Eco-friendly synthesis of indo dyes mediated by a bacterial laccase

Article abstract of DOI:10.1039/c6gc02050j

Several aminoindamine and indoaniline dyes were obtained in good to excellent yields (64-98%) by oxidative cross-coupling between 1,4-phenylenediamine (1), 4-aminophenol (2) or 2,5-diaminotoluene (3) and several meta- and meta,para-substituted couplers (4a-j) using a green biocatalyst, the bacterial enzyme CotA-laccase from Bacillus subtilis, in water and under mild conditions of pH and temperature. Our results show that the enzymatic route described represents an efficient and sustainable alternative to the chemical synthesis of indo dyes, with a potentially high impact in the cosmetic and hair dye industries.

Full text of DOI:10.1039/c6gc02050j

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Eco-friendly synthesis of indo dyes mediated by a
bacterial laccase†
Cite this: DOI: 10.1039/c6gc02050j
Ana Catarina Sousa,a,b M. Fátima M. M. Piedade,b,c Lígia O. Martinsd and
M. Paula Robalo*a,b
Several aminoindamine and indoaniline dyes were obtained in good to excellent yields (64–98%) by oxi-
dative cross-coupling between 1,4-phenylenediamine (1), 4-aminophenol (2) or 2,5-diaminotoluene
(3) and several meta- and meta,para-substituted couplers (4a–j) using a green biocatalyst, the bacterial
enzyme CotA-laccase from Bacillus subtilis, in water and under mild conditions of pH and temperature.
Our results show that the enzymatic route described represents an efficient and sustainable alternative to
the chemical synthesis of indo dyes, with a potentially high impact in the cosmetic and hair dye industries.
Received 25th July 2016,
Accepted 16th August 2016
DOI: 10.1039/c6gc02050j
www.rsc.org/greenchem
aminoindamine and indoaniline dyes have been suggested as
oxidation products, based on the kinetic studies using the
Introduction
Oxidative dyeing of human hair has been practiced for over chemical oxidant ferricyanide.3a,4–6
100 years and as a result of increasing aging and thus greying,
The oxidative process is expected to remain dominant and
the global population demand for these products has been the development of biotechnological systems, such as the
increasing rapidly, a trend that will likely continue in the exploitation of enzymatic oxidation processes to replace con-
future.1 Oxidative hair dyes require three major components in ventional oxidative processes is viewed as an attractive means
the chemical process. The first step is the oxidation of a of addressing potential environmental concerns.7,8
primary intermediate, an oxidation base or a developer, an
Laccases
(benzenodiol : oxygen
oxidoreductases,
EC
o- or p-substituted phenol or aniline derivative; 1,4-phenyl- 1.10.3.2) belong to the multi-copper family of oxidases, with
enediamine (1,4-PDA) and other related derivatives have been four copper ions in two catalytic centers and oxidise a wide
extensively used as oxidation bases in permanent hair colour- range of electron-rich aromatic substrates using oxygen as an
ation processes.1–3 The second component, the coupler, is electron acceptor.9 Considering that they operate under mild
commonly a meta-disubstituted aromatic compound with reaction conditions and water is the only by-product formed,
electron donor groups, including m-phenylenediamines, resor- they are the ideal biocatalysts for sustainable chemical and oxi-
cinol and naphthols and their derivatives; these compounds doreductive biotechnological applications.10 Although the use
by themselves will not produce colour when oxidised, but they of laccases from different origins has been extensively studied
can react with the products from oxidation of the primary with phenols, the substrates of excellence for these enzymes,
intermediates to form dyes. The final component is the their use in the oxidation of aniline derivatives and other aro-
oxidant, almost exclusively hydrogen peroxide under alkaline matic amines has been much less explored.11,12
conditions provided usually by ammonia.1 Structures like
Enzymes particularly of the laccase type have been claimed
to be well suited for use in cosmetic applications. Although
the laccase-based hair dyes have been considered less irritant
and easier to handle than current hair dyes,13,14 most of the
proposed ready-to-use formulations, using laccases of plant or
fungi origins, produce colours which do not fulfill the require-
ments concerning the issues such as the homogeneity of
colour distribution in the hair fibres and the chromaticity of
the dyeing powder.14
Recently, we reported the use of CotA-laccase for the oxi-
dation of aromatic amines looking forward to the sustainable
synthesis of dyes11 and the formation of different heterocyclic
cores.15 The recombinant bacterial CotA-laccase from Bacillus
subtilis, an intrinsically highly thermostable enzyme,16 showed
aÁrea Departamental de Engenharia Química, ISEL-Instituto Superior de Engenharia
de Lisboa, Instituto Politécnico de Lisboa, Rua Conselheiro Emídio Navarro, 1,
1959-007 Lisboa, Portugal. E-mail: mprobalo@deq.isel.ipl.pt; Fax: +351218317267;
Tel: +351218317163
bCentro de Química Estrutural, Complexo I, Instituto Superior Técnico,
Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal
cDepartamento de Química e Bioquímica, Faculdade de Ciências,
Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal
dInstituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de
Lisboa, Av. da República, 2780-15 Oeiras, Portugal
†Electronic supplementary information (ESI) available: X-ray crystallographic
data, NMR and MS spectra. CCDC 1476627. For ESI and crystallographic data in
CIF or other electronic format see DOI: 10.1039/c6gc02050j
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optimal pH values around neutrality for the substrates
tested,11 illustrating the potentiality of this enzyme to use
aromatic amines as substrates. The bacterial origin of the
enzyme, the broad substrate range and its ability to operate at
the alkaline pH range, which promotes hair swelling and the
penetration of colour pigments into the fibres, prompted us to
extend our studies to cross coupling reactions involving these
aromatic derivatives.
