Towards the rational biosynthesis of substituted phenazines and phenoxazinones by laccases

Article abstract of DOI:10.1039/c4gc00901k

Laccases are multi-copper oxidases that oxidise a wide range of substrates including phenol and aniline derivatives, which could be further involved in coupling reactions leading to the formation of dimeric and trimeric structures. This paper describes the enzyme-mediated dimerisation of several ortho and meta,para-disubstituted aromatic amines into phenazine ("head-to-tail" dimers) and phenoxazinone chromophores. The redox properties of substituted aromatic amines were studied by cyclic voltammetry and the kinetic constants of CotA and Trametes versicolor laccases were measured for selected aromatic amines. The structure of novel enzymatically synthesised phenazine and phenoxazinone dyes using CotA laccase was assessed by NMR and MS. Overall our data show that this enzymatic green process is an efficient alternative to the classic chemical oxidation of aromatic amines and phenols, with an impact on the broad field of applications of these heterocyclic compounds. the Partner Organisations 2014.

Full text of DOI:10.1039/c4gc00901k

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Towards the rational biosynthesis of substituted
phenazines and phenoxazinones by laccases†
Cite this: DOI: 10.1039/c4gc00901k
a,b
b
c
Ana Catarina Sousa, M. Conceição Oliveira, Lígia O. Martins* and
M. Paula Robalo*
a,b
Laccases are multi-copper oxidases that oxidise a wide range of substrates including phenol and aniline
derivatives, which could be further involved in coupling reactions leading to the formation of dimeric and
trimeric structures. This paper describes the enzyme-mediated dimerisation of several ortho and meta,
para-disubstituted aromatic amines into phenazine (“head-to-tail” dimers) and phenoxazinone chromo-
phores. The redox properties of substituted aromatic amines were studied by cyclic voltammetry and the
kinetic constants of CotA and Trametes versicolor laccases were measured for selected aromatic amines.
The structure of novel enzymatically synthesised phenazine and phenoxazinone dyes using CotA laccase
was assessed by NMR and MS. Overall our data show that this enzymatic green process is an efficient
alternative to the classic chemical oxidation of aromatic amines and phenols, with an impact on the broad
field of applications of these heterocyclic compounds.
Received 16th May 2014,
Accepted 5th June 2014
DOI: 10.1039/c4gc00901k
www.rsc.org/greenchem
growing since many enzyme-catalysed reactions meet the
criteria of green chemistry. The enzyme phenoxazinone
Introduction
1
5
Phenazines and phenoxazinones are heterocyclic nitrogen synthase is widely distributed in nature and catalyses the oxi-
containing compounds that are broadly distributed in natural dative condensation of two molecules of substituted ortho-
16
and synthetic products. Substituted phenazine and phenoxazi- aminophenols to the phenoxazinone chromophore. This
none cores are important biological active motifs of antibiotics enzyme is a member of the multicopper oxidase family and
1
,2
3,4
5
and antibacterial agents, anti-tumour agents, pesticides,
performs the one-electron oxidation of substrates coupled to
6
–8
9
dyestuffs, biosensors, and are the building blocks for the the four-electron reduction of oxygen to water. Phenoxazinone
synthesis of organic semiconductors or electrical–photo- synthases show an overall fold, structural details in the cata-
chemical materials. Therefore, the oxidation of amines or lytic copper centres and oxidation–reduction mechanisms
aminophenols to phenazine or phenoxazinone derivatives, identical to laccases. Laccase-catalysed reactions are per-
respectively, is of great interest from the biological, chemical formed in aqueous solvent systems under mild reaction pH
1
0
1
1
1
7
and technological points of view.
The production of phenazine
and temperature conditions and the only waste product
formed during the course of their reactions is water. There-
1
2,13
14
18
and phenoxazinone
cores has been assessed through a number of chemical fore, laccases have drawn considerable attention from syn-
methods. However, most of the methods used suffer from a thetic chemists in recent years and have been successfully
number of disadvantages, such as the use of organic solvents, used for the oxidation of several functional groups, such as
the requirement for harsh reaction conditions and, despite ortho- and para-diphenols, methoxyphenols, aminophenols
1
8
some progress, low production yields. The interest in enzyme- and lignin-related molecules. Most of these transformations
catalysed transformations is on the other hand steadily are based on the oxidation of activated C–H bonds to C–O
1
8,19
bonds and of C–O bonds to CvO bonds.
catalyse phenolic oxidative couplings
Laccases can also
and they can initiate
the generation of ortho- and para-quinones followed by
1
8,20
a
Área Departamental de Engenharia Química, Instituto Superior de Engenharia de
Lisboa, Rua Conselheiro Emídio Navarro, 1, 1959-007 Lisboa, Portugal.
E-mail: mprobalo@deq.isel.ipl.pt; Fax: +351218317267; Tel: +351218317163
1
8,21
addition of different nucleophiles.
b
Centro de Química Estrutural, Complexo I; Instituto Superior Técnico, Universidade
In a previous study we reported the “green” production of
dyes from para-substituted aromatic amines, a synthetic
approach, based on the use of CotA-laccase as a biocatalyst
de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal
c
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da
22a
República, 2780-15 Oeiras, Portugal. E-mail: lmartins@itqb.unl.pt;
Fax: +3514411277; Tel: +351214469534
and there are also other reports in the literature related to the
use of laccases for the synthesis of phenoxazinone and related
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
8
,23
c4gc00901k
heterocycles.
However, in spite of the observed chemo-
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selectivity and the relatively good yields obtained, these parameter for predicting the performance of the enzymes
studies were restricted to particular substrate substitution under study.
8
,23
patterns.
