Laccase-catalysed homocoupling of primary aromatic amines towards the biosynthesis of dyes

Article abstract of DOI:10.1002/adsc.201300501

Coloured disubstituted benzoquinonimine trimeric structures are obtained as main reaction products of the oxidation of p-electron donor pri-mary aromatic amines using two different laccases, CotA-laccase from Baccilus subtilus and TvL from Trametes versicolor. These orange-red to purple products, presenting high molar extinction coeffi-cients, presumably result from oxidative homocou-pling reactions, through the formation of N-C bonds at positions 2 and 5, of the laccase oxidised inter-mediate as showed in the proposed oxidative path-way. The product of 1,4-phenylenediamine is shown to be the trimer known as Bandrowski's base which has an established role in hair and fur dyeing. Our results also show that the occurrence and/or rates of oxidation of aromatic amines are strongly dependent on the presence of p-electron releasing substituents in the aromatic ring and are independent on the properties of the enzyme used. Overall our data con-tribute for (i) understanding key features of laccase reactivity with p-substituted aromatic amines and (ii) establishing enzymatic processes that lead to the syn-thesis of coloured bio-products under mild condi-tions with potential impact in the cosmetic and dye industries.

Full text of DOI:10.1002/adsc.201300501

FULL PAPERS
DOI: 10.1002/adsc.201300501
Laccase-Catalysed Homocoupling of Primary Aromatic Amines
towards the Biosynthesis of Dyes
Ana Catarina Sousa,a,b Lꢀgia O. Martins,c,* and M. Paula Robaloa,b,*
a
ꢁrea Departamental de Engenharia Quꢀmica, Instituto Superior de Engenharia de Lisboa, Rua Conselheiro Emꢀdio
Navarro 1, 1959-007 Lisboa, Portugal
Fax : (+35)-1-2183-17267; phone: (+35)-1-2183-17163; e-mail: mprobalo@deq.isel.ipl.pt
Centro de Quꢀmica Estrutural, Complexo I; Instituto Superior Tꢂcnico, Universidade Tꢂcnica de Lisboa, Av. Rovisco
b
Pais, 1049-001 Lisboa, Portugal
Instituto de Tecnologia Quꢀmica e Biolꢃgica, Universidade Nova de Lisboa, Av. da Repfflblica, 2780-15 Oeiras, Portugal
c
Fax : (+35)-14-411-277; phone: (+35)-12-1446-9534; e-mail: lmartins@itqb.unl.pt
Received: June 7, 2013; Revised: September 5, 2013; Published online: October 9, 2013
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201300501.
Abstract: Coloured disubstituted benzoquinonimine results also show that the occurrence and/or rates of
trimeric structures are obtained as main reaction oxidation of aromatic amines are strongly dependent
products of the oxidation of p-electron donor pri- on the presence of p-electron releasing substituents
mary aromatic amines using two different laccases, in the aromatic ring and are independent on the
CotA-laccase from Baccilus subtilus and TvL from properties of the enzyme used. Overall our data con-
Trametes versicolor. These orange-red to purple tribute for (i) understanding key features of laccase
products, presenting high molar extinction coeffi- reactivity with p-substituted aromatic amines and (ii)
cients, presumably result from oxidative homocou- establishing enzymatic processes that lead to the syn-
À
pling reactions, through the formation of N C bonds thesis of coloured bio-products under mild condi-
at positions 2 and 5, of the laccase oxidised inter- tions with potential impact in the cosmetic and dye
mediate as showed in the proposed oxidative path- industries.
way. The product of 1,4-phenylenediamine is shown
to be the trimer known as Bandrowskiꢄs base which Keywords: aromatic amines; biocatalysis; dyes; lac-
has an established role in hair and fur dyeing. Our case; oxidation; radical coupling
Introduction
benign and low cost reactions.[3,4] Their use in the bio-
synthesis of fine chemicals has been fairly explored
Eco-friendly enzymatic bioprocesses represent an at- due to the high specificity, e.g., phenolic substruc-
tractive and important alternative to the conventional tures, such as o- and p-diphenols, methoxyphenols,
chemical synthetic processes. The potential benefits of aminophenols and lignin-related molecules.[2–6] Gener-
utilising biocatalysts, such as enzymes, compared to ally, laccases react with phenolic substrates, which
inorganic or organic catalysts, arise from their activity loose one electron and one proton, generating the
under mild conditions of temperature, pH and pres- corresponding free radicals (phenoxy radicals) that
sure. Moreover, higher reaction rates and specificities are stabilised by resonance into the respective qui-
are usually obtained when compared with reactions none structures or covalently coupled to oligo- or
using chemical catalysts.
