Synthesis of nitrogenated lignin-derived compounds and reactivity with laccases. Study of their application in mild chemoenzymatic oxidative processes

Article abstract of DOI:10.1039/c7ra10497a

The chemical synthesis of a series of lignin-derived nitrogenated compounds was performed in high yields (73-99%) through simple conventional chemical transformations starting from natural monomers such as vanillin, syringaldehyde or 3,4-dihydroxybenzaldehyde. The study of the vanillin-derived compounds as substrates for commercially available laccases from Trametes versicolor and Myceliophthora thermophila in oxidative transformations, generally led to the isolation of several dimeric species in high to excellent conversions (>70%), while for hydrazone derivatives a more rapidly oxidative coupling was evidenced by the formation of oligomers and/or polymers. Remarkably, vanillin was obtained due to the hydrolysis of some of the nitrogenated functional groups, such as the hydrazone or the hydrazono tetrazole. The three families of lignin-derived compounds can provide a great source of new laccase-mediator systems (LMS), the possibility of employing them for lignin modification being particularly attractive. Preliminary experiments showed promising levels of activity towards the oxidation of a monomer (veratryl alcohol, up to 70% conversion) and a dimer (adlerol, up to 22% conversion) lignin models, higher than those achieved with the natural vanillin and syringaldehyde (up to 7% conversion with veratryl alcohol and almost negligible conversion with adlerol), these processes being also highly influenced by the pH of the reaction medium.

Full text of DOI:10.1039/c7ra10497a

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PAPER
Synthesis of nitrogenated lignin-derived
compounds and reactivity with laccases. Study of
their application in mild chemoenzymatic oxidative
processes†
Cite this: RSC Adv., 2017, 7, 50459
Jesus Albarran-Velo,
Marıa Lopez-Iglesias, Vicente Gotor, Vicente Gotor-
´ ´
´
´
*
*
Fernandez
and Ivan Lavandera
´
´
The chemical synthesis of a series of lignin-derived nitrogenated compounds was performed in high yields
(73–99%) through simple conventional chemical transformations starting from natural monomers such as
vanillin, syringaldehyde or 3,4-dihydroxybenzaldehyde. The study of the vanillin-derived compounds as
substrates for commercially available laccases from Trametes versicolor and Myceliophthora thermophila in
oxidative transformations, generally led to the isolation of several dimeric species in high to excellent
conversions (>70%), while for hydrazone derivatives a more rapidly oxidative coupling was evidenced by the
formation of oligomers and/or polymers. Remarkably, vanillin was obtained due to the hydrolysis of some
of the nitrogenated functional groups, such as the hydrazone or the hydrazono tetrazole. The three
families of lignin-derived compounds can provide a great source of new laccase-mediator systems (LMS),
the possibility of employing them for lignin modification being particularly attractive. Preliminary
experiments showed promising levels of activity towards the oxidation of a monomer (veratryl alcohol, up
to 70% conversion) and a dimer (adlerol, up to 22% conversion) lignin models, higher than those achieved
with the natural vanillin and syringaldehyde (up to 7% conversion with veratryl alcohol and almost negligible
conversion with adlerol), these processes being also highly influenced by the pH of the reaction medium.
Received 21st September 2017
Accepted 24th October 2017
DOI: 10.1039/c7ra10497a
rsc.li/rsc-advances
smaller compounds,2 biocatalytic methodologies being
currently booming.3
Introduction
Thus, it has been found that some bacterial and fungal
microorganisms containing potent oxidative enzymes, have the
capability to degrade the structure of lignin by promoting the
cleavage of certain connections as the b-O-4-aryl ether bonds or
the oxidation at Ca positions,3c these predominant linkages
being illustrated in the dimeric species of Fig. 1 (le). Particu-
larly, some peroxidases, peroxygenases and laccases have been
applied in enzyme-catalyzed delignication processes.4
Lignin is a highly abundant and irregular biopolymer which
constitutes lignocellulosic biomass together with cellulose and
hemicellulose. During the last decades, the scientic commu-
nity has deeply studied the usability of these polymers as
sources of energy and high-added value compounds. On the
opposite, the complex structure of lignin has overshadowed its
value as a renewable alternative to the fuel industry, since high
amounts of energy could be involved to break down its cross-
linked structure. Nevertheless, the use of lignin as feedstock
has become an important eld of research in recent years,
nding multiple applications in the production of bio-based
polymers such as adhesives, bioplastics or resins due to its
polyphenolic composition.1 The lignin valorization is also
moving to cleavage processes focused on degradation into
Organic and Inorganic Chemistry Department, Biotechnology Institute of Asturias
´
´
(IUBA), University of Oviedo, Avenida Julian Claverıa 8, 33006 Oviedo, Spain.
E-mail: vicgotfer@uniovi.es; lavanderaivan@uniovi.es
† Electronic supplementary information (ESI) available: This le includes
extensive enzymatic optimization studies, analytical data for the oxidation of
adlerol and copy of 1H, 13C and DEPT NMR spectra for novel synthesized
compounds. See DOI: 10.1039/c7ra10497a
Fig. 1 Lignin-derived model compounds, monomeric and dimeric
species.
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Furthermore, lignin has attracted an increasing interest not derived monomers due to the presence of a phenolic function
only as source of energy but also because of the valuable low- within their structure,13 but still the product formation was not
molecular weight aromatic compounds contained in its struc- studied in many of those cases.
ture, which can also serve as building blocks for synthetic
Herein, we aim to combine the use of compounds derived
processes.5 Within this area, many efforts have been intended to from renewable resources (e.g. lignocellulose) and the benets
investigate the behavior of lignin-related compounds towards that biocatalysis offers due to the capability of enzymes to
cleavage or oxidation processes as representative and handier selectively work under environmentally friendly conditions.
models in which the most characteristic linkages are present. Thus, we rstly report the chemical modication of vanillin and
Among them, some dimeric species such as veratrylglycerol b- syringaldehyde towards the synthesis of nitrogenated deriva-
guaiacyl ether (adlerol, 2, Fig. 1) or similar b-1-type lignin model tives whose broad structure is depicted in Fig. 1 (right). Later
compounds have found wide application, for instance, for these compounds will be tested as suitable substrates in
studying the mechanism of lignin C–C linkage cleavage by laccase-catalyzed processes. Overall, the synthetic strategies will
pyrolytic strategies6 or metal catalysis.2a Also Biocatalysis has provide novel systems that will be applied in the oxidation of
been applied in lignin valorization by studying the cleavage or lignin model compounds, which is the underlying purpose of
oxidation of b-O-4-linkages in dimeric species.7
the present research.
Laccases (EC 1.10.3.2) are multi-copper oxidative enzymes that
consume molecular oxygen as electron acceptor during the
oxidation of a substrate producing water as the only by-product,
which makes them an interesting and suitable tool working
under mild reaction conditions.8 These enzymes have been
employed in lignin degradation,4,9 and also in the opposite
repolymerization processes or related lignin-surface modica-
tions, in order to functionalize it obtaining valuable biomaterials,
for example by single molecule graing or copolymerization
processes.10 Despite these enzymes present a suitable redox
potential to abstract electrons from phenolic compounds, this is
not high enough to transform other types of substrates such as
aliphatic alcohols, so the use of laccase-mediator systems (LMS)
is necessary to allow their oxidation.8c,11 Many synthetic
compounds are commonly employed in these systems, such as
TEMPO, HOBt or ABTS, but also some natural compounds ob-
tained from lignin can be used. Among them, vanillin (3a, Fig. 1),
syringaldehyde (3b, Fig. 1) and their derivatives are some of the
most common lignin-derived monomers that have been exten-
sively employed in different ways, for example as natural LMS.9a,12
In fact, laccases have proven to accept a large number of lignin-
Results and discussion
Chemical modication of natural LMS
Vanillin (3a) and syringaldehyde (3b) were selected as starting
materials for further modications due to the presence of these
compounds in high quantities when degrading lignin. As both
substrates have a similar structure but a different number of
methoxy groups in the aromatic ring, the choice of these two
lignin-type monomers will allow us to compare their capacities
in LMS. Moreover, anticipating the importance of both the
methoxy and the hydroxyl groups in the substrate–laccase
interaction, and in order to complete the study, we decided to
include also 3,4-dihydroxybenzaldehyde (3c), which was
synthesized starting from vanillin via demethylation in 85%
yield as described in the literature (Scheme S1, see the ESI†).14
In the interest of practical designs for the potential media-
tors, we focused on simple and easy synthetic procedures.
Moreover, the rationale for the synthesis of the proposed
compounds is based on the possibility of stabilizing the
oxidized radical intermediates by aromatic systems via electron
Scheme 1 An oxime derivative presenting an increasing number of reacting points.
