On the factors affecting product distribution in laccase-catalyzed oxidation of a lignin model compound vanillyl alcohol: Experimental and computational evaluation

Article abstract of DOI:10.1039/c3ob40783g

Laccases (EC 1.10.3.2) are multicopper oxidases, which can oxidize phenolic substrates by the concomitant reduction of oxygen to water. The phenolic substructures of lignin are also oxidized by laccases, resulting mainly in various polymerized products. Several model compound studies indicate that variations in the reaction media, such as the pH and the enzyme dosage used, have an impact on the observed product distribution of laccase promoted oxidation, but no detailed study has been reported to explain these results. In the present study, a monomeric lignin model compound, vanillyl alcohol, was oxidized in laccase-catalyzed reactions by varying the pH, enzyme dosage and temperature. The energies of all the observed products and potential intermediates were calculated by applying density functional theory (DFT) and the polarizable continuum solvation model (PCM). The observed predominant product at pH 4.5 to 7.5 was clearly the 5-5′ dimer, although the thermodynamic product according to the calculated free energies was vanillin, the difference being 5.6 kcal mol^-1. The hydrogen bonding is shown to give an additional stabilizing effect on the transition state leading to the 5-5′ dimer, but also a kinetic barrier reduces the formation of vanillin. Based on the calculated pK_a-values of the proposed intermediates we suggest that the rearomatization reactions of the quinones formed in the radical reactions under mildly acidic and neutral conditions would preferentially occur through deprotonation rather than through protonation. The Royal Society of Chemistry.

Full text of DOI:10.1039/c3ob40783g

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On the factors affecting product distribution in laccase-
catalyzed oxidation of a lignin model compound
vanillyl alcohol: experimental and computational
evaluation†
Cite this: Org. Biomol. Chem., 2013, 11,
5454
Maarit Lahtinen,*a Petri Heinonen,a Mikko Oivanen,a Pirkko Karhunen,a
Kristiina Kruusb and Jussi Sipiläa
Laccases (EC 1.10.3.2) are multicopper oxidases, which can oxidize phenolic substrates by the concomi-
tant reduction of oxygen to water. The phenolic substructures of lignin are also oxidized by laccases,
resulting mainly in various polymerized products. Several model compound studies indicate that vari-
ations in the reaction media, such as the pH and the enzyme dosage used, have an impact on the
observed product distribution of laccase promoted oxidation, but no detailed study has been reported to
explain these results. In the present study, a monomeric lignin model compound, vanillyl alcohol, was oxi-
dized in laccase-catalyzed reactions by varying the pH, enzyme dosage and temperature. The energies of
all the observed products and potential intermediates were calculated by applying density functional
theory (DFT) and the polarizable continuum solvation model (PCM). The observed predominant product
at pH 4.5 to 7.5 was clearly the 5-5’ dimer, although the thermodynamic product according to the cal-
culated free energies was vanillin, the difference being 5.6 kcal mol−1. The hydrogen bonding is shown
to give an additional stabilizing effect on the transition state leading to the 5-5’ dimer, but also a kinetic
barrier reduces the formation of vanillin. Based on the calculated pKa-values of the proposed intermedi-
ates we suggest that the rearomatization reactions of the quinones formed in the radical reactions under
mildly acidic and neutral conditions would preferentially occur through deprotonation rather than
through protonation.
Received 18th April 2013,
Accepted 27th June 2013
DOI: 10.1039/c3ob40783g
www.rsc.org/obc
coupled through several types of interunit linkages, such as
Introduction
β-O-4, β-5, β–β, 5–5, 5–5/β-O-4 (dibenzodioxocin3), 5-O-4 and
Laccases (EC 1.10.3.2) are multicopper oxidases catalyzing the β-1.4,5 The most abundant linkage in native lignin is the β-O-4
oxidation of various types of substrates using oxygen as a co- linkage, with a frequency of 45–60% depending on the
substrate. The electrons of the substrates, which include source.6 However, when the monolignol coniferyl alcohol is
several types of mono- and polyphenols, methoxy- and amino- oxidized in vitro to dehydropolymers (DHPs), other linkages,
phenols, diamines, aromatic amines and thiols, and some in- especially the β-5 and β-β linkages are dominating in the
organic compounds, are ultimately transferred through the product.7 The relative amounts of different linkages in DHPs
four coppers of the enzyme to reduce the oxygen to water.1,2
have been shown to be dependent on several factors: the
One of the natural substrates of laccases is lignin, which is supply rate of the monomers, the concentration of the mono-
the second most abundant biopolymer on earth. Lignin is a lignol radicals, the pH, the presence of polysaccharides and
complex polymer composed of phenylpropane units, which are the presence of the growing DHP polymer.4,7,8 Attempts to
understand the biosynthesis of lignin have also produced
many theoretical studies comparing the different structures of
lignin.9–11 It has been shown that the most frequent linkages,
