bonds in lignin macromolecules, and
replace natural methoxy groups with hydroxyl groups. The demethylation efficiency of AL
in this work is about 59.3%, which is better than many currently available demethylation
reagents. Although demethylation efficiency needs to be further improved, [EOA][GA]
is less toxic than metal halides, in more benign conditions, and can be recycled, which is
more cost-effective.

Figure 5. (a) DAPIL recycling and (b) demethylation efficiency of different methods. (Lihong Hu.
2013 [24], Qingqing Mei. 2017 [32], Yu Chen. 2023 [65], Balaji. 2020 [66], Jiongjiong Li. 2017 [67], Yilin
Wang. 2023 [68]).

3. Materials and Methods

3.1. Materials
Alkali lignin was obtained from Shandong Longli Bio-technology Co. of China
(Dezhou, China). Succinic acid was purchased from Zancheng (Tianjin, China) Technology
Co. (Tianjin, China). Ethanolamine (EOA, 99%), dioxane (AR, 99%), ethanol (CH3 CH2 OH,
99%), sodium hydroxide (NaOH, 99%), succinic acid (SA, 99%), glutaric acid (GA, 99%),
L-aspartic acid (Asp, 99%), and L-glutamic acid (Glu, 99%) were obtained from Shanghai
Meryer Biochemical Technology Co. (Shanghai, China). Hydrochloric acid (HCl, 37%) was
purchased from Shanghai McLean Biochemical Technology Co. (Shanghai, China). All raw
materials are used directly, except for lignin, which requires further purification.

3.2. Lignin Purification Treatment

In a typical procedure, an appropriate amount of crude lignin was dispersed in a
30 wt% NaOH solution and stirred for 2 h. Then vacuum filtration was carried out using
10 cm of qualitative medium-speed filter paper (Newstar, Hangzhou Special Paper Industry
Co., Ltd., Hangzhou, China), the supernatant was adjusted to pH = 2 with 6 M HCl and
continuously stirred for 3 h at 50 ◦ C. Finally, the mixed system was centrifuged at 8000 rpm
for 5 min. The lignin precipitate was separated by centrifugation, and the purified lignin
was obtained and recorded as AL.
Molecules 2025, 30, 2445 11 of 16

3.3. Preparation of DAPILs

To synthesize the corresponding 8 DAPILs, the dibasic acid (i.e., Asp, Glu, SA, and
GA) and organic base (EOA) were mixed according to a certain stoichiometric ratio with the
ratios of base to acid in the range of 1:1 to 2:1. In a typical preparation, 6.655 g of aspartic
acid was added to 6.1083 g of ethanolamine with 5 mL of deionized water and stirred at
80 ◦ C until the solution was clear to equilibrate proton exchange.

3.4. Demethylation of Purified Lignin in ILs and Recovery of DAPILs

The water content in ILs significantly impacts lignin solubility. Based on previous
literature, we add an appropriate amount of deionized water to the synthesized viscous
ionic liquid to lower its viscosity [67,68]. In total, 0.5 g AL, 2 g IL, and 0.5 mL deionized
water were added into a 15 mL sealed high-pressure glass tube and heated at different
temperatures (70, 90, and 110 ◦ C) for 2 h. After the reaction, the system was cooled to
room temperature. Lignin dissolved in DAPILs was precipitated using an anti-solvent
method. Specifically, 5 g of ethanol was added to the system, followed by an operation of
ultrasonic vibration for 5 min. Then, excess water was added to precipitate the modified
lignin from the system. When the suspension had been transferred to a centrifuge tube, it
was conducted by rotating at 8000 rpm for 5 min to separate the lignin from the DAPILs.
The lignin was centrifuged and washed with 10 mL of deionized water. This was repeated
three times. The separated lignin was freeze-dried at −81 ◦ C and 10 Pa for 12 h. The DAPIL
in the washing solution containing DAPIL was recovered by using a rotary evaporator
(60 ◦ C, 2 h). The recovered DAPIL was denoted as RDAPIL.