We report herein the successful use of 1 U ml−1 of CotA-
laccase, a lower value than the usually reported,14 to mediate
the oxidation of selected aromatic amines, 1,4-phenyl-
enediamine (1,4-PDA), 4-aminophenol (4-AP) and 2,5-dia-
minotoluene
(2,5-DAT)
into
the
corresponding
benzoquinoneimines, which will be further involved in hetero-
coupling reactions with selected couplers. These cross coup-
ling reactions were directed towards the production of indo
dyes, widely used in permanent hair and leather
colouration.1,2
The proposed enzymatic oxidation process leads to the syn-
thesis of a wide range of indo dye structures in good to
excellent yields, showing a comparable performance to the
traditional chemical routes concerning colour development
and the production of indo dyes. Our approach, avoiding the
use of hydrogen peroxide and alkali, which due to their
bleaching effect degrade the hair fibers, rendering them
coarse and fragile,1 can be viewed as a more environmentally
friendly alternative synthetic route.
Fig. 1 Chemical structures of the aromatic amines and phenols used in
the
heterocoupling
reactions
mediated
by
CotA-laccase.
(3-Aminophenol (3-AP), resorcinol (Res), 1,3-phenylenediamine (1,3-
PDA), 3-aminobenzonitrile (3-ABN), 3-aminobenzoic acid (3-ABA),
3-aminosulfonic acid (3-ABSA), 1-naphthol (1-Nol), 1-naphthylamine
(1-NA), 5-amino-o-cresol (5-A-o-cresol), 2,4-diaminotoluene (2,4-DAT).)
Table 1 Electrochemical data of substituted aromatic amines and
phenols used as couplers vs. NHE in buffered solutions at a scan rate of
100 mV s−1
Results and discussion
Epa (V)
The heterocoupling reactions mediated by the bacterial CotA-
laccase were conducted using 1,4-PDA (1), 4-AP (2) or 2,5-DAT (3)
as the oxidation bases and a range of aromatic compounds
as couplers (4a–j) (Fig. 1). The selection of couplers was based
on their use for indo dye synthesis and also on their oxidation
potentials at the reaction pH.
Thus, the knowledge of the redox potential of the com-
pounds involved in the enzyme-catalysed reaction provides
useful information in the prediction and understanding of the
reaction pathway. The electrochemical behaviour of the cou-
plers presented in Fig. 1 was studied by cyclic voltammetry
(Table 1).
Compound
pH = 4
pH = 5
pH = 6
pH = 7
4a (3-AP)
4b (Res)
1.09
1.06
0.91
0.91
0.84
1.33
1.23
1.19
0.93
0.80
0.92
1.06
0.89
0.79
0.83
1.28
1.21
1.16
0.92
0.78
0.87
1.06
0.80
0.76
0.79
1.24
1.19
1.14
0.87
0.79
0.80
1.00
0.79
0.71
0.77
1.22
1.12
1.07
0.85
0.77
4c (1,3-DA)
4d (5A-o-cresol)
4e (2,4-DAT)
4f (3-ABN)
4g (3-ABA)
4h (3-ABSA)
4i (1-Nol)
4j (1-NA)
Experiments were performed at different scan rates and pH
values in (1 : 9) MeOH : aqueous buffer media (pH range 4–7)
using a platinum disk as the working electrode and the the introduction of a methyl group in the ortho-position
Ag/AgCl electrode as the reference electrode and corrected by (4d and 4e) relative to the corresponding disubstituted com-
+0.197 V to the NHE. All the tested couplers showed an ir- pound (4a and 4c) slightly decreases the oxidation potential in
reversible oxidation at positive potentials and the determined the tested pH range.
Epa values (Table 1) decrease as the pH increases, in accord-
ance with our previous results using a diverse group of pounds are most likely resistant to a direct monoelectronic oxi-
aromatic amines.11,15a
dation by CotA-laccase, since the Epa values (from +0.71 V to
The obtained results indicate that all the studied com-
As expected, the redox potentials for meta-donor substituted +1.33 V) are higher than the redox potential of the T1 copper
aromatic amines (4a and 4c) are higher than those previously centre of CotA-laccase (0.55 V).18
determined for the equivalent para-isomers,11 most likely due
The cross coupling reactions between 1,4-PDA (1) and
to the ring activation effect in the para-isomers.17 In contrast, couplers 4a–4e were performed at 37 °C in Britton & Robinson
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Table 2 Heterocoupling reactions between 1,4-PDA and couplers
equivalents of the coupler. The increase of pH and the
reduction of the number of equivalents of the coupler led to
enhanced yields (Table 2, entries 1–4), with the highest yield
(95%) achieved at pH 8, using 0.5 equivalents of the coupler
(Table 2, entry 4). The alkaline conditions most likely promote
the deprotonation of the hydroxyl group in 3-AP (pKa 9.9),
leading to the formation of a phenolate ion and consequently
promoting the electrophilic attack on the para-position.4b
The commercial enzyme TvL from Trametes versicolor was
also used in the heterocoupling between 1,4-PDA (1) and 3-AP
(4a) resulting in significantly lower yields for the indo dye (5)
when compared with CotA-laccase under all the conditions
tested (data not shown) and for this reason it was not further
used in this study.
Representative results of other heterocoupling reactions
between 1,4-PDA (1) and different couplers are also reported in
Table 2 (entries 5–10) and Fig. 2. For m-substituted couplers
(4a–c and 4f–h), the efficiency in the oxidation towards hetero-
coupled dinuclear or trinuclear dyes strongly depends on the
nature of the substituents on the aromatic ring. The use of
m-donor substituted couplers (4b–c) led to the heterocoupling
products (6 and 7) in good to excellent yields (Table 2, entries
7 and 8, Fig. 2), at pH 8 in the presence of 0.5 equivalents of
the coupler. The reactions between the 1,4-PDA and m-substi-
tuted couplers with electron withdrawing groups (3-ABN (4f),
3-ABA (4g) and 3-ABSA (4h)) yielded in all cases the brown dye
Bandrowski’s base (BB) (10) (see Fig. 2) as the only reaction
product (70–100%), independent of the reaction conditions,
the pH variation and the stoichiometric quantity of the
coupler (data not shown). The coupler was always recovered at
the end of reactions and BB is known to result from the homo-
coupling reaction of the oxidation base 1,4-PDA,11 which
means that the couplers 4f, 4g and 4h are most probably
weaker nucleophiles than 1,4-PDA, thus preventing the electro-
philic attack on the coupler.