Scheme 2 Synthesis of 4-hydroxy-3-methoxybenzonitrile (4a) and oximes 5a–c.
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Table 1 Synthesis of new nitrogenated compounds derived from vanillin and syringaldehyde
Entry
1
Substrate
Reactant
Product
Acid
t (h)
Yielda (%)
3a
N2H4$H2O (10)
AcOH
0.83
90 (6a)
2
3b
N2H4$H2O (10)
N2H4$H2O (10)
AcOH
0.83
80 (6b)
3
4
3c
3a
AcOH
AcOH
1
78 (6c)
98 (7a)
1.5
5
3b
AcOH
2
89 (7b)
6
7
3c
3a
AcOH
2.7
3
73 (7c)
99 (8a)
H2SO4 conc.
8
3b
H2SO4 conc.
7.5
99 (8b)
9
3c
3a
3b
H2SO4 conc.
HCl conc.
HCl conc.
6.5
4.5
5
96 (8c)
94 (9a)
97 (9b)
10
11
12
3c
HCl conc.
1.75
80 (9c)
a
Yield obtained aer isolation of the products by solvent evaporation (for 6a–c and 7a–c) or product ltration (for 8a–c and 9a–c).
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Table 2 Laccase-catalyzed oxidation of the new synthesized vanillin-derived compoundsa
Substrate,
concentration
Entry
Cosolvent
Enzyme
t (h)
T (ꢀC)
Product
cb (%)
1
2
3a, 20 mM
3a, 20 mM
EtOH
—
LTv
2
2
30
50
94
93
Novozym 51003
3
4
4a, 10 mM
4a, 10 mM
—
—
LTv
2
2
30
50
>99
>99
Novozym 51003
5
6
5a, 50 mM
5a, 50 mM
EtOH
EtOH
LTv
16
16
30
50
>99
>99
Novozym 51003
7
8
6a, 50 mM
6a, 50 mM
DMSO
DMSO
LTv
16
16
30
50
3ac
3ac
51
69
Novozym 51003
9
7a, 20 mM
—
LTv
16
30
72
10
11
12
7a, 20 mM
8a, 20 mM
8a, 20 mM
—
EtOH
EtOH
Novozym 51003
LTv
Novozym 51003
2
2
2
50
30
50
17 + polymerization
25
93
95
13
14
9a, 10 mM
9a, 10 mM
EtOH
EtOH
LTv
2
2
30
50
3ad
3ad
69
97
Novozym 51003
a
Reaction conditions: substrate 3–9a, cosolvent (10% v/v), acetate buffer pH 4.7 (total volume: 3 mL) saturated with O2, laccase from Trametes
versicolor (LTv, 25 U) or Novozym 51003 (31.4 U). Conversion values were measured by 1H-NMR analysis of the reaction crudes. Vanillin and
b
c
a mixture of dimeric species were obtained. d Vanillin and a mixture of unidentied products were attained.
delocalization or by increasing the number of additional reac- hydroxylammonium sulfate and sodium acetate in ethanol
tive points as in the case of an oxime derivative (Scheme 1), (Scheme 2). By varying the solvent to acetonitrile and using
resembling in this particular case N-oxyl oxidants such as hydroxylamine hydrochloride, dehydration of the aldoxime
TEMPO.
intermediate took place yielding the nitrile derivative 4a from 3a
In a rst set of experiments, we evaluated the use of hydroxyl aer 4 h.
ammonium reagents to introduce both oxime and nitrile
On the other hand, a panel of nitrogenated substituents was
functions. Thus, oximes 5a–c were prepared in good to excellent introduced through the aldehyde function by tuning the
yields following a described methodology15 in the presence of
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strength of the acid catalyst and adjusting the reaction time vanillin itself (3a), observing it was susceptible to dimerization
(Table 1). in a very high extent (entries 1 and 2). Similarly, dimeric species
Firstly, the reaction between hydrazine monohydrate (10) 15–18 were isolated and properly characterized from the reac-
and 3a was performed under acidic conditions, observing full tion crudes when starting from nitrile 4a, oxime 5a, semi-
disappearance of the starting material by TLC analysis aer carbazone 7a, and triazole 8a (entries 3–6, 9, 11 and 12).
50 min. Hydrazone 6a was isolated in 90% yield aer removing Particularly, 4a reacted at 10 mM concentration, achieving
the solvent by distillation (entry 1). The procedure was extended a quantitative conversion with LTv and Novozym 51003 (entries
to the dimethoxylated and dihydroxylated substrates 3b and 3c, 3 and 4). Oxime 5a also led to full conversions at 50 mM (entries
leading to 6b and 6c also in good yields (entries 2 and 3). The 5 and 6), observing with both biocatalysts the formation of
reaction time was increased in the synthesis of semicarbazones a minimum amount of 3a (3%) which was no detected in the
7a–c in the presence of semicarbazide hydrochloride (11), absence of the enzyme. Semicarbazone 7a was converted at
obtaining the desired products in good to excellent yields aer 20 mM concentration in the presence of LTv conducting to
solvent evaporation (entries 4–6).
dimer 17 (72% conversion, entry 9). However, the product
To prepare the triazole derivatives 8a–c, longer reaction formation differed in the case of Novozym 51003 (entry 10), in
times and harsher acidic conditions were needed, so concen- which the presence of a polymeric species, even at short reac-
trated H2SO4 was employed as catalyst.16 The reaction between tion time, was detected due to the broad signals observed in the
3b and 3c with 4H-1,2,4-triazol-4-amine (12) was completed 1H-NMR experiments. The generation of polymeric products in
aer 7.5 and 6.5 h, respectively (entries 8 and 9), while starting laccase-catalyzed reactions has already been reported in the
from 3a only 3 h were necessary (entry 7), recovering 8a–c with literature for this type of lignin monomer.13b,20 Finally, dimer 18
excellent yields. Concentrated HCl was used in the reactions was obtained as a precipitate from the reaction crude when
with tetrazole 13, obtaining 9a–c in 80–97% yield (entries 10– starting from triazole 8a (20 mM, entries 11 and 12) in almost
12). In these cases, the isolation was performed by ltration full conversion with both enzymes. These results can be related
from the reaction crude, since both families of poly- with the stability of the radical species-type A (Scheme 1). If this
nitrogenated compounds 8a–c and 9a–c were insoluble in the intermediate is stable enough, the dimerization process will be
reaction medium. In the latter reactions, it was necessary to favored. Nitrogenated electron-withdrawing groups enhanced
synthesize the reagent 13 through a two-step process (see the stability of this radical as the C–C dimerization reaction was
Experimental section), as it was not commercially available.
prominent.
The hydrazone bond of 6a was hydrolyzed during the enzy-
matic process, so vanillin was detected in signicant amounts
in the H-NMR of the reaction crude (entries 7 and 8), a 4% of
the hydrolysis product 3a being also formed in the absence of
Laccase-catalyzed oxidation reactions of the synthesized
derivatives
1
To develop an efficient LMS with these compounds, rst we enzyme. Moreover, aer aldehyde formation also dimerization
investigated if they could react with laccases. For this, we chose took place, so a mixture of starting material, 3a and several
commercially available laccases from Trametes versicolor (LTv)17 dimeric species were detected in the reaction crude in
and Myceliophthora thermophila (Novozym 51003),18 and the a proportion varying depending on the biocatalyst used. Despite
enzymatic experiments were conducted with the family of of the presence of unreacted hydrazone in the crude, it was
vanillin-modied derivatives, in order to illustrate the oxidative noticed a fast reaction as the mixture acquired a dark purple
processes that could take place. This study would verify the color just aer the addition of the enzyme. When the N-tetrazole
validity of the new synthesized compounds as potential laccase substituted hydrazone derivative 9a (10 mM) was submitted to
mediators. Following previous described protocols with these oxidation, the hydrolysis of the C]N bond was also observed.
biocatalysts, enzymatic reactions were carried out in acetate Thus, a mixture of products was extracted from the reaction
buffer at pH 4.7, varying the substrate concentration from 10 to crude when LTv or Novozym 51003 were used, in which the
50 mM. Due to solubility issues, 10% (v/v) of EtOH or DMSO19 starting material, vanillin and other unidentied products were
were added as water-miscible organic solvents in some cases. already present at 2 h. Nevertheless, polymerization might also
The control reactions in the absence of enzyme led to no be occurring, as a signicant loss of weight in the crude
conversion in all cases, proving the direct role of the laccase as recovered by extraction was observed.
the only catalyst.