aLaboratory of Organic Chemistry, Department of Chemistry, University of Helsinki,
β-O-4, β-5, and β-β, are indeed the thermodynamically favoured
structures, but other factors in the lignifying cell wall must
also have an essential role.11
Because of the complex chemical structure of lignin, reac-
tions are usually studied with model compounds. Laccases
catalyze the oxidation of the phenol to a phenoxy radical,
P.O. Box 55, FIN-00014 HU, Finland. E-mail: maarit.lahtinen@helsinki.fi;
Fax: +3589 1915 0366; Tel: +3589 1915 0372
bVTT, Technical Research Centre of Finland, P.O. Box 1000, FIN-02044, VTT, Finland
†Electronic supplementary information (ESI) available: Computational energy
values for all stuctures and reaction energy changes in gas phase and in water
(PCM) are provided. 1H and 13C NMR spectra of 4-hydroxymethyl-2-(4′-hydroxy-
methyl-2′-methoxyphenoxy)-6-methoxyphenol (3). See DOI: 10.1039/c3ob40783g
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which can either couple to form dimeric to polymeric pro- has a wide pH-optimum range with guaiacol (5–7.5),25 which
ducts, or degrade to phenylpropane units.12–20 According to has a similar structure (methoxyphenol) compared to vanillyl
the present view, however, the degradative pathways are minor alcohol. The products were quantified with HPLC using auth-
reactions in laccase-catalyzed oxidations of lignin, unless a entic compounds as an external reference. In order to under-
suitable redox mediator, which can transfer electrons from stand the effect on product distribution, the thermodynamics
lignin to the laccase, is present.21
of the reactions was evaluated in detail by using computational
It has been shown that laccases follow the outer-sphere methods. Taking into account all the relevant intermediates of
mechanism and thus the radical reactions should not be con- the reactions the energies were obtained at the DFT((U)B3LYP)/
trolled enzymatically.22 The product distributions in laccase- 6-311++G(2df,2p) level of theory from the geometries obtained
catalyzed oxidations of lignin model compounds and also the at the DFT((U)B3LYP)/6-31+G(d) level.
degree of polymerization of isolated lignins have been shown
to vary according to the pH of the reaction media.13,19,23 In
addition, a comparison of separate studies performed using
the same model compound has led to the speculation that
Results and discussion
other factors such as enzyme dosage could have an impact on Laccase catalyzes the oxidation of a phenolic compound to a
the product distribution.18 For example, for the laccase-cata- phenoxy radical, which then reacts further in non-enzymatic
lyzed oxidation of vanillyl alcohol (1) three variable sets of pro- reactions.1 Based on earlier studies18,19,24 performed with
ducts have been reported (Fig. 1). Areskogh et al. detected vanillyl alcohol (1) it was assumed that three different products
5-5′ (2) and 5-O-4′ (3) dimers when Trametes villosa and could be formed initially from two phenoxy radicals of vanillyl
Myceliophthora thermophila laccases were used for oxidation at alcohol (1): 5-5′ dimer (2), 5-O-4′ dimer (3) and vanillin (4). The
pH values 5 and 7.5.19 The product distribution was found to tentative reaction pathways for the formation of each product
vary according to the pH: the 5-5′ product (2) was formed pre- are depicted in Fig. 2 and 3. It is generally accepted that coup-
ferably at the more acidic pH. On the other hand, we found ling of two phenoxy radicals leads to the formation of a
5-5′-coupled dimers (2 and analogous structures) as the main quinone, which is rearomatized to the final product. However,
products and vanillin as a minor product (4) with laccases the mechanisms of the rearomatization and oxidation of the
Trametes hirsuta and Melanocarpus albomyces at pH 4.5.18 The hydroxymethyl side chain of phenols have not yet been veri-
5-5′ dimer (2) was oxidized further to 5-5′ dimers with aldehyde fied.26 We decided to study all reaction pathways by comparing
functionalities as well as to dibenzodioxepin-type trimers. In the observed product distributions with the calculated thermo-
addition, Crestini et al. obtained vanillin (4) and 4,4′-di- dynamic parameters of the ionic structures that may be pro-
hydroxy-3,3′-dimethoxybenzophenone (5) when Trametes versicolor posed to be involved. The reaction pathways considered are
laccase was used to catalyze the oxidation at pH 5.24
shown in Fig. 2 and 3 through tentative formal ionic inter-
The present study aimed at evaluating the combined effect mediates. Free energies and pKa-values were calculated for
of the pH, enzyme dosage and temperature on the product dis- each ion and final product. It is clear that all of these formal
tribution of laccase-catalyzed oxidation of vanillyl alcohol (1). ions do not represent actual accumulating intermediates as
The thermostable M. albomyces laccase was used; this enzyme such. Nevertheless, the calculated pKa-values of these limiting
structures allow us to draw conclusions on the predominant
ionic forms under the reaction conditions, which is an essen-
tial factor for deducing the possible mechanisms. Further-
more, the state of equilibrium may be estimated over the
sequential ions. Thus, the data are valuable for the discussion
of the factors steering the competition between the pathways.