polymeric products. The competence of laccases to
Laccases represent an interesting group of multi- promote homo- or heteromolecular coupling reactions
copper oxidoredutive biocatalysts (EC 1.10.3.2) with has been described, namely in reactions between phe-
great potential for industrial and environmental appli- nols or quinonoid systems and primary amines, with
cations.[1,2] Laccases are widely distributed in fungi, the formation of new C O, C N or N N prod-
higher plants and bacteria showing a broad spectra of ucts.[7–12] Aromatic amines have been mainly used as
low-molecular weight substrates either of natural or nucleophiles in phenol reactions catalysed by lac-
À
À
À
synthetic origin. Laccases catalyse their oxidation
cases.[8,13,14] The coupling of a typical laccase substrate,
ACHTUNGTRENNUNG
using dioxygen as co-substrate which is reduced to such as a substituted hydroquinone, with primary
water in aqueous solutions, resulting in environmental amines usually occurs as nucleophilic amination on
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Adv. Synth. Catal. 2013, 355, 2908 – 2917

Laccase-Catalysed Homocoupling of Primary Aromatic Amines towards the Biosynthesis of Dyes
the aromatic ring by the substitution of hydrogen,[7,8]
halides or alkyl groups.[13] The substituted quinonoid
products formed are often very reactive and can un-
dergo further amination, leading to para-diaminated
compounds. Also, extended reaction periods, high
temperatures or reactions in the presence of an excess
of amine promote the formation of diaminated prod-
ucts.[8]
More recently, however, the use of aromatic amines
as substrates for laccase was described in the oligo-
merisation of aniline for the production of conducting
polyaniline[15] or in the polymerisation of aniline de-
rivatives.[16]
Figure 1. Representative structures of the p-substituted aro-
matic amines.
aminobenzoic acid (4-ABA), 4-aminobenzonitrile (4-
1,4-Phenylenediamine (1,4-PDA) or its substituted ABN) and 4-nitroaniline (4-NA) (Figure 1). These
derivatives and 4-aminophenol (4-AP) are extensively measurements were performed at different pH values,
used in the cosmetic industry as dye precursors or as since pH affects both the properties of enzymes and
oxidation bases for the permanent dyeing of furs and substrates with a strong impact in enzymatic reactivi-
hair. These colourless or weakly coloured compounds ty.
give rise to coloured compounds by oxidative conden-
The aromatic amines show their oxidation process-
sation at the highest alkaline pH range in the pres- es in the potential range 0.40 to 1.30 V at a scan rate
ence of hydrogen peroxide.[17–19] The development of of 100 mVsÀ1 (Table 1) and the electrochemical be-
less aggressive processes for the production of hair haviour showed to be dependent on the electronic
dyes is of great interest due to the involvement of nature of the p-substituent group. The aromatic
direct human contact and laccases are excellent alter- amines with p-electron-withdrawing groups (4-ABA,
native candidates for the production of biocolourants 4-ABSA, 4-ABN and 4-NA) were characterised by
under mild conditions.
well-defined irreversible oxidation peaks (Epa) in the
Therefore, the present work focuses on the charac- pH range. The absence of reduction peaks (Epc) indi-
terisation of homocoupling reactions of p-substituted cates that the electrochemically generated species are
aromatic amines using laccases. The aim is the pro- not stable and rapidly decay to non-reducible counter-
duction of biocolourants, through enzymatic reactions parts. Compounds with p-electron-donor groups, in
better tailored for oxidative synthesis. Four out of particular 1,4-PDA and 4-ADA, presented quasi-re-
eight primary p-substituted aromatic amines were se- versible redox processes at lower pHs (4 and 5),
lected as the enzymeꢄs substrates and orange-red to which become irreversible for higher pHs.
purple coloured diaminated quinonimine products
Independently of the electronic nature of the p-sub-
were identified and characterised by NMR and mass stituent, the oxidation potentials decrease as the pH
spectrometry. The kinetic constants and conversion increases and differences up to 230 mV (for 4-AP)
yields were determined and the results are discussed were obtained. Based on the obtained results, the
in relation with the properties of enzymes used: the amines can be split in two groups: the first group
bacterial CotA-laccase from Bacillius subtilis and comprising compounds with p-electron-donor groups,
TvL-laccase from the fungi Trametes versicolor. A showing oxidation potentials between 0.40 and 0.87 V
pathway for the laccase oxidation of p-substituted ar- (1,4-PDA, 4-ADA, 4-AP and 4-APA) and more prone
omatic amines is proposed.
to be oxidised, and the second group comprising sub-
strates harbouring p-electron-withdrawing groups,
with significantly higher oxidation potentials, between
1.07 and 1.30 V (4-ABA, 4-ABSA, 4-ABN and 4-
NA). Considering the redox potential of the T1
copper centres of CotA and TvL laccases, 0.55 V and
0.79 V (vs. NHE)[24,25] respectively, only the first group
Results and Discussion
Electrochemical Studies of Substrates
The difference in the redox potential between the oxi- of compounds is expected to be oxidised by the lac-
doreductases and their substrates is a critical parame- case systems.
ter in determining the occurrence and/or rates of en-
zymatic reactions.[20–23] Therefore, the redox potential
was determined by cyclic voltammetry for eight aro- Enzymatic Oxidation of p-Substituted Aromatic
matic amine substrates as follows: 1,4-phenylenedi- Amines
ACHTUNGTRENNUNGamine (1,4-PDA), 4-aminodiphenylamine (4-ADA),
4-aminophenol (4-AP), 4-aminophenylacetamide (4- The first indication of reaction between p-substituted
APA), 4-aminobenzenesulfonic acid (4-ABSA), 4- aromatic amines and laccases is the formation of
Adv. Synth. Catal. 2013, 355, 2908 – 2917
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Ana Catarina Sousa et al.
Table 1. Electrochemical data of p-substituted aromatic amines vs. NHE in buffered solutions at a scan rate of 100 mVsÀ1.