Since laccases are not expected to hydrolyze hydrazones, we
When the laccases and the vanillin derivatives were mixed, evaluated if unspecic catalysis of this protein or other impu-
three processes were mainly observed. In most cases the C–C rities that could be present in the commercial laccase prepa-
dimerization reaction of species-type A (Scheme 1) was favored, ration might be responsible of this transformation. So the LTv
but also the formation of vanillin (via hydrolysis) and the was denatured by a heat treatment (for details, see the ESI†).
polymerization/oligomerization of these substrates were The conversion towards 3a formation was just 4%, demon-
observed (Table 2). From now on, no distinction between strating the role of the biocatalytic system in the hydrolysis of
polymerization and oligomerization will be made, since this 6a. While it has been described the hydrolysis of hydrazones
was not the main goal of this research, so the polymeric struc- under oxidative conditions,21 there is only one precedent in the
tures were not analyzed such depth to make that differentiation. literature showing a similar behavior of LMS, the authors
Firstly, the enzymatic experiments were conducted with the observing bio-deoximation processes in the presence of TEMPO
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Table 3 Selected results in the laccase-catalyzed veratryl alcohol oxidation in the presence of different natural or nitrogenated lignin-derived
compoundsa
Entry
Compound
Laccase
T (ꢀC)
pH
Conversionb (%)
1
2
3
4
5
6
7
8
3a
3b
3a
3b
4a
4a
5a
5b
5c
5a
5b
5c
6a
6b
6c
6a
6b
6c
7a
7b
7c
7a
7b
7c
8a
8b
8c
8a
8b
8c
9a
9b
9c
9a
9b
9c
LTv
LTv
30
30
50
50
30
50
30
30
30
50
50
50
30
30
30
50
50
50
30
30
30
50
50
50
30
30
30
50
50
50
30
30
30
50
50
50
5
6
6
6
4.7
6
6
4.7
5
6
7
6
5
5
5.5
6
6
5
6
4.7
4.7
6
7
5
4.7
4.7
4.7
6
6
6
6
5
5
6
6
6
4
6
6
7
3
6
Novozym 51003
Novozym 51003
LTv
Novozym 51003
LTv
37
11
70
34
35
21
39
<1
8
54
3
10
14
23
8
16
23
5
8
5
4
9
16
2
LTv
LTv
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
Novozym 51003
Novozym 51003
Novozym 51003
LTv
LTv
LTv
Novozym 51003
Novozym 51003
Novozym 51003
LTv
LTv
LTv
Novozym 51003
Novozym 51003
Novozym 51003
LTv
LTv
LTv
Novozym 51003
Novozym 51003
Novozym 51003
LTv
LTv
LTv
39
36
11
24
32
3
Novozym 51003
Novozym 51003
Novozym 51003
a
Reaction conditions: natural or nitrogenated derivative (16 mmol), buffer (2 mL), veratryl alcohol (0.9 mL, 6 mmol), laccase from Trametes versicolor
(LTv, 25 U, 30 ꢀC) or Novozym 51003 (31.4 U, 50 ꢀC), 72 h. Buffers used: acetate buffer 100 mM pH 4.7; citrate buffer 50 mM pH 5; citrate buffer
50 mM pH 5.5; citrate buffer 50 mM pH 6; phosphate buffer 100 mM pH 7. b Conversion values were measured by GC analysis of the reaction crudes.
as mediator.11a In our experiments, we assume that the laccase employed as representative monomeric and dimeric structures
is able to catalyze the synthesis of unstable radical intermedi- of lignin units,22 respectively, the alcohol moiety being in both
ates which can undergo hydrolysis releasing vanillin.
cases at benzylic position (Fig. 1). Control experiments in the
absence of laccase and/or nitrogenated compound revealed that
both must be present for an efficient catalysis.
New LMS applied to the oxidation of lignin-model compounds
Beginning with the veratryl alcohol oxidation, we selected
3 mM as starting substrate concentration according to some
reports found in the literature,22d evaluating both commercial
laccases. A deep optimization was performed in order to
determine the best reaction conditions, including the reaction
Aer checking that these compounds were suitable substrates
for the studied laccases, they were tested as potential mediators
to oxidize two different lignin model compounds. The syringyl
and 3,4-dihydroxybenzaldehyde families were also included in
this study. Thus, veratryl alcohol (1) and adlerol (2) were
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medium, temperature, amount of catalyst and concentration of syringyl oximes 5a and 5b, the process being promoted to 37%
the nitrogenated derivative, and the use of different cosolvents conversion at pH 6 with 5a and LTv, and 34% when Novozym
(see Tables S1–S18 in the ESI†). Among them, the pH was 51003 was employed under the same pH conditions at 50 ꢀC
identied as key parameter, and depending on the phenolic (entries 7 and 10). In an attempt to increase those values, the
compound employed, the optimum value varied. Similarly, use of different cosolvents was tested but resulted ineffective
changes in the amount of the co-oxidant implied important (see Table S4 in the ESI†), and longer reaction times led to the
variations in the conversion degree, detecting the higher values disappearance of the product signal in the GC chromatogram.
at 8 mM concentration. The rst experiments were stopped at We hypothesized that the product was degraded, partially to
48 h but, aer observing that the laccase was still active, the form the veratryl acid, which was identied in the reaction
reactions were followed until 72 h. The best results were ob- analysis.
tained in the acidic pH range of 4.7–7, as described for these
The presence of an additional methoxy group in oxime 5b
laccases,17,18 and are summarized in Table 3. LTv works better did not affect signicantly the performance of Novozym 51003,
under acidic conditions while Novozym 51003 accepts higher obtaining a conversion of 35% at pH 7 (entry 11), while the
pHs.
reaction was disfavored with LTv (entry 8), especially at higher
The process was also carried out in the presence of vanillin pHs (see Table S9 in the ESI†).
and syringaldehyde (entries 1–4), to compare the behavior of
When the vanillin hydrazone 6a was studied, good values of
these natural derivatives with the chemically modied ones, conversion were observed (entries 13 and 16), obtaining 54% in
nding an improvement in the conversion values for several of combination with Novozym 51003 at pH 6 (entry 16). On the
the newly nitrogenated synthesized compounds. Still some of opposite, 6b led to low or null conversions with both enzymes
them displayed very low activity even at these long reaction (entries 14 and 17). The results were slightly better but still poor
times, as was the case of the nitrile derivative 4a, which led to with the dihydroxylated derivative 6c (entries 15 and 18), not
less than 10% conversion with both enzymes LTv and Novozym improving in the presence of an organic cosolvent (see Table
51003 (entries 5 and 6). On the contrary, oximes 5a–c exhibited S15 in the ESI†). Conversely to the hydrazones, the semi-
remarkable activities, 5c providing with LTv the highest value of carbazone derived from syringaldehyde 7b (entries 20 and 23)
all derivatives tested with 70% conversion (entry 9). The reac- showed higher activities than the corresponding analogue from
tion exceeded 30% conversion when employing vanillin and vanillin 7a (entries 19 and 22). As for the hydrazone family, the
Fig. 2 Relative activity representing micromoles of alcohol 1 converted into aldehyde 18 at different substrate concentrations, using the new
synthesized compounds as co-oxidants, and Novozym 51003 (up) or LTv (down) as biocatalyst. 100% relative activity corresponds to 0.42 mmol
18 per mL mgꢁ1 of enzyme (entry 9, Table 3). Reactions with relative activities higher than 200% were performed by duplicate, showing similar
results.
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dihydroxylated semicarbazone 7c displayed the lowest conver- other cases, resulting in an enhancement of the relative activity
sions (entries 21 and 24).
for all derivatives tested (Fig. 2).