Three different mechanisms were considered for the for-
mation of vanillin (4, Fig. 3). The mechanisms for the oxi-
dation of the hydroxymethyl side chain of phenols have not yet
been verified.26 Firstly, it has been shown that phenolic
mediators (ArOH) or mediators containing a hydroxylamine
(–NOH) group act through transferring a hydrogen atom from
the benzylic position of
a
non-phenolic substrate.26,27
Secondly, laccase-catalyzed reaction of a syringylic β-1 model
compound in the presence of H218O has been shown to
produce degradation products containing the 18O, which has
been explained by a cationic intermediate.15 It was also recently
shown that with phenolic syringyl-type mediators C–O coupled
products can be detected, especially in the early stages of
laccase-catalyzed reactions.28 The mechanism through the 1-O-4′
intermediate has been also suggested earlier by Kirk et al.12
Fig. 1 Previously found products from the laccase-catalyzed oxidation of vanillyl
alcohol.18,19,24
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Fig. 2 Tentative mechanisms for the formation of 5-5’ (2) and 5-O-4’ (3) dimers.
Although the 1-O-4′ dimer has not been detected in the oxi-
At the lowest enzyme dosage applied, only the 5-5′ product
dation of vanillyl alcohol (1), the structure has been identified 2 was formed, except in one experiment (pH 7.5, at room
from the oligomeric products of 1 and vanillic acid.13,19 Thus, temperature). The 5-O-4′ product 3 and vanillin (4) were
mechanisms for the formation of vanillin (4) were proposed observed as minor products at the higher enzyme dosages.
based (1) on hydrogen atom transfer from the benzylic posi-
With the lowest enzyme dosage applied at room temp-
tion by a phenoxy radical, (2) on the formation of a quinonoid erature, the optimum pH range for the formation of 5-5′
1-O-4′ dimer, which could then be degraded to vanillin (4) and product 2 was 5–7, which is similar compared to guaiacol
the phenol and (3) on a disproportionation leading to a cation, (5–7.5).25 Under all other conditions, the highest amount of
which could deprotonate to form vanillin.
5-5′ product 2 was formed at the lowest pH 4.5, and the yield
was decreased with increasing pH (Fig. 4). Similarly, the total
amount of oxidation products decreased with increasing pH.
This result is in accordance with the earlier report on the
decreasing activity of laccases as the pH increases.29
The limits of the applied quantitative HPLC prevented a
very accurate evaluation of the amounts of 5-O-4′ dimer (3) and
vanillin (4). These were formed at pH ≥ 6.0 at room tempera-
ture, and at higher temperature they were formed over the pH-
range studied. The absence of 5-O-4′ product 3 at pH 7.5 may
be an indication of lability of this compound under the more
basic conditions, although no degradation products were
detected. The slight loss of recovered material at higher
enzyme dosages, which were required for the formation of pro-
ducts 3 and 4, could also be due to increased coupling of free
Yields of different products from laccase-catalyzed oxidation of
vanillyl alcohol in varying reaction conditions
The assumption that at the initial stages of oxidation catalyzed
by laccase only products 2–4 would form was seemingly
correct: these products were the only ones detected in all
experiments after the 2 hour reaction time. The yields of oxi-
dation products and unreacted vanillyl alcohol are shown in
Table 1. The highest dosage of 50 nkat ml−1 was applied only
in one experiment, because the product distribution was very
similar compared to 10 nkat ml−1 dosage at higher tempera-
tures. In other words, the increase of temperature had a
similar effect as the increase of enzyme dosage on product
formation.
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Fig. 3 Tentative mechanisms for the formation of vanillin (4) by several possible reaction routes.
radicals with the aromatic amino acids of laccase,30 as no (1R) were calculated (Fig. 5) and compared with the observed
other products formed from vanillyl alcohol (trimers etc.) were product distribution. The values were similar compared to
detected.
Concluding the results of product distribution, the amount
those calculated previously.24
The O4 of phenoxy radical 1R had the highest spin density,
of 5-5′ product 2 and total yield of the oxidation products followed by C1, C3 and C5. The spin density of the benzylic
decreased as the pH increased. As a consequence, the relative position was very low. Thus, based on spin densities, coupling
amounts of 5-O-4′ (3) and vanillin (4) increased with increasing through O4 would be most probable, but the observed product
pH and enzyme dosage. At higher pH-values the ratio of the distribution showed that coupling through C5 was more
products was possibly distorted because of the decrease of the favourable. It is noteworthy that the spin density of C5 was
total amount of all compounds and the possible lability of the lower than that of C1 and C3, which were occupied by hydroxy-
5-O-4′ compound (3).
