Enzymatic Specific Production and Chemical Functionalization of Phenylpropanone Platform Monomers from Lignin

Article abstract of DOI:10.1002/cssc.201601235

Enzymatic catalysis is an ecofriendly strategy for the production of high-value low-molecular-weight aromatic compounds from lignin. Although well-definable aromatic monomers have been obtained from synthetic lignin-model dimers, enzymatic-selective synthesis of platform monomers from natural lignin has not been accomplished. In this study, we successfully achieved highly specific synthesis of aromatic monomers with a phenylpropane structure directly from natural lignin using a cascade reaction of β-O-4-cleaving bacterial enzymes in one pot. Guaiacylhydroxylpropanone (GHP) and the GHP/syringylhydroxylpropanone (SHP) mixture are exclusive monomers from lignin isolated from softwood (Cryptomeria japonica) and hardwood (Eucalyptus globulus). The intermediate products in the enzymatic reactions show the capacity to accommodate highly heterologous substrates at the substrate-binding sites of the enzymes. To demonstrate the applicability of GHP as a platform chemical for bio-based industries, we chemically generate value-added GHP derivatives for bio-based polymers. Together with these chemical conversions for the valorization of lignin-derived phenylpropanone monomers, the specific and enzymatic production of the monomers directly from natural lignin is expected to provide a new stream in “white biotechnology” for sustainable biorefineries.

Full text of DOI:10.1002/cssc.201601235

Accepted Article
Title: Enzymatic specific production and chemical functionalization of
phenylpropanone platform monomers from lignin
Authors: Yukari Ohta, Ryoichi Hasegawa, Kanako Kurosawa, Toshio
Koizumi, Allyn H. Maeda, Hiroshi Nishimura, Hitomi Okada,
Chen Qu, Kaori Saito, Takashi Watanabe, and Yuji Hatada
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content of this Accepted Article.
To be cited as: ChemSusChem 10.1002/cssc.201601235
Link to VoR: http://dx.doi.org/10.1002/cssc.201601235
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ChemSusChem
10.1002/cssc.201601235
Enzymatic specific production and chemical functionalization of
phenylpropanone platform monomers from lignin
Dr. Yukari Ohta,*[a] Ryoichi Hasegawa,[a] Kanako Kurosawa,[a] Dr. Allyn H. Maeda,[a] Prof. Toshio
Koizumi,[b] Dr. Hiroshi Nishimura,[c] Dr. Hitomi Okada,[c] Dr. Chen Qu,[c] Dr. Kaori Saito,[c]
Prof.Takashi Watanabe,[c] and Prof.Yuji Hatada[d]
*
[
a] Dr. Y. Ohta, R. Hasegawa., K. Kurosawa, Dr. A. H. Maeda
Research and Development Center for Marine Biosciences
Japan Agency for Marine-Earth Science and Technology (JAMSTEC)
2
-15 Natsushima, Yokosuka, Kanagawa, 237-0061 (JAPAN)
FAX: +81-46-867-9595
URL: http//www.jamstec.go.jp/
E-mail: ohtay@jamstec.go.jp
b] Prof. Dr. T. Koizumi
[
Department of Applied Chemistry
National Defense Academy
1
-10-20 Hashirimizu Yokosuka, Kanagawa, 239-8686, (JAPAN)
[
c] Dr. H. Nishimura, Dr. H. Okada, Dr. C. Qu, Dr. K. Saito, Prof. Dr. T. Watanabe
Research Institute for Sustainable Humanosphere (RISH), Kyoto university
Gokasho, Uji, Kyoto, 611-0011 (JAPAN)
[d] Prof. Dr. Y. Hatada
Department of Life Sciences and Green Chemistry
Saitama Institute of Technology
1690 Fusaiji, Fukaya, Saitama, 369-0293 (JAPAN)
1
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ChemSusChem
10.1002/cssc.201601235
Abstract: Enzymatic catalysis is an eco-friendly strategy for the production of high-value
low-molecular-weight aromatic compounds from lignin. Although well-definable aromatic monomers
have been obtained from synthetic lignin-model dimers, enzymatic-selective production of platform
monomers from natural lignin has not been accomplished. In this study, we successfully achieved
highly specific production of aromatic monomers with a phenylpropane structure directly from natural
lignin using a cascade reaction of β-O-4 cleaving bacterial enzymes in one pot.
Guaiacylhydroxylpropanone (GHP) and the GHP/syringylhydroxylpropanone (SHP) mixture are
exclusive monomers from lignin isolated from softwood (Cryptomeria japonica) and hardwood
(Eucalyptus globulus), respectively. The intermediate products in the enzymatic reactions show the
capacity to accommodate highly heterologous substrates at the substrate-binding sites of the enzymes.
To demonstrate the applicability of GHP as a platform chemical for bio-based industries, we chemically
generate value-added GHP derivatives for bio-base polymers. Together with these chemical conversions
for the valorization of lignin-derived phenylpropanone monomers, the specific and enzymatic production
of the monomers directly from natural lignin is expected to provide a new stream in “white
biotechnology” for sustainable biorefineries.
Entry for the Table of Contents
Enzymatic one-pot production of specific aromatic monomers from lignin; the marine bacterial enzymes
recognize and cleave various lignin substructures near β-O-4 linkages and produce two
phenylpropanones, selectively. These monomers are promising platforms for producing bio-plastics and
other functional chemicals in medical and cosmetic industries. This study provides a new stream for
“white-biotechnology”.
