Selective laccase-catalyzed dimerization of phenolic compounds derived from lignin: Towards original symmetrical bio-based (bis) aromatic monomers

Article abstract of DOI:10.1016/j.molcatb.2015.12.006

A laccase-catalyzed process was developed to prepare, selectively, in high yield, dimers of lignin-based phenolic compounds without any purification. The influence of experimental parameters such as laccase loading, nature of solvent and the presence of oxygen on the conversion of vanillin was investigated. After the dimerization, the product obtained as a precipitate is filtered off and the solution containing the enzyme can be re-used several times, which improves the process economics. A phenolic-substrate screening reveals that such process enables to dimerize regioselectively, six ortho-methoxy-para-substituted phenols (vanillin, 4-hydroxy-3-methoxybenzonitrile, acetovanillon, methyl vanillate, 2-methoxy-4-methylphenol, and eugenol) with yields ranging from 87% to 96% and one ortho-disubstituted phenol (2,6-dimethoxyphenol) with 80% yield.

Full text of DOI:10.1016/j.molcatb.2015.12.006

Accepted Manuscript
Title: Selective laccase-catalyzed dimerization of phenolic
compounds derived from lignin: Towards original symmetrical
bio-based (bis) aromatic monomers
´
Author: Audery Llevot Etienne Grau Stephane Carlotti
´
Stephane Grelier Henri Cramail
PII:
S1381-1177(15)30127-2
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2015.12.006
MOLCAB 3296
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Journal of Molecular Catalysis B: Enzymatic
Received date:
Revised date:
Accepted date:
1-10-2015
13-12-2015
15-12-2015
´
Please cite this article as: Audery Llevot, Etienne Grau, Stephane Carlotti,
´
Stephane Grelier, Henri Cramail, Selective laccase-catalyzed dimerization of
phenolic compounds derived from lignin: Towards original symmetrical bio-
based (bis) aromatic monomers, Journal of Molecular Catalysis B: Enzymatic
http://dx.doi.org/10.1016/j.molcatb.2015.12.006
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Selective laccase-catalyzed dimerization of phenolic
compounds derived from lignin: towards original
symmetrical bio-based (bis)aromatic monomers
Audery Llevot,1,2 Etienne Grau,1,2 Stéphane Carlotti,1,2 Stéphane Grelier*1,2 and Henri
Cramail*1,2
1Université de Bordeaux, Laboratoire de Chimie des Polymères Organiques, ENSCBP,
16 Avenue Pey-Berland, Pessac Cedex, F 33607 France.
2CNRS, Laboratoire de Chimie des Polymères Organiques, F 33607 Pessac Cedex, France.
*grelier@enscbp.fr, cramail@enscbp.fr
Corresponding Author
* grelier@enscbp.fr, cramail@enscbp.fr
1

Graphical Abstract
Laccase-catalyzed process was developed to prepare, in high yield and with a high selectivity,
dimers of lignin-based phenolic compounds without any purification.
2

Highlights
A selective enzymatic process of vanillin dimerization is described.
The divanillin is easily recovered as a precipitate.
The selective formation of dimers by C-C coupling was extended to six ortho-methoxy-para-
substituted phenols
3

Abstract
A laccase-catalyzed process was developed to prepare, selectively, in high yield, dimers of
lignin-based phenolic compounds without any purification. The influence of experimental
parameters such as laccase loading, nature of solvent and the presence of oxygen on the
conversion of vanillin was investigated. After the dimerization, the product obtained as a
precipitate is filtered off and the solution containing the enzyme can be re-used several times,
which improves the process economics. A phenolic-substrate screening reveals that such process
enables to dimerize regioselectively, six ortho-methoxy-para-substituted phenols (vanillin, 4-
hydroxy-3-methoxybenzonitrile, acetovanillon, methyl vanillate, 2-methoxy-4-methylphenol,
and eugenol) with yields ranging from 87% to 96% and one ortho-disubstituted phenol (2,6-
dimethoxyphenol) with 80% yield.
KEYWORDS: Laccase, lignin, vanillin, dimerization, enzymatic catalysis.
4