methyl and methoxyl groups. High spin densities have been
previously calculated also for the substituted sites of mono-
lignols (p-coumaryl, coniferyl and sinapyl alcohols), and it was
postulated that reaction through an occupied site would be
Total spin densities of vanillyl alcohol’s phenoxy radical
compared to the observed product distribution
The rate-determining step in the laccase-catalyzed oxidation of unfavourable due to steric hindrance.11
a phenol is the removal of the electron to form a phenoxy
radical cation, which is then rapidly deprotonated.31 The spin
Whereas the radical coupling involves bond formation
between two atoms bearing a high spin density, the spin
densities of the phenoxy radical formed from vanillyl alcohol density of the H-atom abstractor is transferred to the H-atom
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Table 1 Yields of vanillyl alcohol (1) and different oxidation products, 5-5’
dimer (2), 5-O-4’ dimer (3) and vanillin (4) obtained from laccase-catalyzed oxi-
dations under varying reaction conditions
Yields of compounds 1–4 (%)
Dosage (nkat ml−1
)
t (°C) pH
1
2
3
4
Total
1
1
1
1
1
1
1
1
rt
rt
rt
rt
4.5
5.0
6.0
7.0
7.5
4.5
5.0
6.0
7.0
7.5
4.5
5.0
6.0
7.0
7.5
4.5
5.0
6.0
7.0
7.5
4.5
89.5
95.1
92.0
98.6
96.4
97.7
87.0
96.5
95.2
97.9
7.7
8.9
9.4
8.6
4.0
9.1
8.3
6.9
6.7
2.2
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
97.2
104.0
101.4
107.2
rt
1.7 102.1
—
—
—
—
—
0.5
—
0.6
0.7
0.6
0.8
0.6
0.8
0.5
0.6
1.0
45
45
45
45
45
rt
rt
rt
rt
rt
45
45
45
45
45
rt
106.8
95.3
103.4
101.9
100.1
Fig. 5 Total spin densities for the phenoxy radical of vanillyl alcohol (1R).
1
1
thermodynamic corrections for gas phase energies. All opti-
10
10
10
10
10
10
10
10
10
10
50
73.3 24.5
83.8 17.6
71.2 15.4 1.7
69.5 15.5 2.0
98.3 mized geometries were proven to be real minima having no
101.4
88.9
91.0
91.0 through deprotonation and protonation. The energy changes
96.9
97.6
87.7
imaginary frequencies. The changes in reaction energies were
calculated for rearomatization of the quinonoid intermediates
74.7 15.7
—
60.3 33.7 2.1
61.0 33.9 2.1
67.8 17.6 1.5
68.7 17.3 1.6
71.9 15.5
57.2 31.6 2.1
based on the PCM model correlated better with the observed
experimental results and thus these results are presented
88.1 below. The gas-phase energy changes can be found from the
88.0
91.9
—
ESI.† Although one can argue that DFT energies are not accu-
rate to more than approximately a few kcal, it is generally sup-
posed that inaccuracies cancel and the reaction energies
reflect reality well.
Free energy changes of different initial radical couplings
At the first stage after radical formation three competing path-
ways may be considered. Two radicals could couple to form
different quinones (5-5′, 5-O-4′ or 1-O-4′), one radical could
abstract a hydrogen from the benzylic position of the other, or
the two radicals could disproportionate to form a cation and a
phenolate anion. The formation of 5-5′ quinone (2QQ) may
lead to the formation of two stereocenters, resulting in
three stereoisomers: a pair of enantiomers (RR and SS) and
a meso form.
The relative free energy for the formation of quinone 4Q
Fig. 4 Yield of the 5-5’ dimer as a function of pH under varying reaction con-
through H-atom transfer (routes v and vi), leading to the for-
mation of vanillin, was clearly the most favourable (Table 2).
On the other hand, disproportionation to an anion and a
cation was not favourable, showing a positive ΔG-value.
ditions. Diamond: enzyme dosage 1 nkat ml−1, room temperature (rt); square: 1
nkat ml−1, 45 °C; triangle: 10 nkat ml−1, rt; multiplication symbol: 10 nkat ml−1
45 °C.
,
The ΔG difference between the two dimers (2QQ and 3Q)
was small. The calculated ΔG-values for the formation of 2QQ
and 3Q from two radicals were slightly negative and in the
donor.32 These two different processes, radical coupling and
H-atom abstraction most probably have different energy bar-
riers, which might affect the product distribution through
different reaction kinetics. Clearly, the calculated spin den-
sities could not explain the observed product distribution.
Table 2 Changes of free energy for different possible initial radical coupling
Reaction
ΔG (kcal mol−1
)
Comparison of energy changes
1R + 1R → 2QQ (RR, SS)
1R + 1R → 2QQ (meso)
1R + 1R → 3Q
1R + 1R → 4Q (v, vi)
1R + 4R → 4Q (v, vi)
1R + 1R → (1 + 1)Q
1R + 4R → (1 + 4)Q
1R + 1R → 4CAT + 1P−
1R + 4R → 4CAT + 4P−
−2.2
−2.6
−3.8
−28.8
−33.3
−7.0
−10.7
32.8
The energies for each product and intermediate were deter-
mined using the DFT((U)B3LYP)/6-311++G(2df,2p) level of
theory and the solvent effects were taken into account applying
the polarizable continuum solvation model (PCM). The geo-
metries were optimized by DFT((U)B3LYP)/6-31+G(d) cal-
culations which also provided the zero point energies and the
21.8
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same range as the calculated free energies for the coupling of
monolignol p-coumaryl alcohol to 5-5′ and 5-O-4′ products.11
Thus, the relative free energies at this stage could not explain
the observed dominance of product 2.