FULL PAPERS
Substrate
pH 4
pH 5
pH 6
pH 7
Epa (V)
Epc (V)
E1/2 (V)
Epa (V)
Epc (V)
Epa (V)
Epc (V)
Epa (V)
1,4-PDA
4-ADA
4-AP
4-APA
4-ABA
4-ABSA
4-ABN
4-NA
0.60
0.52
0.63
0.87
0.48
0.44
0.43
–
0.54[a]
0.52[b]
0.49
0.53
0.83
–
–
–
–
0.43
0.31
0.32
–
–
–
0.47
0.48
0.48
0.82
1.20
1.18
1.26
1.30
0.37[c]
–
0.26
–
–
–
–
–
0.41
0.44
0.40e)
0.79
1.07
1.16
1.23
1.21
0.48[d]
–
–
–
–
–
–
[f]
[f]
–
–
1.22
–
–
–
[f]
[f]
–
–
–
–
[f]
[f]
[a]
DE=120 mV, Ipc/Ipa =1.0.
[b]
[c]
[d]
[e]
[f]
E
1/2 =0.475 V, quasi-reversible wave, DE=90 mV, Ipc/Ipa =0.9.
DE=100 mV, Ipc/Ipa =0.5, irreversible wave.
DE=80 mV, Ipc/Ipa =1.0.
Epc =0.23 V.
Not detected. Potentials out of the experimental range.
colour when the enzyme was added to reaction mix- tive optimal pH values (Table 2). CotA in general ex-
tures. According to our prediction, the aromatic hibited higher specificity (kcat/KM) values than TvL.
amines with p-electron-withdrawing groups were not No major differences in KM are observed except for
oxidised, while the compounds bearing p-electron- 4-APA where 4-fold higher KM values were calculated
donor groups were all oxidised by both laccases, high- as compared with the other substrates. In contrast,
lighting the need for a good match between the redox the oxidation rates (kcat) showed major differences;
features of the enzyme and the substrate. We have the electron-richer amines 1,4-PDA, 4-ADA and 4-
tested the optimal pH for the four substrates selected AP are oxidised faster, while the 4-APA reacts slower
(1,4-PDA, 4-ADA, 4-AP and 4-APA) and have con- similarly to what was previously observed for the oxi-
cluded that, as shown previously to occur with phe- dation of different phenolic substrates.[23,26,32]
nolic substrates,[26] TvL shows maximal rates at pH 4–
The parameter kcat is reflective of the enzymatic
5, whereas CotA shows a preference for pH values rate-limiting step, assumed to be the substrate oxida-
close to neutrality, pH 5–7 (data not shown). This dif- tion and accordingly, the kcat values followed the
ference relates to the presence of a negatively trend of redox potential of the substrates.
charged residue close to the active site in TvL and
The higher redox potential of TvL would appear to
also in other fungal laccases, proposed to have a role favour an increased reaction rate, as the electron-
in facilitating the formation and stabilisation at low transfer rate (kET) is a major component of the pa-
pH values of the radical intermediate formed during rameter kcat. However, the oxidation rates for the sub-
the catalytic reaction.[27–29] In the case of CotA, or any strates tested are lower for TvL, except for 4-ADA,
bacterial laccase identified so far, no negatively as compared with CotA (Table 2). According to the
charged residue in the vicinity of the substrate bind- Marcus theory, two additional factors apart from the
ing site[30] is present and the efficiency of the oxida- redox potential (Eo) affect the kET in proteins: the
tion of phenols or aromatic amines rely mostly on the donor-acceptor electronic coupling (HAD), where the
protonation/deprotonation state equilibria of the sub- exact geometry of the protein matrix has an impor-
strates themselves.[26,31]
tant role and the reorganisation energy (l) which also
The kinetic constants of CotA and TvL laccases for depends on the structure and dynamics of the pro-
the selected substrates were determined at the respec- tein.[33] In the present case, the reasons for the small
Table 2. Kinetic parameters of oxidation reactions catalysed by CotA and TvL laccases. Reactions were performed at the op-
timal pH (Britton–Robinson buffer) for each substrate.
CotA
KM
[mM]
TvL
KM
[mM]
Substrate Optimal
pH
kcat [sÀ1]
kcat/KM
Substrate Optimal
pH
kcat [sÀ1] kcat/KM
[mMÀ1 sÀ1]
[mMÀ1 sÀ1]
1,4-PDA
4-ADA
4-AP
7
5
6
7
0.6Æ0.1 14.3Æ0.5 22.6Æ4.1
0.4Æ0.1 14.3Æ0.3 35.0Æ4.0
1,4-PDA
4-ADA
4-AP
5
4
5
5
1.3Æ0.1 6.0Æ0.1 4.8Æ0.2
1.4Æ0.2 29.5Æ2.0 21.3Æ1.0
0.5Æ0.1 0.8Æ0.1 1.6Æ0.4
3.9Æ0.4 0.4Æ0.1 0.10Æ0.04
0.5Æ0.2 2.2Æ0.2
4.6Æ0.8
4-APA
1.3Æ0.1 0.20Æ0.04 0.10Æ0.02
4-APA
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Laccase-Catalysed Homocoupling of Primary Aromatic Amines towards the Biosynthesis of Dyes
differences on the overall rate of reactions of the two ly. The central iminic skeleton defined at C-2 and C-5
enzymes must rely in the electronic tunnelling and/or positions was identified, in all cases, by the presence
the reorganisation energy of the two electron transfer of characteristic singlets for protons H-3 and H-6 in
processes (from the substrate to the T1 centre and the aromatic region (5.68–7.64 ppm) and the corre-
from this to the trinuclear site) in TvL and CotA. sponding carbon signals in the 90–100 ppm range in
However, none of these possibilities was investigated the 13C NMR spectra. The multiplicity of these pro-
here.
