Laccase-mediated multi-step homo- and heteromolecular reactions of ortho-dihydroxylated aromatic compounds and mono- or diaminated substances resulting in C-C, C-O and C-N bonds

Article abstract of DOI:10.1016/j.molcatb.2015.08.011

Three types of reactions involving oxidation, inter- and intramolecular Michael-addition as well as homo-intermolecular dimerization, catalyzed by laccase [E.C. 1.10.3.2] of Pycnoporus cinnabarinus in the presence of oxygen, resulted in the formation of dimeric, trimeric and cyclic products with yields up to 44% (non-optimized reactions). For heteromolecular reactions a number of different aminated five- and six-membered aromatic compounds were used. The first reaction type involved the oxidation of catechol, 3- or 4-methylcatechol to o-quinone and subsequent Michael-addition of the amino compounds with C-N bond formation. Conclusive analytical data (UV-vis data, MS spectra) for the respective o-quinone are provided. The second type of reaction included a homo-intermolecular dimerization probably with C-C and C-O bond formation to the proposed dibenzofuran derivatives. The heteromolecular reaction with the amino compounds yielded trimers consisting of homomolecular dimers and the amino compound connected via a C-N bond. The third reaction type started from epinephrine (adrenaline) which was oxidized by laccase and underwent an intramolecular Michael-addition to adrenochrome. The subsequent heteromolecular reaction resulted in the unusual substance class of cyclooctenes (diazocines). Differences between the reaction types in regard to the kind of o-hydroquinone, amino compound used and the types of product recovered are discussed.

Full text of DOI:10.1016/j.molcatb.2015.08.011

Accepted Manuscript
Title: Laccase-mediated multi-step homo- and
heteromolecular reactions of ortho-dihydroxylated aromatic
compounds and mono- or diaminated substances resulting in
C-C, C-O and C-N bonds
Author: Veronika Hahn Annett Mikolasch Cornelius Kuhlisch
Frieder Schauer
PII:
S1381-1177(15)30049-7
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2015.08.011
MOLCAB 3220
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date:
Revised date:
Accepted date:
28-5-2015
7-8-2015
15-8-2015
Please cite this article as: Veronika Hahn, Annett Mikolasch, Cornelius Kuhlisch,
Frieder Schauer, Laccase-mediated multi-step homo- and heteromolecular reactions
of ortho-dihydroxylated aromatic compounds and mono- or diaminated substances
resulting in C-C, C-O and C-N bonds, Journal of Molecular Catalysis B: Enzymatic
http://dx.doi.org/10.1016/j.molcatb.2015.08.011
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Laccase-mediated multi-step homo- and
heteromolecular reactions of ortho-dihydroxylated
aromatic compounds and mono- or diaminated
substances resulting in C-C, C-O and C-N bonds
Veronika Hahna* veronikahahn@gmx.at, Annett Mikolascha, Cornelius Kuhlischb, Frieder
Schauera
aInstitute of Microbiology, Ernst-Moritz-Arndt-University of Greifswald, Friedrich-Ludwig-
Jahn Str. 15, 17487 Greifswald, Germany
bMax Planck Institute for Chemical Ecology, Hans-Knoell-Str. 8, 07745, Jena, Germany
*Corresponding author: Tel: +49 3834 864229; fax: +49 3834 864202.
1

Graphical Abstract
NH2
N
NH2
+
N
H
O
O
HO
N
OH
H
laccase
(Pycnoporus
cinnabarinus)
OH
H
4-aminoimidazole-5-
carboxamide (3c)
N
N
O
O
O
N
air, pH5, rt
N
H
N
OH
OH
N
H
O
8a
yield = 15 %
epinephrine (1d)
7

Highlights
- The laccase-mediated multi-step reactions resulted in homo- or heteromolecular cyclization
products as well as monoaminated quinonoid compounds.
- The mild and environmentally friendly reaction conditions are advantagous properties for
different applications with laccase as green catalyst.
3

