A Sustainable Synthesis of Asymmetric Phenazines and Phenoxazinones Mediated by CotA-Laccase

Article abstract of DOI:10.1002/adsc.201701228

An efficient and sustainable one-step procedure for the synthesis of new asymmetric phenazines and phenoxazinones from commercially available ortho-substituted diamines and ortho-substituted hydroxyamines is reported. In this study we have expanded the substrate scope of CotA-laccase-catalyzed aerobic oxidations through the use of aromatic amines presenting variable functional groups, including N-substitution, contributing to the rational synthesis of different heterocyclic scaffolds. The transformations proceed smoothly through a cascade of oxidative reactions to the benzoquinonediimine intermediates followed by nucleophilic addition, intramolecular cyclization and aromatization, all performed in mild conditions. (Figure presented.).

Full text of DOI:10.1002/adsc.201701228

Title: A Sustainable Synthesis of Asymmetric Phenazines and
Phenoxazinones Mediated by CotA-Laccase
Authors: Ana Catarina Sousa, Maria Conceição Oliveira, Ligia Martins,
and Maria Paula Robalo
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To be cited as: Adv. Synth. Catal. 10.1002/adsc.201701228
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10.1002/adsc.201701228
Advanced Synthesis & Catalysis
UPDATE
DOI: 10.1002/adsc.201((will be filled in by the editorial staff))
A Sustainable Synthesis of Asymmetric Phenazines and
Phenoxazinones Mediated by CotA-Laccase
Ana Catarina Sousaa,b, M. Conceição Oliveira b, Lígia O. Martinsc and M. Paula
Robaloa,b*
a
Área Departamental de Engenharia Química, ISEL- Instituto Superior de Engenharia de Lisboa, Instituto
Politécnico de Lisboa, R. Conselheiro Emídio Navarro, 1, 1959-007 Lisboa, Portugal.
(e-mail: mprobalo@deq.isel.ipl.pt; phone: +3518317163; fax: +3518317267)
Centro de Química Estrutural, Complexo I; Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais,
b
1049-001 Lisboa, Portugal .
c
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av da República, 2780-
157 Oeiras, Portugal.
Received: ((will be filled in by the editorial staff))
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201######.((Please
delete if not appropriate))
Abstract. An efficient and sustainable one-step procedure The transformations proceed smoothly through a cascade of
for the synthesis of new asymmetric phenazines and oxidative reactions to the benzoquinonediimine interme-
phenoxazinones from commercially available ortho- diates followed by nucleophilic addition, intramolecular
substituted diamines and ortho-substituted hydroxyamines is cyclization and aromatization, all performed in mild
reported. In this study we have expanded the substrate scope conditions.
of CotA-laccase-catalyzed aerobic oxidations through the
use of aromatic amines presenting variable functional
groups, including N-substitution, contributing to the rational
synthesis of different heterocyclic scaffolds.
Keywords: Biocatalysis; Homocoupling reactions;
Laccase; Nitrogen heterocycles; Oxidation
affect several properties such as the redox potential
and solubility with consequences in their biological
activity and thus potential applications.[17-19]
Introduction
Phenazines are multifunctional versatile compounds
with antioxidant, antibiotic, anticancer and
antimicrobial activities,[1-5] which is mainly
associated with their redox activity and their capacity
to intercalate between DNA subunits. Furthermore,
the heteroaromatic phenazine π-core is present in a
vast array of heterocyclic compounds such as
The most traditional reported methods for the
preparation of phenazines[15,18] are based in the
assembly of the central heterocyclic ring and showed
several disadvantages like harsh reaction conditions,
in the presence of organic solvents and aggressive
oxidants, and limitation in the substrate scope related
with the localization and electronic character of the
ring substituents.[15]
pharmaceuticals,[6]
sensors
and
biosensors,[7]
OLEDS,[8] chemical switches,[9] photovoltaic cells for
renewable energy and in nanotechnology.[10] The
phenoxazinone core is also an important building
block present in compounds displaying significant
biological activities and redox properties.[11-13]
Considering their broad applications, there is a
growing interest in finding alternative chemical
routes for the formation of new phenazines or the
modification of those obtained from natural biologic
sources.[14-16] The nuclear phenazine core can be
substituted at different positions leading to symmetric
or asymmetric derivatives and these modifications
The formation of asymmetric phenazines can be
reached by cross-coupling reactions of aromatic
amines or in substitution reactions from symmetric
cores. Despite the efforts made in the development of
new sustainable methods,[18,20,21] the regioselective
synthesis of derivatives substituted in both rings is
still considered a challenge.