tons (H-3, H-6) and their correlation with carbons C-
1
The conversion yields of CotA for the different p- 2 and C-5 (153–156 ppm), in the H-13C HMBC spec-
substituted aromatic amines after 24 h of reaction are tra, confirmed the attributed quinonoid character of
1
presented in Table 3. The orange-red to purple reac- the structures. For compound 1a, the H NMR data
were in accordance with the literature values.[34]
The oxidation product of 1,4-PDA, the trimer 1,4-
diamino-2,5-benzoquinonediimine (1) was isolated in
two forms 1a and 1b (Figure 2), showing distinct col-
ours (orange and dark purple, respectively) and dis-
tinct water solubilities. We have analysed both forms
by UV-Vis spectrophotometry and have observed a re-
versible interconversion upon pH change in accord-
ance with the pKa of the trimer.[35] The two absorption
bands, near 335 and 450 nm observed for 1a
(Figure 3), arise from p-p* electronic transitions
within the benzoquinonoid ring and are shifted to 350
and 540 nm, respectively in form 1b. This red-shift,
more pronounced for the lowest energy band, may
result from the delocalisation of the conjugated p-
system and the shift values found are within the range
of related benzoquinonediimine derivatives.[35,36]
Therefore, the appearances of 1a and 1b are ex-
plained by the protonation/deprotonation at the
Table 3. Products formed after 24 h of reaction using 5 mM
substrate, 1 UmLÀ1 CotA laccase, at 378C in methanol:buff-
er (1:10).
Substrate pH Reaction products
(Yield [%])[a]
Colour l [nm] (e
[MÀ1 cmÀ1])
1,4-PDA
7
1b (90)
dark
510 (3,467)
purple
8
6
6
7
1a (95)
2 (97)
5 (75)
3 (31)
4 (20)
orange 450 (3,408)[b]
4-ADA
4-AP
4-APA
violet
570 (1,046)[c]
brown 400 (9,551)[c]
orange 360 (8,335)
yellow
–
[a]
Isolated yields.
Acetone:buffer (1:10).
Methanol.
[b]
[c]
tion products, with high molar extinction coefficients, iminic nitrogens and formation of dicationic cyanine
were isolated by solid-phase extraction (products subunits stabilised by intramolecular delocalisation in
soluble in buffer solution) or filtration (insoluble 1b.
products) in amounts considered sufficient for struc-
This trimer is known as the Bandrowskiꢄs base and
tural characterisation. Higher conversion yields were is traditionally obtained from 1,4-PDA oxidation in
obtained for 4-ADA 1,4-PDA and 4-AP when com- aqueous ferrocyanide solution[35] or by permanent
pared with 4-APA, in agreement with the kinetic hair colouration processes with alkaline hydrogen per-
data. Identical products and similar conversion yields oxide.[18] Taking this into account, the present work
were obtained for the TvL enzyme (data not shown). shows an alternative bioprocess for the production of
Overall our results suggest that the electronic this product at high yields (90 to 95%) and milder
nature of the p-substituent plays a crucial role in the conditions.
oxidation efficiency of aromatic amines by laccase.
In the case of 4-APA, two products are formed, the
trimer 3[37] and an additional yellow azo product (4)[38]
resulting from the coupling of the enzymatic oxidised
1
Identification of the Biotransformation Products
4-APA intermediates (Figure 2). The H NMR spec-
trum of 4 showed a multiplet centered at 7.80 ppm in-
The oxidation of 1,4-PDA, 4-ADA and 4- APA, i.e., dicating the formation of a symmetric structure and
those bearing amine or amide electron-releasing a low field shift for all the aromatic protons, consis-
groups, result in the formation of trimers with a 1,4- tent with the formation of an N=N bond. Further-
substituted-2,5-benzoquinonediimine skeleton (1–3, more, the respective mass spectrum shows peaks at
Figure 2). The oxidation of 4-AP, with a hydroxy m/z=295 and 297 in negative and positive modes, re-
group results in the formation of the trimeric struc- spectively, as well as a major peak at m/z=319 ([M+
ture 5, with a 1,4-benzoquinonemonoimine skeleton Na]+) which are consistent with a molecular mass of
substituted at positions 2 and 5.
296 gmolÀ1. Fragmentation of the positive ion (m/z=
The aromatic nature of the benzoquinonediiminic 297) generated the major ions with m/z of 255, 162
structures (1, 2 and 3) was confirmed by the presence and 134 (base peak), which can be explained by cleav-
À
of multiple signals in the range 5.5–7.8 ppm and 90- age of the C N amide bound (m/z=255) and by the
1
160 ppm in both H and 13C NMR spectra, respective- cleavage at the central azo group followed by loss of
Adv. Synth. Catal. 2013, 355, 2908 – 2917
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Ana Catarina Sousa et al.
FULL PAPERS
Figure 2. Structures of main products formed in laccase reactions with p-substituted aromatic amines.