Abstract
Three types of reactions involving oxidation, inter- and intramolecular Michael-addition as
well as homo-intermolecular dimerization, catalyzed by laccase [E.C.1.10.3.2] of Pycnoporus
cinnabarinus in the presence of oxygen, resulted in the formation of dimeric, trimeric and
cyclic products with yields up to 44% (non-optimized reactions). For heteromolecular
reactions a number of different aminated five- and six-membered aromatic compounds were
used. The first reaction type involved the oxidation of catechol, 3- or 4-methylcatechol to o-
quinone and subsequent Michael-addition of the amino compounds with C-N bond formation.
Conclusive analytical data (UV–vis data, MS spectra) for the respective o-quinone are
provided. The second type of reaction included a homo-intermolecular dimerization probably
with C-C and C-O bond formation to the proposed dibenzofuran derivatives. The
heteromolecular reaction with the amino compounds yielded trimers consisting of
homomolecular dimers and the amino compound connected via a C-N bond. The third
reaction type started from epinephrine (adrenaline) which was oxidized by laccase and
underwent an intramolecular Michael-addition to adrenochrome. The subsequent
heteromolecular reaction resulted in the unusual substance class of cyclooctenes (diazocines).
Differences between the reaction types in regard to the kind of o-hydroquinone, amino
compound used and the types of product recovered are discussed.
Keywords: oxidoreductase; quinone; benzene-1,2-diol; cascade / domino reaction;
aminobenzoic acid
4

1. Introduction
The formation of novel heterocyclic compounds is becoming more and more important not
only as a source of building blocks for synthetic organic chemistry but also for the
development of new drugs. In this respect laccases [E.C. 1.10.3.2, benzenediol:dioxygen
oxidoreductase] possess numerous advantages. Laccases use oxygen as co-factor, water is the
only by-product and mild environmentally friendly reaction conditions (atmospheric pressure,
pH usually at 5-7, room temperature) enable a “green” synthesis of various compounds.
Laccases contain four copper atoms responsible for the catalysis of the oxidation reaction
resulting in radicals which may undergo non-enzymatic reactions.
So far, laccase-mediated reactions have been used for the synthesis of dimers and trimers as
well as of higher polymers. The dimeric products can be homomolecular consisting of two
identical molecules connected via a C-C bond (such as salicylic esters) [1] or a C-O bond as
described for 2-hydroxydibenzofuran [2]. For heteromolecular products - formed between two
or more different reaction partners C-C [3,4], C-O [5] and also C-N [6,7] or C-S [8,9] bonds
are possible. Some of the products may be biologically active e.g. antibacterial [10,11],
antifungal [12], antioxidative [13,14] or anticancer [9].
In recent years the synthesis of heterocyclic compounds by laccase-mediated domino
reactions has been described and this is a very promising approach since it permits a “one-
pot” reaction without the need for protecting groups. Different types of heterocycles may be
formed during laccase-catalyzed reactions. In this way five-membered rings such as
benzofurans [15,16], dibenzofurans [17-21], thiazoles [22] as well as six-membered rings
such as phenoxazinone chromophores [23-25] have been synthesized.
Recently, we used laccase to produce additional heterocycles including cycloheptenes
(diazepines), cyclooctenes (diazocines), diazaspiro cyclohexenes, and phenazines using para-
dihydroxybenzoic acid derivatives and aromatic or heteroaromatic amines [26].
The aim of this study was to describe the product formation - in particular of the cyclic
products - generated using the ortho-dihydroxylated substances catechol, 3-methylcatechol, 4-
methylcatechol, and epinephrine (adrenaline). For heteromolecular reactions a number of
aminated azoles and benzoic acid derivatives were used. The formation of diazocines was of
particular interest since the chemical synthesis of these substances involves harsh reaction
conditions as which involve heating at 110°C with ethyl acetate [27] as well as the use of
acids or metals such as palladium [28].
5

In contrast, the laccase based reaction is suitable for a more environmentally friendly
synthesis of these compounds. To the best of our knowledge this is the first study concerning
the laccase-mediated synthesis of such epinephrine derivatives.
2. Experimental
2.1 Chemicals
Catechol and 2-aminobenzoic acid were purchased from Merck (Hohenbrunn, Germany). 3-
Methylcatechol, 4-methylcatechol, epinephrine, 2-aminobenzamide and 4-aminoimidazole-5-
carboxamide hydrochloride were from Sigma-Aldrich Chemie GmbH (Steinheim, Germany).
3-Aminopyrazole-4-carboxamide hemisulfate was purchased from Tyger Scientific Inc.
(Ewing Township, New Jersey, USA).
2.2 Enzymes
The laccase used was obtained from Pycnoporus cinnabarinus SBUG-M 1044. This white rot
fungus was isolated from an oak tree in northern Germany, and is deposited at the strain
collection of the Department of Biology of the University of Greifswald (SBUG).
Cultivation of Pycnoporus cinnabarinus SBUG-M 1044 and the preparations of the crude
laccase were carried out as reported previously [2]. This enzyme preparation contains
isoenzymes of laccase, but no other enzymes and was always used in 20 mM sodium acetate
buffer (SAB) pH 5.0, at its pH optimum [2,29].
2.3 Measurement of laccase activity
The activity of laccase was determined spectrophotometrically at 420 nm with ABTS
(2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) as substrate [30]
using the method described by Jonas et al. [2]. 1 U is defined as the turnover of 1 µmolml-
1min-1.
6