3.5. Measurement of Ph-OH in Lignin Species by Ultraviolet Differential Photometry

According to the method reported in the literature [69–71], the solution with pH 6 and
0.2 mol/L NaOH was firstly prepared. Specifically, a standard solution with a pH of
6 (solution 1) was made by volumes of 3.368 g of KH2 PO4 and 0.113 g of NaOH in a 500 mL
volumetric flask. Then, a 0.2 mol/L NaOH solution (solution 2) was prepared by adding
4 g NaOH into 500 mL DI water. A total of 5~10 mg of modified lignin was dissolved in
10 mL of aqueous dioxane (9:1, dioxane/water) (solution 3). Two 50 mL volumetric flasks
were taken and 2 mL of solution 3 was added to each of them and then fixed to scale with
solution 1 and solution 2, respectively. The instrument used was the ultraviolet-visible
spectrophotometer (Hitachi U-3900, Hitachi High—Tech Science Corporation, Shizuoka,
Japan), and the cuvette path length (L) was 1 cm. With solution 1 as the reference, the
absorbance curve of solution 2 in the range of 200~400 nm was measured. The phenolic
hydroxyl content (PHC) of lignin was calculated by the peak intensity at 300 nm and
360 nm in the absorbance curve. The PHC calculation formula is as follows:

250 × ( A1 × 0.182+ A2 × 0.425)

PHC = (1)
ms × L

In the above Equation (1), A1 represents the intensity of the absorption peak near
360 nm, A2 represents the intensity of the absorption peak near 300 nm, ms represents the
mass of the weighed sample in mg, L denotes path length in cm.

3.6. Characterization of Lignin by Fourier Transform Infrared Spectroscopy

Fourier transform infrared spectra (FTIR) (IRTracer-100, Shimadzu, Kyoto, Japan) were
scanned with the frequency region of 4000~400 cm−1 . The lignin was characterized by FTIR
using the KBr pellet method. The KBr powder and lignin powder were mixed and ground
(100/1, mKBr /mlignin ), pressed into tablets for infrared characteristic peak characterization,
and parallel measurements were made three times.
Molecules 2025, 30, 2445 12 of 16

3.7. Gel Permeation Chromatography (GPC) Analysis of Lignin

GPC was performed using Waters 1515 GPC (Waters Corporation, Milford, MA, USA).
Firstly, the raw lignin sample was acetylated to increase its solubility. Typically, 100 mg
of lignin, 2 mL of pyridine and 2 mL of acetic anhydride were placed in a 15 mL vial and
stirred in a dark environment for 24 h. At the end of the reaction, to maintain structural
integrity, the acetylated lignin was precipitated using DI water as an anti-solvent.
Before the test, the Waters 1515 GPC system (GPC Breeze System; Milford, MA, USA)
was calibrated using the universal calibration method. After that, 4 mg acetylated lignin
was dissolved in 8 mL THF at a mobile phase elution rate of 1 mL/min.

3.8. Elemental Analysis

Lignin was analyzed for elements using an organic elemental analyzer (Leeman
EA3000, LEEMAN LABS INC, Hudson, NH, USA). A totla of 30 mg of lignin was taken for
C, H, and N contents and O content was obtained by difference calculation.

3.9. 1 H NMR of Lignin and DAPILs

The lignin samples were acetylated to better dissolve the lignin in the system, and the
strong solvent DMSO-d6 was used as the system solvent. 1 H NMR samples were prepared
by weighing appropriate amounts of acetylated lignin and p-nitrobenzaldehyde (NBA)
(4/1, wLignin /wNBA ) dissolved in DMSO-d6 . The experimental tests were performed using
a Bruker 400 MHz with 128 scans at room temperature, and the relative content of the
specific functional groups in the lignin samples was calculated according to the formula.
The specific formula for calculating the relative content of functional groups is given below:

F = ( Mi /Ii × A)/M × 100% (2)

In the above equation, F: relative content of specific functional groups (mmol/g); Mi :

weight of the inner label NBA (mg); Ii : peak area of internal standard NBA; A: peak area of
functional groups; M: weight of lignin samples.