Electrochemical studies of the aromatic amines were per-
The rational production of new substituted phenazine and formed by cyclic voltammetry with a platinum disk working
phenoxazinone scaffolds is highly desirable and has been electrode in 100 mM of Britton–Robinson (pH 4–5) or phos-
mainly prevented by the absence of general methodologies for phate (pH 6–7) buffer.
the production of diversely substituted phenazines and
In general the tested compounds show similar electro-
phenoxazines. Herein, we report the enzymatic oxidation of chemical behaviours generating well-defined irreversible oxi-
aromatic amines, such as ortho-phenylenediamines, sub- dation peaks (Epa) (Table 1). The absence or the presence of
stituted para-phenylenediamines and ortho-aminophenols, by small reductive peaks (Epc) for most of the substrates tested
2
4
CotA-laccase from Bacillus subtilis for the production of sub- indicates that the oxidation process probably generates a
stituted phenazine and phenoxazinone derivatives, extending highly reactive quinone-diimine or quinone-imine species that
the scope of laccase-catalysed transformations. We show that undergoes further non-enzymatic reactions. The only exception
this enzymatic approach constitutes a promising method for was for ANP, where quasi-reversible redox processes were
the green production of phenazine and phenoxazinone frame- observed, with E1/2 in the range 0.20–0.29 V.
works with an impact on a variety of applications.
The aromatic amines exhibited oxidation peaks in the
potential range of 0.24 V (for ANP at pH = 7) to 0.71 V (for
−1
2-NPDA and 2-AP at pH = 4) at a scan rate of 50 mV s . The
oxidation potential is influenced by the additional presence of
Results and discussion
Electrochemical studies of substrates
a substituent group (R ) on the meta position (see Table 1); the
2
presence of an electron donor group, OH in 2,5-DAP, resulting
in a lower oxidation potential and is expected to favour the
A series of compounds derived from 1,4-phenylenediamine
enzymatic oxidation, while the electron acceptors SO H
(1,4-PDA) and 4-aminodiphenylamine (4-ADA) were studied,
3
(
2
in 2,5-DABSA and 4-ADASA) and NO (in 2-NPDA) groups
together with 1,2-substituted aromatic derivatives, in order to
draw trends in the type of compounds that are susceptible for
efficient oxidation by laccases (Fig. 1). It is known that the
nature and position of the substituents in the amine aromatic
ring affect the redox potential of the substrates and conse-
resulted in higher oxidation potentials and a loss of reversibil-
ity of the redox processes. Moreover, the increased oxidation
potentials measured for 1,2-PDA and 2-AP showed that ortho-
substituted derivatives are more difficult to oxidise than the
related para-substituted derivatives.
19d,22
quently the rate of electron transfer to the enzyme.
The
Differences of up to 200 mV were found in the substrate oxi-
dation potentials within the pH range tested. Increasing the
pH from 4 to 7 resulted in an almost linear decrease in the oxi-
dation potential consistent with our previous results with
laccase’s activity is enhanced by the presence of electron-
donating groups at the benzene ring, which by decreasing the
2
2
electrochemical potential make them more easily oxidisable.
The pH is also a factor that plays a pivotal role in the laccase’s
enzymatic activity as it affects both the enzyme’s performance
and the redox potential of their putative substrates, i.e. their
susceptibility for oxidation. Therefore, the information of the
2
2a
p-substituted aromatic amines.
redox potential of each substrate at a particular pH is a critical Table 1 Electrochemical data of substituted aromatic amines vs. NHE
in buffered solutions at a scan rate of 50 mV s−
1
pH = 4
pa (V)
pH = 5
pa (V)
pH = 6
pa (V)
pH = 7
pa (V)
Substrate
E
E
pc (V)
E
Epc (V)
E
Epc (V)
E
1
,2-PDA
0.69
—
0.71
0.60
0.52
0.62
—
—
—
0.48
0.44
0.53
0.69
—
—
—
—
0.47
0.33
0.64
0.88
0.58
0.52
0.49
0.59
0.76
0.63
0.83
0.69
0.32
—
—
—
—
0.43
0.31
0.45
0.70
—
—
—
—
—
0.61
0.88
0.56
0.47
0.48
0.58
—
0.63
0.82
0.69
0.30
—
—
—
—
0.37
—
—
—
—
—
—
—
—
0.22
0.58
0.85
0.51
0.41
0.44
0.55
—
0.52
0.82
0.64
—
2
1
4
4
-AP
,4-PDA
2
2a
2
2a
-ADA
-ADASA
a
b
b
0
.74
,5-DABSA 0.65
.83
-NPDA
,5-DAP
2
0
0.71
0.41
2
2
c
0.55
—
0.24
d
e
f
g
ANP
0.43
0.32
0.26
0.31
a
4
-ADA = 4-aminodiphenylamine. ΔE = 90 mV, Ipc/Ipa = 0.5, irreversible
b
d
c
wave.
Ipc/Ipa = 0.5, irreversible wave. E1/2 = 0.51 V, ΔE = 80 mV, Ipc/Ipa
e
=
6
1.0. ΔE = 100 mV, Ipc/Ipa = 0.4, irreversible wave.
E
1/2 = 0.29 V, ΔE =
f
g
0 mV, Ipc/Ipa = 1.0.
1/2
E1/2 = 0.265 V, ΔE = 90 mV, Ipc/Ipa = 1.0. E =
Fig. 1 Representative structures of the substituted aromatic amines.
0.205 V, ΔE = 70 mV, Ipc/Ipa = 1.0.
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Overall, the oxidation potentials measured are slightly mation of phenazines with a “head-to-tail” framework. The
higher or lower than the redox potential of CotA (0.55 V vs. ability of the enzyme to produce the tricyclic heterocycle core
25
26
NHE ) and TvL enzymes (0.79 V vs. NHE ) respectively, indi- was confirmed using 1,2-phenylenediamine as the model com-
cating the absence of redox constraints for their oxidation by pound. The oxidation of 1,2-PDA resulted in the formation of
both enzymatic systems.