1. Instroduction
Nowadays, the partial replacement of fossil feedstocks by renewable resources attracts a thriving
interest due to the petrol depletion and the growth of environmental concerns.[1, 2] In this
purpose, new molecules and monomers issued from available biomass have to be developed.[3-
6] Lignin is the main source of aromatic bio-based substrates. Despite extensive researches on
efficient ways of recovering aromatic products from lignin,[7] nowadays, the only commercial
process is the production of vanillin and vanillic acid by hydrothermal pretreatment under
alkaline conditions of lignosulfonates, by-products of the sulfite paper industry.[8-12]
Vanillin can be derived into divanillin, also called dehydrodivanillin, which is used mainly as
flavoring[13] and antioxidant agent in food, cosmestic and pharmaceutical industry but can also
be employed in microlithography[14] and in polymer synthesis.[15, 16] Over the years,
divanillin has been synthesized by different methods. It is commonly produced by oxidative
phenol-coupling using iron(III) chloride (FeCl3) or iron(II) sulfate (FeSO4).[17-22] These non-
sustainable processes require a high amount of iron catalyst, long reaction times and are not fully
selective, thus generating a mixture of products and a difficult work up. In order to avoid the use
of inorganic salts and toxic agents (sodium persulfate), enzymatic pathways were developed. In
1972, the formation of divanillin was observed for the first time after oxidation of vanillin in
aqueous solution with peroxidase in the presence of hydrogen peroxide.[23] In 2004, Dordick
and coworkers studied the structural diversity of peroxidase-catalyzed oxidation products of o-
methoxyphenols, leading to oligomers in the case of vanillin.[24] Further improvements of the
conditions were needed to reach a rather good selectivity in dimer formation.[25]
Laccase is another very well-known class of oxidative enzyme studied since 1883.[26] The latter
were identified in several plants, insects, bacteria and fungus, where they have different
5

biological functions.[27-29] Contrary to peroxidases, laccases employ dioxygen as oxidant.
Currently, a lot of studies report the use of laccases as biocatalysts for the oxidation of functional
moieties or the oxidative coupling of phenolic substrates.[30-37] Laccases generate radical
intermediates on phenolic compounds, which can undergo self-coupling reactions generally
resulting in the formation of a mixture of products from dimers to higher oligomers. The
selectivity of the coupling and the size of the oligomers depend on a broad range of parameters
such as laccase source, pH, temperature, substitution of the phenolic compound, solvents, etc.
The use of laccases is limited due to their lack of selectivity. Recently, Beifuss and coworkers
described a method on the coupling of vanillidene derivatives catalyzed by laccase from
Trametes versicolor which provided the best result, in terms of yield/selectivity, of dimer
synthesis by laccase catalysis.[38] Some specific substrates selectively led to one dimer in yield
of over 80% but the authors did not investigate further the coupling reaction.
This study extends previous works in laccase-catalyzed dimer formation of ortho-methoxy-para-
substituted phenols by improving dimerization process, product yields and extending the range
of molecules studied for this reaction.Indeed, different reaction parameters such as reaction time,
laccase loading and type of solvent were investigated on the example of vanillin. A refill
procedure was also developed in order to recycle the catalyst solution. Afterwards, the same
reaction conditions were applied on several phenolic substrates and the structures of the resulting
products investigated.
6

2. Results and discussion
2.1 Coupling process development and optimization on the example of vanillin
Vanillin dimerization, catalyzed by laccase from Trametes versicolor, was performed at room
temperature in a solution saturated in oxygen (Scheme 1). Prior to the addition of the acetate
buffer (90 vol%, pH = 5), vanillin was dissolved into acetone (10 vol%). Hence, the reactant
stays in solution while the resulting product precipitated. After addition of the laccase, the
colorless solution turned yellow, which either indicate the formation of radicals or quinone
structures. After few minutes, a brown solid precipitated. The first reactions were performed on
1.5 g scale of vanillin, employing 100 U of laccase, in 200 mL solvent, for 24 h. The precipitate
was filtered off, washed with water and analyzed by mass spectrometry, NMR and HPLC
(Figures S1 to S4, SI). These analyses revealed the selective formation of a symmetric dimer,
divanillin 1 (Scheme 1). Particularly, the NMR spectroscopy analyses were in agreement with
the study of Eswaran and coworkers.[14]
The selectivity and yield of coupling reactions catalyzed by laccase depend on the reaction
conditions. [39] In this work, the influence of various parameters (enzyme loading, solvent, pH
and saturation in oxygen) was investigated. The enzyme loading can be decreased down to 20 U
without affecting the yield of divanillin, which after 24 h, remained over 80% (Figure 1). Below
this value, the yield decreased drastically to 50%. Thus, the quantity of laccase for the following
reactions was set at 20 U for 1.5 g of substrate.
1
Vanillin conversion under different reaction conditions was followed by H NMR spectroscopy
(Figure 2).
7