Laccases (EC 1.10.3.2) are copper-containing
metalloenzymes that have received increasing
attention over the past few decades in the field of
organic synthesis for being environmentally benign
catalysts.[22] They oxidize, in aqueous medium, a
1
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10.1002/adsc.201701228
Advanced Synthesis & Catalysis
range of different substrates including aromatic
amines but the vast majority of synthetic reactions
reported involved radical-coupling reactions of
phenolic monomers and cross-coupling reactions of
substituted catechols and hydroquinones with
nitrogen based[23] and carbon derived nucleophiles[24]
via in situ generated ortho- and para-quinones.
Concerning the laccase oxidation of aromatic amines
and further radical-coupling reactions involving the
generated ortho and para-benzodiimine or
benzoquinoneimine intermediates, few reports were
found.[25-28]
Our previous studies[25b,c] showed the potential of the
bacterial CotA-laccase to mediate the formation of
heterocyclic cores through homocoupling reactions
involving substituted aromatic amines. The formation
of symmetric phenazines as well as asymmetric
phenoxazinones was observed[25b] and this have
encouraged the extension of our studies to the use of
additional ortho-substituted aromatic amines looking
forward the formation of new asymmetric phenazines.
In this study we report the enzymatic preparation of
asymmetric phenazines by oxidation of aromatic
amines with different substitution patterns. This
process generated the corresponding ortho-
benzodiimine intermediates followed by in situ
intramolecular electrophilic aromatic substitution and
aromatization. This approach also prompted us to
synthetize N-substituted phenazines, extending the
scope of the enzymatically obtained heterocyclic
cores.
Figure 1. Chemical structures of the aromatic ortho-
diamines and ortho-hydroxyamines used as CotA-laccase
substrates.
(4-methyl-1,2-benzenediamine
(1a),
4-
methoxybenzene-1,2-diamine (1b), 4-bromobenzene-1,2-
diamine (1c), 3,4-diaminobenzoic acid (1d), 3,4-diami-
nobenzonitrile (1e), 2-N-phenylbenzene-1,2-diamine (2),
2-amino-4-methylphenol (3) and 2-aminopyridin-3-ol (4))
the range between 0.57 V (for 4-MB-1,2-DA, 1b) and
1.02 V (for 4-BB-1,2-DA, 1c) (see table S1,
Supplementary material). The absence of reductive
waves indicated that the oxidation process most
probably generated highly reactive quinoneimine or
quinone-diimine species which undergoes further
spontaneous reactions. The oxidation potential was
found to be influenced by the presence of the
substituent group (R) (see Figure 1 and Table S1);
lower oxidation potentials for EDG groups which is
expected to favour the enzymatic oxidation and
higher oxidation potentials for the EWG groups.
Considering that the redox potential for CotA-
laccase is only 0.535 V vs NHE,[29] slightly lower
than most of the selected substrates, the enzymatic
reactions were carried out in the absence of
exogeneous redox mediators.
The enzymatic reactions were performed in a
phosphate buffer (20 mM) or in a mixture containing
90 % of buffer and 10 % of ethanol, to guarantee the
complete solubility of aqueous-insoluble substrates,
in the presence of catalytic amounts of CotA-laccase
(1U.mL-1) at 37 °C with stirring. The time course of
reactions was followed by the formation of color in
the reaction mixtures and by TLC. The oxidative
processes occurred in aqueous media and in the
presence of air; oxygen is converted to water by the
enzyme and no toxic byproducts are formed. The
time course of reactions was reduced to 2h, a
significantly shorter period compared to our earlier
studies in the oxidation of aromatic amines mediated
by laccases,[25b,c] where the products were recovered
after 24h of reaction. Furthermore, the possibility of
recyclability of the enzymatic catalyst was also
examined using substrate 4 as an example. After
completion of the homocoupling reaction in
The dimerization of aromatic o-hydroxyamines with
subsequent formation of phenoxazinones was also
achieved,
highlighting
the
inclusion
of
heteroaromatics rings in the scope of aromatic
substrates used by CotA-laccase.