In general, the triazole family was poorly active, affording the
Noteworthy, it was possible to rise the substrate concentra-
dimethoxylated derivative 8b the highest conversion value in tion until 15 mM and 20 mM, maintaining good levels of
the reaction with Novozym 51003 at pH 6 (entry 29). Interest- catalytic activity and, therefore, improving the productivities.
ingly, tetrazole derivatives 9a and 9b showed enhanced For instance, relative activity values of 186–295% were found
conversions (entries 31–32 and 34–35). As for the case of semi- with oxime 5b, hydrazone 6a and tetrazole 9b within the
carbazones 7a–b and triazoles 8a–b, Novozym 51003 exhibited Novozym 51003-catalyzed reactions (Fig. 2, top). In general, LTv
higher acceptance to the syringyl-derived substrate than to the showed less alcohol concentration tolerance, but even so oxime
corresponding vanillin-derived one (entries 34 and 35), 5c displayed an excellent activity in the presence of this enzyme
behaving positively the additional methoxy group. On the (286%, Fig. 2, bottom).
contrary, when the reaction was carried out in the presence of
Finally, the methodology was extended to the oxidation of
the dihydroxylated derivative 9c, the conversions decayed the benzylic position of the dimeric lignin model compound
signicantly, especially for Novozym 51003 acting as catalyst adlerol 2 using the derivatives that displayed the higher activi-
(entry 36).
ties with the laccases for the oxidation of 1 (Fig. 3). The reaction
As the next step, we decided to increase the veratryl alcohol conditions were taken in analogy to the previously shown for
concentration for those cases where higher conversions were veratryl alcohol.
found. This would allow us to study the effect of this parameter
Despite the conversions were lower than for the case of 1,
in the reaction outcome and also to obtain a higher productivity signicant improvements were accomplished with some deriv-
of the aldehyde 18 in the process. Remarkably, at 10 mM atives, obtaining the best results (22% conversion) for the
substrate concentration, conversions suffered just a slight combination of oxime 5b with Novozym 51003, which is
decrease in some cases, maintaining the previous values in a remarkable value when considering the examples reported in
the literature using vanillin or syringyl-type natural mediators
in this kind of process.22 In order to verify the improvement over
the natural compounds, 3a and 3b were tested, observing no
formation of hydroxy ketone 19.
Oxime 5c showed signicant conversions in the LTv-
catalyzed process, as well as tetrazoles 9b and 9c (Fig. 3b). On
the contrary, the use of Novozym 51003 as catalyst resulted in
high conversion values with derivatives 6a, 9a and 9b (Fig. 3a).
We gure out that the lower conversion values may be caused by
a fast reaction of the phenolic compound with the laccase,
leading to the formation of inactive species and disabling the
oxidative capacity in the adlerol transformation. As a nal
remark, it must be mentioned that coupling between the
synthesized nitrogenated derivatives and the substrates
(veratryl alcohol and adlerol) was never observed.
Conclusions
The chemical modication of some lignin-type monomers such
as vanillin and syringaldehyde has been carried out through
simple methodologies toward the synthesis of several families
of nitrogenated compounds. These derivatives were envisaged
as suitable candidates for LMS due to the stabilization of the
phenoxy radicals formed. Their combination with two
commercially available laccases, the one from Trametes versi-
color and the one from Myceliophthora thermophila, allowed
their dimerization or oligomerization, showing that they could
act as substrates for laccases.
The application of these compounds in oxidative processes
of lignin model units in laccase-mediator systems has been
investigated employing these commercial laccases, showing
much better conversions than the natural compounds vanillin
and syringaldehyde. Remarkably, both the substitution pattern
of the aromatic ring and the pH have shown a dramatic inu-
ence in the reactivity behavior, conducting to higher
Fig. 3 Oxidation of adlerol (2) using natural or nitrogenated lignin-
derived compounds with: (a) Novozym 51003; or (b) LTv. All the
experiments were performed by triplicate.
50466 | RSC Adv., 2017, 7, 50459–50471
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conversions according to the presence of additional methoxy under reux for 4 h, cooled at room temperature, resuspended
and/or hydroxyl groups. On the one hand, veratryl alcohol was in CH2Cl2 (10 mL), ltered and washed with CH2Cl2 (2 ꢂ 2 mL).
transformed into veratraldehyde in high conversions at 3 mM, The solvent of the ltrate was evaporated under reduced pres-
and the increase in the substrate concentration up to 20 mM led sure obtaining pure oxime 5a. Oxime 5b was submitted to
to higher relative activity values (up to 295%). On the other column chromatography (eluent: 30% EtOAc/hexane) for
hand, lower conversions were obtained in the oxidation of the further purication.
lignin model dimer adlerol, but these results are a good starting
For the isolation of oxime 5c, the solvents were evaporated
point for future studies in the enzymatic modication of lignin and the residue was redissolved in H2O. The mixture was
surfaces.
extracted with EtOAc (3 ꢂ 5 mL), the organic layers were
combined, dried over Na2SO4 and concentrated in vacuo,
affording pure 5c.
Experimental section
General methods
4-Hydroxy-3-methoxybenzaldehyde oxime (5a).15 Light brown
solid (550 mg, >99% yield). Rf (50% EtOAc/hexane): 0.67. Mp:
110–112 ꢀC. 1H NMR (300.13 MHz, MeOD-d4): d 3.83 (s, 3H), 6.79
Chemical reagents employed in the synthesis of starting and
reference materials were purchased from Sigma-Aldrich.
Solvents for chemical reactions were used without further
treatment.
3
3
4
(d, JHH ¼ 8.1 Hz, 1H), 6.95 (dd, JHH ¼ 8.1 Hz, JHH ¼ 1.7 Hz,
1H), 7.23 (d, JHH ¼ 1.7 Hz, 1H), 8.00 (s, 1H). 13C NMR (75.5
4
MHz, MeOD-d4): d 56.2 (CH3), 109.6 (CH), 116.0 (CH), 122.4
(CH), 126.0 (C), 149.1 (C), 149.3 (C), 150.6 (CH). HRMS (ESI+, m/
z): calcd for (C8H10NO3)+ (M + H)+ 168.0655, found: 168.0656.
4-Hydroxy-3,5-dimethoxybenzaldehyde oxime (5b). Light
brown solid (590 mg, 91% yield). Rf (50% EtOAc/hexane): 0.46.
Mp: 126–128 ꢀC. 1H NMR (300.13 MHz, MeOD-d4): d 3.82 (s, 6H),
6.87 (s, 2H), 7.99 (s, 1H). 13C NMR (75.5 MHz, MeOD-d4): d 56.6
(2CH3), 105.1 (2CH), 124.9 (C), 138.2 (C), 149.2 (2C), 150.7 (CH).
HRMS (ESI+, m/z): calcd for (C9H11NNaO4)+ (M + Na)+ 220.0580,
found: 220.0582.
Laccase from Trametes versicolor (2.5 U mgꢁ1) was purchased
from Sigma, while the laccase from Myceliophthora thermophila
(Novozym 51003, 3.14 U mLꢁ1) was acquired from Novozymes.
Enzymatic activities were measured using the ABTS assay.23
Thin-layer chromatography (TLC) was conducted using
Merck Silica Gel 60 F254 precoated plates and visualized with
UV and potassium permanganate stain. Column chromatogra-
1
phies were performed using silica gel 60 (230–400 mesh). H-
NMR, 13C-NMR, and DEPT experiments were registered using
different Bruker spectrometers (1H, 300.13 MHz and 13C, 75.5
MHz). The chemical shis (d) are given in ppm and the coupling
constants (J) in Hertz (Hz). High resolution mass spectra
(HRMS) experiments were carried out by ESI+ or ESIꢁ using an
Impact II ESI-Q-Tof spectrometer.
3,4-Dihydroxybenzaldehyde oxime (5c). Light brown solid
(448 mg, 89% yield). Rf (50% EtOAc/hexane): 0.42. Mp: decom-
poses at 160–162 ꢀC. 1H NMR (300.13 MHz, DMSO-d6): d 6.72 (d,
3JHH ¼ 8.1 Hz, 1H), 6.81 (dd, 3JHH ¼ 8.1 Hz, 4JHH ¼ 2.0 Hz, 1H),
4
7.04 (d, JHH ¼ 2.0 Hz, 1H), 7.91 (s, 1H), 9.18 (br s, 2H), 10.79
(br s 1H). 13C NMR (75.5 MHz, DMSO-d6): d 112.6 (CH), 115.5
(CH), 119.3 (CH), 124.5 (C), 145.5 (C), 146.9 (C), 148.2 (CH).
HRMS (ESI+, m/z): calcd for (C7H8NO3)+ (M + H)+ 154.0499,
found: 154.0498.
Synthesis of 4-hydroxy-3-methoxybenzonitrile (4a)24
Hydroxylamine hydrochloride (82 mg, 1.18 mmol) was added to
a solution of vanillin (3a, 150 mg, 0.986 mmol) in acetonitrile
(1 mL) and the mixture was stirred under reux for 4 h. Aer
cooling the mixture at room temperature, H2O was added and
the reaction was extracted with Et2O (3 ꢂ 10 mL). The organic
layers were combined, washed with an aqueous saturated
NaHCO3 solution (15 mL) and dried over Na2SO4. The solvent
was removed by distillation under reduced pressure and the
reaction crude was puried by column chromatography on
silica gel (eluent: 30% EtOAc/hexane) yielding the nitrile deriv-
ative 4a in 85% isolated yield. White solid. Rf (50% EtOAc/
General procedure for the synthesis of hydrazones 6a–c
Hydrazine monohydrate (10, 207 mL, 6.57 mmol) and glacial
acetic acid (10 drops) were successively added to a solution of
3a–c (6.57 mmol) in ethanol (10 mL). The mixture was stirred
under reux for 50 min or 1 h (see Table 1), and the solvent was
removed by distillation under reduced pressure isolating 6a–c
in 78–90% yield.