(4a–4e) mediated by CotA-laccase (1 U mL−1 a
)
Entry
Coupler
Conditions (pH, coupler)
Yieldb (%)
Product
1
2
3
4
5
6
7
8
9
4a
4a
4a
4a
4b
4b
4b
4c
4d
4e
6, 1.0 eq.
7, 1.0 eq.
7, 0.5 eq.
8, 0.5 eq.
7, 1.0 eq.
7, 0.5 eq.
8, 0.5 eq.
8, 0.5 eq.
7, 1.0 eq.
7, 1.0 eq.
40
50
5
5
5
5
6
6
6
7
8
9
80
93c
20
27
64c
82c
83c
98c
10
a Reaction conditions: phosphate buffer (10% of ethanol); substrate
concentration: 5 mM; reaction time: 24 h; temperature: 37 °C. b Yield
of products was determined by 1H NMR spectroscopy. c Isolated yields.
(B&R) or phosphate buffer (10 mM, in the pH range between
6 and 8) containing 10% of ethanol, using 0.5 or 1.0 equivalent
of the coupler and 1 U mL−1 of CotA-laccase (Table 2). The use
of 10% of the organic solvent increases the solubility of
substrates and intermediates leading to improved results.
The formation of the coloured products occurs immediately
after the addition of the CotA-laccase. For most of the reac-
tions, the product formation is complete within 30 min to 1 h
(data not shown). The yields presented in Table 2 were deter-
mined after 24 h; for prolonged reactions a considerable
amount of by-products, most probably in the form of poly-
meric compounds, were observed. Control reactions confirmed
that only small amounts of products (0–20%) were detected in
the absence of the enzyme.
From the heterocoupling reactions between 1,4-PDA (1) and
the m,p-disubstituted couplers 5-A-o-cresol (4d) and 2,4-DAT
(4e) the indoaniline dyes (8) and (9) were respectively obtained
in good to excellent yields (Table 2, entries 9 and 10).
The heterocoupling reaction between 1,4-PDA (1) and 3-AP
(4a) results in the trinuclear indo dye (5) (Table 2, Fig. 2). To
increase the efficiency of the process, experiments were
conducted at different pHs and in the presence of different
The favorable results obtained with m-substituted and
m,p-disubstituted couplers prompted us to extend our studies
to naphthalene couplers (1-Nol (4i) and 1-NA (4j)), where the
presence of electron donor groups promotes the hetero-
coupling reactions with 1,4-PDA catalysed by CotA-laccase
(Table 3, entries 1 and 2). Surprisingly, the main product iso-
lated in both reactions was identified to be the naphthol-
derived indoaniline 11 (Fig. 3). This could be explained by the
hydrolysis of the terminal amino/imino group in accordance
with the previous reports.5,6,19 The cross-coupling reaction
between 1,4-PDA and 4j additionally leads to the secondary
product 12 (Fig. 3).
To expand the scope of the biocatalysed route, additional
oxidation bases 4-AP (2) and 2,5-DAT (3) were used with m,p-di-
substituted couplers under optimised reaction conditions
(Table 3, entries 3–5). The heterocoupling reactions of 4-AP
with 4d and 4e resulted in the production of the respective
Fig. 2 Dinuclear and trinuclear compounds obtained in the biocata-
lysed reactions of 1,4-PDA and couplers (4a–e and 4f–h) and
Bandrowski’s base (BB).
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Table 3 Heterocoupling reactions between 1,4-PDA, 4-AP or 2,5-DAT
Experimental section and the ESI†). Insoluble products were
isolated from the reaction media by filtration, while the
soluble products were extracted with acetyl acetate and isolated
by solvent evaporation. For the reactions where two products
were obtained, the isolation process includes a step of puri-
fication by silica gel preparative or column chromatography.
The structures proposed for the products 5–9 and 13–15
have been previously suggested by Corbett and coworkers,3a,4–6
based on the chemical oxidation of several primary intermedi-
ates in the presence of different couplers. Nevertheless, with
the exception of 5, 8 and 13, for which the proton NMR data
were reported, the full spectroscopic characterization for these
compounds was never reported.
and couplers mediated by CotA-laccase (1 U mL−1 a
)
Entry
Oxidation base
Coupler
Yieldb (%)
Product
1
2
3
4
5
1 (1,4-PDA)
1 (1,4-PDA)
2 (4-AP)
2 (4-AP)
3 (2,5-DAT)
4jc
4jc
4d
4e
4d
74
56 + 29
98
52 + 28
60 + 27
11
11 + 12
13
14 + 15
16 + 17
a Reaction conditions: phosphate buffer (10% of ethanol); pH 7; sub-
strate concentration: 5 mM; 1 equivalent of coupler; reaction time:
24 h; temperature: 37 °C. b Isolated yields. c 0.5 equivalents.
The CotA-laccase mediated reactions between 1,4-PDA and
the couplers 3-AP, 1,3-PDA and resorcinol yield the trimers 5
and 6 and the dimer 7 respectively (see Fig. 2). The presence of
a quinone–imine central structure for the trimers and a quino-
nediimine pattern for the dimer 7 was confirmed by the com-
bined analysis of the NMR and ESI/MS data. The 1H and
13C NMR data of trimers 11 and 12, obtained from 1,4-PDA
and naphthalene derived couplers, confirm the presence of a
substituted naphthalene central core. The symmetric structure
proposed for 12 points out the loss of the amino group of the
naphthalene coupler during the course of the reaction.