N2, a typical fragmentation of aromatic azo com- presence of the hydroxy group on C-1, from which
pounds.[39]
a quinonic structure could be easily obtained, should
The formation of an azo compound as product of be the responsible for the formation of the 1,4-benzo-
the biotransformation of aromatic amines was previ- quinonemonoimine skeleton. This trimeric structure is
ously observed by Vanhulle et al.[10] in the degrada- different from the one reported before for 4-AP oxi-
tion of the anthraquinonic dye, Acid Blue 62 by lac- dation[40,41] leading to the formation of the 1,4-substi-
cases. We showed later that this azo product is tuted-2,5-benzoquinonediimine trimer.
formed through the creation of an azo bond by cou-
pling between the amine radical intermediates of the
enzymatic reaction.[11]
Proposed Pathway for the Oxidation of p-Substituted
The oxidation of 4-AP leads to formation of Aromatic Amines by Laccases
a brown solid (5) with a 2,5-diaminated 1,4-quinone-
monoiminic structure. The multiplicity of H-3 and H- Electron-donor p-substituted aromatic amines can be
6 protons, the presence of two signals at low field, used as substrates for laccases with their oxidation
177.6 and 161.6 ppm (characteristic from C=O and C= being initiated by one electron abstraction by the T1
N bonds respectively), in the 13C NMR spectrum and Cu(II) ion of laccases. This leads to the formation of
the HMBC correlations between the H-3, H-6 and aminyl radicals in a pathway similar to the one previ-
the carbonyl signal at 177.6 ppm, are indicative of the ously described for the oxidation of phenol deriva-
diaminated 1,4-quinonemonoiminic structure. The tives.[1,4,6,42]
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Laccase-Catalysed Homocoupling of Primary Aromatic Amines towards the Biosynthesis of Dyes
Conclusions
Herein, we characterise the ability of laccase to gener-
ate colour “in situ” with potential application in, e.g.,
hair permanent dyeing from originally non-coloured
aromatic amines. The use of oxidoreductive enzymes
represents a convenient and eco-friendly way of syn-
thesising new molecules of complex structure. There-
fore, laccases can be used in alternative bioprocesses
for the cosmetic and dye industries.
Coloured homocoupling products from 1,4-PDA, 4-
ADA, 4-APA with trimeric 1,4-substituted-2,5-benzo-
quinonediimine structures were unambiguously iden-
tified and characterised, together with a trimeric 1,4-
quinonemonoimine structure for 4-AP, where the
presence of the hydroxy group allowing easy oxida-
tion to quinone, is responsible for the formation of
the substituted 1,4-quinonemonoimine trimer.
The rates of reactions, conversion yields and prod-
ucts formed are independent of the enzymatic system
used. The enzymeꢄs efficiency is mostly dependent on
the redox features of substrates and consequently on
the electronic nature of the p-substituent. Our data
show that the presence of a p-electron-donor group
Figure 3. UV-Vis spectra of forms 1a and 1b (5”10À4 M) in
Britton–Robinson buffer (pH 4 or 10) with 10% of
(CH3)2CO.
Scheme 1 shows the proposed pathway that leads to converts recalcitrant amine molecules into laccase
the formation of the disubstituted benzoquinonedii- substrates by increasing the electronic density on the
mine trimers. The initial step of the laccase oxidation amine group.
process should be the abstraction of an electron fol-
lowed by deprotonation of the substrate. The end
products are expected to be two short-lived inter-
mediates: an aminium cation radical (A+C) or a neutral
radical species (AC), the aminyl radical, in accordance
with species previously detected in the course of the
chemical oxidation of 1,4-PDA by ferricyanide[19] and
as intermediates of the amineꢄs oxidation by cyto-
chrome P450 enzyme.[43,44] Thus, electron-donor sub-
Experimental Section
General Procedures
All reagents and solvents are commercially available
(Sigma–Aldrich Co) and were used without further purifica-
tion. The UV-visible spectra of compounds 1a, 1b, 2, 3 and 5
stituents in the p-position, by stabilising the radical were obtained in B&R buffer or methanol in a Cecil Instru-
ments CE2041 spectrophotometer. The molar extinction co-
efficients for compounds (1, 2, 3 and 5) were determined in
the concentration range (1: 3”10À5–4.5”10À4 M, 2: 1”10À4–
5.5”10À4 M, 3: 3”10À5–4.5”10À4 M and 5: 3”10À5–2.5”
10À4 M) in a Nicolet Evolution 300 spectrophotometer. The
product characterisation was performed by 1D NMR
(1H,13C) and 2D NMR (COSY, HSQC, HMBC and
NOESY) spectra, obtained in DMSO-d6, (CD3)2CO, or
CD3OD on a Bruker Advance 400 MHz spectrometer.
Chemical shifts are reported in ppm relative to the solvent
cation, are expected to lower the transition state
energy and speed up the enzymatic oxidation, where-
as electron-withdrawing substituents do retard it.
Therefore, starting from the radical intermediates,
which are also susceptible to sequential self-conjuga-
tion, the reaction proceeds through the formation of
the benzoquinonediimine intermediate and N C cou-
pling in the activated ortho position (C-5) to the
amino group to form a homomolecular dimeric struc-
À
ture. After the first coupling, the second addition on peaks and coupling constants (J) are reported in Hertz. FT-
IR spectra were obtained in KBr pellets on a Bruker Vertex
70 FT-IR spectrometer. Mass spectra (MS) were recorded
on a 500-MS LC ion trap mass spectrometer (Varian, Inc.,
Palo Alto, CA), operated in the positive and negative elec-
trospray ionisation (ESI) modes. The optimised operating
parameters were: ion spray voltage: Æ5 kV; capillary volt-
age: 20 V and RF loading: 90%. HR-MS (EI) were recorded
on a Micromass Autospec at Unidade de Spectrometrꢀa,
Universidade de Santiago de Compostella. Recombinant
CotA-laccase from Bacillus subtilis (1 UmLÀ1 defined as the
amount of enzyme that transformed 1 mmol of ABTS per
the aromatic ring will be performed in the para posi-
tion (C-2) relative to the first covalent C N bond site
À
and the central ring is stabilised by resonance.