Results and Discussion
Our previous studies[25b] using CotA-laccase and the
model substrates 1,2-phenylenediamine (1,2-PDA)
and 2-aminophenol (2-AP) in aqueous medium
resulted in the formation of the 2,3-diaminephenazine
and 2-amino-3-phenoxazinone with 66 % and 83 %
yields, respectively. Following a similar strategy
under optimized reaction conditions,[25b] in this study
a set of substituted aromatic ortho-diamines with
different electron donor groups (EDG) and electron
withdrawing groups (EWG) (1a-e, 2 Figure 1) was
used to perform the enzymatic oxidative reactions.
Two aromatic ortho-hydroxyamines (3 and 4) (Figure
1) were also used to generate oxidative coupling
products at good to excellent yields.
In order to appraise the redox requirements for the
enzymatic oxidation, we have determined the redox
potential of the substrates by cyclic voltammetry
using a platinum wire working electrode in 20 mM
phosphate buffer pH 6.0 (the pH used in the
enzymatic reactions). All substrates showed a very
similar electrochemical behavior generating one or
two well-defined irreversible oxidation waves (Epa) in
2
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10.1002/adsc.201701228
Advanced Synthesis & Catalysis
approximately 2h, the reaction was recharged with an
additional amount of 4 (5 mM). This procedure was
repeated for three cycles without significant changes
on the final product yields.
conversion yields of 96%, 93% and 73%, respectively.
1
Their H-NMR spectra showed a similar pattern with
resonances for the aromatic protons in the range 6.03-
8.64 ppm, typical for the heteroaromatic phenazine
core. For compound 5, the singlet at 3.90 ppm was
indicative of the presence of only one methoxy group
in the molecule. Thus, the formation of the phenazine
core was accompanied by the loss of the methoxy,
bromo and carboxyl groups in one of the substrate
molecules, which is consistent with similar
observations previously reported for the formation of
phenoxazine cores.[25b,30] The elimination of the
methoxy, bromo and carboxyl groups was also
supported by the ESI- MS results. The first-order full
The products 5-13 were isolated at good to excellent
yields (Scheme 1), identified and fully characterized
by spectroscopic methods. Structural assignments
1
were made based on the observed H-13C HSQC
direct and HMBC 3-bond correlations (see the details
in the Supplementary data).
The enzymatic oxidation of ortho-diamines 1b-d
originated the respective water soluble colored
phenazines 5-7 (Scheme 1), as single products. These
compounds were isolated as brown solids with
Scheme 1 – Asymmetric heterocyclic cores obtained from the oxidation of aromatic ortho-diamines and ortho-
hydroxyamines mediated by CotA-laccase
mass spectrum of compound 5 shows the protonated
molecule [M+H]+ at m/z 241 and its fragmentation
yielded fragment ions with m/z 226 and m/z 198, due
to the characteristic loss of the methyl radical and the
3
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10.1002/adsc.201701228
Advanced Synthesis & Catalysis
subsequent loss of CO. For compound 6, the
proposed structure was corroborated by a group of
peaks at m/z 289/291 present in the ESI-mass
spectrum in positive mode, that exhibits the isotopic
distribution expected for the presence of one bromine
in the structure. For compound 7, the [M+H]+ and
[M-H]- ions at m/z 255 and 253 respectively, were
found in the full mass spectra. The MS2 spectrum of
the deprotonated molecule (m/z 253) was
characterized by the loss of CO2 resulting in an
abundant fragment ion with m/z 209.
was the first evidence of the occurrence of a
cyclization without demethylation. The presence of
two doublets at 3.09 and 3.27 ppm attributed to the
methylene protons, prompted us to suggest the
structure 10, similar to the one previously proposed in
the literature for the oxidation of ortho-aminophenols
by human hemoglobin.[31] The proposed structure was
also corroborated by the ESI mass spectrum which
showed in positive mode the protonated molecule
(m/z 242). MS2 fragmentation of the precursor m/z
242 produced two major product ions with m/z 227
and 214, due to the losses of the methyl radical and
CO, respectively.
CotA-laccase mediated oxidation of 3,4-DABN (1e),
containing the electron acceptor cyano group, under
the same reaction conditions, led to the correspondent
phenazine product 8 (48 %) and to a second product
(9) (49 %) (Scheme 1). Furthermore, the isolated
phenazine 8 is not a dimeric structure, but resulted
from the coupling of three substrate molecules as
Looking forward to extend the scope of the present
enzymatic approach to N-substituted phenazines, the
oxidation of the 2-N-phenylbenzene-1,2-diamine (2)
was performed and the phenazine 11 was isolated as a
1
water soluble brown solid. The H NMR spectrum
1
supported by the NMR and ESI-MS data. The H-
showed diverse signals in the aromatic region and the
13C spectrum was consistent with the presence of
twenty four carbons in the proposed structure, with
two resonances at low field (159.1 and 149.5 ppm)
characteristic of a diimine structure. The structure
was corroborated by the ESI-mass spectrum in
positive mode that exhibits a main peak at m/z 363
([M+H]+).