The reactions of 1,4-PDA, 4-AP and 2,5-DAT with the m,p-di-
substituted couplers 4d and 4e yielded two different dyes: the
indoaniline type dyes 8, 13, 16 and 17 and the aminoindamine
dyes 9 and 14 (see Fig. 2 and 3). The position of the new N–C
bonds and the presence of the quinoneimine (for indoani-
lines) and the quinonediimine (for aminoindamines) struc-
tures were unambiguously indicated by the characteristic
resonances of carbonyl and imine quaternary carbons in the
13C NMR spectra.
Fig. 3 Dinuclear and trinuclear compounds obtained in the cross-
coupling reactions of 1,4-PDA (1), 4-AP (2) and 2,5-DAT (3) and couplers
(4d, 4e, 4i and 4j).
dinuclear indoaniline 13 and 2-aminoindamine 14 dyes, with
The structure of compound 8 was obtained by single X-ray
the intramolecular cyclisation of the latter to an asymmetric diffraction. The compound crystallises in the monoclinic
phenazine core 15 (Table 3, entries 3 and 4, Fig. 3). The for- centrosymmetric space group P21/n, with one molecule per
mation of heterocyclic phenazines was previously reported by asymmetric unit. The molecular diagram (Mercury21) with the
Brown and Corbett5,3a,19a for similar oxidative reactions with atomic numbering scheme is presented in Fig. 4.
4-AP, using the chemical oxidant ferricyanide.
Substitution of the 1,4-PDA ring with a methyl group
(2,5-DAT) and coupling with 4d resulted in the production of
the dimeric structures 16 and 17 but in lower yields (Table 3,
entry 5, Fig. 3). This result can be explained by a lower
oxidation rate for 2,5-DAT, when compared with 1,4-PDA. In
fact, Corbett et al.3b,6 showed that the C-methylation of the
diamine 1,4-PDA decreases the rate of chemical oxidation with
ferricyanide, due to the loss of basicity on the diamine,
leading to a decreased amount of protonated species present
at a specific pH. Compound 17 results from a hydrolysis
process of the terminal amino group which could occur either
during the oxidation, the heterocoupling reaction or the purifi-
cation processes, as previously described.5,15a,19,20
The obtained products were isolated in good to excellent
yields (64–98%) directly as crude solids and were fully charac-
Fig. 4 Molecular diagram showing the crystallographic labeling
scheme for compound 8. Thermal ellipsoids are drawn at the 50% prob-
terised by FTIR, NMR (1H, 13C, COSY, HSQC and HMBC) and
UV-Visible spectroscopy and mass spectrometry (see the ability level.
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The compound scaffold is composed of two 6-membered average of 20–35 scans. Tandem mass spectra were obtained
ring systems, a benzenoid and a quinoid ring. The bond dis- with an isolation window of 2 Da, a 30% relative collision
tances [1.405(2) Å to 1.378(1) Å] and bond angles [120.6(1)° to energy and with an activation energy of 30 ms. HR ESI(+/−)
118.0(1)°] for aromatic carbons are consistent within the mass spectra were obtained on a QTOF Impact II™ mass
expected range for a benzene ring. The N1–(C1–C6)–N2 aro- spectrometer (Bruker Daltonics, GMBH, Germany), operating
matic fragment is almost planar, with maximum deviations in the high resolution ion mode. Calibration of the TOF mass
out-of-plane of 0.036(1) Å and 0.168(1) Å in the N1 and N2 analyser was performed with a lock mass calibrant.
atoms, respectively. In the quinoid fragment, the electronic
The recombinant CotA-laccase from Bacillus subtilis (1 U
delocalisation is observed with the short bond lengths C8–C9 defined as the amount of enzyme that transformed 1 µmol of
and C11–C12 [1.368(2) Å and 1.344(1) Å], while C7–C8, C9–C10, ABTS per min at 37 °C) was produced and purified as
C10–C11 and C12–C7 are larger [1.420(2) Å to 1.490(2) Å], described previously.16,24 Trametes versicolor laccase (TvL,
in accordance with the values reported for similar specific activity 20 U mg−1; 1 U defined as the amount of
compounds.20,22 The connection between the benzenoid and enzyme that transformed 1 µmol of catechol per min) was pur-
quinoid fragments is established by the C4–N2–C7 single and chased from Sigma-Aldrich. The protein concentration was
double bonds with 1.400(1) Å and 1.295(1) Å, respectively. The measured by using the Bradford assay using bovine serum
dihedral angle formed between the planes of the two frag- albumin as a standard.16 Both laccases were stored frozen at
ments is 47.42(3)°, denoting the distortion inside the molecule −18 °C prior to use.
(see the ESI†).
The enzyme-catalysed formation of the aminoindamine using an EG&G Princeton Applied Research Model 273A poten-
and indoaniline dyes (see the ESI, Fig. S1†) follows a similar tiostat/galvanostat monitored with personal computer
The redox potentials were measured by cyclic voltammetry
a
pathway to the one previously proposed for chemical oxi- loaded with Electrochemistry PowerSuite v2.51 software from
dations and is reported in the literature.3,23 The first step, the Princeton Applied Research. Cyclic voltammograms were
enzymatic oxidation of the primary intermediate (oxidation obtained using 1 mM of compounds in 9 : 1 buffer : MeOH
base or developer) promotes the formation of the benzo- (phosphate buffer for pH ≥ 6 and Britton–Robinson (B&R)
quinone-monoimine or -diimine intermediates, which are buffer for pH 4 and 5 and 10 mM) using a three-electrode
further involved in cross-coupling reactions with the aromatic configuration cell with a home-made platinum-disk working
or naphthalene couplers, yielding the dinuclear leuco electrode (1.0 mm diameter), a platinum wire counter elec-
derivatives and finally the dinuclear or trinuclear indoaniline trode and an Ag/AgCl reference electrode (purchased from
or aminoindamine dyes.