2.4 Experimental procedures
For analytical experiments the dihydroxylated compounds (1 mM, 2 mM) and amines (1 mM,
2 mM) were incubated with 0.5 U of laccase.
For reactions with epinephrine 16 mM of this compound and 16 mM tartaric acid
(Lachema/Chemapol, Brno/Prague, Czech Republic) were gently mixed in SAB and used
after one day for biotransformation with an end concentration of 1 mM epinephrine.
In controls the respective compounds were incubated in SAB without laccase. Reaction
mixtures were incubated with agitation at 200 rpm at 23 °C in the dark.
2.5 Analytical HPLC (high-performance liquid chromatography)
For routine analysis, the reaction mixtures and isolated products were analyzed using an
HPLC system LC-10AT VP (Shimadzu, Germany) consisting of a FCV-10AL VP pump,
SPD-M10A VP diode array detector, and a SCL-10A VP control unit controlled by Class-VP
version 6.12 SP5. Substances were separated on an endcapped, 5-µm, LiChroCART® 125-4
RP18 column (Merck, Darmstadt, Germany) run at a flow rate of 1 ml/min. The solvent
system used consisted of methanol (eluent A) and 0.1% phosphoric acid (eluent B), starting
from an initial ratio of 10% A and 90% B and reaching 100% methanol within 14 min.
2.6 Structural characterization of products by LC/MS (liquid
chromatography/mass spectrometry) and NMR (nuclear magnetic resonance)
spectrometry
The reaction mixtures and isolated products were characterized using a LC/MS system. The
atmospheric pressure ionization (API) mass spectrometry experiments were performed on an
Agilent Series 1200 HPLC system with diode array detector and an Agilent 6120 quadrupole
mass spectrometer (Waldbronn, Germany). The MS was run with the electrospray ionization
(API-ES) source in positive mode (dry and nebulizer gas: nitrogen; nebulizer pressure: 45
psig; drying gas flow: 10 l/min; drying gas temperature: 350°C; capillary voltage: 4 kV;
fragmentor voltage: 75 V). HPLC separation was performed on a Zorbrax SB-C18
(2.1x50mm, 1.8µm) column (Agilent, Waldbronn, Germany), at a flow rate of 0.08 ml/min
(excepting reactions with epinephrine: 0.12 ml/min). The solvent system consisted of
acetonitril (eluent A) and 0.1% aqueous ammonium formate (eluent B), starting from an
7