2,3-diaminophenazine (1), a planar electron rich heterocyclic
aromatic compound known by its rich organic chemistry and
intense luminescence. The H NMR spectrum (CD OD-d ) of
3 4
Laccase-mediated oxidation of selected substrates
27
1
The biotransformation of the aromatic substrates (Fig. 1) was 1 showed peaks in the 7.00–8.00 ppm region indicating the
initially investigated using two laccases, the bacterial CotA presence of six aromatic protons in a symmetrical tricyclic phe-
2
8
laccase and the commercial fungal TvL. The pH optima for nazine core consistent with data in the literature. The struc-
selected substrates (4-ADASA, 2,5-DABSA and 2,5-DAP) were ture was corroborated by the ESI-mass spectrum in the positive
+
measured (data not shown) and bell-shaped pH activity pro- mode that exhibited a major peak at m/z 211 ([M + H] ).
files with maximal enzymatic rates at neutral pH (around 6–7)
for CotA-laccase and under slightly acidic conditions for the tron-withdrawing sulfonyl substituent in the meta position
TvL laccase (around 4–5) were obtained.
relative to the amino group resulted in the formation of phena-
Laccase oxidation of 4-ADASA and 2,5-DABSA with the elec-
The kinetic constants for 4-ADASA and 2,5-DABSA were zine coloured compounds 2 and 4 as main products. A similar
measured using the two laccases (Table 2), showing the impor- phenazine derivative (6) was obtained from 2-NPDA, but in a
tance of sulfonyl side groups in decreasing the apparent Km lower yield. The structural identification of the phenazine skel-
values of the enzymes. The specificities (kcat/K
laccase and TvL are comparable for the 4-ADASA substrate, ences in the chemical shifts of the aromatic protons. The
however the specificity of CotA for 2,5-DABSA is one order of disappearance of the singlet for H , present in all substrates,
m
) of CotA- eton (for compounds 2, 4 and 6) was based on observed differ-
6
magnitude higher as compared with TvL. The improved speci- and the concomitant presence of resonances in the
ficity of CotA-laccase for both substrates combined with its 8.01–6.38 ppm region corresponding to the two doublets for
optimal pH close to the neutrality encouraged us to pursue the protons H3,8 and H4,9 confirmed the substitution at the ortho
work using the bacterial CotA-laccase.
position relative to the phenazine ring and the symmetry of
the phenazine central core. For compound 2, the signal at
Structural characterisation of the biotransformation products
10.75 ppm corresponds to the sulfonic acid protons, while the
Under the optimal pH conditions, all the tested substrates broad signal at 6.75 ppm of compound 6 was attributed to
were oxidised by CotA-laccase on a preparative scale. To esti- amino groups. In contrast, no signals were found for amino or
mate the yield of product formation, the enzyme (1 U) was sulfonic groups for compound 4. The assignment of the struc-
1
13
added to 10 ml reaction mixtures containing 5 mmol of sub- tures in both rings was confirmed by H– C HSQC and HMBC
strate. In the presence of the enzyme, the disappearance of the correlations.
substrates was completed after 24 h and, in general, the main
products were obtained in good to excellent isolated yields mined by negative-ion ESI mass spectrometry and the pro-
60–90%).
posed structures were confirmed by tandem mass
The obtained products, directly as crude solids or purified spectrometry. The full mass spectra of 2 and 4 displayed deproto-
by silica gel preparative chromatography, were fully character- nated molecular ion signals with different charges, as expected
The molecular weights of compounds 2, 4 and 6 were deter-
(
1
13
ised by IR, NMR ( H, C, COSY, HSQC, HMBC and NOESY), for disulfonated compounds. The mass spectra of 2 and 4
UV-visible spectroscopy and mass spectrometry (Fig. 2).
showed relatively intense peaks at m/z 521 and 369, and at m/z
The enzymatic oxidation of meta,para-disubstituted amine 259.9 and 183.9 corresponding to [M − H] and [M − 2H]
−
2−
,
2
derivatives (2,5-DABSA, 2-NPDA and 4-ADASA) led to the for- respectively. The MS mass spectrum of the precursor ion m/z
Table 2 Kinetic parameters of oxidation reactions catalysed by CotA-laccase and TvL. Reactions were performed at the optimal pH (Britton–
Robinson buffer) for each substrate
CotA
cat (s−
1
)
kcat/K
m
(mM−1 s−1
)
Substrate
Optimal pH
K
m
(mM)
k
4
2
-ADASA
,5-DABSA
6
6
0.21 ± 0.12
0.4 ± 0.12
12.6 ± 2.5
31.7 ± 5
68.1 ± 24
81.8 ± 10
TvL
cat (s−
1
)
kcat/K
m
(mM−1 s−1
)
Substrate
Optimal pH
K
m
(mM)
k
4
2
-ADASA
,5-DABSA
4
5
0.2 ± 0.01
1.6 ± 0.3
13.8 ± 2.5
9.6 ± 0.7
62.3 ± 9
5.9 ± 0.6
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Fig. 2 Structures and yields (%) of the main products formed in laccase-catalysed reactions with substituted aromatic amines.
5
21 presented fragments at m/z 457, 441 and 377 due to losses structure. Compounds 2 and 4 are water soluble, with
−
1
−1
of 64 u (SO ), 80 u (SO ) and 144 u (SO + SO ), respectively, molar extinction coefficients (ε) of 494 M
which is a characteristic of aromatic disulfonates.
cm
and
2
3
2
3
2372 M− cm , respectively.
1
−1
For compound 6, the ESI mass spectrum presents a peak
In the biotransformation of 4-ADASA by CotA-laccase, a sec-
−
at m/z 299 ([M − H] ) consistent with a molecular mass of ondary azo product, compound 3, was formed and isolated
1
1
3
00 g mol−
.
through preparative chromatography. The H NMR spectrum
Fragmentation of the precursor ion m/z 299 yielded the showed a singlet at 8.15 ppm and two doublets at 7.75
2
9
characteristic product ions for nitro compounds, at m/z 282, and 7.36 ppm, with characteristic meta coupling constants,
−
•
−•
2
69 and 239 attributed to [M − H − OH] , [M − H − NO]
6 4 3
attributed to protons of H , H and H , respectively. This is
−
and [M − H − 2NO] , respectively. The fragment ion at m/z 239 indicative that compound 3 has a similar structural pattern to
is in agreement with the presence of two nitro groups in the the substrate. In addition, the shift of all aromatic signals
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for low field is consistent with the presence of a symmetrical
The proposed structure 7 was corroborated with ESI-MS
structure connected by a NvN bond. Furthermore, the data. The full scan spectrum in positive-ion mode exhibits two
−
+
+
respective mass spectrum shows peaks at m/z 523 ([M − H] ), major peaks at m/z 228 ([M + H] ) and 250 ([M + Na] ). The
−
2−
2
5
45 ([M − 2H + Na] ) and 260.9 ([M − 2H] ) which are consis- MS spectrum of the protonated molecule showed a peak at
tent with a molecular mass of 524 g mol . The MS
−
1
2
+
m/z 200 ([M + H − CO] ) due to the characteristic loss of a CO
−
spectrum of the [M − H] ion at m/z 523 was characterised by molecule (−28 u). Additionally the IR spectrum with a band at
resulting in an abundant fragment ion with m/z 1649 cm− , assigned to the carbonyl group, and without the
1
the loss of SO
43, which further led to product ions with m/z 415 (rearrange- wide band at ν = 3200 to 2500 cm , typical of intramolecular
ment loss of N from the group azo) and 247 (cleavage of C–N hydrogen bonds of the phenolic group, corroborates the for-
bound at the central group azo) in the MS experiment. mation of the quinone-imine central ring.