After 8 h, 85% conversion of vanillin into divanillin was achieved for an acetone/acetate buffer
ratio of 10/90 under O2 bubbling (a). In the following samples, the concentration of vanillin in
the solution was too low to be detected (Figure S5, SI). Instead of bubbling O2, the reaction was
carried out in a beaker with a large surface in contact with air, under vigorous stirring. The
vanillin conversion (b) was similar to the conversion obtained in a solution saturated in oxygen
(a). However, if the quantity of O2 in the solution is limited by bubbling N2 into the solution,
after 25 h, the vanillin conversion (d) only reached 25%. The saturation of the solution with O2 is
thus a key parameter to reach high yield in divanillin.
When the acetate buffer is substituted by water, the conversion profile (c) follows the reference
curve (a) until 5 h of reaction. Beyond this time, the reaction speed decreased and, only 75%
conversion was achieved after 24 h. An increase of the pH from 5 to 7 was observed after the
reaction was stopped, that can explain the low conversion in the last hours. Indeed, the optimal
pH zone for laccase ranges from 4 to 6; off this range, the laccase activity decreases.
The amount and nature of solvent also influence the laccase activity. Increasing the
acetone/buffer ratio to 40% dramatically decreased the reaction speed (e). When the reaction was
carried out with 70% of acetone, no conversion of the starting compound was observed (f). It is
thus crucial to use the minimum amount of solvent required to dissolve the starting material in
order to achieve high yields. Acetone can be substituted by other organic solvents provided the
latter do not inhibit the laccase. However, depending on the solvent and quantity, the reaction
yield can be affected.[40, 41] For instance, 10% of DMSO was tested as alternative co-solvent
and led to a yield around 90% after 8 h.
8

In this process, the recovery of divanillin as a precipitate presents three advantages: (i) as vanillin
is soluble into the solution, the purity of the obtained dimer is very high (95% by NMR) and no
purification is needed (ii) the precipitation shifts the reaction equilibrium to divanillin formation
and prevent divanillin from further phenolic coupling, (iii) after filtration, the filtrate can be re-
used for a new reaction. Indeed, after reaction, the precipitate was filtered off. The filtrate, which
still contains the laccase, was saturated again with oxygen and another batch of vanillin was
added. After few seconds, vanillin was dissolved and after few minutes, a brown solid started to
precipitate (Figure S6, SI). Even after 8 runs of recharging the catalyst with the substrat, more
than 80% of product was isolated (Figure 3). The combination of the easy recovery method
without a marginal loss of activity constitutes the main advantage of this process.
2.2 Substrate screening
The dimerization procedure developed previously was applied on several substrates with the
objective to selectively produce dimers. First, the dimerization of ortho-methoxy phenols with
different para substituents was investigated.
The reaction of some of the phenols led to complex mixtures. Vanillic acid led to oligomers up to
5 units, probably due to a decarboxylation reaction (Figure S7, SI). Indeed, in literature,
decarboxylation reactions were reported for the reaction of syringic acid with laccase (Figure S8
SI). The postulated reaction mechanism is explained by the formation of phenoxy radicals which
can couple each other to give the quinoide-type intermediates. The dimer is formed by release of
carbon dioxide from the intermediate. Further couplings lead to the formation of poly(phenylene
oxide).[42] Similarly, under our reaction conditions, poly(phenylene oxide) was produced from
syringic acid (Figure S9, SI). The coupling of vanillyl alcohol resulted in a mixture of dimers,
9