Keywords: Biomass • Enzyme catalysis •Lignin • Molecular recognition • Reaction mechanisms
2
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10.1002/cssc.201601235
Introduction
Biorefineries for obtaining sustainable energy and chemical production from renewable biomass
havebeen the focus of intensive research and development owing to the depletion of world petroleum
reserves together with global warming due to anthropogenic greenhouse gas emissions.[1] In particular,
lignocellulosic biomasses such as woody feedstock, agricultural waste, and perennial grass can be used
as raw materials because they are abundant and not used for food and feed production.[2–4]
The main constituents of lignocellulosic biomasses are cellulose, hemicellulose, and lignin.[5]
Processes for converting cellulose and hemicellulose to bioethanol and chemicals have been developed,
and they play a central role in commercial practices worldwide.[4, 6] Although lignin is considered to be
the major renewable source of high-value aromatic compounds because of its intrinsic polyaromatic
chemical structure, its use has been mostly limited to low-value applications such as solid fuels and
admixtures for concrete.[7–8] Many efforts have been made to develop thermochemical, catalytic, and
enzymatic strategies for the efficient production of high-value low-molecular-weight aromatic
compounds from lignin.[8–12] Although substantial yields have been achieved using synthetic
lignin-model compounds, the yield and product distribution obtained from natural lignin are highly
dependent on the specific lignin structure, which is often modified and repolymerized in complex ways
during preparation.[13] The products of this process are almost always highly heterogeneous and thereby
hinder lignin valorization.[14]
For example, Lancefield et al.[15] reported an isolation method for simple aromatic monomers with a
phenylpropanone structure via selective oxidation of benzylic alcohol at the Cα position, followed by
reductive cleavage of the β-O-4 linkages using zinc as the reductant. Rahimi et al.[16] reported that the
formic-acid-induced preoxidized lignin depolymerization produced aromatic monomers with structural
variations, including diketones, aldehydes, and carboxylic acids. Although these methods offer some
technological applications in definable aromatics production, they require toxic compounds, heavy
metals, and high-temperature reactions. Thus, more environment-friendly methods need to be
developed.
An alternative for the specific production of aromatic monomers from lignin is enzymatic catalysis.
Several enzymes can catalyze the selective cleavage of β-O-4 linkages in lignin model dimers.[17]
Aromatic monomers were produced from lignin model synthetic dimers via a cascade reaction
comprising three steps, which involved the use of multiple enzymes derived from Sphingobium sp.
strain SYK-6. The cascade required six reactions for the conversion of racemic lignin model dimers with
3
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ChemSusChem
10.1002/cssc.201601235
two chiral carbons at the α and β positions into respective monomers. In addition to the enzymes from
the strain SYK-6, a homology-based amino-acid database search for β-O-4 cleaving enzymes were
identified from two Novosphingobium genomes.[18, 19] All six enzymatic reactions in the cascade were
strictly stereospecific.[20-22] Prior to our study, no enzyme capable of efficiently reacting with the
β(S)-isomer in the last step has been identified. Owing to the lack of genetic information regarding the
enzyme responsible for the conversion of the β(S)-intermediates into aromatic monomers, a recombinant
enzymatic process capable of producing aromatic monomers could not be developed.[23] Currently,
aromatic monomer production from lignin preparations using enzymes has not been successful and has
resulted in only a trace amount of aromatics, such as ferulic acid and vanillin from alkali lignin.[23]
Recently, we reported that a combination of six enzymes produced using genes from a marine
Novosphingobium strain, which was isolated from sunken wood in Suruga Bay, Japan,[24] entirely
converted a racemic lignin model dimer into its respective monomer in three steps (Figure 1).[17, 25] Two
short-chain dehydrogenase/reductases (SDRs; EC 1.1.1.--) (SDR3, SDR5) and two
Glutathione-S-transferases (GSTs; EC 2.5.1.18) with β-etherase activity (GST4, GST5) that catalyze the
first and second steps, respectively, were strictly stereospecific. Surprisingly, GST3 catalyzed the third
step by efficiently removing glutathione from both compounds 5 and 6 (Figure 1), which were produced
from a racemic lignin model dimer (Figure 1_a mixture of compounds 2 and 3). The discovery of the
nonstereoselective enzyme GST3 paved the way to convert all stereoisomers of compound 1 to their
respective monomers for the first time. However, a feasible method of processing natural lignin of
biomass origins using these five enzymes remains unknown in terms of reactivity and selectivity for
highly heterologous natural lignin substructures.
In this study, we investigated the optimal reaction conditions for GST3. Using the five enzymes
shown in Figure 1, we enzymatically produced phenylpropanone monomers from isolated natural lignin.
To elucidate the catalytic mechanism of the β-O-4 cleavage of natural lignin, we analyzed the
intermediates produced from lignin. Finally, we assessed the future prospects of these phenylpropanone
monomers as new platform chemicals derived from natural lignin.
Results and Discussion
Optimal pH and temperature for GST3 activity
4
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10.1002/cssc.201601235
We identified the optimal pH values and temperatures for the activity and kinetic parameters of SDRs
SDR3, SDR5) and GSTs (GST4, GST5).[25] In contrast, GST3 characterization has not been directly
performed because the substrates for GST3, glutathione-conjugated GHPs (GS-GHPs) (Figure
_compound 5 and 6), have not been commercially available. Here we enzymatically produced GS-GHPs
from racemic 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-1-propanone
(2-methoxyphenoxy)hydroxypropiovanillone; MPHPV] (Figure 1_compound 2 and 3) using GST4 and
(
1
[
GST5. The reaction products were designated as GS-GHPgst4 and GS-GHPgst5 (Figure 1_compound 5
and 6), respectively. Then, we purified each of the GS-GHPs. Using purified each of GS-GHPs as a
substrate, we investigated the effect of pH on GST3 activity. The optimum pH value and temperatures
for GST3 activity were approximately 8.0 (Figure 2a, b) and 25°C–30°C (Figure 2c, d), respectively, both
for GS-GHPgst4 and GS-GHPgst5. The specific activities in 0.1-M
N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid buffer (TAPS) pH 8.5 at 15°C were 3.9 and
-1
9
.1 U mg protein for GS-GHPgst4 and GS-GHPgst5, respectively.
GHP (Figure 1_compound 7) production via enzymatic cascade reactions from milled wood lignins
(MWLs)
Each enzyme retained more than 80% of the respective maximal activity at pH 8.5.[25] The optimal
temperature of SDR3 was 15°C, lower than those of the other enzymes, indicating the instability of
SDR3 at a higher temperature range. SDR3 was thought to be the limiting reaction step in the
enzymatic cascade reactions. Accordingly, the following enzyme reactions were conducted under
optimal conditions for SDR3 activity (0.1-M TAPS at pH 8.5 at 15°C) using the enzymes simultaneously
in one pot.