According to this pathway, the stability of the radi-
cals seems to be of major importance for the catalytic
efficiency and the presence of electron-donating sub-
stituents on the aromatic ring is found to be a key
factor for this stability.
Adv. Synth. Catal. 2013, 355, 2908 – 2917
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Ana Catarina Sousa et al.
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Scheme 1. A proposed oxidative pathway for the formation of the disubstituted benzoquinonediimine trimers from laccase
and aromatic amines.
min at 378C) was produced and purified as described previ-
ously.[31,45] Trametes versicolor laccase (TvL, specific activity
20 UmgÀ1; 1 UmLÀ1 defined as the amount of enzyme that
transformed 1 mmol of catechol per min) was purchased
from Sigma–Aldrich. Both laccases were stored frozen at
À188C prior to use.
(pH 4–10; 100 mM phosphoric acid, 100 mM boric acid and
100 mM acetic mixture with 0.5M NaOH to the desired pH)
for pH 4 and 5, using a three-electrode configuration cell
with an home-made platinum-disk working electrode
(1.0 mm diameter), a platinum wire counter electrode and
a silver/silver chloride reference electrode (Ag/AgCl) (pur-
chased from Radiometer analytical, SAS, France). The po-
tential was scanned from À0.7 to +1.2 V at a scan rate of
100 mVsÀ1. All measurements were done at room tempera-
ture and the solutions were deaerated with dinitrogen
before use. The measured potentials were corrected by
+0.197 V to the normal hydrogen electrode (NHE).
Electrochemical Measurements
The redox potentials for p-substituted aromatic amines were
measured by cyclic voltammetry using an EG&G Princeton
Applied Research Model 273A potentiostat/galvanostat
monitored with a personal computer loaded with Electro-
chemistry PowerSuite v2.51 software from Princeton Ap-
plied Research. Cyclic voltammograms were obtained using
1 mM of compounds in 1:10 MeOH:buffer (phosphate
buffer for pHꢀ6 and Britton–Robinson (B&R) buffer
Enzymatic Assays
The effect of pH on the oxidation of 1,4-PDA, 4-ADA, 4-
AP and 4-APA (5 mm) by CotA-laccase was performed in
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Adv. Synth. Catal. 2013, 355, 2908 – 2917

Laccase-Catalysed Homocoupling of Primary Aromatic Amines towards the Biosynthesis of Dyes
Britton–Robinson (B&R) buffer by measuring the initial
rate of product fÀo1rmaÀti1on: 510 nm (e=3,467MÀ1 cmÀ1),
570 nm (e=1,046M cm ), 400 nm (e=9,551MÀ1 cmÀ1) and
360 nm (e=8,335MÀ1 cmÀ1), respectively. The kinetic param-
eters for the aromatic amine substrates (0.05–5 mM) were
monitored at 378C at the optimum pH for each substrate.
Kinetic constants (KM and kcat) were fitted directly to the
Michaelis–Menten equation (OriginLab software, North-
ampton, MS, USA). All enzymatic assays were performed at
least in triplicate. The protein concentration was measured
by using the Bradford assay[45] using bovine serum albumin
as a standard.
The laccase biotransformations of p-substituted aromatic
amines (at a final concentration of 5 mM) were first fol-
lowed by colour appearance using microplates (96-well)
during 24 h at 378C in B&R buffer (1:10, MeOH:buffer)
with 1 UmLÀ1 of enzyme. A control without enzyme was
also prepared to check for the auto-oxidation of substrates.
Reactions that resulted in colour formation were selected
for scale-up.
123.3 (C-8, C-12, C-8’, C-12’), 115.5 (C-9, C-11, C-9’, C-11’),
92.3 (C-3,C-6); MS-ESI (positive mode): m/z=319, [M+
H]+; HR-MS(EI): m/z=318.1592, calcd. for C18H18N6:
318.1593.
1b: (protonated form), dark purple solid; yield: 24.0 mg
À
(0.075 mmol, 90%); FT-IR (KBr): n=3463 (N H), 3418
À
À
(N H), 3337 (N H), 3210, 1601, 1541, 1502, 1435, 1403,
1277, 1239, 1166, 1077, 940, 834, 817, 565, 539, 518,
502 cmÀ1; H NMR (CD3OD): d=6.95 (d, 4H, J=8.7 Hz, H-
1
8, H-12, H-8’, H-12’), 6.78 (d, 4H, J=8.7 Hz, H-9, H-11, H-
9’, H-11’), 6.02 (s, 2H, H-3, H-6); 13C NMR (CD3OD): d=
148.3 (C-7, C-7’), 125.9 (C-8, C-12 and C-8’, C-12’), 116.6 (C-
9, C-11 and C-9’, C-11’), 94.2 (C-3, C-6), resonances for C-1–
C-4, C-2–C-5, and C-10, C-10’ were not detected in CD3OD.