Transition states for the quinone intermediates and H-atom
transfer
Computations of the transition states for radical–radical coup-
ling as well as for H-atom abstraction between two radicals
were attempted using 4-hydroxymethylphenoxy radical (6R) as
a model (i.e. phenoxy radical of vanillyl alcohol without the
methoxyl group). The transition states of radical–radical coup-
ling reactions proved to be beyond reach with DFT methods,
but this type of reaction for monolignols is reported to have
only a small energy barrier of approximately 2–5 kcal mol−1 9
.
Although the transition states for radical–radical coupling
were not resolved, it was found that the transition state
leading to the formation of 5-5′ quinone 2QQ was clearly
stabilized by the hydrogen bonding between the two hydroxy-
methyl groups of the forming quinone (Fig. 6).
An estimate of the transition state energy for the hydrogen
atom abstraction from the benzylic position could be obtained
by using 4-hydroxymethylphenol (6) as the H-atom donor
instead of the radical (Fig. 7). The bond distances were 1.25 Å
for the O–H and 1.32 Å for the forming C–H. The energy
barrier for the reaction was found to be 18 kcal mol−1. Assum-
ing that the formation of a biradical would not be significantly
Fig. 7 Transition state of the H-atom transfer from 4-hydroxymethylphenol (6)
to 4-hydroxymethylphenoxy radical (6R).
Table 3 Changes of the free energies for the aromatizations of the quinone
intermediates
more favourable than the formation of benzyl radical at the Aromatization through protonation
transition state, this value (18 kcal mol−1) shows that the
Protonation
Aromatization
Overall aromatization
energy barrier of H-atom abstraction is clearly higher com-
pared to the 2–5 kcal mol−1 for the radical coupling. This Product
ΔG
Product
ΔG
Reaction
ΔG
partly explains the differences between the experimental data
and the calculated energies for product formation. A higher
energy barrier compared to the radical coupling through an
unoccupied site (C5) could also be involved in the formation
of vanillin (4) through the 1-O-4′ dimer due to steric
hindrance.
2QHQ m
2QHQ e
2PQH
3QH
4QH
24.7
26.2
25.0
27.2
13.0
2PQ
2PQ
2
3
4
−44.0
−45.8
−42.4
−48.7
−29.1
2QQ → 2PQ
2QQ → 2PQ
2PQ → 2
3Q → 3
−19.3
−19.6
−17.4
−21.5
−16.1
4Q → 4
Aromatization through deprotonation
Aromatization
Protonation
Product
Overall aromatization
Product
ΔG
ΔG
Reaction
ΔG
2PQ− m
2PQ− e
2PP−
3P−
−0.9
−1.3
2PQ
2PQ
2
3
4
−18.3
−18.3
−5.6
−13.7
−8.9
2QQ → 2PQ
2QQ → 2PQ
2PQ → 2
3Q → 3
−19.4
−19.6
−17.4
−21.5
−16.1
−11.8
−7.8
4P−
−7.2
4Q → 4
Rearomatization of quinones
The overall free energy change for rearomatization was favour-
able by 16–22 kcal mol−1 (Table 3). When the rearomatization
was split into two steps, aromatization and protonation, it was
found that aromatization and following protonation of the
phenolate anions decreased free energies, but protonation of
the quinones increased free energies. These energy changes
are visualized in Fig. 8, showing the relative energies of all
Fig. 6 An attempt to compute the transition state for the formation of 5-5’
quinone (6QQ) using 4-hydroxymethyl (6) as the model, revealing the stabilizing
hydrogen bond between the hydroxymethyl groups.
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The 5-O-4′ (3) was clearly the least favourable product, by
14.0 kcal mol−1 compared to 5-5′ (2). Also, under experimental
conditions, 3 was formed only when higher enzyme dosages
were used. Thus, the computational results were in agreement
with the experimental results when the two products formed
through radical coupling were compared. On the other hand,
this effect is parallel with the effect of hydrogen bonding
found in the formation of 5-5′ dimer (2) providing an
additional stabilization for the transition state.
Vanillin (4) was not produced in a detectable amount at the
lowest enzyme dosage at room temperature. However, accord-
ing to the calculated free energies, the amount of vanillin (4)
should have been much higher compared to the amount of 5-
O-4′ (3), if thermodynamic control was followed. In fact, the
observed amount of vanillin (4) was close to that of the 5-O-4′
dimer (3). For comparison, vanillin (4) was also totally absent
in the product mixture obtained by Areskogh et al.19 A possible
explanation is that the additional steering effect of the hydrogen
bonds at the transition state leading to the formation of 5-5′
dimer (2QQ) may enhance the formation of 2. A kinetic barrier
for the H-atom abstraction or 1-O-4′ dimer formation, whichever
route is followed, may have an effect in the same direction. An
additional proof for the kinetic barrier is that when the product
formation was, in a previous study, monitored as a function of
time under mild reaction conditions (i.e. low enzyme dosage
and room temperature) at pH 4.5, vanillin (4) was clearly
observed at a later time than the 5-5′ dimer (2).18
Fig. 8 Relative changes of free energies of different intermediates for the for-
mation of 5-5’ dimer (2) from vanillyl alcohol (1). Top: rearomatization through
deprotonation; bottom: rearomatization through protonation.