MWLs from softwood and hardwood were prepared from Japanese cedar (Cryptomeria japonica) and
Eucalyptus globulus wood and are here designated C-MWL and E-MWL, respectively The main product
from C-MWL was determined to be GHP according to the retention time (t ) on the LCMS
R
chromatogram, observed molecular mass, calculated elemental composition, fragmentation pattern on
mass spectra, and UV spectrum using authentic GHP as a reference (Figure 3a, c and Figure S5a, c in
-1
the Supporting Information). The yield of GHP was 2.4 ± 0.005 wt % [24 ± 0.05 mg g lignin]. The main
products from in the Supporting Information E-MWL were determined to be GHP and SHP using
authentic GHP and SHP as references (Figure 3b, c and Figure S5b, c in the Supporting Information).
5
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The yields of the two compounds were 1.9 ± 0.012 and 4.7 ± 0.025 wt % [19 ± 0.12 and 47 ± 0.25 mg g-1
lignin], respectively. To the best of our knowledge, this is the first report of enzymatic production of
phenylpropanone monomers from MWL.
There have been numerous studies on the production of aromatic monomers from lignin[7-9]. For
example, pyrolysis and cracking processes have been extensively studied for decades. The major
products from different types of pyrolysis processes are complex mixtures of deoxygenated aromatic
monomers such as vinylphenols, toluene, xylene, and many other aromatics along with gaseous
products, i.e., hydrocarbons of low molecular weight. For the cracking process, a high yield of
approximately 80 wt % was recorded for a mono/oligomeric phenolic mixture. The common drawback of
pyrolysis and cracking processes is the required harsh operating conditions (a high temperature range
between 300–650°C under high pressures) and side reactions that are difficult to control. To overcome
these problems, hydrogenolysis and hydrolysis under subcritical/supercritical conditions with various
catalysts operating at temperatures below 300°C have been attempted and have drawn attention for
their potential to be viable new methods. As an example of a significant study in this field, nickel-based
catalysts were used for the selective production of propylguaiacol and propylsyringol from sawdust with
approximately 50 wt % of lignin conversion at 200°C[26]. The supply of hydrogen and recycling of the
catalysts is a major issue in the process that must be addressed though. Another excellent contribution
in chemical catalysis was reported by Lancefield et al.[15] The production of the phenolic monomers
(GHP and SHP) was conducted at 80°C using a chlorinated oxidizer and zinc as a reductant under
atmospheric pressure in the presence of air. The production yields of GHP and SHP from a birch lignin
preparation were 0.46 and 4.6 wt %, respectively. Although these values depend strongly on the
biomass source and process of lignin preparation,[13] the yields of enzymatically-produced GHP and SHP
in this study were comparable to those obtained by chemical catalysis using toxic halogenated
aromatics and heavy metals. The theoretical yield of these monomers (GHP plus SHP) was calculated to
be approximately 12 wt % based on the content of releasable β-O-4 linkages[15]. The experimental yields
obtained by Lancefield et al. and by us were similar and approximately half of the theoretical value. The
yield limitation in the chemical catalytic process was estimated to be owing to the formation of
by-products that include unknown repolymerized products. We believe that in our ongoing and future
studies it should be possible to increase the yields obtained in our study by improving the catalytic
property of the enzymes such as by improving their catalytic efficiencies and substrate recognition
capacities as well as improving their protein stabilities via protein engineering. Furthermore,
6
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ChemSusChem
10.1002/cssc.201601235
conducting the enzymatic process under mild conditions shows promise for avoiding repolymerization,
which is likely what leads to the formation of unwanted by-products.
Enzymatic production of aromatic monomers is an attractive alternative to thermal and chemical
conversion of lignin. Many microbial enzymes have been reported to have the ability to produce
lignin-related aromatic monomers. Although fungal and bacterial peroxidases can break the linkages of
inter lignin monomers using extracellular radical molecules, the enzyme activity and specificity is low
and can even cause repolymerization during the enzymatic reaction. Other bacterial enzymes that have
been examined to produce aromatic monomers including vanillin, cafeic acid 4-vinylguaiacol, coniferyl
alcohol, and GHP using ferulic acid[27], eugenol[28], and lignin model dimers[22, 23] as starting materials;
however, these enzymes have not successfully been used for natural lignin preparations. In contrast,
there have been a few reports demonstraiting microbial production of aromatic monomer directly from
lignin. For example, metabolically-engineered Rhodococcus jostii RHA1 reportedly produced vanillin up
to 96 mg mL after 6-days cultivation in the media containing lignocellulose [29]. Although this study
-1
demonstrated possibility of biological production of the aromatic monomer, vanillin has already
[9]
produced chemically from lignosulfonate at industrial scale .
In our study, the enzyme reactions were conducted with MWLs suspended in an aqueous buffer with
a small amount of organic solvent. At present, we use N,N-dimethylformamide to solve MWL. However,
in a scaled-up production we propose that a safer and greener solvent should be used instead.
Biomass-based solvents with lower boiling points than the products, such as acetone, butanol, or
tetrahydrofuran[30], are potential candidates to increase the sustainability of the enzymatic production
of GHP and SHP.
In general, the productivity must reach 1–3 g L−1 h−1 for the products to reach economic viability for
bio-based chemicals[31]. To meet this goal, the productivity (approximately 0.005 g L−1 h−1 of the
products in the current experimental setting) must be increased by 2–3 orders magnitude by operating
at higher volumetric concentrations. In addition, it is necessary to enhance the enzymatic activity and
stability by protein engineering and stoichiometric optimization of the multiple reactions[32] to reduce
+
the loadings on the enzymes and improve the yields. In addition, recycling of the cofactors of NAD and
GSH as well as an eco-friendly pretreatment for biomasses suitable for enzymatic reactions are needed
for the cascade presented in this study to become economically sound.