trimers and tetramers (Figure S10, SI). The latter has already been reported in literature and,
depending on the conditions, dimers with three different structures, trimers or tetramers were
produced.[43, 44] In the case of substrates bearing a para substituent containing a double bond
conjugated to the aromatic ring, the radical generated by laccase oxidation is delocalized over the
extended π-system. This conjugation leads to the formation of further coupling products. In this
case, the formation of β-5 dimers, 5,5’ dimer, phenylcoumaran and β-β’ dimers were reported in
literature.[45-48] These dimers further recombine with other radical species, resulting in
complex oligomeric structures. We investigated the coupling of coniferaldehyde (Figure S11,
SI), 2-methoxy-4-vinylphenol (Figure S12, SI) and isoeugenol (Figure S13, SI) which,
respectively, led to a complex mixture of dimers to pentamers, oligomers up to 6 000 g/mol and
mainly dimers (90%) plus oligomers (10%) in which coumaran and β-β’ units were observed
(Table 1).
The selective formation of dimers by C-C coupling was achieved under the above mentioned
conditions with six ortho-methoxy-para-substituted phenols bearing aldehyde, nitrile, ketone,
ester, methyl and alkylene as para substituents. 4-Hydroxy-3-methoxybenzonitrile dimer, 2,
diacetovanillon, 3, methyl vanillate dimer, 4, 2-methoxy-4-methylphenol dimer, 5, and
dieugenol, 6, selectively precipitated with yields ranging from 87% to 96% (Scheme 2).
The HPLC profiles of each reaction product show a single peak and the mass spectrum reveals
the formation of the corresponding dimer (see SI). The symmetry of the molecule was
demonstrated by NMR spectroscopy and fully assigned (Figure 4). The main difference between
substrate and dimer is the disappearance of an aromatic proton.
10

Furthermore, the coupling of 2,6-dimethoxyphenol was also investigated at the aforementioned
conditions. The reaction led selectively to the formation of dimer 8 in 80% yield, contrary to a
previously reported study, where four demethylated products were also observed employing
laccase from Trametes pubescens.[49] During the oxidative coupling of 2,6-dimethoxyphenol,
Q1 is produced by recombination of two para radical species. Further reaction of this compound
with laccase can lead either to its re-aromatization into compound 8 or to the formation of the
very stable quinone 7 (Scheme 3) which show different molar masses. In the literature,
depending on the study, the formation of 8 or 7 was reported.[49, 50] In this experiment, mass
spectroscopy resulted in a molar mass of 304 g/mol attributed to the quinone 7. The nature of the
ortho substituents plays an important role in the coupling selectivity. For instance, the coupling
of 2,6-dimethylphenol led to a mixture of dimers (around 15%) and oligomers of phenylene
oxide with a molar mass of 1 300 g/mol. This difference in selectivity may be attributed to
different inductive effects.
3. Conclusion
A green and easy way to synthesize divanillin in high yield (95%) and purity was developed and
scale up to 15 g. This process presents several advantages: (i) the divanillin formation occurs at
room temperature, under oxygen which could be replaced by air, (ii) the employed (co)solvent,
10% of acetone, shows a low toxicity, (iii) the product extraction is easy and the purity is high
(95%) because the solvent conditions enable the reactant solubility while the so-formed dimer
precipitates, (iv) a low quantity of enzyme is required and the catalyst-containing solution can be
re-used to dimerize a new batch of substrate. Some parameters such as a minimal amount of
11

organic solvent, sufficient oxygen content and the use of a buffer solution appeared to be crucial
to reach a high vanillin conversion. Furthermore, this dimerization procedure was extended to
several substrates. In addition to vanillin, the coupling of 4-hydroxy-3-methoxybenzonitrile,
methyl vanillate, 4-methyl-2-methoxyphenol, 2,6-dimethoxyphenol and eugenol yielded
selectively dimers with yields over 85%, with a high purity and without further purification.
Such a platform of symmetrical and functional aromatic dimers is of high interest for the design
of novel rigid (semi)aromatic bio-based polymers.
4. Experimental
4.1. Material
Laccase from Trametes versicolor, acetic acid, eugenol, isoeugenol, 2,6-dimethoxyphenol,
coniferaldehyde and 2-methoxy-4-vinylphenol were purchased from Sigma Aldrich. Vanillic
acid, vanillin and 2-methyl-4-methoxyphenol were purchased from Alfa Aesar. Acetovanillon
was purchased from Acros Organics. All products and solvents (reagent grade) were used as
received.
4.2. Instrumentations
All NMR experiments were performed at 298 K on a Bruker Avance 400 spectrometer operating
at 400 MHz, in DMSO-d6. Size exclusion chromatography (SEC) analyses were performed in
THF (40°C) on a PL-GPC 50 plus Integrated GPC from Polymer laboratories-Varian with a
series of four columns from TOSOH (TSKgel TOSOH: HXL-L (guard column 6.0mm ID x
4.0cm L); G4000HXL (7.8mm ID x 30.0cm L); G3000HXL (7.8mm ID x 30.0cm L) and
G2000HXL (7.8mm ID x 30.0cm L)). The elution of the filtered samples was monitored using
simultaneous refractive index and UV detection. The elution times were converted to molar mass
12