Calculated pKa-values for the products and quinone
intermediates
The total amount of products decreased with increasing pH
(Table 1), reflecting the decreasing activity of laccases with
increasing pH.29 In addition, however, the observed ratio of
different products also seemed to be affected by the pH as the
amount of the 5-5′ dimer (2) decreased more relative to the
other products. To further investigate the effect of pH, pKa’s
were calculated for the products and intermediates (Table 5).
The applied method is known to cause a systematic error.33
proposed intermediates involved in the formation of dehydro-
divanillyl alcohol (2). A noteworthy difference between the
pathways through deprotonation and protonation is that in
deprotonation the free energy is decreased at all intermediate
stages, whereas protonation results in an increase of energy.
Comparison of the free energy changes between the final
products 2–4
Table 5 Calculated pKa values of different phenolic products and
The changes in the free energies calculated for the formation
of the observed products from 1 are compared in Table 4. The
thermodynamically most favoured product was vanillin (4). In
comparison to the reaction energy leading to vanillin (4) the
changes in the free energies were 5.6 kcal mol−1 and 19.6 kcal
mol−1 less negative for the 5-5′ (2) and 5-O-4′ (3) dimers.
intermediates
Structure
Calculated pKa
Literature pKa
1
2
9.36
6.85
9.7834
6.8734
3
4
8.93
7.71
8.92
5.08
5.17
10.13
2.38
7.4034
9.9935
Phenol
2QQ (RR, SS)
2QQ (meso)
2PQ → 2PQ−
2PQ → 2PP−
3Q
Table 4 Total free energy changes for the reaction products 2–4 formed from
1. The relative ΔG has been obtained by setting the energy of the compu-
tational thermodynamic product (4) to 0.0 kcal mol−1
3.40
Reaction
product
Reaction ΔG
Relative ΔG
4Q
3.56
(kcal mol−1
)
(kcal mol−1
)
2QHQ (RR, SS)
2QHQ (meso)
2PQH
3QH
4QH
−1.32
−0.94
−1.02
−1.58
2.08
2
3
4
−62.9
−48.9
−68.6
5.6
19.6
0.0
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The systematic error for the calculations was corrected based that are affected by the pH, but without the ability to dis-
on the experimental literature values of methoxyphenols.34 tinguish the radical reactions from the proton-transfer reac-
The systematic error for the intermediates might have differed tions, experimental evidence of any such factor is impossible
from the one for methoxyphenols, as it did for the phenol,35 to gain. It has been shown that laccases do not control the
and thus the values can be over- or underestimated. However, radical reactions, and also pH has been shown to affect the
for example, the experimentally determined value for the pKa- product distribution in Ag2O-oxidation of coniferyl
value of the protonated quinone methide of Mitomycin C is alcohol.22,38 If the oxidant does not affect the product ratio as
−1.2, and the calculated values were in a similar range.36
a function of pH or the rearomatization reactions cannot
The calculated pKa-values for protonated quinones revealed explain the observed product distribution, the radical reactions
that they are highly acidic. Thus, in the applied pH range, could be affected by the pH. Possibly, the changes in the reac-
starting from pH 4.5, the quinones are not protonated. The cal- tion environment affect the orientation of the reacting radicals
culated pKa-values for the quinones 3Q and 4Q were below the as they diffuse away from the oxidant or the hydrogen bonding
applied pH-range (3.40 and 3.56), which suggests that the in the transition states is altered as the pH changes.
phenolate anion forms are dominating in all experiments.
However, the calculated pKa-values for the 5-5′ quinone (2QQ)
stereoisomers were 5.08 (RR, SS) and 5.17 (meso). Thus, the
equilibrium of the 2QQ and its anion could change in the
Conclusions
applied pH-range and the phenolate form would start to domi- By a comparison of the observed product distributions of the
nate above pH 5. Likely, the anion could be thought to rearo- laccase-catalyzed oxidation of vanillyl alcohol and the results
matize through the deprotonation pathway, rather than obtained by using computational methods, it seemed that the
through two protonation steps. Although the rearomatization observed product distribution could be explained to some
of quinones is usually depicted as taking place through proto- extent. The observed relative product amounts correlated with
nation, because of the acidic environment, our results suggest the computational free energy changes when the two products
that the pathway through deprotonation could be more likely, formed through radical coupling were compared. Vanillin,
also considering the decreasing energy at different stages of which is formed through a different route, should have been
the reaction (Fig. 8). Of course, the rates of proton transfer are the most favourable product, in contrast to the observed
fast, and thus protonation of the neutral quinone form could amount. Although the energies of the biradical transition
also be possible. The high acidities of protonated quinones states could not be computed using DFT, the stabilizing hydro-
could also explain the formation of 4,4′-dihydroxy-3,3′- gen bonding leading to the 5-5′ dimer and also the kinetic
dimethoxybenzophenone (5) that was detected by Crestini barrier for the formation of vanillin were evident.