Product recovery was conducted in this study by liquid phase extraction using ethyl acetate as an
organic solvent. The other practical product recovery options need to be assessed and optimized because
7
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10.1002/cssc.201601235
the processes accounts for a major part of a total cost and is critical for achieving sustainable production
of bio-based chemicals.[33] In addition, the residual insoluble and soluble lignin fractions have to be
recovered and analyzed for efficient utilization for entire lignin valorization. Although there are many
problems to overcome, further optimization of each process in the upstream and downstream processes
are possible and provide grounds for further study.
Analysis of substrate recognition tolerance of the enzymes for MWL substructures
The bacterial enzymes involved in the cleavage of β-O-4 ether linkages have been considered to show
substantial activity only on dimeric lignin-model synthetic compounds, with low activity on polymeric
substrates.[18] However, our enzyme system produced phenylpropanone monomers. To elucidate the
reaction mechanism of the enzymes displaying substrate recognition tolerance for unknown
substructures present in MWL, we analyzed the reaction products from C- and E-MWL by the four
enzymes (SDR3, SDR5, and GST4–5). The glutathione conjugates produced by the enzymes were
analyzed by LCMS (Figure S6a, b in the Supporting Information). The glutathione conjugates obtained
from authentic guaiacylglycerol-β-guaiacyl ether (GGGE, Figure1_compound 1) by the same enzyme set
were analyzed under the same conditions as the references (Figure S6c in the Supporting Information).
The observed molecular masses in the LCMS analyses and calculated elemental compositions of the
main two reaction products (t
in the Supporting Information) and C20
molecular ion of glutathione-conjugated GHP [GS (elemental composition, C10
on LCMS chromatograms, observed molecular mass, and fragmentation patterns on the mass spectra
R
; 1.69 and 1.97 min) from C-MWL were the same, m/z 500.13 (Figure S7a
10S, in agreement with those of the deprotonated parent
]. The
H
26
N O
3
16
H N
3 6 11
O S)-C10H O
4
t
R
of the major products from C-MLW agreed well with those of the products from the enzyme reactions
using GGGE as a substrate (Figure S7c in the Supporting Information). From these results, the two
peaks were inferred to be isomers. Four major products were produced in the case of E-MWL (Figure
S8b in the Supporting Information). MS data from two peaks (t
those from C-MWL, and the others (t ; 1.86 and 2.03 min) were molecules with m/z 530.14 and
calculated compositions of C21 11S, in agreement with those of the deprotonated parent ion of
glutathione-conjugated SHP (GS-C11 ). The MS spectra contained several fragment ions, such as
R
; 1.64 and 1.96 min) were the same as
R
H
28
N O
3
H O
13 5
m/z 272.09 and 254.08 (Figure S8a–c in the Supporting Information), in common with the MS spectrum
8
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ChemSusChem
10.1002/cssc.201601235
obtained from authentic glutathione (Figure S7d in the Supporting Information). These fragment ions
were speculated to have been derived from the conjugated glutathione.
LCMS/MS analyses revealed that the reactions using C- and E-MWL as the substrates produced
numerous molecules with different molecular size, providing deprotonated molecular ions ranging from
m/z 500.13 to 964.30 (Figure S8 in the Supporting Information). The observed molecular masses,
fragment ions, and calculated elemental compositions are listed in Table 1. These results suggested that
these enzymes could accommodate not only lignin model dimers but also lignin oligomers consisting of
approximately 10–30 carbon atoms as their substrates and cleave the β-O-4 linkages in natural lignin
with highly heterologous substructures.
GHP transformation into various derivatives
We investigated the chemical conversion of GHP to show the feasibility of effective lignin use with a
wide range of applications (Figure 4).
1
8
-Guaiacyl-1,3-propanediol (GPD, 1-(4-hydroxy-3-methoxyphenyl)-1,3-propanediol, Figure 4_compound
) synthesis from GHP
The simple and convenient GPD synthesis was achieved by the reaction of GHP with NaBH (Figure 4a).
4
The reaction proceeded completely at room temperature in aqueous NaOH to afford GPD in 79%. The
pH adjustment to approximately 6–9 was important for the efficient separation of GPD from the
reaction mixture. Some methods for synthesizing GPD have been reported, but multi-step processes are
required.[34, 35]
3
,3-Bisguaiacyl-1-propanol (BGP, 3,3-bis(4-hydroxy-3-methoxyphenyl)-1-propanol, Figure 4_compound
) synthesis from GPD under acidic conditions
9
We found that GPD shows high reactivity with both acids and alkalis. In the presence of an acid
catalyst, its reactivity leads to the formation of BGP (Figure 4b, Figure 2a, b in the Supporting
Information), a bisphenol that may be used as a raw material for epoxy resins; as a hardener for epoxy
and urethane resins; and as a raw material for new functional polyesters and polycarbonates. In the
9
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ChemSusChem
10.1002/cssc.201601235
earlier study,[36] the reaction of GPD with an excess of phenol was performed using hydrochloric acid to
give 3-(hydroxyphenyl)-3-(4-hydroxy-3-methoxyphenyl)-1-propanol, an unsymmetry compound and
possibly a mixture, in view of the two reaction sites of phenol.
Coniferyl alcohol (Figure 4_compound 10) synthesis from GPD under basic conditions
Coniferyl alcohol is a monolignol, which is a precursor of lignin biosynthesis,[37] and has important
applications in research areas as an artificial lignin. Coniferyl alcohol can be converted to valuable
aromatic compounds, such as medicines for cancers and diabetes; functional foods and cosmetics with
antioxidizing activity; and pesticides.[38] We achieved the formation of coniferyl alcohol from GPD under
heating conditions using triethylamine as the base and isopropanol as the solvent (Figure 4c). The
choice of base and solvent was crucial because coniferyl alcohol tends to polymerize under basic
conditions, particularly in aqueous solvents. The yield was higher with an organic base such as
triethylamine. Methanol or ethanol as the solvent led to the formation of
3
-(4-hydroxy-3-methoxyphenyl)-3-alkoxy-1-propanol (an alkoxylated compound) as the main product.