Radiometer analytical, SAS, France). The potential was
scanned from −0.7 to 1.2 V at a scan rate of 100 mV s−1. All
measurements were performed at room temperature and the
solutions were deaerated with dinitrogen before use. The
measured potentials were corrected by +0.197 V to the normal
hydrogen electrode (NHE).
Experimental
General procedures
Commercial reagents and solvents were used as received
without additional purification. The aromatic compounds
used as starting materials (Fig. 1) were commercially available
(Sigma-Aldrich Co.). TLC: Merck 60 F254 plates. Preparative
chromatography: Silicagel Fischer 60A (200 micron). The
General procedure for laccase mediated heterocoupling
reactions
Procedure A – for compounds 5–9 and 11–17. Laccase
UV-Visible data for compounds were obtained in methanol on (1 U mL−1) was added to a stirred solution of the primary inter-
a Nicolet Evolution 300 spectrophotometer. The product mediate (1,4-PDA, 4-AP or 2,5-DAT) (0.5 mmol, 5 mM) in phos-
characterization was performed by 1D-NMR (1H, 13C) and phate buffer : ethanol solution (9 : 1) (10 mL, 10 mM) at the
2D-NMR (COSY, HSQC and HMBC) spectra, obtained in selected pH. The solution was stirred at 37 °C in the presence
CD3OD on a Bruker Advance 400 MHz spectrometer with a of an adequate amount of the coupler and the reaction was
5 mm probe. Chemical shifts are reported in ppm relative to monitored by TLC. After 24 h, for the soluble products, the
the solvent peaks and coupling constants (J) are reported in aqueous solution was extracted with ethyl acetate. The col-
hertz. FTIR spectra were obtained in KBr pellets on a Bruker lected organic phases were dried over Na2SO4, filtered, concen-
Vertex 70 FT-IR spectrometer. LRESI mass spectrometry and trated in a vacuum and characterised. In the cases of
tandem mass experiments were carried out on a LCQ Fleet compounds 5, 6 and 11, insoluble in the reaction medium, the
mass spectrometer operated in the ESI positive/negative ion final aqueous phase was filtered and the residue was directly
modes (Thermo Scientific). The optimised parameters were as lyophilised and characterised. In the case of mixtures, the pro-
following: ion spray voltage, 4.5 kV; capillary voltage, +16 and ducts were isolated by chromatography (column or preparative)
−20 V; tube lens offset, −63 and +82 V; sheath gas (N2), using an acetyl acetate/ethanol 3 : 1 (v/v) solution as an eluent.
80 arbitrary units; auxiliary gas, 5 arbitrary units; capillary Control reactions in the absence of the enzyme were also con-
temperature, 250 °C. The spectra were recorded in the range of ducted to check for the auto-oxidation of substrates and after
100–1000 Da. The spectrum typically corresponds to the 24 h, the same work-up procedure was followed.
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Procedure B. Laccase (1 U mL−1) was added to a stirred solu- ESI/MS positive mode: m/z 250 (37%, [M + Na]+), 228 (100,
tion of 1,4-PDA (0.5 mmol, 5 mM) in phosphate : ethanol solu- [M + H]+); MS2 m/z 212 (100%, [M + H − CH4]+); HR-ESI/MS:
tion (9 : 1) (10 mL, 10 mM) at the selected pH. The solution m/z calcd for C13H14N3O [M + H]+: 228.1131; found 228.1126.
was stirred at 37 °C in the presence of an adequate amount of
9: dark blue solid (10.3 mg, 98%); UV/Vis: λmax(MeOH)/nm
couplers (3-ABN, 3-ABA, and 3-ABSA) and the reaction was 600 (ε/dm3 mol−1 cm−1 22 899); FTIR (KBr) νmax/cm−1: 3461,
monitored by TLC. After 24 h, the dark blue solution was 3343, 3202, 1634, 1603, 1544, 1498, 1455, 1327, 1282, 1170,
extracted with ethyl acetate. The collected organic phases were 1081, 983, 866, 715, 610, 526. 1H NMR (400 MHz, CD3OD):
dried over Na2SO4, filtered, concentrated in vacuo and analysed δ (ppm) = 7.12 (d, 1H, J = 8.7 Hz, H8, H12), 7.00 (s, 1H, H5),
1
by H NMR spectroscopy. In all the cases, the coupler and the 6.78 (d, 2H, J = 8.7 Hz, H9, H11), 6.00 (s, 1H, H2), 2.16 (s, 3H,
trinuclear dye BB were found to be the final products.
CH3); 13C{H} NMR (400 MHz, CD3OD): δ (ppm) = 165.2 (C1),
5: brown solid (6.8 mg, 93%); UV/Vis: λmax(MeOH)/nm 400 159.6 (C3), 153.2 (C10), 145.4 (C4), 139.5 (C7), 134.1 (C6), 129.3
(ε/dm3 mol−1 cm−1 4845); FTIR (KBr) νmax/cm−1: 3482, 3441, (C8, C12), 125.9 (C5), 116.0 (C9, C11), 92.9 (C2), 17.4 (CH3); ESI/
3342, 3217, 1627, 1576, 1511, 1431, 1410, 1362, 1269, 1231, MS positive mode: m/z 227 (100%, [M + H]+); MS2 m/z 211
1
1207, 1170, 1125, 1013, 831, 489; H NMR (400 MHz, CD3OD): (100%, [M + H − CH4]+); HR-ESI/MS: m/z calcd for C13H15N4
δ (ppm) = 6.90 (d, 2H, J = 8.7 Hz, H8, H12), 6.77 (d, 2H, J = [M + H]+: 227.1291; found 227.1297.