4-(Hydrazonomethyl)-2-methoxyphenol (6a). Yellow solid
(983 mg, 90% yield). Rf (50% EtOAc/hexane): 0.55. Mp: 177–
179 ꢀC. 1H NMR (300.13 MHz, MeOD-d4): d 3.93 (s, 3H), 6.88 (d,
3JHH ¼ 8.1 Hz, 1H), 7.24 (dd, 3JHH ¼ 8.3, 4JHH ¼ 1.9 Hz, 1H), 7.52
(d, 4JHH ¼ 1.9, 1H), 8.53 (s, 1H). 13C NMR (75.5 MHz, MeOD-d4):
56.4 (CH3), 110.8 (CH), 116.3 (CH), 125.3 (CH), 127.2 (C), 149.5
(C), 151.4 (C), 162.7 (CH). HRMS (ESI+, m/z): calcd for
(C8H11N2O2)+ (M + H)+ 167.0815, found: 167.0810.
1
ꢀ
hexane): 0.80. Mp: 86–88 C. H NMR (300.13 MHz, MeOD-d4):
3
d 3.87 (s, 3H), 4.91 (br s, 1H), 6.87 (d, JHH ¼ 8.1 Hz, 1H), 7.18
(dd, 3JHH ¼ 8.1, 4JHH ¼ 1.9 Hz, 1H), 7.21 (d, 3JHH ¼ 1.7 Hz, 1H).
13C NMR (75.5 MHz, MeOD-d4): d 56.6 (CH3), 103.1 (C), 115.7
(CH), 117.0 (CH), 120.5 (C), 127.7 (CH), 149.3 (C), 152.7 (C).
HRMS (ESI+, m/z): calcd for (C8H8NO2)+ (M + H)+ 150.0550,
found: 150.0548.
4-(Hydrazonomethyl)-2,6-dimethoxyphenol
yellow solid (1.03 g, 80% yield). Rf (50% EtOAc/hexane): 0.24.
(6b).
Light
General procedure for the synthesis of oximes 5a–c
Hydroxylamine sulfate (650 mg, 3.95 mmol) and sodium acetate Mp: 212–214 ꢀC. 1H NMR (300.13 MHz, DMSO-d6): d 3.82 (s, 6H),
(447 mg, 3.29 mmol) were successively added to a solution of 7.16 (s, 2H), 8.59 (s, 1H), 9.08 (br s, 1H). 13C NMR (75.5 MHz,
3a–c (3.29 mmol) in ethanol (3.3 mL). The mixture was stirred DMSO-d6): 56.0 (2CH3), 105.8 (2CH), 124.3 (C), 139.9 (C), 148.1
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4
(2C), 162.9 (CH). HRMS (ESI+, m/z): calcd for (C9H13N2O3)+ (M + (d, JHH ¼ 1.9, 1H), 8.03 (br s, 1H), 9.00 (s, 1H), 9.66 (s, 2H). 13C
H)+ 197.0920, found: 197.0915.
NMR (75.5 MHz, DMSO-d6): d 55.7 (CH3), 110.2 (CH), 115.9 (CH),
4-(Hydrazonomethyl)benzene-1,2-diol (6c). Light orange 122.4 (C), 125.5 (CH), 139.4 (2CH), 148.3 (C), 152.1 (C), 162.5 (CH).
solid (780 mg, 78% yield). Rf (50% EtOAc/hexane): 0.16. Mp: HRMS (ESI+, m/z): calcd for (C10H11N4O2)+ (M + H)+ 219.0877,
decomposes at 200 ꢀC. 1H NMR (300.13 MHz, DMSO-d6): d 6.81 found: 219.0878.
(d, 3JHH ¼ 8.1 Hz, 1H), 7.10 (dd, 3JHH ¼ 8.3, 4JHH ¼ 2.0 Hz, 1H),
4-(((4H-1,2,4-Triazol-4-yl)imino)methyl)-2,6-dimethoxyphenol
4
7.32 (d, JHH ¼ 2.0, 1H), 8.45 (s, 1H), 9.42 (br s, 2H). 13C NMR (8b). White solid (161 mg, 99% yield). Rf (10% MeOH/EtOAc):
(75.5 MHz, DMSO-d6): 113.9 (CH), 115.6 (CH), 121.9 (CH), 125.6 0.25. Mp: decomposes at 160 ꢀC. 1H NMR (300.13 MHz, DMSO-
(C), 145.7 (C), 148.9 (C), 160.5 (CH). HRMS (ESI+, m/z): calcd for d6): d 3.84 (s, 6H), 6.44 (br s, 1H), 7.16 (s, 2H), 8.99 (s, 1H), 9.48 (s,
(C7H9N2O2)+ (M + H)+ 153.0659, found: 153.0658.
2H). 13C NMR (75.5 MHz, DMSO-d6): d 56.1 (2CH3), 106.3 (2CH),
121.6 (C), 139.1 (2CH), 140.6 (C), 148.2 (2C), 160.8 (CH). HRMS
(ESI+, m/z): calcd for (C11H13N4O3)+ (M + H)+ 249.0982, found:
249.0982.
General procedure for the synthesis of semicarbazones 7a–c
Semicarbazide hydrochloride (11, 733 mg, 6.57 mmol) and
glacial acetic acid (10 drops) were successively added to a solu-
tion of 3a–c (6.57 mmol) in ethanol (10 mL). The mixture was
stirred under reux for 1.5–2.7 h (see Table 1) and the solvent
was removed by distillation under reduced pressure, isolating
semicarbazones 7a–c in good to excellent yields (73–98%).
2-(4-Hydroxy-3-methoxybenzylidene)hydrazine-1-carboxamide
(7a).25 Yellow solid (1.3 g, 98% yield). Rf (5% MeOH/EtOAc): 0.51.
Mp: 228–230 ꢀC. 1H NMR (300.13 MHz, DMSO-d6): d 3.81 (s, 3H),
6.45 (br s, 2H), 6.75 (d, 3JHH ¼ 8.1 Hz, 1H), 6.96 (dd, 3JHH ¼ 8.1,
4JHH ¼ 1.9 Hz, 1H), 7.97 (d, 4JHH ¼ 1.9, 1H), 7.71 (s, 1H), 9.31 (br s,
1H), 10.03 (br s, 1H). 13C NMR (75.5 MHz, DMSO-d6): d 55.7
(CH3), 109.0 (CH), 115.2 (CH), 121.1 (CH), 126.4 (C), 139.9 (CH),
147.9 (C), 148.0 (C), 157.0 (C). HRMS (ESI+, m/z): calcd for
(C9H12N3O3)+ (M + H)+ 210.0873, found: 210.0872.
2-(4-Hydroxy-3,5-dimethoxybenzylidene)hydrazine-1-carboxa-
mide (7b). Light orange solid (1.4 g, 89% yield). Rf (5% MeOH/
EtOAc): 0.42. Mp: 148–150 ꢀC. 1H NMR (300.13 MHz, DMSO-
d6): d 3.79 (s, 6H), 6.49 (br s, 2H), 6.97 (s, 2H), 7.70 (s, 1H), 8.65
(br s, 1H), 10.09 (s, 1H). 13C NMR (75.5 MHz, DMSO-d6): d 56.1
(2CH3), 104.2 (2CH), 125.2 (C), 136.9 (C), 139.9 (CH), 148.1 (2C),
157.0 (C). HRMS (ESI+, m/z): calcd for (C10H14N3O4)+ (M + H)+
240.0979, found: 240.0975.
4-(((4H-1,2,4-Triazol-4-yl)imino)methyl)benzene-1,2-diol (8c).
White solid (129 mg, 96% yield). Rf (5% MeOH/EtOAc): 0.37. Mp:
decomposes at 224–226 ꢀC. 1H NMR (300.13 MHz, DMSO-d6):
d 4.29 (br s, 2H), 6.89 (d, 3JHH ¼ 8.1 Hz, 1H), 7.16 (dd, 3JHH ¼ 8.2,
4
4JHH ¼ 2.0 Hz, 1H), 7.32 (d, JHH ¼ 2.0, 1H), 8.88 (s, 1H), 9.38 (s,
2H). 13C NMR (75.5 MHz, DMSO-d6): d 114.1 (CH), 116.0 (CH),
122.9 (C), 123.4 (CH), 139.2 (2CH), 146.1 (C), 150.8 (C), 161.0 (CH).