Guaiacylvinylketone (GVK, 1-(4-hydroxy-3-methoxyphenyl)-2-propene-1-one, Figure 4_compound 12)
synthesis from GHP
GVK synthesis (Figure 3d) was performed according to a method used for
1
1
-(4-hydroxyphenyl)-2-methyl-2-propene-1-one synthesis from
-(4-hydroxyphenyl)-3-hydroxy-2-methyl-1-propanone.[39] The reaction of GHP with hydrochloric acid
was conducted at approximately 70°C to provide a high yield of
-chloro-1-(4-hydroxy-3-methoxyphenyl)-1-propanone (96%)( Figure 4_compound 11). The chlorinated
3
product was treated with sodium ethoxide in ethanol to GVK in 61%. We found that this
dehydrochlorination reaction also proceeded in NaOH aqueous solution under mild conditions, between
room temperature and approximately 60°C.In our preliminary polymerization experiment of GVK using
azobisisobutyronitrile and tetrahydrofuran as the radical initiator and solvent, respectively (Figure 4e),
a solid was obtained. The 1H-NMR spectrum of the compound confirmed the presence of aliphatic
methylene and methine protons and the absence of vinyl protons (Figure S4 in the Supporting
Information), indicating polymerization via the vinyl group.
Conclusions
1
0
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An enzymatic strategy for the highly selective production of aromatic monomers with phenylpronanone
structure from MWL isolated from hardwood and softwood was demonstrated using five enzymes in a
one-pot. The recognition capacity of the broad substrate range by these enzymes may shed light on the
mechanism by which the enzymes cleave β-O-4 linkages in natural lignin preparations that show high
structural heterogeneity. The selectivity of the enzymatic one-pot reaction under mild conditions is
appealing. Still, the process presents problems to overcome, including the costs of the enzyme and
cofactor production, wastewater treatment, and utilization of the remaining lignin fraction. All the
derivatives obtained in this study retain the aromatic rings, phenolic hydroxyl groups, and methoxy
groups in their structure. This finding suggests that these compounds have various applications as
lignin-derived materials. The chemical conversions in this study demonstrated that the
enzymatically-produced phenylpropanone monomers will be the platform aromatic chemicals for
sustainable industries based on woody biomasses.
Experimental Section
General methods
1
Commercially available compounds were purchased and used as received unless otherwise stated. H
and 13C NMR spectra were recorded on a Varian Inova 400- and 500-MHz spectrometer. Multiplicities
were described using the following abbreviations: s, singlet; d, doublet; t, triplet; and m, multiplet; the J
couplings were reported in Hz. IR spectra were obtained on a JASCO FT/IR-6200 type A Fourier
transform IR spectrophotometer from KBr discs; only characteristic peaks were reported. LCMS data
were generated using a Waters Xevo G2 quadrupole time-of-flight mass spectrometer operated in
negative ion ESI mode. The inlet system was a Waters Acquity H-class UPLC system and was operated
at a flow rate of 0.4 mL min−1 using a BEH C18 reverse-phase column (1.8-μm particle size, 100 × 2.1
mm; Waters) using the mobile-phase gradients A (2-mM sodium acetate and 0.05% formic acid) and B
(
95% acetonitrile/H O) under the following conditions: 0–6 min, 95%–5% A with B as the remaining
2
eluent; and from 6–7 min, 100% B. The eluate was monitored at 270 nm using a Waters photo diode
array (PDA) eλ detector. Data were acquired over the mass range of 100–1000 Da with a 0.45-s scan
time using a desolvation temperature of 500°C, source temperature of 150°C, and cone voltage of 30 V.
1
1
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ChemSusChem
10.1002/cssc.201601235
HPLC analysis was performed using a Waters Alliance 2796 liquid chromatography (LC) system
equipped with an Xbridge C18 reversed-phase column (3.5-μm particle size, 100 × 4.6 mm; Waters)
-1
operated at a flow rate of 1.2 mL min using the mobile-phase gradients A (2-mM sodium acetate and
0
.05% formic acid) and C (95% methanol/H O) under the following conditions: 0–1 min, 90% A and 10%
2
C; 1–8 min, a decreasing gradient of 90%–10% A with C as the remaining eluent; followed by 8–10 min
00% C. The eluate was monitored at 270 nm using a Waters 2998 PDA detector. Chiral
1
chromatography of the enzymatic reaction mixtures was performed using an Alliance 2796 LC system
equipped with a CHIRALPAK IE-3 column (4.6 × 250 mm; Daicel Chemical Industries). A acetonitrile
-1
and H
concentration of the mobile phase was adjusted as follows (the remaining eluent was H
0% acetonitrile; 10–15 min, gradient from 20% to 30% acetonitrile; and 15–30 min, 30% acetonitrile.
2
O mixture was used as the mobile phase at a flow rate of 1.0 mL min . The acetonitrile
2
O): 0–10 min,
2
The eluate absorbance was monitored at 270 nm using a Waters 2998 PDA detector. The recombinant
enzymes [SDR3 (accession; GAM05523), SDR5 (accession; GAM05547), GST3 (accession; GAM05529),
GST4 (accession; GAM05530), and GST5 (accession; GAM05531)] were prepared as previously
reported.[25]
Lignin model dimer synthesis
GGGE (Figure 1_compound 1) and MPHPV (Figure 1_compound 2, 3) were synthesized as previously
described.[25]
Biochemical characterization of GST3
A glutathione-conjugated intermediate (GS-GHP) was enzymatically produced by the reaction of either
GST4 and GST5 with 2-mM MPHPV using 4-mM glutathione as a cofactor at 20°C for 16 h. Each of the
products (GS-GHPgst4 and GS-GHPgst5) was then purified by solid-phase extraction from the
respective reaction mixture, as follows. The reaction mixture was diluted 10-fold with methanol and
applied to a phenomenex Strata extra clean NH column. After the column was washed with distilled
2
water, GS-GHP was eluted with 10-mM NaCl. GS-GHP concentration was calculated based on GHP
concentration determined by HPLC after complete removal of conjugated glutathione by addition of
−1
excess GST3 (0.2 mg mL ) and incubation at 20°C for 16 h. GST3 was reacted with 1-mM GS-GHPgst4
1
2
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ChemSusChem
10.1002/cssc.201601235
or GS-GHPgst5 as a substrate and 2-mM glutathione as a cofactor at 25°C for 30 min. The formation of
the reaction product GHP was measured by HPLC. The determination of the pH optimum for GST3
activity was performed using the following buffers (0.1 M): 2-(N-morpholino)ethanesulfonic acid (MES)
(pH 5.5–7.0), 3-[N-morpholino]propanesulfonic acid(MOPS) (pH 7.0–8.0), TAPS (pH 8.0–9.0),
N-cyclohexyl-2-aminoethanesulfonic acid (CHES) (pH 9.0–10.0), and
N-cyclohexyl-3-aminopropanesulfonic acid (CAPS) (pH 10.0–11.0). The optimal temperature was
determined by quantifying the reaction product (GHP) after a 30-min reaction in 0.1-M TAPS, pH 8.5.