NMR spectrum of compound 8 clearly showed the
presence of more resonances than the expected for a
basic dimeric phenazine core. Indeed, three additional
resonances at 7.52, 7.07 and 6.28 ppm with the same
multiplicity as the substrate, suggested a very similar
spin system pattern (D ring). The presence of one
singlet for proton H7 described the A ring, while the
C ring was outlined by two duplets and one singlet
for protons H1, H2 and H4, respectively. In the 13C
spectrum, all resonances were identified as expected
in the range 160 to 95 ppm. The ESI-MS spectrum, in
negative-mode shows the [M-H]- species at m/z 375
while its sequential fragmentation resulted in the
rearrangement ion at m/z 348 due to the loss of HCN.
The substitution of the ortho-amino group by a
hydroxyl group led to the formation of
phenoxazinone derivatives, which confirms the use of
CotA-laccase for the preparation of compounds based
on structural variations of the 2-aminophenoxazin-3-
one scaffold. Moreover, the introduction of a
substituted pyridine ring as substrate extends the
scope of the type of substrates used by this enzyme.
The enzymatic oxidation of 2-amino-4-methylphenol
(3) resulted in the formation of a brownish yellow
compound 3-amino-1,4a-dihydro-4a,8-dimethyl-2H-
phenoxazin-2-one (12) (Scheme 1), with a very good
yield (88 %) in a short time (ca 2h). In accordance
1
For compound 9, the H-NMR spectrum showed a
similar pattern of the precursor molecule, with one
singlet at 8.17 ppm and two doublets at 7.03 and 7.45
ppm. This observation together with the deshielding
effect observed for all aromatic resonances is
consistent with the presence of a symmetrical
structure connected by a N=N bond, as previously
1
with the structure proposed to compound 10, the H
observed.[25a,b] Moreover, the respective MS spectrum, NMR spectrum of the compound exhibits two singlet
in negative mode shows a peak at m/z 261 ([M-H]-),
which generated in the MS2 analysis a fragment with
m/z 233 that results from the loss of N2 from the azo
group.
methyl signals at 1.05 and 1.26 ppm and two isolated
methylene signals at 3.4 and 3.6 ppm (J = 15.6 Hz).
All other proton resonances were consistent with the
proposed structure, which resulted from deamination
by an hydrolysis process during the reaction, in
agreement with the observed with other
substrates.[25b]
Overall these results indicate that the reaction course,
leading to different type of products, can be affected
by the electronic character of the R substituent. While
the presence of an electron donor group or a medium
electron acceptor group (COOH) in the meta(para)
position allowed the formation of asymmetric
phenazines with excellent yields, the presence of the
stronger acceptor CN group originated two different
products, a trimer based on the phenazine core and an
azo compound.
The enzymatic oxidation of 2-APy (4), a pyridine
derivative, resulted in the formation of the
pyridyloxazinone 13 (Scheme 1), a planar electron
rich heterocyclic aromatic compound, for which the
1H NMR spectrum showed resonances in the 6.55–
8.54 ppm region correspondent to the four aromatic
protons
of
the
asymmetrical
tricyclic
The enzymatic oxidation of 4-M-1,2-BDA (1a)
resulted in the water soluble dark red phenazine 10,
obtained with an excellent yield (91 %). Based on the
1H NMR spectrum, it was evident the presence of two
resonances at 1.24 and 2.25 ppm for the aliphatic
methyl groups, which indicates the presence of two
different groups in the final product. This observation
pyridyloxazinone core. The structure was also
confirmed by the ESI-MS spectrum in the positive
mode that exhibited a major peak at m/z 215 assigned
to the protonated molecule of 13.