3
−
1
4
2
3
The formation of azo compounds was previously observed as
The synthesis of phenoxazine derivatives using laccases and
laccase biotransformation products derived from para- ortho-aminophenol substrates was also confirmed through the
2
2a
substituted aromatic amines
Blue 62 dye.
or the anthraquinonic Acid use of 2-AP and ANP as model compounds. Laccase-mediated
3
0,31
conversion of 2-AP yielded the phenoxazine 8, a brown product
The biotransformation of 2-NPDA led also to the formation with identical physicochemical features to the 2-amino-3H-
3
2
of two products, the previously described yellow phenazine 6 phenoxazin-3-one. The structure was confirmed by ESI-MS in
and an additional dark red product (5), with a 2,5-diaminated positive-ion mode, which produced two main ions with m/z
+
+
1
,4-benzoquinonediimine skeleton, resulting from the sequen- 213 ([M + H] ) and 235 ([M + Na] ). The precursor ion m/z 213
tial self-coupling of the 2-NPDA intermediate (Fig. 2) at posi- exhibited a prominent fragment at m/z 185 corresponding to
tions 2 and 5. The multiplicity and resonances of the signals the loss of a CO molecule.
1
13
found in H and C-NMR spectra are in accordance with pre-
The oxidation of ANP yields also a phenoxazine compound
viously reported data for similar benzoquinonediimine struc- (9) for which the presence of six aromatic signals (four doublets
2
2a
1
tures.
The coupling must be performed through the amino and two triplets) in the region 7.25–8.42 ppm in the H NMR
group in the meta position to the nitro group, as suggested by spectrum showed identical multiplicity to those of the substrate
the correlations found in the NOESY-NMR spectrum between ANP, suggesting the occurrence of very similar spin system
NH protons and H and H . The full ESI mass spectrum of pattern for naphthalene ring C. The presence of two pairs of
2
11
17
−
5
shows a base peak at m/z 452 ([M − H] ) which is in agree- related doublets and triplets for protons H
ment with a molecular mass of 453 g mol . Fragmentation of naphthalene A ring. Although many attempts were made to
the precursor ion m/z 452 generated a predominant product detect the quaternary carbons in the C spectrum, these were
1
4
to H defines the
−
1
1
3
ion with m/z 405 attributed to the loss of HNO
2
.
not observed, by reasons that are not clear at the present time.
Substitution of the meta electron-withdrawing groups by an
The ESI mass spectra of 9 show intense peaks at m/z 316
hydroxyl group led to the formation of a phenoxazine deriva- and 338, and at m/z 314 assigned to the protonated molecule
+
+
tive, which highlights the potential use of laccases for the [M + H] and its sodiated adduct [M + Na] , and to the deproto-
−
preparation of compounds based on structural variations of nated molecule [M − H] , respectively. The even nominal mass
the 2-aminophenoxazin-3-one scaffold.
found for the (de)protonated ions dictates an odd number of
The conversion of 2,5-DAP yielded a purple product 7 for nitrogen atoms in the structure. In order to confirm the pro-
which the specific pattern of a phenoxazine ring was identi- posed structure CID experiments were performed. The major
2
fied. Downfield shielding was observed for all the proton fragmentation channel observed in the MS spectrum of 9 is
signals of this heterocycle relative to their respective parent due to the loss of 28 u (CO) yielding ion at m/z 288. Further
compound. Furthermore, the three aromatic signals at fragmentation of these ions gave rise to ions at m/z 270 (loss of
7
.44 (H ), 6.77 (H ) and 6.63 ppm (H ) possess identical multi- a water molecule) and 260 (loss of a CO molecule), and other
9 8 6
plicity to those of the substrate 2,5-DAP, suggesting the occur- low-abundance products, as summarized in the fragmentation
rence of a very similar spin system pattern for ring C. The routes proposed for 9 (see ESI†).
presence of two singlets for protons H and H defines the A
The structural characterization of the orange compound 9
1
4
1
3
ring. In the C spectrum, resonances at low field regions, and the MS results in particular indicated the presence of two
namely 181.5 and 151.2 ppm, characteristic of carbonyl and hydroxyl groups (naphthalene ring A). This suggests the hydro-
imine quaternary carbons (C and C ) confirmed the presence lysis of the imine group in the final reduced phenoxazine inter-
3
1
11
13
of a quinone-imine ring. The H– C HMBC spectrum also pro- mediate which could occur during the oxidation or the
8c
vided crucial information for the attribution of the quaternary purification processes, as previously described by Bruyneel et al.
carbons and in particular confirmed the substitution at
C
C
7
(ring C). The connectivities between H
together with the coupling constants of the protons H
6
and H
8
with
, H
Proposed pathway for laccase’s oxidation of substituted
aromatic substrates
7
6
8
and H support the substitution pattern of ring C. For ring A,
9
the substitution pattern was proposed based on the corre- An enzymatic mechanistic pathway for the production of sub-
lations between H
C3, C11 and C12.
1
and C
2
, C11 and C12 and between H
4
and stituted heterocycles with phenazine or phenoxazine scaffolds
is proposed based on our experimental results and on previous
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results on the oxidative cross-coupling of substituted o-amino- phenoxazine, which is spontaneously air-oxidised to produce
3
3,34
33,34
phenols
The ortho-diamine or ortho-aminophenol precursor is en-
zymatically processed through two successive one-electron oxi- placement of an amino group in the 2,5-DAP, in the formation
(Scheme 1).
the desired heterocycle.