8.7 Hz, H15, H17), 6.71 (d, 2H, J = 8.7 Hz, H14, H18), 6.67 (d, 2H, J =
11: purple solid (1.2 mg, 74%); UV/Vis: λmax(MeOH)/nm 505
8.7 Hz, H9, H11), 6.01 (s, 1H, H5), 5.63 (s, 1H, H2); 13C{H} NMR (ε/dm3 mol−1 cm−1 8634); 1H NMR (400 MHz, CD3OD):
(400 MHz, CD3OD): δ (ppm) = 182.1 (C1), 159.1 (C3), 153.1 (C4), δ (ppm) = 8.44 (dd, 1H, J = 7.1 Hz, J = 1.6 Hz, H8), 8.19 (dd, 1H,
146.3 (C13), 143.7 (C6, C10), 130.9 (C7), 124.8 (C8, C12), 124.2 J = 7.8, J = 1.6 Hz, H5), 7.74 (t, 1H, J = 8.8 Hz, J = 1.6 Hz, H7),
(C15, C17), 117.1 (C9, C11), 116.7 (C14, C18), 97.4 (C2), 89.9 (C5); 7.75 (dt, 1H, J = 8.8 Hz, J = 1.6 Hz, H6), 6.96 (d, 2H, J = 8.8 Hz,
ESI/MS positive mode: m/z 342 (34%, [M + Na]+), 320 (100, H18, H22), 6.77 (m, 4H, H12, H13, H15, H16), 6.69 (d, 2H, J =
[M + H]+); MS2 m/z 303 (27%, [M + H − NH3]+), 292 (100, 8.8 Hz, H19, H21), 6.54 (s, 1H, H3); 13C{H} NMR (400 MHz,
[M + H − CO]+), 212 (20, [M + H − C6H8N2]+); HR-ESI/MS: m/z CD3OD): δ (ppm) = 180.3 (C1), 170.3 (C4), 134.2 (C7), 131.2 (C6),
calcd for C18H18N5O [M + H]+: 320.1506; found 320.1505.
127.1 (C5), 125.7 (C8), 125.2 (C18, C22), 124.1 (C13, C15), 117.1
6: brown solid (4.6 mg, 64%); UV/Vis: λmax(MeOH)/nm 420 (C19, C21), 116.9 (C12, C16), 97.1 (C3); ESI/MS negative mode:
(ε/dm3 mol−1 cm−1 3962); FTIR (KBr) νmax/cm−1: 3335, 3207, m/z 353 (100%, [M − H]−); ESI/MS positive mode: m/z 377
1650, 1541, 1510, 1347, 1287, 1168, 826, 502; 1H NMR (100%, [M + Na]+), 355 (33, [M + H]+); MS2 m/z 237 (100%,
(400 MHz, CD3OD): δ (ppm) = 7.06 (d, 4H, J = 8.6 Hz, H8, H12, [M + H − C7H8N2]+), 248 (18, ½M þ H ꢀ C6H7N• ꢁ•þ), 262 (13,
2
H14, H18), 6.72 (d, 4H, J = 8.6 Hz, H9, H11, H15, H17), 6.68 (s, [M + H − C6H7N]+), 328 (8, [M + H − HCN]+); HR-ESI/MS: m/z
1H, H2), 6.03 (s, 1H, H5), 5.42 (s, NH2); 13C{H} NMR (400 MHz, calcd for C22H19N4O [M + H]+: 355.1553; found 355.1539.
CD3OD): δ (ppm) = 176.1 (C1), 155.2 (C3, C4), 149.6 (C10, C16),
12: red solid (4.5 mg, 29%); UV/Vis: λmax(MeOH)/nm 505
127.1 (C6), 126.9 (C7, C13), 126.3 (C8, C12, C14, C18), 118.8 (C2), (ε/dm3 mol−1 cm−1 5780); 1H NMR (400 MHz, CD3OD):
116.2 (C9, C11, C15, C17), 85.8 (C5); ESI/MS positive mode: m/z δ (ppm) = 8.44 (m, 2H, H4, H4′), 7.63 (m, 2H, H5, H5′), 7.04 (s,
321 (100%, [M + H]+); MS2 m/z 293 (15%, [M + H − CO]+), 2H, 2H, H1, H1′), 6.80 (m, 8H, H7, H8, H10, H11, H7′, H8′, H10′
,
213 (100, [M + H − C6H8N2]+); HR-ESI/MS: m/z calcd for H11′); 13C{H} NMR (400 MHz, CD3OD): δ (ppm) = 180.3
C18H17N4O2 [M + H]+: 321.1346; found 321.1356.
(C2, C2′), 170.3 (C9, C9′), 147.0 (C6, C6′), 134.7 (C3, C3′), 131.2
7: purple solid (8.0 mg, 82%); UV/Vis: λmax(MeOH)/nm 560 (C5, C5′), 126.5 (C1, C1′), 125.7 (C4, C4′), 124.2 (C7, C11, C7′, C11′),
(ε/dm3 mol−1 cm−1 12 745); 1H NMR (400 MHz, CD3OD): 116.8 (C8, C10, C8′, C10′); ESI/MS positive mode: m/z 339 (100%,
δ (ppm) = 7.23 (d, 1H, J = 9.2 Hz, H5), 7.17 (d, 2H, J = 8.9 Hz, [M + H]+); MS2 m/z 322 (8%, [M + H − NH3]+), 233 (100,
H8, H12), 6.77 (d, 2H, J = 8.9 Hz, H9, H11), 6.65 (dd, 1H, J = [M + H − C6H6N2]+); HR-ESI/MS: m/z calcd for C22H19N4 [M +