HRMS (ESI+, m/z): calcd for (C9H9N4O2)+ (M + H)+ 205.0720, found:
205.0721.
General procedure for the synthesis of hydrazono tetrazoles
9a–c
5-Hydrazinyl-1H-tetrazole (13, 227 mg, 2.27 mmol) and an
aqueous concentrated HCl solution (5 drops) were successively
added to a solution of 3a–c (2.27 mmol) in ethanol (4 mL). The
mixture was stirred under reux for 1.75–5 h (see Table 1) and
the resulting precipitate was ltrated from the reaction media,
yielding the corresponding tetrazoles 9a–c in 80–97% yield.
4-((2-(1H-Tetrazol-5-yl)hydrazono)methyl)-2-methoxyphenol
(9a). White solid (500 mg, 94% yield). Rf (10% MeOH/EtOAc):
1
ꢀ
0.72. Mp: 197–198 C. H NMR (300.13 MHz, DMSO-d6): d 3.84
3
(s, 3H), 6.75 (br s, 2H), overlapped with 6.81 (d, JHH ¼ 8.1 Hz,
2-(3,4-Dihydroxybenzylidene)hydrazine-1-carboxamide (7c).
Orange solid (936 mg, 73% yield). Rf (5% MeOH/EtOAc): 0.49.
1H) and 7.04 (dd, 3JHH ¼ 8.1, 4JHH ¼ 1.9 Hz, 1H), 7.48 (d, 4JHH
¼
1.9, 1H), 7.93 (s, 1H), 11.61 (br s, 1H). 13C NMR (75.5 MHz,
DMSO-d6): d 55.9 (CH3), 109.2 (CH), 115.4 (CH), 121.6 (CH),
125.9 (C), 143.8 (CH), 148.2 (C), 148.5 (C), 156.6 (C). HRMS (ESI+,
m/z): calcd for (C9H11N6O2)+ (M + H)+ 235.0938, found: 235.0936.
4-((2-(1H-Tetrazol-5-yl)hydrazono)methyl)-2,6-dimethoxyphenol
(9b). White solid (582 mg, 97% yield). Rf (10% MeOH/EtOAc): 0.66.
Mp: decomposes at 197–199 ꢀC. 1H NMR (300.13 MHz, DMSO-d6):
d 3.82 (s, 6H), 7.07 (s, 2H), 7.93 (s, 1H), 8.09 (br s, 2H), 11.71 (br s,
1H). 13C NMR (75.5 MHz, DMSO-d6): d 56.2 (2CH3), 104.5 (2CH),
124.8 (C), 137.3 (C), 143.7 (CH), 148.2 (2C), 156.5 (C). HRMS (ESI+,
1
ꢀ
Mp: decomposes at 210 C. H NMR (300.13 MHz, DMSO-d6):
d 6.30 (br s, 2H), 6.72 (d, 3JHH ¼ 8.1 Hz, 1H), 6.92 (dd, 3JHH ¼ 8.1,
4
4JHH ¼ 2.0 Hz, 1H), 7.09 (d, JHH ¼ 2.0, 1H), 7.66 (s, 1H), 8.98
(br s, 1H), 9.31 (br s, 1H), 9.98 (s, 1H). 13C NMR (75.5 MHz,
DMSO-d6): d 113.2 (CH), 115.5 (CH), 119.1 (CH), 126.3 (C), 140.2
(CH), 145.5 (C), 146.9 (C), 156.9 (C). HRMS (ESI+, m/z): calcd for
(C8H10N3O3)+ (M + H)+ 196.0717, found: 196.0709.
General procedure for the synthesis of triazoles 8a–c
4-Aminotriazole (12, 55 mg, 0.657 mmol) and an aqueous m/z): calcd for (C10H13N6O3)+ (M + H)+ 265.1044, found: 265.1038.
concentrated sulfuric acid solution (5 drops) were successively 4-((2-(1H-Tetrazol-5-yl)hydrazono)methyl)benzene-1,2-diol (9c).
added to a solution of 3a–c (0.657 mmol) in ethanol (2 mL). The White solid (400 mg, 80% yield). Rf (10% MeOH/EtOAc): 0.70. Mp:
mixture was stirred under reux for 3–7.5 h (see Table 1) and the decomposes at 250 ꢀC. 1H NMR (300.13 MHz, DMSO-d6): d 6.77 (d,
resulting precipitate was ltrated from the reaction media, 3JHH ¼ 8.1 Hz, 1H), 6.99 (dd, 3JHH ¼ 8.1, 4JHH ¼ 2.0 Hz, 1H), 7.20 (d,
yielding 8a–c in 96–99% yield.
4JHH ¼ 2.0, 1H), 7.86 (s, 1H), 8.99 (br s, 1H), 9.44 (br s, 1H), 11.45 (s,
4-(((4H-1,2,4-Triazol-4-yl)imino)methyl)-2-methoxyphenol (8a).16 1H), 15.23 (br s, 1H). 13C NMR (75.5 MHz, DMSO-d6): d 113.5 (CH),
White solid (142 mg, 99% yield). Rf (5% MeOH/EtOAc): 0.40. Mp: 115.5 (CH), 119.6 (CH), 125.9 (C), 144.1 (CH), 145.6 (C), 147.4 (C),
198–200 ꢀC. 1H NMR (300.13 MHz, DMSO-d6): d 3.84 (s, 3H), 6.97 156.5 (C). HRMS (ESI+, m/z): calcd for (C8H9N6O2)+ (M + H)+
(d, 3JHH ¼ 8.2 Hz, 1H), 7.33 (dd, 3JHH ¼ 8.2, 4JHH ¼ 1.9 Hz, 1H), 7.44 221.0781, found: 221.0783.
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d 56.5 (2CH3), 108.4 (2CH), 124.9 (2CH), 125.4 (2C), 126.3 (2C),
146.7 (2C), 149.5 (2C), 150.6 (2CH). HRMS (ESI+, m/z): calcd for
(C16H17N2O6)+ (M + H)+ 333.1081, found: 333.1086.
Synthesis of 5-hydrazinyl-1H-tetrazole 13
5-Aminotetrazole monohydrate (5 g, 48.51 mmol) was dissolved
in a 15% aqueous NaOH solution (50 mL). The mixture was
heated at 100 ꢀC and KMnO4 (7 g) was slowly added by portions
until the colour of the solution no longer changed. Aer stirring
30 min, ethanol was added to eliminate the excess of KMnO4.
The warm solution was ltered and concentrated under reduced
pressure to 1/3 of the initial volume. Sodium 5,50-azotetrazolate
was crystallized as a yellow solid (6.2 g, 61% yield).
To a suspension of sodium 5,50-azotetrazolate (6.2 g,
29.51 mmol) in water (62 mL), an aqueous HCl 6 N solution
(13 mL) was added. The mixture was warmed until the gas
evolution nished, then evaporated to dryness from water three
times to ensure the removal of HCl. The residue was dissolved
in the minimum volume of hot water and a hot solution of
NaOAc (6.2 g, 75.58 mmol) in water (6.2 mL) was added. The
apparatus was ushed out with carbon dioxide and the solution
was allowed to cool to give 5-hydrazinyl-1H-tetrazole (13) as
a white solid (987 mg, 20% yield). Mp: 192–194 ꢀC. 1H NMR
(300.13 MHz, DMSO-d6): d 8.17 (s, 4H). 13C NMR (75.5 MHz,
DMSO-d6): d 161.4 (C).
2,20-((6,60-Dihydroxy-5,50-dimethoxy-[1,10-biphenyl]-3,30-diyl)-
bis(methanylylidene))bis(hydrazine-1-carboxamide) (17).29 Dark
1
brown solid. H NMR (300.13 MHz, DMSO-d6): d 3.89 (s, 6H),
6.47 (br s, 4H), 6.96 (s, 2H), 7.39 (s, 2H), 7.75 (s, 2H), 8.76 (br s,
2H), 10.04 (s, 2H). HRMS (ESI+, m/z): calcd for (C18H20N6NaO6)+
(M + Na)+ 439.1337, found: 439.1339.
5,50-Bis((E)-((4H-1,2,4-triazol-4-yl)imino)methyl)-3,30-dime-
thoxy-[1,10-biphenyl]-2,20-diol (18). White solid. 1H NMR
(300.13 MHz, DMSO-d6): d 3.88 (s, 6H), 7.33 (d, 4JHH ¼ 1.9 Hz,
2H), 7.39 (d, 4JHH ¼ 1.9 Hz, 2H), 8.94 (s, 2H), 9.08 (s, 4H). 13C
NMR (75.5 MHz, DMSO-d6): d 55.9 (2CH3), 108.1 (2CH), 115.6
(2C), 121.5 (2C), 125.6 (2C), 126.4 (2CH), 138.9 (4CH), 148.8
(2C), 158.4 (2CH). HRMS (ESI+, m/z): calcd for (C20H19N8O4)+
(M + H)+ 435.1524, found: 435.1527.