All experiments were performed in triplicate.
MWL preparation and enzyme reaction with MWLs
The wood meal was extracted with a toluene and ethanol (2:1, v/v) mixture using a Soxhlet extractor for
10 h. The wood meal was dried at 105°C for 12 h and finely divided in a vibratory ball mill for 48 h
under a nitrogen atmosphere with constant cooling using running water. The wood meal was extracted
with 96% aq. dioxane for 24 h at room temperature. The extract was evaporated and then freeze-dried.
The crude MWL was dissolved in 90% aq. acetic acid and then precipitated from distilled water. The
precipitates were dissolved in a 1,2-dichloroethane and ethanol (2:1, v/v) mixture and added to diethyl
ether. The precipitate was washed with petroleum ether and evaporated to give the MWL fractions. C-
or E-MWL was dissolved at 10% w/v in N,N- dimethylformamide and then suspended at 0.2% w/v in
0
.1-M TAPS, pH 8.5. The mixture was incubated with enzymes (SDRs, 50 mU mL−1 and GSTs, 5 mU
−1
mL ) at 15°C for 24 h in the presence of cofactors (10-mM NAD sodium salt and 20-mM glutathione).
Parallel reactions were conducted without enzymes to assess the non-enzymatic production of
GHP/SHP. For quantification of the reaction products, the reaction mixtures were extracted three times
with four volumes of ethyl acetate. The extract was dried under N gas at room temperature and then
2
dissolved in 20% acetonitrile and analyzed with LCMS coupled with a UV detector. The GHP/SHP
concentrations in samples were calculated from the peak area under the UV chromatogram at 270 nm
using authentic GHP/SHP as standards. All samples were normalized to ethyl ferulate as an internal
standard. All reactions were performed in triplicate.
Chemical synthesis of GHP derivatives
1
3
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10.1002/cssc.201601235
1
-Guaiacyl-1,3-propanediol (GPD, 1-(4-hydroxy-3-methoxyphenyl)-1,3-propanediol) synthesis from GHP
GHP (2.05 g, 10.5 mmol) was dissolved in a 200-mL Erlenmeyer flask containing 100 mL water and 1-N
aqueous sodium hydroxide (11.2 g, 11.2 mmol). Sodium borohydride (0.48 g, 12.7 mmol) was slowly
added over approximately 10 min at room temperature (26°C); the mixture was allowed to react for 24 h
at 26°C. After confirming the complete conversion of GHP by HPLC, 10% aqueous hydrochloric acid (6.1
g) was slowly added dropwise to adjust the pH to 9.0. The mixture was extracted twice with 70 mL of
1
-butanol and then the combined 1-butanol layers were washed with 30 mL of 1.5% aqueous sodium
bicarbonate and further with 30 mL of water. 1-butanol was removed by reduced-pressure distillation to
provide 1.63 g of transparent, viscous GPD in a 79% yield. The 1H-NMR spectrum (Figure S1 in the
Supporting Information) and HRMS of synthetic GPD were as follows.
1H-NMR (D
2 2 2 3
O,  ppm): 1.93–2.12 (m, 2H, -CH -), 3.57–3.70 (m, 2H, -CH -O-), 3.90 (s, 3H, -OCH ), 4.78
(
(
dd, J = 7.6 Hz, J = 6.4 Hz, 1H, -CH=), 6. 90–6.95 [m, 2H, Ar-H(-5, -6)], and 7.06 [d, 1H, Ar-H(-2)]. HRMS
−
ESI): m/z = [M–H] , 197.0810 (calcd mass: 198.0892, formula: C10
H
14
O )
4
BGP synthesis from GPD
Water (15 mL) and GPD (0.4 g, 2.0 mmol) were placed in a 50-mL round-bottomed flask; the GPD was
dissolved at room temperature. Methanesulfonic acid (44 mg, 0.46 mmol) was added; the mixture was
allowed to react for 22.5 h at 65°C. Aqueous NaOH (1 N, 0.46 g) was added to neutralize the acid
catalyst, methanesulfonic acid, and then the target product was extracted twice (10 and 5 mL) with
ethyl acetate to provide 0.32 g of a viscous liquid. The HPLC purity of the liquid,
3,3-bis(4-hydroxy-3-methoxyphenyl)-1-propanol, was 68% (determined by area normalization without
sensitivity correction). This liquid was chromatographed on a silica-gel column (Wakogel C-200) with
ethyl acetate:toluene (2:3) to provide 0.15 g of purified product (yield: 49%). The NMR spectra (Figure
S2a, b in the Supporting Information), HRMS, and IR of synthetic BGP were as follows.