3.10. 2D HSQC NMR

2D Heteronuclear single quantum coherence (HSQC) NMR was performed on lignin
1H NMR and 13 C NMR spectra using BRUKER AVANCE (Bruker Corporation, Karlsruhe,
Germany) 400 at 25 ◦ C. A total of 500 mg of lignin sample was dissolved in 500 µL of
DMSO-d6 . To obtain more accurate results, the experimental parameters should be set as
follows: spectral widths of 5000 Hz for HSQC on 1 H NMR and 18,000 Hz on 13 C NMR,
respectively. The DS and NS were 16 and 110, respectively, and a relaxation delay of 2.0 s.
The contents of different types of lignin (G, S, and H) were determined by a semiquantitative
computational method, and the plots were based on the center peak of DMSO-d6 (δC /δH at
39.5 ppm/2.49 ppm) as an internal reference.

3.11. DAPILs Conductivity Test

Measure the conductivity of DAPILs with a conductivity meter (Shanghai Remco
DDSJ-308F, Shanghai, China). A standard solution of conductivity (1408 µs/cm) was first
prepared. A total of 0.744g KCl was put into a 1000 mL volumetric flask, and then fixed to
the scale, which was used as the standard solution. The conductivity of eight DAPILs was
then determined.

3.12. Determination of the pH Value of DAPILs

pH measurements of the DAPIL were performed using a pH meter (LeiCi PHS-3C,
Shanghai Yidian Science Instrument Co., Ltd., Shanghai, China). Prior to use, the electrode
Molecules 2025, 30, 2445 13 of 16

was rinsed with deionized water and gently dried with filter paper. Calibration was carried
out using standard buffer solutions at pH 6.86 and pH 4.00. After calibration, the electrode
was cleaned again and immersed into the DAPIL sample. After the reading stabilizes,
record the pH value, repeat twice, and calculate the average.

3.13. Meyer Bond Orders

The Gaussian 16 suite of programs was used to optimize the configurations with the
B3LYP functional and the 6-311G (d,p) basis set [72]. Meyer bond orders of all compounds
were analyzed using Multiwfn software (gaussian 16 A03) [58].

4. Conclusions
In summary, a DAPIL [EOA][GA] with dual active sites was developed in this paper.
Through the demethylation of the DAPIL system, the demethylation efficiency reached
59.3%, and the lignin polyphenol content was increased by 1.58 times. Through a series of
characterization methods, it was proved that demethylation of lignin was accompanied
by effective cleavage of β-O-4′ bond, and both ways increased the content of the phenol
hydroxyl group. This method is simple to synthesize, has a short reaction time and mild
reaction conditions, and further verifies the feasibility of customized ILs system-modified
lignin. DAPIL-modified lignin is a promising method for lignin modification. It not only
broadens the application field of DAPILs, but also provides a new way for the appreciation
of lignin. The improvement of lignin polyphenol properties has great application potential
in the pharmaceutical, chemical, construction, and other fields.

Supplementary Materials: The following supporting information can be downloaded at: https://www.
mdpi.com/article/10.3390/molecules30112445/s1.

Author Contributions: Conceptualization, C.L. and J.S.; Methodology, C.L., X.X., Q.L., W.Z. and
L.-P.X.; Software, Q.L.; Validation, W.X.; Formal analysis, C.L. and X.X.; Investigation, C.L., W.Z. and
W.X.; Resources, S.Z.; Data curation, C.L. and X.X.; Writing—original draft, C.L.; Writing—review &
editing, L.-P.X., S.Z. and J.S.; Visualization, S.Z.; Supervision, L.-P.X., Y.T. and J.S.; Project administra-
tion, J.S.; Funding acquisition, S.Z. and J.S. All authors have read and agreed to the published version
of the manuscript.

Funding: This work was supported by the National Natural Science Foundation of China (22078023,
22178037, U22A20424), Startup Foundation of Beijing Institute of Technology (3160011181808
and 3160011182007).

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement: Data are contained within the article and Supplementary Materials.

Acknowledgments: The authors thank the Biological and Medical Engineering Core Facilities of
Beijing Institute of Technology for supporting experimental equipment.

Conflicts of Interest: The authors declare no conflict of interest.

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