using a calibration curve based on low dispersity polystyrene standards. HPLC was performed
using a Spectra system instrument fitted with a Phenomenex Luna 5µ C18 100A column and
compounds were detected with a Sedere Sedex 85 LT ELSD detector at 40°C (G=4, filter OFF).
These analyses were performed in acetonitrile at a flow rate of 1 mL/min. Mass (FD) spectra
were performed by the Centre d’Etudes Structurales et d’Analyses des Molécules, CESAMO
(Bordeaux, France). The measurements were carried out on a TOF mass spectrometer AccuTOF
GCv using an FD emitter with an emitter voltage of 10 kV. One to two microliters solution of the
compound is deposited on a 13mm emitter wire.
4.3. Experimental procedure
4.3.1. Synthesis of methylvanillate
Vanillic acid (15.0 g, 0.09 mol) was dissolved in methanol (75 mL). Sulfuric acid (2.1 mL) was
added and the mixture was stirred and warmed to reflux for 8 h. After evaporation of methanol,
the solid was dissolved in ethylacetate (60 mL), washed with a NaHCO3 solution (30 mL), water
(2 times) and brine (1 time). The organic phase was evaporated under reduced pressure. Yield:
90%.
1H NMR (400 MHz, CDCl3, δ (ppm)): δ 7.45 (m, 2H, Ar), 6.88 (d, 1H, Ar), 3.81 (s, 3H, OCH3),
3.79 (s, 3H, OCH3 ester).
13C NMR (400 MHz, CDCl3, δ (ppm)): δ 166,03 (OCH3 ester), 151.22 (Ar-C), 147.20 (Ar-C),
123.38 (Ar-C), 120.30 (Ar-C), 115.13 (Ar-C), 112.42 (Ar-C), 55.27 (OCH3), 51.60 (OCH3 ester).
13

4.3.2. 4-Hydroxy-3-methoxybenzonitrile synthesis
Vanillin (750 mg, 5 mmol) was dissolved in acetic acid (15 mL). NH2OH.HCl (520 mg,
7.5 mmol) was added and the mixture was stirred and warmed at 110 °C for 2 h. The reaction
was stopped by adding H2O; the organic product extracted using CH2Cl2, dried and purified by
flash chromatography (Ethyl acetate/cyclohexane 3/7).
1
Yield: 80%. H NMR (400 MHz, DMSO, δ (ppm): δ 7.34 (1H, s, Ar), 7.27 (d, 1H Ar), 6.88 (d,
1H Ar) 3.81 (s, OCH3).
13C NMR (400 MHz, DMSO, δ (pm)) δ 151.79 (Ar-C), 148.41 (Ar-C), 126.92 (Ar-C), 120.06
(CN), 116.68 (Ar-C), 115.56 (Ar-C); 101.1 (Ar-C)1, 56.39 (OCH3).
4.3.3. Enzyme activity
The activity of commercial laccase from Trametes versicolor was determined
spectrophotometrically by monitoring the oxidation of 2,2’-azino-bis-(3-ethylbenzthiazoline-6-
sulfonic acid) (ABTS, ε = 36 000 mol-1.cm-²). The reaction mixture contained 0.04 mM of
ABTS, 50 mM of acetate buffer (pH 5.0) and laccase. The absorbance change was monitored at
414 nm for 5 min at room temperature.[51] The amount of laccase that generated 1 µmol of
ABTS radical cation per minute was defined as one unit, U. The activity of laccase batch used in
this study was evaluated at 1.6 U/mg.
4.3.4. General procedure for dimerization: synthesis of dimers 1 to 7
A solution of phenol substrate (1-7) (1.5 g) in acetone (20 mL) was added to NaOAc buffer
(180 mL, 0.1 M, pH 5.0). O2 was bubbled into the solution for 5 min. Laccase from Trametes
versicolor (20 U, 12.4 mg) was added and the reaction was stirred at room temperature for 24 h.
14