2.4 Hz, J = 9.2 Hz, H6) and 5.99 (dd, 1H, J = 2.4 Hz, H2); 13C{H} H]+: 339.1604; found 339.1609.
NMR (400 MHz, CD3OD): δ (ppm) = 170.4 (C1), 139.5 (C7),
13: dark orange solid (10.2 mg, 98%); UV/Vis: λmax(MeOH)/
135.6 (C10), 129.8 (C8, C12), 128.0 (C5), 125.3 (C6), 116.0 (C9, nm 460 (ε/dm3 mol−1 cm−1 9340); FTIR (KBr) νmax/cm−1: 3548,
C11), 92.3 (C2); ESI/MS positive mode: m/z 213 (100%, 3475, 3417, 1639, 1618, 1573, 1540, 1498, 1434, 1384, 1268,
[M + H]+); HR-ESI/MS: m/z calcd for C12H13N4 [M + H]+: 1236, 1101, 988, 879, 853, 795, 619, 528; 1H NMR (400 MHz,
213.1135; found 213.1126.
CD3OD): δ (ppm) = 6.86 (m, 4H, H8, H9, H11, H12), 6.75 (s, 1H,
8: brown solid (8.7 mg, 83%); UV/Vis: λmax(MeOH)/nm 460 H5), 5.66 (s, 1H, H2), 1.91 (s, 3H, CH3); 13C{H} NMR (400 MHz,
(ε/dm3 mol−1 cm−1 10 438); FTIR [KBr] νmax/cm−1: 3427, 3370, CD3OD): δ (ppm) = 188.3 (C1), 157.1 (C10), 156.1 (C3), 153.2
3311, 3213, 3161, 2942, 2930, 1650, 1599, 1582, 1502, 1444, (C4), 143.4 (C6), 141.9 (C7), 124.4 (C8, C12), 122.8 (C5), 116.7
1385, 1361, 1287, 1256, 1214, 1168, 1132, 1003, 885, 853, 819, (C9, C11), 100.2 (C2), 16.6 (CH3); ESI/MS positive mode: m/z 229
731, 706, 686, 636, 615, 550. 1H NMR (400 MHz, CD3OD): (100%, [M + H]+); MS2 m/z 212 (100%, [M + H − OH•]•+);
δ (ppm) = 6.87 (d, 2H, J = 8.7 Hz, H8, H12), 6.84 (s, 1H, H5), HR-ESI/MS: m/z calcd for C13H13N2O2 [M + H]+: 229.0972;
6.76 (d, 2H, J = 8.7 Hz, H9, H11), 5.65 (s, 1H, H2), 1.91 (s, 3H, found 229.0964.
CH3); 13C{H} NMR (400 MHz, CD3OD): δ (ppm) = 188.5 (C1),
156.7 (C4), 151.3 (C3), 149.2 (C10), 142.4 (C6), 140.1 (C7), 125.6 nm 320 (ε/dm3 mol−1 cm−1 24 459); FTIR (KBr) νmax/cm−1
(C8, C12), 123.2 (C5), 116.2 (C9, C11), 100.1 (C2), 16.6 (CH3); 3428, 2923, 1639, 1581, 1391, 1138, 994, 855, 620, 534;
14: dark orange solid (5.4 mg, 52%); UV/Vis: λmax(MeOH)/
:
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1H NMR (400 MHz, CD3OD): δ (ppm) = 7.06 (d, 2H, J = 8.8 Hz, and a full sphere of data was obtained. All the data were cor-
H8, H12), 6.91 (d, 2H, J = 8.6 Hz, H9, H11), 6.85 (s, 1H, H5), 6.04 rected for Lorentzian, polarization, and absorption effects
(s, 1H, H2), 2.16 (s, 3H, CH3); 13C{H} NMR (400 MHz, CD3OD): using SAINT 25 and SADABS 26 programs. SIR97 27 and
δ (ppm) = 165.8 (C1), 159.3 (C10), 150.6 (C4), 141.4 (C7), 136.8 SHELXL-97 28 were used for structure solution and refinement,
(C6), 126.7 (C8, C12), 125.4 (C5), 117.5 (C9, C11), 92.8 (C2), 17.4 respectively. Calculations were performed using the package of
(CH3); ESI/MS positive mode: m/z 228 (100%, [M + H]+); MS2 programs WINGX-Version 1.80.05.29 A full-matrix least-squares
m/z 211 (100%, [M + H − OH•]•+).
refinement on F2 was performed on the non-hydrogen atoms
15: dark orange solid (2.8 mg, 28%); 1H NMR (400 MHz, with anisotropic thermal parameters. The hydrogen atoms
CD3OD): δ (ppm) = 7.90 (d, 1H, J = 9.4 Hz, H9), 7.73 (s, 1H, were inserted in the idealised positions (d(Cmethyl–H) = 0.96 Å,
H6), 7.30 (d, 1H, J = 9.2 Hz, H8), 7.16 (s, 1H, H1), 7.00 (s, 1H, d(Crings–H) = 0.93 Å) and were allowed to refine riding on
H4), 2.44 (s, 3H, CH3); 13C{H} NMR (400 MHz, CD3OD): the parent carbon atom, with Uiso(H) = 1.5 Ueq. (Cmethyl) and
δ (ppm) = 131.0 (C9), 129.3 (C6), 123.8 (C8), 107.0 (C1), 102.1 1.2 Ueq. (other C atoms); an exception for H atoms bonded to
(C4), 18.2 (CH3); ESI/MS positive mode: m/z 226 (100%, nitrogen whose positions were found in the electronic density
[M + H]+); MS2 m/z = 211 (45%, ½M þ H ꢀ CH•3ꢁ•þ), 198 (28%, map and were allowed to refine freely. The graphics were made
[M + H − CO]+); HR-ESI/MS: m/z calcd for C13H12N3O [M + H]+: using MERCURY 3.0.21 PLATON 30 was used for determining
226.0975; found 226.0965.
the hydrogen bond and intermolecular interactions.