The proposed molecular pathway (Scheme 2)[25b, 28e,32]
that led to the formation of asymmetric phenazines in
CotA-laccase mediated reactions, using aromatic
4
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10.1002/adsc.201701228
Advanced Synthesis & Catalysis
Conclusion
amines as substrates, initiates with the abstraction of
one electron from the more liable amino group in the
In this work the formation of asymmetric phenazines
by a one-step sustainable and environmental friendly
synthesis from commercially available starting
materials was described. The CotA-laccase enzymatic
system was shown to be selective towards the
synthesis of phenazine derivatives using ortho-
diamine derivatives with electron-rich and electron-
poor substituents, as substrates. Importantly, the
enzymatic process showed attractive advantages
including mild reaction conditions and short reaction
times with excellent yields when compared to the
established chemical oxidative methods.
The identification and characterization of products
provided a basis for the proposed oxidative enzymatic
pathway of substituted aromatic amines. The first
oxidative step generates the electrophilic ortho-
quinonediimine (or ortho-quinoneimine) intermedia-
te, which further reacts with a nucleophilic partner (a
precursor molecule that has not been oxidized)
followed by intramolecular cyclization processes.
The scope of aromatic substrates that can be
transformed by this enzymatic approach was further
extended with the introduction of N-substituted
ortho-diamines and the heterocyclic pyridine
derivative, allowing the formation of N-substituted
phenazine and a pyridyloxazinone. The formation of
these products also contributes to increase the variety
of heterocyclic cores that can be obtained by this
methodology establishing this enzymatic route as a
valuable alternative and a promising approach for the
rational synthesis of different heterocyclic scaffolds.
substrate
molecule,
generating
the
ortho-
quinonediimine key intermediate (A). Under the
reaction conditions, this electrophilic species (A)
suffer rapid nucleophilic addition by other substrate
molecule in the para-position to an imine group
forming the (B) intermediate. The substrate’s amino
group responsible for the nucleophilic addition to the
intermediate is dependent on the electronic nature of
the other substituent group on the ortho-diamine ring
(EDG or EWG). Therefore, for substrates with EDG
(4-M-1,2-BDA, 4-MB-1,2-DA and 4-BB-1,2-DA),
the nucleophilic attack is performed by the para
amino group, while for substrates with EWG (3,4-
DABA and 3,4-DABN) the nucleophilic attack is
attributed to the meta amino group. After a second
oxidation step, also expected to be mediated by the
laccase, the process continues by an intramolecular
cyclization with the concomitant displacement of the
R group (OCH3, COOH and Br), followed by
hydrogen shift giving rise to the asymmetric
phenazines. The loss of R group in one of the two
substrate molecules is consistent with similar
observations on the formation of the phenoxazine
cores. [25b,30a,33]
The tricyclic phenazine nuclear core of compound
(10) is also obtained by the intramolecular Michael
addition, but no further demethylation occurs and the
presence of the methyl group at C12 blocks the final
dehydrogenation, as already suggested by Tomoda et
al.[31a]
For the diamine substituted with a cyano group (3,4-
DABN), the final phenazine 8 is obtained from the
intermediate (B’) following a different pattern. The
structural characterization of the product 8, showed a Experimental Section
trimeric structure with the presence of three cyano
groups on the molecule, which is not compatible with
the loss of the R group as mentioned above. In this
case, a third precursor molecule is inserted by
nucleophilic attack before intramolecular cyclization.
Several positions on the diimine (B’) are susceptible
to nucleophilic attack and the selected site depends
on the nucleophile and the structure of the diimine.
For electronic and steric reasons, the nucleophilic
attack occurs on the C1 position rather than C4,
followed by an intramolecular cyclization involving
the imino group and the carbon bearing the amino
group. The displacement of the NH2 group and
rearomatization yields the final product. The
formation of N-substituted phenazine 11 from the
precursor 2 follow the pathway presented for the
formation of phenazines (5-7).
All reagents were from analytical grade, acquired from
commercial suppliers and used without further purification.
1D (1H and 13C) and 2D (COSY, HSQC and HMBC) NMR
spectra were recorded on a Bruker Avance (400 MHz)
spectrometer in CD3OD-d4, (CD3)2CO-d6 and CD2Cl2-d2 as
solvents. Chemical shifts are reported in ppm relative to
the solvent peaks and coupling constants (J) are reported in
hertz. Resonance and structural assignments were based on
the analysis of coupling patterns, including the 13C−1H
coupling profiles obtained in bidimensional heteronuclear
multiple bond correlation (HMBC) and heteronuclear
single quantum coherence (HSQC) experiments,
performed with standard pulse programs. Low resolution
mass spectra were recorded on a LCQ Fleet, Thermo
Scientific Ion Trap mass spectrometer, operated in the
electrospray ionization (ESI) positive/negative ion modes.