The precipitate was filtered off the solution and the product dried overnight at 80 °C under
vacuum.
This procedure was adapted on 15g in the case of vanillin.
Divanillin (1): MW = 302 g/mol, yield: 96%.
1H NMR (400 MHz, CDCl3, δ (ppm)): δ 9.85 (s, CHO), 7.50 (s, 2H Ar), 4.00 (s, OCH3).
13C NMR (400 MHz, CDCl3, δ (ppm)): δ 191.04 (CHO), 150.70 (Ar-C), 147.95 (Ar-C), 128.30
(Ar-C), 127.69 (Ar-C),124.52 (Ar-C), 109.10 (Ar-C), 55.88 (OCH3).
4-Hydroxy-3-methoxybenzonitrile dimer (2): MW = 296 g/mol, yield: 95%.
1H NMR (400 MHz, CDCl3, δ (ppm)): δ 9.91 (s, 2H, HO), 7.57(s, 2H, Ar), 7.42 (s, 2H, Ar), 3,93
(s, 6H, OCH3).
13C NMR (400 MHz, CDCl3, δ (ppm)): δ 148.63 Ar-C), 147.85 (Ar-C), 128.05 (Ar-C), 124.56
(Ar-C), 119.45 (Ar-C), 114.03 (Ar-C), 100.30 (CN), 56.15 (OCH3).
Diacetovanillon (3): MW = 330 g/mol, yield: 92%.
1H NMR (400 MHz, CDCl3, δ (ppm)): δ 7.49 (s, 4H, Ar), 3.93 (s, 6H, OCH3), 2.56 (s, 6H,
C=OCH3).
13C NMR (400 MHz, CDCl3, δ (ppm)): δ 196.07 (OCH), 149.22 (Ar-C), 147.06 (Ar-C), 127.81
(Ar-C), 124.23 (Ar-C), 124.04 (Ar-C), 109.03 (Ar-C), 55.76 (OCH3), 26.25 (CH3).
Dimethyl divanillate (4): MW = 362 g/mol, yield: 90%.
1H NMR (400 MHz, CDCl3, δ (ppm)): δ 9.60 (s, 2H, HO), 7.46 (s, 4H, Ar), 3.90 (s, 6H, OCH3),
3.80 (s, 6H, OCH3 ester).
13C NMR (400 MHz, CDCl3, δ (ppm)): δ 166,04 (OCH3 ester), 148.60 (Ar-C), 147.27 (Ar-C),
125.25 (Ar-C),123.93(Ar-C), 119.21 (Ar-C), 110.89 (Ar-C), 55.97 (OCH3), 51.75(OCH3 ester).
2-Methoxy-4-methyl phenol dimer (5): MW = 274 g/mol, yield: 92%.
15