This intramolecular Michael addition might lead to the dis-
8
d
dations, generating the ortho-quinone-diimine or ortho- of the phenoxazine moiety. The loss of the NH group in one
2
quinone-imine intermediates (A). These species are further of the two substrate molecules is consistent with similar
involved in a 1,4-conjugated Michael addition, on its most observations on the formation of the phenoxazine core
electrophilic carbon atom (in the para position to the quinonic accompanied by the loss of COOH or CHO groups, previously
3
5
36
group), together with a nucleophilic partner (the amino group reported by Suzuki et al. and Hughes et al.
of another substrate molecule). This second step, followed by a For the meta,para-disubstituted aromatic amines (2,5-DABSA,
proton shift, yields the first coupling intermediate (B). The 2-NPDA and 4-ADASA), the first benzoquinonediimine inter-
second 2e− oxidation is also expected to be mediated by mediate is formed through laccase oxidation in a similar
3
3
laccase as suggested by Bruyneel et al.
Intramolecular manner to that described above. However, the sequential non-
Michael addition of the amino group (or phenol) to the C5 enzymatic step followed a different pattern, where the inter-
atom leads to the aminophenazine or fully reduced amino- mediate and the nucleophilic amino group of another sub-
Scheme 1 Proposed oxidative pathway for the formation of the phenazine and phenoxazinone frameworks from laccase and substituted aromatic
amines.
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strate molecule are involved. In contrast to the ortho disub- Electrochemistry PowerSuite v2.51 software from Princeton
stituted derivatives, the nucleophilic attack at the para position Applied Research. Cyclic voltammograms were obtained using
relative to the amino group is hampered by the presence of a 1 mM of compounds in 1 : 9 MeOH: 100 mM buffer (phos-
substituent. Instead, the nucleophilic attack occurs at the phate buffer for pH ≥ 6 and Britton–Robinson (B&R) buffer for
ortho position, adjacent to the sulfonyl or nitro side groups. pH 4–5) solutions using a three-electrode configuration cell
The subsequent proton shift aromatises the first oxidised with a home-made platinum-disk working electrode (1.0 mm
−
intermediate as above. A 2e oxidation followed by an intra- diameter), a platinum wire counter electrode and a silver/silver
molecular Michael addition of the aromatic amino group chloride reference electrode (Ag/AgCl) (purchased from Radio-
again yields a tricyclic phenazine framework.
meter analytical, SAS, France). The potential was scanned from
−
0.7 to 1.2 V at a scan rate of 50 mV s− . All measurements
1
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
Experimental
General procedures
(
NHE).
All reagents and solvents are commercially available (Sigma-
Aldrich Co) and were used without further purification. The
Enzymatic assays
1
13
product characterisation was performed by 1D-NMR ( H, C)
and 2D-NMR (COSY, HSQC, HMBC and NOESY) in DMSO-d
D O, CD CN-d or CD OD-d on a Bruker Advance 400 MHz
The effect of pH on the oxidation of 2,5-DABSA, 4-ADASA, and
6
,
2,5-DAP (1 mM) by CotA-laccase and TvL was analysed in B&R
2
3
3
3
4
buffer (pH 3–10; 100 mM phosphoric acid, 100 mM boric acid
and 100 mM acetic mixture with 0.5 M NaOH to the desired
pH) by measuring the initial rate of substrate oxidation at
spectrometer. Chemical shifts are reported in ppm relative to
the solvent peaks and coupling constants (J) are reported in
hertz. FTIR spectra were obtained in KBr pellets on a Bruker
Vertex 70 FT-IR spectrometer.
4
60 nm. The kinetic parameters for the aromatic amine sub-
strates (0.05–5 mM) were monitored at 37 °C at the optimum
pH for each substrate. Kinetic constants (K and kcat) were
ESI-MS spectra were acquired on a 500-MS LC ion trap mass
spectrometer (Varian, Inc., Palo Alto, CA), operated in the posi-
tive and negative ion modes. The optimised operating para-
meters were: ion spray voltage: ±5 kV; capillary voltage: 20 V
and RF loading: 90%. Nitrogen was used as nebulizing and
drying gas, at pressures of 25 and 10 psi, respectively; the
drying gas temperature was set at 300 °C. The spectra were
recorded in the range 100–1000 Da. Spectra typically corres-
pond to the average of 20–35 scans. Tandem mass spectra were
obtained with an isolation window of 2.0 Da, excitation energy
m
fitted directly to the Michaelis–Menten equation (OriginLab
software, Northampton, MS, USA). All enzymatic assays were
performed at least in triplicate. The protein concentration was
measured using the Bradford assay using bovine serum
albumin as a standard.
2
4
CotA laccase mediated reactions: general procedure
Preparative scale reactions were performed under the following
conditions: in a 25 ml round-bottom flask, the aromatic sub-
strate 4-ADASA, 2,5-DABSA, 2-NPDA, 1,2-PDA, 2,5-DAP, 2-AP, or
ANP (0.05 mmol) dissolved in 1 ml of methanol was added to
values between 1.2 and 2.2 V, and an excitation time of 10 ms
3
(
CID up to MS ).
High resolution mass measurements were performed on a
9
(
ml of 100 mM phosphate buffer (pH 6–7). Then, the laccase
1 U ml− ) was added and the reaction mixture was stirred at
1
MicrOTOF equipped with an ESI source (Bruker, Daltonics) at
Unidade de Spectrometria, Universidade de Santiago de
Compostela.
The UV-Visible spectra of the substrates and the molar
extinction coefficients were obtained on a Nicolet Evolution
00 and an Agilent Technologies Cary 60 spectrophotometer.
Recombinant CotA-laccase from Bacillus subtilis (1 U
defined as the amount of enzyme that transformed 1 µmol of
ABTS per min at 37 °C) was produced and purified as
37 °C under aerobic conditions. The conversion was followed
by thin layer chromatography (TLC) on aluminium sheet
Silicagel 60 F254 (Merck). After 24 h, the insoluble 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 extraction with methanol
and solvent evaporation. Whenever necessary the crude resi-
dues were purified by preparative chromatography.