et al.24 The work-up of the oxidation products before analysis
with the strong acid HCl could enable the formation of the function of pH were mainly affected by the rate-limiting oxi-
benzophenone. dation of the substrate by laccase, as the enzyme activity
It was also clear that the amounts of different products as a
As the kinetic barrier for the radical coupling leading to the decreases with increasing pH. However, the observed change
two dimers, 5-5′ (2) and 5-O-4′ (3) is very low and the reaction in the product ratio as a function of pH was not evidently
could be reversible, the acid–base equilibrium of the quinone explained by the computed thermodynamics of the rearomati-
intermediates or kinetic barriers of the proton-transfer steps zation reactions. It is possible that at different pH-values
could affect the product distribution. Incubation of the 5-5′ either the geometry of the reacting radicals or hydrogen
dimer (2) in D2O–d6-DMSO at 45 °C did not result in the for- bonding is affected. Although the computed pKa-values could
mation of the other two products, and thus there seemed to be not explain the product ratios, it seemed likely that rearomati-
no thermodynamic equilibrium between the different quinone zation proceeds through deprotonation rather than through
forms. The kinetic barrier for the C–H bond cleavage was protonation under the applied pH-range of 4.5–7.5.
found to be only 5.6 kcal mol−1, calculated from the transition
According to these results, the differences in the reaction
state of internal proton transfer from 2PQ− to 2PP−, which media, which may affect the solubility of the substrate and pre-
excluded also the possibility that kinetic barriers at the proton ferences of laccases, have also a significant effect on the
transfer stages could affect the product distribution. Thus, the formed products because of the intrinsic chemical properties
proton transfer reactions seemed not to be able to explain the of lignin substructures. Possibly, modification of lignin by
different product ratios at different pH-values.
laccases under varying reaction conditions could also result in
As the rate-limiting step of a laccase-catalyzed reaction is different chemical properties of the formed products.
the oxidation of the substrate,37 the decreasing yield of the 5-5′
product (2) could mainly be due to the decreasing activity of
laccase as a function of increasing pH. However, neither the
decreasing laccase activity nor the thermodynamics or kinetics
of the rearomatization reactions can explain the observed
Experimental
Reagents, equipment and analysis
decreasing amount of 5-5′ dimer relative to the other products All reagents and solvents were used as received unless other-
as the pH increased. Thus, there must be some other factors wise mentioned. A Varian Mercury 300 MHz spectrometer was
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used for NMR measurements, which were performed at 27 °C 170.79 (–OCOCH3); HR-TOF-ESI-MS (pos) m/z 450.1754
in CDCl3. For the high-resolution mass spectra (HR-ESI-MS) [M + NH4]+ (C22H28NO9 requires 450.1759).
the Bruker Daltonics microTOF ESI-TOF was used; aceto-
Reaction procedure for the oxidation of vanillyl alcohol by
laccase and preparation of HPLC samples
nitrile–water was used as the solvent and Agilent ES Tuning-
Mix (G2421A) diluted in acetonitrile–water was used for the
calibration.
HPLC was performed using a reverse-phase column (Agilent
The variables in the reaction conditions were enzyme dosage,
temperature and pH. The used enzyme dosages were 1, 10 and
50 nkat ml−1 based on activity determination with ABTS, and
the last mentioned dosage of 50 nkat ml−1 was used in
only one experiment. Reactions were carried out at room temp-
erature (rt) and 45 °C. Reactions were performed using
5 different buffers: 25 mM Na-succinate, pH 4.5; 25 mM Na-
acetate, pH 5.0; 25 mM Na-citrate, pH 6.0; 25 mM Na-citrate,
pH 7.0; McIlvaine buffer, pH 7.5 (0.2 M Na2HPO4 and 0.1 M
citric acid).
Vanillyl alcohol (1) (20 mg, 1.3 mmol) was dissolved in
dioxane (0.2 ml, distilled over sodium). Buffer was added, fol-
lowed by a suitable dilution of the enzyme, to a total volume of
0.8 ml of buffer and enzyme dilution. The mixture was stirred
for 2 h, and a 20 μl sample was taken to a vial containing NaN3
(to inhibit laccase,18 Riedel-de Haën), water and methanol.
The volume of the final sample was 2 ml and it contained
2.5 mM of NaN3 in methanol–water (1 : 1). The samples were
filtrated prior to analysis using a syringe filter (0.45 μm).
ZORBAX Eclipse XDB-C8, 4.6 mm × 15 cm, 5 μm), methanol–
water as the eluent and an Agilent 1200 series HPLC. Gradient
elution was applied for 5 minutes starting from 35% methanol
(v/v); the flow rate was 0.3 ml min−1. The unreacted vanillyl
alcohol and the oxidation products were quantified from the
samples using external calibration. For the external cali-
bration, a linear three-point calibration curve was produced for
each compound using the software of the HPLC (Agilent
ChemStation). All authentic compounds were found to be
stable during the HPLC runs. The results of the quantitative
analysis of the starting material and products are shown in
Table 1.