1H-NMR (CDCl
,  ppm): 1.34 (m, H, -OH(-CH
-OH)), 2.23 (ddd, J = 8.0 Hz, J = 6.4 Hz, J = 6.4 Hz, 2H,
), 3.98 (t, J = 8.0 Hz, 1H, -CH=), 5.53 (s, 2H, Phenol-
3
2
-
CH -), 3.61 (m, 2H, -CH
2
2
-O-), 3.82 (s, 6H, -OCH
3
OH), 6.70 [d, J = 2.0 Hz, 2H, Ar-H(-2)], 6.76 [dd, J = 8.4 Hz, J = 2.0 Hz, 2H, Ar-H(-6)], and 6.84 [d, J = 8.4
Hz, 2H, Ar-H(-5)]. 13C-NMR (CDCl
,  ppm): 38.76 (-CH -), 46.75 (-CH=), 55.92 (-OCH ), 61.23 (-CH O-),
3
2
3
2
1
4
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ChemSusChem
10.1002/cssc.201601235
110.65, 114.29, 120.20 (ArC protonated), 136.82, 144.11, and 146.58 (ArC substituted). HRMS (ESI):
−
−1
m/z = [M–H] , 303.1227 (calcd mass: 304.1311, formula: C17
H
20
O ). IR (cm ): 3530, 3451, 3139, 3010,
5
2962, 2936, 2894, 2842, 1604, 1514, 1275, 1034, and 817
Coniferyl alcohol synthesis from GPD
GPD (164 mg, 0.83 mmol), isopropanol (2 g, 33.3 mmol), and triethylamine (0.4 g, 4.0 mmol) were placed
in a 10-mL test tube and stirred for 48 h at 85–90°C, after which the reaction mixture was transferred
to a 50-mL round-bottomed flask and 20 g of water was added. Volatiles were removed by
reduced-pressure distillation in a 45°C bath until the reaction mixture weighed 7 g, after which 60 mg
of 10% HCl was added. The reaction mixture turned cloudy at pH 5. The mixture was transferred to a
separatory funnel and extracted twice with 2 ml of ethyl acetate. Evaporation of the ethyl acetate
provided a viscous liquid product, coniferyl alcohol (118 mg, 79%; HPLC purity, 90%). The crude
coniferyl alcohol (118 mg) was chromatographed on a glass column (inner diameter: 23 mm, packed
length: 100 mm) packed with silica gel (Wakogel C-200) using a 2:3 ratio of ethyl acetate:toluene. The
purified coniferyl alcohol (83 mg) was obtained as white needle crystals (HPLC purity: 97%, melting
point: 72–74°C).
GVK synthesis from GHP
GHP (2.0 g, 10.2 mmol) was dissolved in 24 g of concentrated hydrochloric acid (36% HCl) in a 100-mL
Erlenmeyer flask, after which the reaction mixture was placed in an oil bath and the reaction was
performed for 0.5 h at 90°C. The flask was cooled to room temperature. The resulting crystals were
filtered off, washed with water, and dried in vacuo to yield 2.1 g of
3
-chloro-1-(4-hydroxy-3-methoxyphenyl)-1-propanone (2.1 g, 96%). The HPLC purity was 96% and the
impurity included unreacted GHP.
-Chloro-1-(4-hydroxy-3-methoxyphenyl)-1-propanone (1.41 g, 6.5 mmol) was dissolved in a 100-ml
Erlenmeyer flask containing 20 mL of ethanol at room temperature, after which sodium ethoxide (0.9 g,
3.2 mmol) was added at room temperature. After 1.5 h, the reaction mixture was neutralized by
3
1
addition of 10 ml of water and 2.4 g of 10% HCl. Removal of ethanol by reduced-pressure distillation
resulted in the separation of a brown, oily substance, which was extracted with 10 mL of ethyl acetate
1
5
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ChemSusChem
10.1002/cssc.201601235
and washed with water. The ethyl acetate was removed by reduced-pressure distillation, forming 1.22 g
of crude GVK with a HPLC purity of 93%. The crude GVK was chromatographed on silica gel (Wakogel
1
C-200) using toluene. The purified product yield was 61% and its HPLC purity was 99%.The H-NMR
spectrum (Figure S3 in the Supporting Information) and HRMS were as follows.
1H-NMR (acetone-d
J = 16.8 Hz, J = 2.0 Hz, 1H, C=CH
H, -CH=), 7.61 [d, J = 2.0 Hz, 1H, Ar-H(-2)], 7.65 [dd, J = 8.4 Hz, J = 2.0 Hz, 1H, Ar-H(-6)]. HRMS
,  ppm): 3.93 (s, 3H, -OCH
), 5.85 (dd, J = 10.4Hz, J = 2.0Hz, 1H, C=CH ), 6.36 (dd,
6
3
2
2
), 6.95 [d, J = 8.0 Hz, 1H, Ar-H(-5)], 7.39 (dd, J = 16.8 Hz, J = 10.4Hz,
1
−
(
ESI): m/z = [M–H] , 177.0550 (calcd mass: 178.0630, formula: C10
H
10
O )
3
Radical polymerization of GVK
Crude GVK (160 mg) obtained above, 1 g of tetrahydrofuran dried for 2 days over a 3Å molecular sieve,
and 7 mg of 2,2’-azobisisobutyronitrile as a radical initiator were placed in a 10-mL test tube, sealed
with a silicone rubber stopper, and purged with nitrogen, after which the tube was placed in a silicone
oil bath at 60°C and left to react for 45 h. The solvent was removed by reduced-pressure distillation to
yield a pale-yellow powder (151 mg). The polymerization product (38 mg) was dissolved in 5 g water; 8
mg of aqueous 1-M NaOH was added with agitation at room temperature. HCl (10%) was added to
lower the pH to approximately 6; the deposited precipitant was filtered and washed with water. Drying
in a desiccator formed a 38-mg solid. The NMR spectra of the compound are shown in Figure S4a, b in
the Supporting Information.
Acknowledgements
This research was funded in part by the Funding Program for Next Generation World-Leading
Researchers (number GS031) of the Japan Society for the Promotion of Science (JSPS) awarded to Y.O.,
a JSPS KAKENHI Grant (number 15K00649) awarded to Y.O., a research grant for Exploratory
Research on Sustainable Humanosphere Science from Research Institute for Sustainable
Humanosphere (RISH), Kyoto University and the Advanced Low Carbon Technology Research and
Development Program (ALCA) of the Japan Science and Technology Agency (JST) awarded to Y.O. and
H.N. We thank Mr. Makoto Roppongi (University of Utsunomiya) for technical assistance.