3
2
0e,24
described previously.
Trametes versicolor laccase (TvL,
−
1
Oxidation of 1,2-phenylenediamine (1,2-PDA)
specific activity 20 U mg ; 1 U defined as the amount of
enzyme that transformed 1 µmol of catechol per min) was pur-
chased from Sigma-Aldrich. Both laccases were stored frozen
at −18 °C prior to use.
At pH 7, a dark yellow solid was collected by filtration, washed
with diethyl ether and dried under vacuum to afford com-
pound 1.
1
: Yellow-brownish solid; yield: 3.2 mg (0.0152 mmol, 66%);
Electrochemical measurements
−1
FTIR (KBr) ν(cm ): 3433, 3335, 3177, 1642, 1493, 1467, 1406,
The redox potentials for all aromatic amines and amino- 1336, 1261, 1224, 1101; H NMR (CD OD-d ): δ (ppm) = 7.95
1
3
4
phenols were measured by cyclic voltammetry using an EG&G (m, 2H, H6, H9), 7.62 (m, 2H, H7, H8), 7.03 (s, 2H, H1, H4);
1
3
Princeton Applied Research Model 273A potentiostat/galva-
3 4
C{H} NMR (CD OD-d ): δ (ppm) = 146.4 (C2, C3), 143.7 (C11,
nostat monitored with a personal computer loaded with the C12), 141.2 (C13, C14), 128.5 (C7, C8), 128.1 (C6, C9) and 103.2
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−
1
cm−1 (MeOH); HR-ESI/MS: m/z calcd for C12
−
(
C1, C4); UV/Vis: 440 nm, ε = 18 387 M
H
9
N
4
O
6
S
2
[M − H] : 368.9964;
+
ESI-MS positive mode: m/z = 211.1 [M + H] .
found 368.9958.
a
3 2 6
Referenced to the (CD ) CO-d solvent.
Oxidation of 4-aminodiphenylamine-2-sulfonic acid (4-ADASA)
Oxidation of 2-nitro-1,4-phenylenediamine (2-NPDA)
At pH 6, a brown solution was obtained and after solvent
evaporation, the residue was extracted with methanol. The At pH 6, the dark red solid was collected by filtration and was
final residue was purified by silicagel column chromatography purified by silicagel column chromatography (mobile phase:
(
mobile phase: MeOH–CH Cl 1 : 3) to afford two fractions. C H O –CH (CH ) CH 3 : 1) to afford two fractions. The first
2 2 4 8 2 3 2 4 3
The first eluted pink fraction and the second yellow fraction eluted red fraction and the second yellow fraction were dried
were dried under vacuum to afford compounds 2 and 3, under vacuum to afford compounds 5 and 6, respectively.
respectively.
5: Dark red solid; yield: 3.9 mg (0.008 mmol, 52%); FTIR
: pink solid; yield: 9.1 mg (0.017 mmol, 70%); FTIR (KBr) (KBr) ν(cm− ): 3469, 3354, 2958, 2923, 2853, 1721, 1629, 1568,
1
2
−1
1
ν(cm ): 3436, 2925, 1627, 1597, 1545, 1509, 1420, 1386, 1331, 1508, 1469, 1431, 1337, 1243, 1162, 1083, 821; H NMR
1
1
198, 1173, 1112, 1043, 823, 793, 796, 710, 625; H NMR (CD OD-d ) δ(ppm) = 7.71 (d, 1H, J = 2.4 Hz, H8), 7.56 (d, 1H,
3 4
6 3
(DMSO-d ): δ(ppm) = 10.75 (s, 2H, SO H), 7.98 (d, 2H, J = J = 2.4 Hz, H14), 7.19 (dd, 1H, J = 2.4 Hz and J = 9.2 Hz, H12),
9
7
.2 Hz, H4, H9), 7.70 (d, 2H, J = 9.2 Hz, H3, H8), 7.40 (t, 4H, J = 7.11 (dd, 1H, J = 2.4 Hz and J = 8.8 Hz, H18), 7.04 (d, 1H, J =
.8 Hz, H17, H17′, H19, H19′), 7.22 (d, 4H, J = 7.8 Hz, H16, 8.8 Hz, H11), 6.93 (d, 1H, J = 8.8 Hz, H17), 5.95 (s, 1H, H3);
1
3
1
H16′, H20, H20′), 7.17 (t, 2H, J = 7.2 Hz, H18, H18′), C{H}
NMR (DMSO-d ): δ(ppm) = 141.2 (C2, C7), 141.1 (C15, C15′), 1H, NH), 7.69 (s, 1H, H8), 7.59 (s, 1H, H14), 7.20 (d, 1H, J =
39.7 (C11, C13), 138.1 (C12, C14), 131.4 (C4, C9), 129.6 (C17, 9.2 Hz, H12), 7.12 (d, 1H, J = 8.8 Hz, H18), 7.04 (d, 1H, J = 8.8
3 3
H NMR (CD CN-d ): δ(ppm) = 8.58 (sbr, 1H, NH), 8.25 (sbr,
6
1
C17′, C19, C19′), 123.2 (C18, C18′), 122.9 (C3, C8), 121.4 (C16, Hz, H11), 6.93 (d, 1H, J = 8.8 Hz, H17), 6.56 (sbr, 4H, NH
2
),
C16′, C20, C20′), 120.2 (C3, C8); UV/Vis: 460 nm, ε = 494 M− cm
1
−1
5.96 (s, 1H, H3), 5.84 (sbr, 2H, NH iminic ring). C{H} NMR
13
−
(
2
MeOH); ESI-MS negative mode: m/z 521.1 [M − H] , m/z (CD OD-d ): δ(ppm) = 153.0 (C1), 152.8 (C4), 152.7 (C2), 145.3
3
4
2
−
2
59.9 [M − 2H] ; MS (m/z, (relative intensity)): 457 (100%) (C10), 144.9 (C16), 140.5 (C7), 139.9 (C13), 132.6 (C12), 132.2
−
−
[(M − H) − SO
2
] , 441 (45%) [(M − H) − SO
3
] , 377 (10%) (C15), 132.0 (C9), 130.5 (C18), 120.8 (C11), 120.6 (C17), 119.7
−
[
C
(M − H) − (SO2 + SO ) ] ; HR-ESI/MS: m/z calcd for (C5), 118.3 (C6), 118.0 (C8), 115.6 (C14), 91.9 (C3); UV/Vis:
3
−
−1
−1
24
H
17
N
4
O
6
S
2
[M − H] : 521.0595; found 521.0574.