The optimized parameters were as following: ion spray
voltage, ±4.5 kV; capillary voltage, +16 and -20 V; tube
lens offset, -63 and +82 V; sheath gas (N2), 80 arbitrary
units; auxiliary gas, 5 arbitrary units; capillary temperature,
The
dihydrophenozaxinone
12
and
the
pyridyloxazinone 13 can also be obtained by a similar
pathway to the one reported for the formation of
products 5-7 and 10 respectively and in agreement
with previously reported results.[25b]
5
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10.1002/adsc.201701228
Advanced Synthesis & Catalysis
Scheme 2 – Proposed pathways to asymmetric phenazines through the homocoupling oxidative process of ortho
substituted diamines mediated by CotA-laccase.
6
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10.1002/adsc.201701228
Advanced Synthesis & Catalysis
250 ᵒC. The spectra were recorded in the range 100 – 1000
Da. Spectrum typically corresponds to the average of 20–
35 scans. Sequential mass spectra was obtained with an
isolation window of 2 Da, a 25-30% relative collision
energy and with an activation energy of 30 msec. High
Resolution ESI(+/-) mass spectra were obtained on a
QTOF Impact IITM mass spectrometer (Bruker Daltonics,
GMBH; Germany), operating in the high resolution ion
mode. Calibration of the TOF analyser was performed with
a 10 mM sodium formate calibrant solution. Data was
processed using Data Analysis 4.2 software (Bruker
Daltonics, Bremen, Germany).
The redox potentials were measured by cyclic voltammetry
using an EG&G Princeton Applied Research (PAR) Model
273A potentiostat/galvanostat monitored with the
Electrochemistry PowerSuite v2.51 software from PAR.
Cyclic voltammograms were obtained using 1 mM of
substrates in phosphate buffer (pH 6, 20 mM):ethanol (9:1)
using a three-electrode configuration cell with an home-
made platinum disk working electrode (1.0 mm diameter),
a platinum wire counter electrode and an Ag/AgCl
reference electrode (purchased from 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
done at room temperature and the solutions were deaerated
with dinitrogen. The measured potentials were corrected
by +0.197 V to the normal hydrogen electrode (NHE).
Oxidation of 4-methoxybenzene-1,2-diamine (4-MB-
1,2-DA):
After 2h of reaction, the brown solution was extracted with
acetyl acetate (3 x 10 mL) and after solvent evaporation, a
pure brown solid (5) was obtained.
1
5: yield: 5.8 mg (0.006 mmol, 96 %); H NMR (CD3OD-
d4) δ(ppm) = 7.84 (d, 1H, J = 9.2 Hz, H9), 7.29 (dd, 1H, J
= 2.4 Hz and J = 9.2 Hz, H8), 7.22 (dd, 1H, J = 2.4 Hz,
H6), 7.02 (s, 1H, H4), 6.97 (s, 1H, H1), 3.97 (s, 3H,
CH3).13C{H} NMR (CD3OD-d4) δ(ppm) = 161.4 (C7),
147.7 (C2, C11), 144.9 (C3, C12), 141.8 (C13), 136.9
(C14), 129.3 (C9), 122.6 (C8), 104.1 (C4), 103.8 (C6),
101.2 (C1) and 56.3 (CH3). ESI-MS positive mode: m/z
• •+
241 [M+H]+; MS2 m/z 226 [M+H-CH3 ] : MS3 m/z 198
•
[M+H-CH3 -CO]•+.
ESI-HRMS:
m/z
calcd.
for
[C13H12N4O+H]+: 241.1084; found 241.1077.
Oxidation of 4-bromobenzene-1,2-diamine (4-BB-1,2-
DA):
After 2h of reaction, the green olive solution was
evaporated under reduced pressure and the crude extracted
with ethanol to yield a pure olive green solid (6).
6: yield: 5.2 mg (0.018 mmol, 73 %); 1H NMR (Acetone-
d6) δ(ppm) = 7.36 (s, 1H, H1), 6.97 (d, 1H, J = 2.1Hz, H6),
6.73 (dd, 1H, J = 8.4 Hz and 2.1 Hz, H8), 6.57 (d, 1H, J =
8.4 Hz, H9), 6.03 (s, 1H, H4), 5.89 (bs, 2H, NH2) and 4.71
(bs, 2H, NH2). ESI-MS positive mode: m/z 289/291
[M+H]+. ESI-HRMS: m/z calcd. for [C12H9BrN4+H]+:
289.0083; found 289.0075.