Oxidation of 4-Aminodiphenylamine (4-ADA)
At pH 6, the dark red residue was collected by filtration and
purified by silica gel column chromatography [mobile
phase: (CH3)2CO:C6H14 1:2] to afford a violet solid (2).
2: violet solid; yield: 44.0 mg (0.08 mmol, 97%); FT-IR
(KBr): n=3488 (N-H), 3369 (N-H), 3279, 3029, 1595, 1548,
1505, 1444, 1401, 1315, 1237, 1213, 1171, 1112, 1076, 1027,
General Procedure for CotA-Laccase Oxidation of
the p-Substituted Aromatic Amines
1
997, 879, 831, 748, 693, 518, 500 cmÀ1; H NMR (DMSO-d6):
d=8.45 (s, 1H, NH), 8.13 (s, 2H, NH), 7.33–7.19 (m, 8H,
H-8, H-9, H-11, H-12, H-21, H-21’, H-23, H.23’), 7.19–6.95
(m, 9H, H-10, H-15 H-15’, H-17, H-17’, H-20, H-20’, H-24,
H-24’), 6.88 (dd, 4H, J=8.0 Hz, H-14, H-14’, H-18, H-18’),
6.79 (t, 2H, J=7.2 Hz, H-22, H-22’), 6.31 (s, 2H, NH2), 6.17
(s, 1H, H-3), 5.83 (s, 1H, H-6); 13C NMR (DMSO-d6): d=
153.6 (C-5), 153.3 (C-2), 148.9 (C-1), 144.0 (C-7, C-19, C-
19’), 143.9 (C-4), 143.9 and 142.8 (C-13, C-13’), 139.5 and
139.3 (C-16, C-16’), 129.1 (C-21, C-21’, C-23, C-23’), 126.0
(C-9, C-11), 123.0 (C-10), 122.5 and 122.3 (C-14, C-14’, C-18,
C-18’), 121.6 (C-8, C-12), 119.2 (C-22, C-22’), 117.9 (C-15,
C-15’, C-17, C1-7’), 116.0 (C-20, C-20’, C-24, C-24’), 91.2 (C-
3), 90.7 (C-6); MS-ESI (positive mode): m/z=547, [M+H]+;
(negative mode) m/z=545, [MÀH]À; HR-MS (EI): m/z=
546.2534, calcd. for C36H30N6: 546.2532.
Preparative scale reactions were performed under the fol-
lowing conditions: in a 100-mL round-bottom flask, the aro-
matic amine 1,4-PDA, 4-ADA, 4-APA or 4-AP
(0.025 mmol) dissolved in 5 mL of methanol was added to
45 mL of 100 mM phosphate buffer (pH 5–7). Then, the lac-
case (1 UmLÀ1) was added and the reaction mixture was
stirred at 378C under aerobic conditions. The conversion
was followed by thin layer chromatography (TLC) on alumi-
nium sheet silica gel 60 F254 (Merck). After 24 h, the insolu-
ble products were separated by filtration and dried. For
soluble products, the solvent was evaporated under reduced
pressure and the products were isolated by solid-phase ex-
traction with methanol and solvent evaporation. The crude
residues were purified by preparative column chromatogra-
phy with silica gel Fischer 60 A (200 micron).
Oxidation of 1,4-Phenylenediamine (1,4-PDA)
Oxidation of 4-Aminophenylacetamide (4-APA)
The final reaction colour showed to be pH dependent. At
pH 8, an orange solution with a solid residue was observed.
After filtration, an orange solid (1a) was obtained as a pure
compound. For pH 7 a dark purple solution was obtained
and the final residue was purified by silica gel column chro-
matography (mobile phase: MeOH:CHCl3 1:3) to afford
a dark purple solid (1b).
At pH 7, an orange solid that was collected by filtration and
purified by silica gel column chromatography [mobile
phase: (CH3)2CO:C6H14 1:1] to afford two fractions. The
first eluted orange fraction and the yellow fraction were
dried under vacuum to afford compounds (3) and (4), re-
spectively.