450 nm, ε = 6626 M cm (MeOH); ESI-MS negative mode:
−
2
3
: Yellow solid; yield: 3.5 mg (0.007 mmol, 27%); FTIR (KBr) m/z 452.2 [M − H] ; MS (m/z, (relative intensity)): 405 (100%)
−
1
−
ν(cm ): 2925, 2855, 1636, 1535, 1468, 1321, 1084, 1024, 934, [(M − H) − HNO ] ; HR-ESI/MS: m/z calcd for C H N O
2
18 16 9 4
1
−
5
38; H NMR (CD
H6′), 7.81 (dd, 2H, J = 9.2 Hz, J = 2.4 Hz, H4, H4′), 7.37 (d, 2H,
J = 9.2 Hz, H3, H3′), 7.35 (t, 2H, J = 7.6 Hz, H9, H11, H9′, H11′), ν(cm− ): 3418, 2924, 2853, 1641, 1522, 1384, 1101; H NMR
3
OD-d
4
), δ(ppm) = 8.38 (d, 2H, J = 2.4 Hz, H6, [M − H] : 452.1073; found 452.1067.
6: Yellow solid; yield: 2.1 mg (0.007 mmol, 28%); FTIR (KBr)
1
1
7
7
.27 (d, 4H, J = 7.6 Hz, H8, H12, H8′, H12′), 7.08 (t, 2H, J = (CD
3
CN-d
3
): δ(ppm) = 7.91 (d, 2H, J = 9.6 Hz, H4, H9), 7.46
); UV/Vis:
13
.6 Hz, H10, H10′). C{H} NMR (CD
3
OD-d
4
): δ(ppm) = 145.8 (d, 2H, J = 9.6 Hz, H3, H8) and 6.75 (sbr, 4H, NH
2
−
1
−1
(C5), 145.0 (C7), 142.2 (C2), 131.5 (C1), 130.48 (C9, C9′, C11, 350 nm, ε = 3998 M cm (MeOH); ESI-MS negative mode:
−
2
C11′), 126.3 (C4), 124.6 (C6), 124.32 (C10), 122.44 (C8, C8′, m/z 299.1 [M − H] ; MS (m/z, (relative intensity)): 282 (100%)
C12, C12′), 115.24 (C3); ESI-MS negative mode: m/z 523.1 [(M − H) − OH ] , 269 (42%) [(M − H) − NO ] , 239 (25%)
•
−•
• −•
−
−
2−
• −
[M − H] ; m/z 545.1 [M − 2H + Na] and m/z 260.9 [M − 2H]
,
[(M − H) − 2 × NO ] ; HR-ESI/MS: m/z calcd for C H N O4
1
2
7
6
6
2
−
−
MS (m/z, (relative intensity)): 459 (20%) [(M − H) − SO
2
] , 443 [M − H] : 299.0534; found 299.0521.
−
3
(100%) [(M − H) − SO
3
] ; MS (m/z, (relative intensity)): 415
−
−•
Oxidation of 2,5-diaminophenol·2HCl (2,5-DAP)
(15%) [(M − H) − SO3 − N ] , 247 (100%) [C H NO S] ;
2
12
−
9
3
HR-ESI/MS: m/z calcd for C24
found 523.0712.
H
19
N
4
O
6
S
2
[M − H] : 523.0746; At pH 7, the dark solid was collected by filtration, washed with
diethyl ether and dried under vacuum to afford compound 7.
7
: Purple solid; yield: 3.2 mg (0.014 mmol, 62%); FTIR (KBr)
Oxidation of 2,5-diaminobenzene sulphonic acid (2,5-DABSA)
At pH 6, a dark magenta solution was obtained and after 1236, 1204, 1128, 840, 810, 705; H NMR (CD
solvent evaporation, the final residue affords a dark magenta = 7.44 (d, 1H, J = 8.8 Hz, H9), 6.77 (dd, 1H, J = 2.4 Hz and J =
solid 4. 8.8 Hz, H8), 6.63 (sd, 1H, J = 2.4 Hz, H6), 6.48 (s, 1H, H1), 6.33
: dark magenta solid; yield: 8.0 mg (0.02 mmol, 90%); (s, 1H, H4); C{H} NMR (CD
−1
ν(cm ): 3427, 3334, 3212, 1649, 1571, 1504, 1481, 1424, 1326,
1
3
4
OD-d ): δ(ppm)
1
3
4
3
OD-d
4
): δ(ppm) = 181.5 (C3)
−
1
FTIR (KBr) ν(cm ): 3435, 1631, 1498, 1439, 1316, 1186, 1128, 153.6 (C13), 151.2 (C11), 147.2 (C12), 146.8 (C14), 143.5 (C2),
2
O) : δ(ppm) = 130.3 (C9), 127.9 (C7), 115.1 (C8), 103.9 (C1), 101.3 (C4), 99.3
.04 (d, 2H, J = 9.5 Hz, H4, H9); 6.38 (d, 2H, J = 9.5 Hz, H3, (C6); UV/Vis: 520 nm, ε = 8950 M cm (MeOH); 460 nm, ε =
H8); C{H} NMR (D O) : δ(ppm) = 146.6 (C2, C7); 139.5 (C11, 5455 M cm (MeOH). ESI-MS positive mode: m/z 228.1
C13); 137.9 (C12, C14); 132.9 (C4, C9); 127.2 (C3, C8); 111.3 [M + H] , m/z 250.0 [M + Na] ; MS (m/z, (relative intensity)):
2
C1, C6); UV/Vis: 520 nm, ε = 2372 M cm (H O); ESI-MS 200 (100%) [(M + H) − CO] . HR-ESI/MS: m/z calcd for
1
a
1
7
046, 960, 877, 799, 710, 682, 648; H NMR (D
−1
−1
1
3
a
−1
−1
2
+
+
2
−1
−1
+
(
−
2−
+
negative mode: m/z 369.1 [M − H] , m/z 183.9 [M − 2H]
;
C H N O [M + H] : 228.0773; found: 228.0762.