General procedure for CotA laccase mediated reactions
Recombinant CotA-laccase from Bacillus subtilis was
produced and purified as previously described.[34-36]
Oxidation of 3,4-diaminobenzoic acid (3,4-DABA):
After 2h of reaction, the brown solution was evaporated
under reduced pressure and crude residue extracted with
ethanol to afford a pure brown solid (7).
Reactions were performed in 10 mL of phosphate buffer
(pH 6, 20 mM) in the presence or absence of 10% ethanol
with 5 mM of the precursor molecules (1a-1e, 2-4) (4-M-
1,2-BDA, 4-MB-1,2-DA, 4-BB-1,2-DA, 3,4-DABA, 3,4-
DABN, N-P-1,2-DA, 2-AMP and 2-APy), at room
temperature with stirring and started by adding 1U.mL-1 of
purified CotA-laccase. 1U is defined as the amount of
enzyme that transformed 1μmol of ABTS per min 37°C.
The oxidation of ABTS was followed by an absorbance
increase at 420 nm (ε= 3.6 x104 M-1.cm-1).[34] The protein
concentration was measured using the Bradford assay.[37]
Reactions were monitored by thin layer chromatography
(TLC) on aluminum-backed Merck silica gel 60 F254. The
insoluble products (8 and 9) were separated by filtration
and dried under vacuum. For the soluble products (6, 7 and
11) the solvent was evaporated under reduced pressure and
the products were isolated by solid-phase extraction with
ethanol and solvent evaporation. For the other soluble
products (5, 10, 12 and 13), the aqueous solution was
extracted with ethyl acetate and the combined organic
phases were collected, dried over anhydrous Na2SO4,
filtered and concentrated under vacuum. The product
mixtures were purified by silica preparative or column
chromatography. Control reactions in the absence of
enzyme were also performed to check for the auto-
oxidation of substrates.
1
7: yield: 5.9 mg (0.023 mmol, 93 %); H NMR (CD3OD-
d4) δ(ppm) = 8.64 (s, 1H, H9), 8.15 (d, 1H, J = 9.2 Hz, H7),
7.95 (d, 1H, J = 9.2 Hz, H6), 7.03 (s, 1H, H4), 7.01 (s, 1H,
1
H1). H NMR (D2Oa) δ(ppm) = 7.46 (dd, 1H, J = 2.0 Hz,
H9), 7.44 (dd, 1H, J = 2.0 Hz and J = 8.4 Hz, H7), 7.44 (d,
1H, J = 8.4 Hz, H6), 6.89 (s, 1H, H4), 6.87 (s, 1H, H1).
13C{H} NMR (D2Oª) δ(ppm) = 173.7 (COOH), 139.3 (C11,
C12), 129.6 (C13, C14), 125.4 (C7), 124.3 (C6), 119.9
(C9), 116.3 (C1, C4); ESI-MS positive mode: m/z 255
[M+H]+; MS2 m/z 211 [M+H-CO2]+. ESI-HRMS: m/z
calcd. for [C13H10N4O2+H]+: 255.0877; found 255.0873.
a referenced to (CD3)2CO-d6 solvent.
Oxidation of 3,4-diaminobenzonitrile (3,4-DABN):
After 3h of reaction, a dark orange solid was isolated by
filtration and purified by preparative chromatography
(ethyl acetate:hexane 2:1 v/v) affording two isolated
fractions. The first eluted yellow fraction and the second
eluted orange fraction were concentrated under vacuum to
afford compounds 9 and 8 respectively.
8: dark orange solid ; yield: 3.0 mg (0.008 mmol, 48 %);
1H NMR (Acetone-d6) δ(ppm) = 8.57 (s, 1H, H4), 8.26 (d,
1H, J = 8.8 Hz, H2), 7.92 (d, 1H, J = 8.8 Hz, H1), 7.62 (s,
1H, H7), 7.52 (d, 1H, J = 9.2 Hz, H18), 7.07 (d, 1H, J =
9.2 Hz, H17), 6.91 (bs, 2H, NH2), 6.28 (s, 1H, H20), 5.96
(bs, 2H, NH2). 13C{H} NMR (Acetone-d6) δ(ppm) = 158.3
(C12), 157.8 (C11), 151.1, 150.4, 143,9 (C16), 140.5 (C6),
136.2, 135.2 (C4), 134.3 (C18), 133.5 (C7), 132.2 (C2),
7
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10.1002/adsc.201701228
Advanced Synthesis & Catalysis
128.6, 127.2 (C1), 123.7, 120.0, 118.8, 117.4, 116.6, 115.2
(C17), 98.9 (C8), 95.8 (C20). ESI-MS negative mode: m/z
375 [M-H]-; MS2 m/z 348 [M-H-HCN]-. ESI-HRMS: m/z
calcd. for [C21H12N8-H]-: 375.1112; found 375.1123.