16: purple solid (7.0 mg, 60%); UV/Vis: λmax(MeOH)/nm 510 Crystallographic and experimental details are summarised in
(ε/dm3 mol−1 cm−1 21 217); 1H NMR (400 MHz, CD3OD): Table S1 in the ESI and in Tables S2 and S3,† the bond
δ (ppm) = 6.84 (d, 1H, J = 7.6 Hz, H8), 6.79 (s, 1H, H12), 6.68 (d, lengths, bond angles and torsion angles, respectively.
1H, J = 7.6 Hz, H9), 6.55 (s, 1H, H5), 5.66 (s, 1H, H2), 2.17 (s, CCDC 1476627 contains the cif file for this compound.
3H, CH3 aromatic ring), 1.90 (s, 3H, CH3 iminic ring); 13C{H} NMR
(400 MHz, CD3OD): δ (ppm) = 188.6 (C1), 170.2 (C10), 151.0
(C4), 142.5 (C3), 139.6 (C7), 134.9 (C8), 126.4 (C6), 123.0
(C12), 118.3 (C9), 116.2 (C11), 117.6 (C5), 100.1 (C2), 18.3
Conclusions
(CH3 aromatic ring), 16.5 (CH3 iminic ring); ESI/MS positive mode: We have demonstrated the ability and efficacy of CotA-laccase
m/z 242 (100%, [M + H]+); MS2 227 (100%, ½M þ H ꢀ CH•3ꢁ•þ); to oxidise, in water, three oxidation bases (1,4-PDA, 4-AP and
HR-ESI/MS: m/z calcd for C14H16N3O [M + H]+: 242.1288; 2,5-DAT) to the corresponding benzoquinonediimine or
found 242.1285.
benzoquinoneimine species and promote the selected cross-
17: yellow solid (3.2 mg; 27%); UV/Vis: λmax(MeOH)/nm 440 coupling reactions with different aromatic couplers,
(ε/dm3 mol−1 cm−1 3962); 1H NMR (400 MHz, CD3OD): which resulted in the formation of dimeric and trimeric indo
δ (ppm) = 7.10 (d, 1H, J = 8.1 Hz, H8), 6.64 (s, 1H, H12), 6.34 dyes. The dinuclear and trinuclear aminoindamine, indo-
(s, 1H, H5), 6.29 (d, 1H, J = 8.1 Hz, H9), 5.66 (s, 1H, H2), 2.20 aniline and indonaphthol compounds were obtained in good
(s, 3H, CH3
ring), 1.88 (s, 3H, CH3
ring); 13C{H} to excellent yields and the results showed that the electron
aromatic
iminic
NMR (400 MHz, CD3OD): δ(ppm)
= 188.1 (C1), 170.5 character and position of the substituents in the coupler have
(C10), 156.9 (C4), 149.1 (C3), 144.1 (C7), 132.0 (C8), 123.7 fundamental importance in the occurrence of the hetero-
(C6), 122.9 (C12), 112.7 (C9), 108.6 (C11), 108.2 (C5), 99.8 (C2), coupling reactions.
20.2 (CH3
ring), 15.9 (CH3
ring); ESI/MS negative
iminic
The proposed mechanistic pathway for the dye formation
aromatic
mode: m/z = 241 (100%, [M − H]−); MS2 m/z = 226 (100, [M − H involves an initial oxidation of the primary intermediates by
− CH4]−), 223 (41, [M − H − H2O]−), 199 (29, [M − H − laccase and further reactions with amine or phenolic couplers
CH2O]−); ESI/MS positive mode: m/z = 243 (100%, [M + H]+); to give the leuco-indo dyes and, upon oxidation, the corres-
HR-ESI/MS: m/z calcd for C14H15N2O2 [M + H]+: 243.1128; ponding indamine or indoaniline derivatives.
found 243.1128.
Thus, the laccase-based approach developed herein depicts
a greener alternative route for the preparation of indo dyes and
contributes to improved sustainability for the synthesis of this
Crystal structure determination of compound 8
Single crystals of compound 8 were obtained by slow evapor- important class of dyes.
ation of the phosphate buffer saturated solution.
Crystal data. C13H13N3O, M = 227.26, monoclinic, a =
13.551(1) Å, b = 3.9004(3) Å, c = 20.986(2) Å, β = 94.725(4)°,
Acknowledgements
V = 1105.5(2) Å3, T = 150(2) K, space group P2(1)/n, Z = 4,
10 590 reflections measured, 2710 unique (Rint = 0.0295), This work was supported by FCT- Fundação para a Ciência e
which were used in all calculations. The final R and wR(F2) Tecnologia through projects PTDC/BIO/72108/2006, RECI/
were 0.0399 and 0.1021, respectively.
QEQ-QIN/0189/2012 and REM2013 and R&D units UID/QUI/
The X-ray diffraction data were collected using a Bruker 00100/2013 and UID/Multi/04551/2013 (GreenIT). The authors
AXS-KAPPA APEX II diffractometer with graphite-monochro- thank Joana Bernardo for help in preliminary studies and
mated radiation (Mo Kα, λ = 0.71069 Å) at 150 K. The X-ray Dr M. Conceição Oliveira for the helpful discussions about the
data were collected using the omega scans of 0.5 per frame ESI-MS results.
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14 (a) B. Lalleman, P. Choisy and A. Lagrange, (L’Oreal), WO
2010057854A2, 2010; (b) G. Lang and J. Cotteret, (L’Oreal),
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(L’Oreal), US 7175673B2, 2007; (d) G. Lang and J. Cotteret,
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