Enzyme and determination of enzyme activity
The M. albomyces laccase was overproduced in Trichoderma
reesei and purified as described earlier.39
Laccase activity was determined using ABTS [2,2′-azino-bis-
(3-ethylbenzothiazoline)-6-sulphonate] (Sigma) as a substrate
in 25 mM Na-succinate buffer, pH 4.5, at 22 °C.40 Absorbance
of the oxidation product was measured at 436 nm (ε = 29 300
M−1 cm−1). The activity was determined from the slope of the
linear curve and expressed as katals.
Computational methods
A conformational analysis of all studied structures was done at
PM3 semiempirical level using in house made perl script and
GAMESS ab initio program to scan systematically conformers.
The best conformers were optimized with the DFT B3LYP
method using 6-31+G(d) basis set and unrestricted wave func-
tion in the case of radical species. Optimized geometries were
proven to be real minima by evaluating vibrational analysis in
the same level of theory, the calculation providing also the
thermodynamic corrections which were used without scaling.
The single point energies were calculated at the DFT B3LYP
level using a larger 6-311++G(2df,2p) basis set. Energies in the
solvent (water) were obtained using the PCM continuum sol-
vation model with 6-311++G(2df,2p) basis set and gas phase
geometries. All calculations were done with GAMESS ab initio
program.
To calculate the free energy change of protonation in a reac-
tion, the computed energy change for deprotonation of a
phenol was used, from which the experimental value was sub-
tracted from (ΔGcomp − ΔGexp). The experimental values used
were (at 298.15 K) 344.5 kcal mol−1 for the gas phase43 and
13.62 kcal mol−1 in water (calculated from the pKa of phenol,
9.99).35
Synthesis of vanillyl alcohol and reference compounds
Vanillin (4) was purchased from Fluka. Vanillyl alcohol (1) was
reduced from vanillin with NaBH4 (Fluka) in ethanol and 5-5′
dimer (2) was synthesized according to the method reported
by Adler and Hernestam.41
4-Hydroxymethyl-2-(4′-hydroxymethyl-2′-methoxyphenoxy)-
6-methoxyphenol (3). The 5-O-4′ dimer was obtained as the
monoaldehyde through oxidation of vanillyl alcohol with Ag2O
as described by Ralph et al.42 The monoaldehyde (120 mg,
0.39 mmol) was dissolved in ethanol (15 ml) and NaBH4
(70 mg, 1.9 mmol) was added to the resulting solution. After
stirring (2 h), water was added (10 ml) followed by 2 M HCl
(2 ml). The product was extracted with ethyl acetate; the
organic phase was washed with water, fresh 10% NaHCO3 and
brine and dried with Na2SO4. The product was found to
degrade without the NaHCO3 treatment. Evaporation of the
solvent yielded 90 mg (0.29 mmol, 75%) of product (3).
1H NMR (300 MHz, CDCl3), δH = 2.06, 2.11, 2.24 (9H, each s,
–OCOCH3), 3.83, 3.85 (6H, each s, –OCH3), 4.95, 5.07 (4H, each
s, –CH2–), 6.42 (1H, d, J 1.8, ArH), 6.69 (1H, d, J 1.8, ArH),
6.86–6.97 (3H, m, ArH); 13C NMR (75 MHz, CDCl3), δC = 20.31,
20.90, 20.98 (–OCOCH3), 56.06, 56.20 (–OCH3), 65.83, 66.00
(–CH2), 106.63, 110.37, 112.98, 120.59, 121.11 (ArC–H), 130.14,
The pKa-values were calculated using the total free energies
in water from PCM calculations using a formula modified
from Yu et al.:33
ΔGdeprot
pKaðHAÞ ¼ pKaðH2OÞ þ
ln 10 RT
132.75, 134.22 (ArC–CH2OCOCH3, ArC–OCOCH3), 144.95, here, ΔGdeprot is the computational free energy change in water
150.10, 150.99, 152.62 (ArC–OCH3, ArC–OAr), 168.36, 170.60, for the deprotonation of the acid by a hydroxide ion. For the
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pKa of water, a value of 13.995 was used.35 Based on a compari- 17 K. Rittstieg, A. Suurnakki, T. Suortti, K. Kruus, G. Guebitz
son of the literature values,34 all values were corrected by −1.5.
and J. Buchert, Enzyme Microb. Technol., 2002, 31, 403;
A. Marjasvaara, M. Torvinen, H. Kinnunen and
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18 M. Lahtinen, K. Kruus, P. Heinonen and J. Sipilä, J. Agric.
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Acknowledgements
The Finnish Bioeconomy Cluster (FIBIC) is gratefully acknow- 19 D. Areskogh, J. Li, P. Nousiainen, G. Gellerstedt, J. Sipilä
ledged for financial support.
and G. Henriksson, Holzforschung, 2010, 64, 21.
20 M.-L. Mattinen, P. Maijala, P. Nousiainen, A. Smeds,
J. Kontro, J. Sipilä, T. Tamminen, S. Willför and L. Viikari,
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