General procedure for the laccase-catalyzed oxidation of
lignin model compounds in the presence of natural or
nitrogenated compounds
The corresponding derivative (16 mmol) was placed in a test
tube. The buffer (2 mL) saturated with O2 prior to the experi-
ment (pH 4.7: acetate buffer 100 mM; pH 5–6: citric
buffer 50 mM; pH 7: KPi buffer 100 mM) and the veratryl alcohol
(1, 6 mmol) or adlerol (2, 6 mmol) were added. Aer stirring, the
commercial laccases from Trametes versicolor (25 U) or the lac-
case from Myceliophthora thermophila (Novozym 51003, 31.4 U),
were added and the reaction stirred at 30 or 50 ꢀC, respectively,
during 72 h. Then, the reaction was acidied with an aqueous
HCl 2 N solution (4 drops) and extracted with EtOAc (2 ꢂ 1 mL).
The organic layers were combined and dried with Na2SO4. For
the oxidations of veratryl alcohol, an aliquot was taken and
analyzed by GC. In the case of the oxidation of adlerol, the
solvent of the organic layers was evaporated and the reaction
General procedure for the laccase-catalyzed oxidation of the
new synthesized compounds
In a test tube, the corresponding derivative was dissolved in an
organic cosolvent (EtOH or DMSO, 300 mL). Then, the corre-
sponding buffer saturated with O2 prior to the experiment
(2.7 mL) and the commercial laccases from Trametes versicolor
(25 U) or the laccase from Myceliophthora thermophila (Novozym
51003, 31.4 U), were successively added. A balloon that was l-
led with oxygen was attached to the test tube and the mixture
was stirred at 30 ꢀC or 50 ꢀC for the time shown in Table 2. Then,
the reaction was nished by extraction with EtOAc (3 ꢂ 2 mL),
and the organic layers were combined and dried with Na2SO4.
Aer solvent evaporation under reduced pressure, the reaction
crude was analyzed by 1H-NMR. In those cases in which an
abundant precipitate was detected in the reaction medium, this
1
crudes were analyzed by H-NMR. Thus, the conversions were
calculated.
1
was separated by decantation and analyzed by H-NMR.
6,60-Dihydroxy-5,50-dimethoxy-[1,10-biphenyl]-3,30-dicarbal-
Conflicts of interest
1
dehyde (14).26 Brown solid. H NMR (300.13 MHz, DMSO-d6):
d 3.92 (s, 6H), 7.41 (d, 4JHH ¼ 1.9 Hz, 2H), 7.44 (d, 4JHH ¼ 1.9 Hz,
2H), 9.80 (s, 2H), 10.11 (br s, 2H). 13C NMR (75.5 MHz, DMSO-
d6): d 56.0 (2CH3), 108.9 (2CH), 124.9 (2C), 127.4 (2C), 128.3
(2CH), 148.4 (2C), 151.3 (2C), 191.1 (2CH). HRMS (ESIꢁ, m/z):
calcd for (C16H13O6)ꢁ (M ꢁ H)ꢁ 301.0718, found: 301.0706.
6,60-Dihydroxy-5,50-dimethoxy-[1,10-biphenyl]-3,30-dicarboni-
trile (15).27 White solid. 1H NMR (300.13 MHz, DMSO-d6): d 3.89
(s, 6H), 7.23 (d, 4JHH ¼ 1.9 Hz, 2H), 7.41 (d, 4JHH ¼ 1.9 Hz, 2H),
9.85 (br s, 2H). 13C NMR (75.5 MHz, DMSO-d6): d 56.4 (2CH3),
100.3 (2C), 114.1 (2CH), 119.5 (2C), 124.6 (2C) 128.1 (2CH), 147.9
(2C), 148.9 (2C). HRMS (ESI+, m/z): calcd for (C16H13N2O4)+ (M +
H)+ 297.0870, found: 297.0870.
There are no conicts to declare.
Acknowledgements
This work was supported by the Spanish Ministry of Economy
and Competitiveness and the European Union (Project OXY-
POL, PCIN-2014-134 from an ERA-NET ERA-IB 2 Call).
Notes and references
1 (a) O. Y. Abdelaziz, D. P. Brink, J. Prothmann, K. Ravi,
6,60-Dihydroxy-5,50-dimethoxy-[1,10-biphenyl]-3,30-dicarbal-
dehyde dioxime (16).28 Light brown solid. 1H NMR (300.13 MHz,
MeOD-d4): d 3.92 (s, 6H), 6.99 (d, 4JHH ¼ 1.6 Hz, 2H), 7.27 (d, 4JHH
¼ 1.5 Hz, 2H), 8.01 (s, 2H). 13C NMR (75.5 MHz, MeOD-d4):
M. Sun, J. Garcıa-Hidalgo, M. Sandahl, C. P. Hulteberg,
´
´
C. Turner, G. Liden and M. F. Gorwa-Grauslund,
Biotechnol. Adv., 2016, 34, 1318–1346; (b) S. Sen, S. Patil
and D. S. Argyropoulos, Green Chem., 2015, 17, 4862–4887;
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 50459–50471 | 50469

View Article Online
RSC Advances
Paper
(c) K. Wang, H. Yang, S. Guo, X. Yao and R.-C. Sun, J. Appl. 10 (a) L. Munk, A. M. Punt, M. A. Kabel and A. S. Meyer, RSC
Polym. Sci., 2014, 131, 39673.
Adv., 2017, 7, 3358–3368; (b) M. D. Cannatelli and
A. J. Ragauskas, Appl. Microbiol. Biotechnol., 2016, 100,
8685–8691; (c) D. Huber, A. Pellis, A. Daxbacher,
G. S. Nyanhongo and G. M. Guebitz, Polymers, 2016, 8, 280/
1–280/10; (d) H. Wu, J. Noro, Q. Wang, X. Fan, C. Silva and
A. Cavaco-Paulo, RSC Adv., 2016, 6, 90427–90434; (e)
´
2 (a) X. Huang, O. M. M. Gonzalez, J. Zhu, T. I. Koranyi,
M. D. Boot and E. J. M. Hensen, Green Chem., 2017, 19,
175–187; (b) H. Yang, J. Li, J. Xu and L. Mo, BioResources,
2017, 12, 344–361; (c) H. Zhu, J. P. Max, C. L. Marcum,
¨
H. Luo, M. M. Abu-Omar and H. I. Kenttamaa, J. Am. Soc.
¨
¨
˘
Mass Spectrom., 2016, 27, 1813–1823; (d) M. D. Karkas,
I. F. Fi¸tigau, C. G. Boeriu and F. Peter, Macromol. Symp.,
B. S. Matsuura, T. M. Monos, G. Magallanes and
2015, 352, 78–86.
´
´
´
´
´
C. R. J. Stephenson, Org. Biomol. Chem., 2016, 14, 1853– 11 (a) J. Gonzalez-Sabın, N. Rıos-Lombardıa, I. Garcıa,
˜ ´
N. M. Vior, A. F. Brana, C. Mendez, J. A. Salas and
1914; (e) S. Huang, N. Mahmood, M. Tymchyshyn, Z. Yuan
and C. Xu, Bioresour. Technol., 2014, 171, 95–102.
´
´
F. Morıs, Green Chem., 2016, 18, 989–994; (b) A. Dıaz-
´ ´
Rodrıguez, L. Martınez-Montero, I. Lavandera, V. Gotor
3 (a) G. de Gonzalo, D. I. Colpa, M. H. M. Habib and
M. W. Fraaije, J. Biotechnol., 2016, 236, 110–119; (b)
´
and V. Gotor-Fernandez, Adv. Synth. Catal., 2014, 356,
´
´
´ ´
2321–2329; (c) K. K˛edziora, A. Dıaz-Rodrıguez,
F. Asina, I. Brzonova, K. Voeller, E. Kozliak, A. Kubatova,
B. Yao and Y. Ji, Bioresour. Technol., 2016, 220, 414–424; (c)
L. Zhang, T. You, T. Zhou, L. Zhang and F. Xu,
´
I. Lavandera, V. Gotor-Fernandez and V. Gotor, Green
Chem., 2014, 16, 2448–2453.