1H NMR (400 MHz, CDCl3, δ (ppm)): δ 6.73 (s, 2H, Ar) , 6.53 (s, 2H, Ar), 3.79 (s, 6H, OCH3),
2.23 (s, 6H, CH3).
13C NMR (400 MHz, CDCl3, δ (ppm)): 147.52 (Ar-C), 140.99 (Ar-C), 126.92 (Ar-C), 125.68
(Ar-C), 123.04 (Ar-C), 111.61 (Ar-C), 55.85 (OCH3), 20.65 (CH3).
Dieugenol (6): MW = 326 g/mol, yield: 87%.
1H NMR (400 MHz, CDCl3, δ (ppm)): δ 6.74 (s, 2H Ar), 6.52 (s, 2H Ar), 5.94 (q, 2H CH-CH2),
5.03 (d, 4H CH-CH2), 3.79 (s, OCH3), 3.27 (d, 2H CH2).
13C NMR (400 MHz, CDCl3, δ (ppm)): δ 147.80 (Ar-C), 141.62 (Ar-C), 138.38 (CH-CH2),
129.57 (Ar-C), 125.67 (Ar-C), 122.62 (Ar-C), 115.28 (CH-CH2), 105.56 (Ar-C), 55.64 (OCH3),
39.19 (CH2).
2,6-Dimethoxyphenol dimer (7): MW = 304 g/mol, yield: 80%.
1H NMR (400 MHz, CDCl3, δ ppm): δ 8.32 (s, 2H, HO), 6.82 (s, 4H, Ar), 3,84 (s, 12H, OCH3).
4.3.5. Refill procedure
After filtration of the dimer, the solution is kept, refilled with 1.5 g of vanillin O2 again. After
24h, the precipitate is filtered and the refill procedure followed again up to 9 times.
4.3.6 Conversion investigation
Dioxane was used as an internal reference. 0.4 mL of solution is sampled regularly, filtered and
1
diluted in acetone-d6. The samples are analyzed by H NMR spectroscopy. The vanillin
conversion is extracted from the ratio of CHO peak integration at 9.81 ppm and the dioxane peak
integration at 3.63 ppm.
Details experiments Figure 3:
(a) 10% acetone-90% acetate buffer/O2: general procedure described above.
16

(b) 10% acetone-90% acetate buffer/air: the solution is not bubbled with O2 but is carried
out under high stirring in an open beaker.
(c) 10% acetone-90% water/O2: the acetate buffer is replaced by distilled water.
(d) 40% acetone-60% acetate buffer/O2: the solvent ratio is modified.
(e) 10% acetone-90% acetate buffer/N2: the solution is not bubbled with O2 but with N2.
(f) 70% acetone-30% acetate buffer/O2: the solvent ratio is modified.
ACKNOWLEDGMENT
The authors thank the University of Bordeaux, which financially supported the work. They
also thank the Centre d’Etudes Structurales et d’Analyses des Molécules, CESAMO (Bordeaux,
France) for the mass spectra, the ANR-10-EQPX-16 XYLOFOREST, Bordeaux INP and CNRS.
17

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19

Figure 1. Divanillin yields depending on laccase quantity for 1.5 g of vanillin, in 200 mL of
solvent, after 24 h.
1
Figure 2. Vanillin conversion versus time using data extracted from H NMR spectra (aldehyde
signal): different conditions: (a) 10% acetone-90% acetate buffer/O2, (b) 10% acetone-90%
acetate buffer/air, (c) 10% acetone-90% water/O2, (d) 40% acetone-60% acetate buffer/O2, (e)
10% acetone-90% acetate buffer/N2, (f) 70% acetone-30% acetate buffer/O2..
20

Figure 3. Divanillin yield, after 24 h of reaction under optimized conditions (20 U, acetate
buffer) for the first reaction plus 8 refills.
21

Figure 4. 1H NMR spectra of the dimers obtained by selective oxidative coupling catalyzed by
laccase of vanillin (1), acetovanillon (2), methylvanillate (3), 4-hydroxy-3-methoxybenzonitrile
(4), 2-methoxy-4-methyl phenol (5), eugenol (6) and of 2,6-dimethoxyphenol (7).
22

Scheme 1. Laccase-catalyzed vanillin dimerization, in acetone/acetate buffer 10/90, under
oxygen, at room temperature.
Scheme 2. Products of laccase-catalyzed selective dimerization of vanillin, 4-hydroxy-3-
methoxybenzonitrile, acetovanillon, methylvanillate, 2-methoxy-4-methyl phenol and eugenol.
23

Scheme 3. Laccase-catalyzed dimerization of 2,6-dimethoxyphenol.
24

Table 1. Laccase-catalyzed oxidative coupling of ortho mono substituted phenol.
Yielda
(%)
Starting compound
Final product
Vanillic acid
Oligomers up to 5 units
40
85
Vanillyl alcohol
Mixture of dimers and tetramers
Several dimers, trimers and
tetramers
Coniferaldehyde
92
2-Methoxy-4-vinylphenol
Oligomers up to 6 000 g/mol
Mainly dimers and oligomers
83
85
Isoeugenol
a Yield of the isolated precipitate
25