12 10 3 2
Green Chem.
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Oxidation of 2-aminophenol (2-AP)
Overall these results show that the enzyme-catalysed oxi-
dative sequence, a green chemistry process, constitutes a valu-
able alternative to the chemical oxidative coupling of aromatic
At pH 7, the brown solid was collected by filtration, washed
with diethyl ether and dried under vacuum to afford com-
pound 8.
amines and phenolic precursors providing
a promising
approach for the rational synthesis of different heterocyclic
scaffolds.
8
: Brown solid; yield: 4.6 mg (0.021 mmol, 83%); FTIR (KBr)
−1
ν(cm ): 3412, 3305, 1657, 1587, 1573, 1495, 1470, 1467, 1425,
401, 1291, 1273, 1204, 1173, 1147, 1116, 894, 844, 762, 584;
H NMR (CD
1
1
2 2 2
Cl -d ): δ(ppm) = 7.74 (d, 1H, J = 8.4 Hz, H6),
7
.45–7.37 (m, 3H, H7, H8, H9), 6.47 (s, 1H, H4), 6.36 (s, 1H,
Acknowledgements
1
3
H1); C{H} NMR (CD Cl -d ): δ(ppm) = 180.6 (C3), 149.8
2
2
2
(
(
C14), 149.2 (C2), 146.2 (C13), 143.1 (C11), 134.4 (C12), 129.9
C9), 129.1 (C6), 125.6 (C8), 116.3 (C7), 104.2 (C1), 101.1 (C4);
The authors thank the FCT-Fundação para a Ciência e Tecno-
logia (Projecto Estratégico PEst-OE/QUI/UI0100/2013 and
REM2013) and the IST-UTL NMR network for facilities.
−1
−1
UV/Vis: 430 nm, ε = 9095 M cm (MeOH); ESI-MS positive
mode: m/z 213.1 [M + H] , m/z 235.0 [M + Na] ; MS (m/z, (rela-
tive intensity)): 185 (100%) [(M + H) − CO] .
+
+
2
+
References
Oxidation of 1-amino-2-naphthol (ANP)
At pH 7, the final residue was purified by silica gel preparative
chromatography (mobile phase: C H O –CH (CH )CH ; 3 : 1)
4 8 2 3 2 3
1
(a) Y. Hosoya, H. Adachi, H. Nakamura, Y. Nishimura,
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1
996, 37, 9227; (b) M. McDonald, B. Wilkinson, C. W. Van’t
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9
: Dark orange solid; yield: 4.5 mg (0.014 mmol, 59%); FTIR
−
1
1
(
KBr) ν (cm ): 3442, 1634, 1524, 1467, 1383, 1278, 1083; H
NMR (CD OD): δ(ppm) = 8.42 (d, 1H, 7.5 Hz, H13), 8.17 (d, 1H,
.0 Hz, H4), 7.90–7.78 (m, 3H, H10, H12, H1), 7.75 (t, 1H,
.5 Hz, H11), 7.63 (d, 1H, 8.5 Hz, H8), 7.44 (t, 1H, 7.8 Hz, H2),
1
3
8
7
7
7
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3
.33 (t, 1H, 7.8 Hz, H3), 7.26 (d, 1H, 9.0 Hz, H9); C{H} NMR
(CD OD-d ) δ(ppm) = 135.3 (C13), 133.1 (C4), 132.7 (C10),
3 4
2 P. B. Gomes, M. Nett, H.-M. Dahse, I. Sattler, K. Martin and
1
1
30.1 (C12), 129.3 (C1), 129.1 (C11), 128.2 (C8), 125.2 (C2),
22.4 (C3), 119.7 (C9); ESI-MS positive and negative modes:
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3
(a) S. Nakaike, T. Yamagishi, K. Nanaumi, S. Otomo and
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and R. Loddo, J. Med. Chem., 2002, 45, 5205;
+
+
−
m/z 316 [M + H] ; m/z 338 [M + Na] and m/z 314 [M − H] ;
MS (m/z, (relative intensity)): 298 (5%) [(M + H) − H
2
+
2
O] , 288
+
+
(100%) [(M + H) − CO] , 246 (10%) [(M + H) − C
3
H
2
O
3
] ,
+
3
1
58 (10%) [C H NO] ; MS (m/z, (relative intensity)): 270
1
0
8
+
(100%) [(M + H) − CO − H
2
O] , 260 (7.5%) [(M + H) − CO −
+
+
+
CO] , 158 (10%) [C10
calcd for C H NO [M − H] : 314.0823; found 314.0817.
H
8
NO] , 115 (8%) [C
9
H
7
] ; HR-ESI/MS: m/z
−
2
0
12
3
(
b) A. Bolognese, G. Correale, M. Manfra, A. Lavecchia,
O. Mazzoni, E. Novellino, V. Barone, P. La Colla and
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Conclusions
5
The efficiency of CotA-laccase to mediate coupling reactions
was demonstrated using several meta,para-disubstituted
phenylamines, ortho-phenylenediamines and substituted
ortho-aminophenols as substrates leading to the synthesis of a
diversity of phenazine and phenoxazinone frameworks. These
were obtained in good to excellent yields and the identification
of the products provided a basis for the proposal of a pathway
for the laccase catalysed oxidation of substituted aromatic sub-
strates. The laccase mediated oxidation of a given ortho-sub-
stituted amine or meta,para-disubstituted amine generates
directly ortho-quinonediimine (or ortho-quinoneimine) or para-
quinonediimine intermediates, respectively. These highly
electrophilic species then react with a nucleophilic partner
following the chemical rules governing the classical 1,4-conju-
gated Michael addition.
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9
R. Pauliukaite, M. E. Ghica, M. M. Barsan and
C. M. A. Brett, Anal. Lett., 2010, 43, 1588.
M. M. Santos, M. P. Robalo and L. O. Martins, J. Biotech-
nol., 2009, 139, 68.
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