´
BioResources, 2016, 11, 3972–3986; (d) D. Salvachua, 12 (a) A. Kirsch, K. Ostendorf, A. Kharazipour and M. Euring,
R. Katahira, E. N. S. Cleveland, P. Khanna, M. G. Resch,
B. A. Black, S. O. Purvine, M. Zink, A. Prieto,
BioResources, 2016, 11, 7091–7101; (b) A. Rico, J. Rencoret,
´
´
´
J. C. del Rıo, A. T. Martınez and A. Gutierrez, Biotechnol.
´
´
¨
M. J. Martınez, A. T. Martınez, B. A. Simmons,
J. M. Gladden and G. T. Beckham, Green Chem., 2016, 18,
6046–6062; (e) T. D. H. Bugg, M. Ahmad, E. M. Hardiman
and R. Rahmanpour, Nat. Prod. Rep., 2011, 28, 1883–1896.
4 (a) Z. Ahmad, M. Asgher, F. Hussain and M. A. Randhawa,
BioResources, 2016, 11, 10511–10527; (b) C. Zhao, S. Xie,
Y. Pu, R. Zhang, F. Huang, A. J. Ragauskas and J. S. Yuan,
Biofuels, 2014, 7, 6; (c) P. Konst, S. Kara, S. Kochius,
D. Holtmann, I. W. C. E. Arends, R. Ludwig and
F. Hollmann, ChemCatChem, 2013, 5, 3027–3032; (d)
T. Rosado, P. Bernardo, K. Koci, A. V. Coelho, M. P. Robalo
and L. O. Martins, Bioresour. Technol., 2012, 124, 371–378;
˜
(e) A. I. Canas and S. Camarero, Biotechnol. Adv., 2010, 28,
694–705.
Green Chem., 2016, 18, 1306–1312; (c) P. Picart, 13 (a) T. Saravanakumar, H.-S. Park, A.-Y. Mo, M.-S. Choi,
´
´
L. Wiermans, M. Perez-Sanchez, P. M. Grande,
D.-H. Kim and S.-M. Park, J. Mol. Catal. B: Enzym., 2016,
134, 196–205; (b) S. Slagman, J. Escorihuela, H. Zuilhof
and M. C. R. Franssen, RSC Adv., 2016, 6, 99367–99375; (c)
R. Reiss, J. Ihssen, M. Richter, E. Eichhorn, B. Schilling
´
´
A. Schallmey and P. Domınguez de Marıa, ACS Sustainable
Chem. Eng., 2016, 4, 651–655.
5 (a) Y. Ohta, R. Hasegawa, K. Kurosawa, A. H. Maeda,
T. Koizumi, H. Nishimura, H. Okada, C. Qu, K. Saito,
¨
and L. Thony-Meyer, PLoS One, 2013, 8, e65633.
T. Watanabe and Y. Hatada, ChemSusChem, 2017, 10, 425– 14 J. Ravindran, G. V. Subbaraju, M. V. Ramani, B. Sung and
433; (b) L. Yang, K. Seshan and Y. Li, Catal. Today, 2017, B. B. Aggarwal, Biochem. Pharmacol., 2010, 79, 1658–1666.
298, 276–297; (c) T. D. Bugg and R. Rahmanpour, Curr. 15 H. E. Master, S. I. Khan and K. A. Poojari, Bioorg. Med. Chem.,
Opin. Chem. Biol., 2015, 29, 10–17. 2005, 13, 4891–4899.
6 (a) X.-Y. Jiang, Q. Lu, X.-N. Ye, B. Hu and C.-Q. Dong, 16 H. S. Patel and K. B. Patel, Phosphorus, Sulfur Silicon Relat.
BioResources, 2016, 11, 6232–6243; (b) M. Akazawa, Y. Kato
and Y. Kojima, J. Anal. Appl. Pyrolysis, 2016, 122, 355–364.
7 (a) L. T. M. Pham, S. J. Kim and Y. H. Kim, Biotechnol.
Biofuels, 2016, 9, 247; (b) E. Rosini, C. Allegretti, R. Melis,
Elem., 2009, 184, 2443–2452.
17 (a) M.-J. Han, H.-T. Choi and H.-G. Song, J. Microbiol., 2006,
¨
43, 555–560; (b) P. Cassland and L. J. Jonsson, Appl.
Microbiol. Biotechnol., 1999, 52, 393–400.
L. Cerioli, G. Conti, L. Pollegioni and P. D'Arrigo, Catal. 18 R. M. Berka, P. Schneider, E. J. Golightly, S. H. Brown,
Sci. Technol., 2016, 6, 2195–2205.
M. Madden, K. M. Brown, T. Halkier, K. Mondorf and
8 (a) M. D. Cannatelli and A. J. Ragauskas, Chem. Rec., 2017,
F. Xu, Appl. Environ. Microbiol., 1997, 63, 3151–3157.
17, 122–140; (b) T. Kudanga, B. Nemadziva and M. L. Roes- 19 Dimethylsulfoxide is known as radical scavenger, but it has
Hill, Appl. Microbiol. Biotechnol., 2017, 101, 13–33; (c)
M. Mogharabi and M. A. Faramarzi, Adv. Synth. Catal.,
been described as a useful organic co-solvent in laccase-
catalyzed reactions. See, for instance ref. 11c.
2014, 356, 897–927; (d) S. Witayakran and A. J. Ragauskas, 20 (a) B. Ramalingam, B. Sana, J. Seayad, F. J. Ghadessy and
Adv. Synth. Catal., 2009, 351, 1187–1209; (e) S. Riva, Trends
Biotechnol., 2005, 24, 219–226.
9 (a) B. Yao, P. Kolla, R. Koodali, Y. Ding, S. Balaranjan,
S. Shrestha and A. Smirnova, New J. Chem., 2017, 41, 958–
964; (b) S. Xie, Q. Sun, Y. Pu, F. Lin, S. Sun, X. Wang,
A. J. Ragauskas and J. S. Yuan, ACS Sustainable Chem. Eng.,
M. B. Sullivan, RSC Adv., 2017, 7, 11951–11958; (b)
N. H. Pham, F. Hollmann, D. Kracher, M. Preims,
D. Haltrich and R. Ludwig, J. Mol. Catal. B: Enzym., 2015,
120, 38–46; (c) K. Rittstieg, A. Suurnakki, T. Suortti,
K. Kruus, G. Guebitz and J. Buchert, Enzyme Microb.
Technol., 2002, 31, 403–410.
2017, 5, 2215–2223; (c) Q. Wang, S. Liu, G. Yang and 21 See, for instance: (a) J. Safaei-Ghomi and A. R. Hajipour, Org.
J. Chen, Bioresour. Technol., 2015, 175, 638–641.
Prep. Proced. Int., 2011, 43, 372–376; (b) D. Enders,
50470 | RSC Adv., 2017, 7, 50459–50471
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
L. Wortmann and R. Peters, Acc. Chem. Res., 2000, 33, 157– 25 R. B. de Oliveira, E. M. de Souza-Fagundes, R. P. P. Soares,
169.
A. A. Andrade, A. U. Krettli and C. L. Zani, Eur. J. Med.
Chem., 2008, 43, 1983–1988.
22 (a) P. Nousiainen, J. Kontro, H. Manner, A. Hatakka and
¨
`
J. Sipila, Fungal Genet. Biol., 2014, 72, 137–149; (b) 26 M. Delomenede, F. Bedos-Belval, H. Duran, C. Vindis,
`
T. M. Larson, A. M. Anderson and J. O. Rich, Biotechnol.
M. Baldas and A. Negre-Salvayre, J. Med. Chem., 2008, 51,
´
´
Lett., 2013, 35, 225–231; (c) M. Dıaz-Gonzalez, T. Vidal and
3171–3181.
T. Tzanov, Appl. Microbiol. Biotechnol., 2011, 89, 1693–1700; 27 S. Grelier, H. Cramail, A. Llevot, S. Carlotti and E. Grau,
(d) R. Bourbonnais and M. G. Paice, FEBS Lett., 1990, 267,
99–102.
Patent WO 2016/050988 A1, 2016.
28 D. R. Kelly, S. C. Baker, D. S. King, D. S. de Silva, G. Lord and
J. P. Taylor, Org. Biomol. Chem., 2008, 6, 787–796.
29 A. M. Costero, S. Gil, M. Parra, P. M. E. Mancini,
M. N. Kneeteman and M. I. Quindt, Tetrahedron Lett.,
2015, 56, 3988–3991.
¨
23 M. L. Niku-Paavola, L. Raaska and M. Itavaara, Mycol. Res.,
1990, 94, 27–31.
24 V. R. Kanetkar, D. D. Zope and Y. V. V. Subrahmanyam,
Indian J. Chem., Sect. B: Org. Chem. Incl. Med. Chem., 2005,
44, 1317–1318.
This journal is © The Royal Society of Chemistry 2017
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