1
6
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10.1002/cssc.201601235
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[39] M. M. Curzu, G. A. Pinna, D. Barlocco, C. Bugatti, G. Cignarella, Synthesis (Stuttg) 1984, 4,
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Captions for Figures
Figure 1. The enzymatic cascade for GHP (compound 7) production from lignin model dimers (GGGE,
compound 1) via MPHPV (compound 2, 3). The responsible enzymes and their required cofactors are
shown. Abbreviations: SDR, short chain dehydrogenase–reductase; GST, glutathione-S-transferase;
GS-GHPgst4 (compound 5), glutathione conjugate of GHP produced by β-O-4 bond cleavage and
removal of guaiacol (compound 4) by GST4; GS-GHPgst5(compound 6), glutathione conjugate of GHP
+
produced by GST5; NAD , oxidized form of nicotinamide adenine dinucleotide (NAD); GSH, reduced
form of glutathione; and GSSG, oxidized form of glutathione. Protein accessions in the
DDBJ/EMBL/GenBank database; SDR3 (GAM05523), SDR5 (GAM05547), GST3 (GAM05529), GST4
(GAM05530), and GST5 (GAM05531).
Figure 2. The pH-activity curves of the purified GST3 using GS-GHPgst4 (a) and GS-GHPgst5 (b) as the
substrates, respectively, are shown. The buffers used were 0.1 M MES (pH 5.5–7.0; closed circles),
MOPS (pH 7.0–8.0; open squares), TAPS (pH 8.0–9.0; closed triangles), CHES (pH 9.0–10.0; open
circles), CAPS (pH 10.0–11.0, closed squares). The activity was measured in the buffers including at
25°C. The values are shown as percentages of the maximal activity of GST3 observed at pH 7 for
GS-GHPgst4 and pH 8 for GS-GHPgst5, which are taken as 100%. The temperature-activity curve of
the purified GST3 using GS-GHPgst4 (c) and GS-GHPgst5 (d) as the substrates, respectively, are shown.
The values are shown as percentages of the maximal activity of GST3 observed at 25°C for GS-GHPgst4
and 30°C for GS-GHPgst5, which are taken as 100%.
Figure 3. One-pot enzymatic production of GHP and SHP from C-MWL (a) and E-MWL(b). Total ion
chromatograms obtained from LCMS analysis of the reaction of five enzymes (SDR3, SDR5, and GST3–
5
) with the MWLs and cofactors are shown (top). The reactions were conducted under the same
conditions without enzymes but with cofactors to assess the non-enzymatic production of GHP/SHP
bottom). The authentic GHP (c; top) and SHP (c; bottom) were analyzed under the same conditions.
(
1
9
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ChemSusChem
10.1002/cssc.201601235
Figure 4. A schematic diagram of the chemical synthesis that yields functional chemicals from GHP, a
key platform compound. Reaction conditions; a) NaBH , 26°C, 24 h, 79% for GPD (compound 8); b)
methanesulfonic acid, 65°C, 22.5 h, 49% for 3, BGP (compound 9); c) isopropanol and trimethylamine,
4
85°C–90°C, 48 h, 79% for coniferyl alcohol (compound 10); d) HCl, 90°C, 96% for
3-chloro-1-(4-hydroxy-3-methoxyphenyl)-1-propanone (compound 11); and e) sodium ethoxide, RT, 1.5 h,
61% for GVK (compound 12).
2
0
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10.1002/cssc.201601235
Figures
Figure 1
Figure 2
2
1
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10.1002/cssc.201601235
Figure 3
2
2
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10.1002/cssc.201601235
Figure 4
2
3
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ChemSusChem
10.1002/cssc.201601235
Table
1
Glutathione-conjugated intermediate products from MWLs
produced by enzymatic reaction (SDR3, SDR5, GST4, and GST5) detected
by LCMS/MS.
Detected
parent
Rete
n-tio
n
Detected
Detected fragment
ion[
ion
M–H] ;
parent ion
b]
Source
−
−
[
time
[min]
[M–H] ;
[m/z]
[
c]
[
5
5
5
HRMS,
m/z]
[HRMS, m/z]
[
a]
482, 470, 464, 272,
1.59,
C
H
20 27
N O
3 10
S
4
00.1342
00.1318
30.1464
C-MWL
E-MWL
E-MWL
254, 210, 179, 143,
1.94
[GS-C10H
11
O
]
128
482, 470, 464, 272,
1.52,
C
H
20 27
N O
3 10
S
4
254, 210, 179, 143,
1.93
[GS-C10H
11
O
]
128
512, 500, 494, 383,
C
[
H
21 29
N O
3 11
S
5
1.95
272, 254, 239, 210,
GS-C11H
13
O
]
179, 143, 128
3
1
63, 306, 288, 272,
37
6
36.1857
78.1962
C-MWL
C-MWL
2.31
2.58
ND
C
[
30
H
37
N
3
13
O S
6
660, 648, 642, 272
GS-C20H
21
7
O ]
678.1972
706.1913
738.2160
856.2609
E-MWL
E-MWL
E-MWL
C-MWL
2.57
2.51
2.43
2.92
660, 648, 272
688, 419, 272, 254
720, 306, 272, 254
855, 838, 306
ND
ND
ND
C
40
H
49
N
H
3
O
17
S
874.2701
C-MWL
2.56
856, 306, 272
[GS-C30
33
O
11
]
8
56, 843, 838, 802,
782, 519, 466, 321,
72, 207, 143, 140
306, 305, 272
C
40
H
49
N
H
3
O
17
S
874.2723
E-MWL
2.56
[GS-C30
33
O
11
]
2
9
32.3058
64.3091
E-MWL
E-MWL
3.06
2.55
ND
ND
9
3
63, 946, 782, 536,
06, 272
9
[a] The MS chromatograms obtained from LCMS/MS analysis after
enzymatic reactions with C- and E-MWLs are shown in Figure S9 in the
Supporting Information. [b] The fragment ions derived from glutathione
(
Figure S8d in the Supporting Information) are underlined. [c] ND, not
determined and GS, conjugated glutathione (elemental composition:
S-).
10 16 3 6
C H N O
2
4
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