3: orange solid; yield: 11.5 mg (0.026 mmol, 32%); FT-IR
À
À
1a: (neutral form), orange solid; yield: 25.2 mg
(KBr): n=3439 (N H), 3371 (N H), 2925, 2853, 1666 (C=
O), 1604, 1503, 1405, 1371, 1315, 1261, 1206, 1109, 1016, 843,
À
(0.079 mmol, 95%); FT-IR (KBr): n=3461 (N H), 3417
1
(N H), 3335, 3033, 1604, 1544, 1501, 1415, 1322, 1275, 1236,
728, 597, 505 cmÀ1; H NMR (DMSO-d6): d=9.98 (NH aro-
À
1208, 1166, 1127, 1008, 939, 857, 834, 728, 691, 607, 560,
matic rings), 9.92 (NH aromatic rings), 9.44 (s, 1H, NH
iminic ring), 7.64 (s, 1H, H-6), 7.62 (d, 4H, J=8.8 Hz, H-9,
H-9’, H-11, H-11’), 6.83 (d, 4H, J=8.8 Hz, H-8, H-8’, H-12,
H-12’), 6.37 (s, 2H, NH2 iminic ring), 5.68 (s, 1H, H-3), 2.09
(s, 3H, CH3), 2.06 (s, 3H, CH3), 2.05 (s, 3H, CH3); 13C NMR
(DMSO-d6): d=169.2 (C=O), 168.1 (C=O), 167.9 (C=O),
154.1 (C-2), 153.3 (C-5), 148.0 (C-4), 145.3 and 144.7 (C-7,
C-7’), 138.1 (C-1), 136.3 and 135.5 (C-10, C-10’), 121.2 (C-8,
C-12), 120.9 (C-8’, C-12’), 119.6 (C-9, C-11), 119.4 (C-9’, C-
11’), 103.3 (C-6), 90.5 (C-3), 24.6 (CH3), 23.97 (CH3), 23.9
(CH3); UV/Vis [CH3OH:B&R pH 6 (1:10)]: l=250 (e=
534 cmÀ1; H NMR (CD3OD): d=6.76 (m, 8H, H-8, H-12,
1
H-8’, H-12’, H-9, H-11, H-9’, H-11’), 5.79 (s, 2H, H-3, H-6);
13C NMR (CD3OD): d=156.0 (C-2, C-5), 123.9 (C-8, C-12,
C-8’, C-12’), 117.1 (C-9, C-11 and C-9’, C-11’), 93.4 (C-3, C-
6), resonances for C-1, C-4, C-7, C-7’ and C-10, C-10’ were
1
not detected in CD3OD; H NMR [(CD3)2CO]: d=6.67 (m,
8H, H-8, H-12, H-8’, H-12’, H-9, H-11, H-9’, H-11’), 5.79 (s,
2H, H-3, H-6), 5.69 (s, 4H, NH2, aromatic ring), 4.52 (s, 4H,
NH2, iminic ring); 13C NMR [(CD3)2CO]: d=154.7 (C-2, C-
5), 149.3 (C-1, C-4), 145.7 (C-7, C-7’), 141.9 (C-10, C-10’),
Adv. Synth. Catal. 2013, 355, 2908 – 2917
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Ana Catarina Sousa et al.
FULL PAPERS
11,170MÀ1 cmÀ1). 360 (e=8,335MÀ1 cmÀ1) 430 nm (should-
er); MS-ESI (positive mode): m/z=467, [M+Na]+, 445,
[M+H]+ ; MS-ESI: (negative mode) m/z=443 [MÀH]À;
HR-MS (EI): m/z=444.1910, calcd. for C24H24N6O3;
444.1910.
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4: yellow solid; yield: 7.2 mg (0.024 mmol, 20%); FT-IR
[7] A. Mikolasch, E. Hammer, U. Jonas, K. Popowski, A.
Stielow, F. Schauer, Tetrahedron 2002, 58, 7589–7593.
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À
À
(KBr): n=3489, 3361 (N H), 3326 (N H), 3192, 3071, 2924,
2853, 1733, 1662 (C=O), 1603, 1543 (NO2), 1501, 1466, 1410,
1370, 1325, 1306, 1271, 1157, 1113, 1075, 1041, 1021, 963,
1
846, 727, 648, 607, 548 cmÀ1; H NMR (DMSO-d6): d=10.26
(s, NH); 7.83–7.79 (m, 8H, H-2, H-2’, H-3, H-3’, H-5, H-5’,
H-6, H-6’), 2.09 (s, 3H, CH3); 13C NMR (DMSO-d6): d=
169.2 (C=O), 148.0 (C-1, C-1’), 142.4 (C-4, C-4’), 123.8 (C-2,
C-2’, C-6, C-6’), 119.6 (C-3, C-3’, C-5, C-5’), 24.6 (CH3); MS-
ESI (positive mode): m/z=319 [M+Na]+, 297 [M+H]+,
255, 162, 134 (MS2); ME-ESI (negative mode): m/z=295
[MÀH]À; HR-MS (EI): m/z=296.1274, calcd. for
C16H16N4O2: 296.1273.
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Oxidation of 4-Aminophenol (4-AP)
At pH 6, a dark brown residue that was collected by filtra-
tion and purified by Silicagel chromatography [mobile
phase: (CH3)2CO:C6H14 (1:1)] to afford a brown solid (5).
5: brown solid; yield: 19.9 mg (0.062 mmol, 75%); FT-IR
(KBr): n=3181 (OH), 1607 (C=O), 1564, 1510, 1372, 1251,
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1168, 1064, 867, 822, 564, 510 cmÀ1 1H NMR (DMSO-d6):
;
d=7.09 (d, 2H, J=8.8 Hz, H-8, H-12), 7.06 (d, 2H, J=
8.8 Hz, H-8’, H-12’), 6.79 (d, 2H, J=8.4 Hz, H-9, H-11), 6.78
(d, 2H, J=8.8 Hz, H-9’, H-11’), 5.91 (s, 1H, H-3), 5.48 (s,
1H, H-6); 13C NMR (DMSO-d6): d=177.6 (C-1), 161.6 (C-
4), 154.8 (C-10), 154.5 (C-10’), 151.1 (C-5), 143.3 (C-2),
131.3 (C-7’), 130.1 (C-7), 125.1 (C-8, C-12), 124.6 (C-8’, C-
12’), 115.5 (C-9, C-11), 115.3 (C-9’, C-11’), 94.5 (C-6), 93.3
(C-3); MS-ESI (positive mode): m/z=322, [M+H]+; ME-
ESI (negative mode): m/z=320, [MÀH]À; HR-MS (EI): m/
z=321.1109, calcd. for C18H15N3O3: 321.1113.
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We thank Luciana Pereira for help in preliminary studies.
This work was partially supported by the grants from Funda-
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BIO/72108/2006) and SOPHIED (FP6-NMP2-CT-2004-
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