[M+H-H2O]+, 216 [M+H-CO]+. ESI-HRMS: m/z calcd. for
[C14H13NO3+H]+: 244.0968; found 244.0961.
Oxidation of 2-aminopyridin-3-ol (2-APy):
After 2h, the reaction was completed and the yellow
solution was extracted with ethyl acetate (3 x 10 mL) and
after solvent evaporation, afforded the pure yellow solid
(13).
9: dark yellow solid ; yield: 3.2 mg (0.012 mmol, 49 %);
1H NMR (Acetone-d6) δ(ppm) = 8.17 (s, 2H, H3, H3’),
7.45 (d, 2H, J = 8.8 Hz, H5, H5’), 7.07 (bs, 4H, NH2), 7.02
(d, 2H, J = 8.8 Hz, H6, H6’). 13C{H} NMR (Acetone-d6)
δ(ppm) = 150.2 (C2, C2’), 136.7 (C1, C1’), 135.2 (C5,
C5’), 127.0 (C3, C3’), 120.2 (C2, C2’), 118.4 (C7, C7’),
98.9 (C4, C4’). ESI-MS negative mode: m/z 261 [M-H]-;
MS2 m/z 233 [M-H-N2]-. ESI-HRMS: m/z calcd. for
[C14H10N6-H]-: 261.0894; found 261.0898.
1
13: yield: 5.2 mg (0.024 mmol, 97 %); H NMR (MeOD-
d4) δ(ppm) = 8.54 (d, 1H, J = 8.4 Hz, H7), 7.93 (d, 1H, J =
8.4 Hz, H9), 7.51-7.47 (m, 1H, H8), 6.55 (s, 1H,
H1).13C{H} NMR (MeOD-d4) δ(ppm) = 147.6 (C7), 121.7
(C9), 125.6 (C8), 106.6 (C1). ESI-MS positive mode: m/z
215 [M+H]+; ESI-HRMS: m/z calcd. for [C10H6N4O2+H]+:
215.0564; found 215.0548.
Oxidation of 4-methyl-1,2-benzenediamine (4-M-1,2-
BDA):
After 1h30m of reaction, the red solution was extracted
with ethyl acetate (3 x 10 mL) and after solvent
evaporation, a dark red solid (10) was obtained.
Acknowledgements
We gratefully acknowledge financial support from FCT
-
1
10: Dark red solid; yied: 5.4 mg (0.022 mmol, 91 %); H
Fundação para a Ciência e Tecnologia (Projecto Estratégico
UID/QUI/00100/2013, PTDC/BBB-EBB/0122/2014, RECI/QEQ-
QIN/0189/2012 and Projeto Infraestruturas 022125) and
Research Unit GREEN-it "Bioresources for Sustainability" at
ITQB (UID/Multi/04551/2013) and the IST-UL NMR Network for
facilities.
NMR (CD3OD-d4) δ(ppm) = 7.39 (d, 1H, J = 8.4 Hz, H9),
6.92 (d, 1H, J = 7.6 Hz, H8), 6.76 (s, 1H, H6), 6.66 (s, 1H,
H1), 3.27 (d, 1H, J = 14.4 Hz, H4a), 3.09 (d, 1H, J = 16.4
Hz, H4b), 2.25 (s, 3H, CH3) and 1.24 (s, 3H, CH3). ESI-
MS positive mode: m/z 242 [M+H]+; MS2 m/z 227 [M+H-
• •+
CH3 ] , 214 [M+H-CO]+. ESI-HRMS: m/z calcd. for
[C14H15N3O+H]+: 242.1288; found 242.1287.
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11: yield: 8.5 mg (0.003 mmol, 94 %); H NMR (Acetone-
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Advanced Synthesis & Catalysis
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10.1002/adsc.201701228
Advanced Synthesis & Catalysis
10
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