initial ratio of 10% A and 90% B and reaching 100% methanol within 7 min. Elution with
methanol was continued for a further 6 minutes.
NMR spectra were obtained at 600 MHz (1H, HSQC, HMBC) and at 150 MHz (13C) in
deuterated methanol or dimethyl sulfoxide on a Bruker Avance 600 instrument (Rheinstetten,
Germany).
2.6.1 2-(3,4-Dihydroxyphenylamino)-benzamide (4a)
Synthesis and isolation as described above. Red solid. Yield 43.5 % (10.7 mg). 1H NMR: δ
(600 MHz, DMSO-d6) 6.42 (m, J = 2.2, J = 8.4, 1H, H-6’), 6.56 (d(s), J = 2.2, 1H, H-2’), 6.62
(m, J = 7.9, 1H, H-5), 6.68 (d, J = 8.4, 1H, H-5’), 6.96 (d, J = 8.5, 1H, H-3), 7.20 (m, J = 8.5,
1H, H-4), 7.64 (d, J = 7.9, 1H, H-6), 9.69 (s, 1H, NH). 13C NMR: δ (150 MHz, DMSO-d6)
110.7 (C-2’), 113.3 (C-3), 113.6 (C-6’), 115.8 (C-5), 116.1 (C-5’), 129.1 (C-6), 132.1 (C-4).
HMBC correlations: (DMSO-d6) NH (C-1, (C-1’)*, (C-2), (C-2’), C-3, C-6’), H-2’ ((C-1’),
(C-3’), C-4’, C-6’), H-3 (C-1, C-5, (C-7)), H-4 (C-2, C-6), H-5 (C-1, ((C-2))**, C-3, ((C-4)),
((C-6))), H-5’ (C-1’, C-3’, (C-4’)), H-6 (C-2, C-4, C-7), H-6’ (C-2’, C-4’). Rf (HPLC) 7.08
min, Rf (LC/MS) 8.773 min, UV-vis λmax 209, 290, 492 nm. MS m/z (rel. intensity) AP-ESI:
pos. ion mode [M+H]+ 243.1 (100), [2M+Na]+ 507.1 (10).
* () signals with low intensity
** (()) signals with very low intensity
2.6.2 Product 5b
Rf (HPLC) 13.26 min, Rf (LC/MS) 18.942 min, UV-vis λmax 205, 398 nm. MS m/z (rel.
intensity) AP-ESI: pos. ion mode [M+H]+ 243.0 (100), [2M+Na]+ 507.1 (5).
2.6.3 Product 6h
Synthesis and isolation as described above. Brown green solid. Yield 9.4 % (16.1 mg). 1H
NMR: δ (600 MHz, DMSO-d6) 1.63 (s), 1.66 (s), 1.67 (s), 1.74 (s), 1.80 (s), 1.89 (s), 1.97 (s),
2.08 (s), 2.11 (s), 6.64 (s), 6.65 (s), 7.23 (s), 7.74 (s), 7.84 (s), 7.94 (s), 8.00 (s), 8.81 (s). 13C
NMR: δ (150 MHz, DMSO-d6) 9.4, 15.8, 15.9, 16.5, 21.5, 102.7, 127.6, 128.4, 130.5, 135.7,
135.8, 137.6. HMBC correlations: (DMSO-d6) 1.63 (109.8, 132.7, 143.5), 1.66 (108.5, 132.7,
143.9), 1.67 (108.5, 132.7, 143.9), 1.74 (128.4, 135.1, 184.2), 1.80 (127.6, 135.1, 184.2), 1.89
(172.1), 1.97 (138.2, 183.1), 2.08 (121.6, 124.8, 143.9), 2.11 (129.9, 137.2, 183.9), 6.64
((15.9)*, (113.1), (145.2), 184.2), 6.65 ((15.9), (113.1), (145.2), 184.2), 7.23 (112.8, 132.5,
143.4), 7.74 (123.4, 147.5), 7.84, 7.94, 8.00 ((144.3)), 8.81. Rf (HPLC) 6.60 min, Rf (LC/MS)
8

14.437 min, UV-vis λmax 232, 267, 293, 463 nm. MS m/z (rel. intensity) AP-ESI: pos. ion
mode [M+H]+ 367.0 (100).
* () signals with low intensity
2.6.4 Epinephrine derivative (8a)
Synthesis and isolation as described above. Yellow solid. Yield 14.5 % (13.0 mg). 1H NMR: δ
(600 MHz, DMSO-d6) 3.06 (s, 3H, H-5), 4.15 (m, J = 18.8, 2H, H-4), 5.04 (s, 1H, H-1), 7.20
(s, 1H, NH), 7.34 (s, 1H, NH, H-16), 7.82 (s, 1H, H-12), 11.09 (s (broad), 1H, OH), 13.31 (s,
1H, NH, H-13). 13C NMR: δ (150 MHz, DMSO-d6) 32.2 (C-5), 60.0 (C-4), 81.3 (C-1), 137.5
(C-12). HMBC correlations: (DMSO-d6) H-1 (C-2, C-3, C-7, C-8, C-9), H-4 (C-2, C-3, C-5,
C-6, C-7), H-5 (C-4, C-6), H-12 (C-11, C-14, C-15), H-13 (C-11), H-16 (C-7, C-8, C-9, C-14,
C-15). Rf (HPLC) 5.18 min, Rf (LC/MS) 1.156 min, UV-vis λmax 204, 272, 296, 354, 414 nm.
MS m/z (rel. intensity) AP-ESI: pos. ion mode [M+H]+ 302.0 (100).
Experimental methods; UV–vis data and MS spectra for all compounds (Tab. S1-34); 1H-
NMR, 13C-NMR, HSQC, HMBC spectra of product 4a-c, 8a,b (Tab. S5-7, S33, 34); 1H-
NMR, HSQC, HMBC spectra of product 6h (Tab. S27) is available in the electronic
supplementary file.
3. Results and discussion
The laccase is able to oxidize substrates to form reactive radicals which can undergo reactions
with each other or with additional reaction partners like amino compounds. In these reactions
multiple bond formations are possible resulting e.g. in cyclic products. We demonstrate the
product formation during homomolecular reaction of o-dihydroxylated aromatic substances as
well as the heteromolecular reaction of these products with amino compounds.
3.1 First reaction type: In the laccase-catalyzed homomolecular reactions catechol (1a), 3-
methylcatechol (1b) or 4-methylcatechol (1c) were completely transformed within 20 minutes
and the corresponding o-quinones (2a-c) were formed (Fig. 1). The HPLC analysis showed
that the retention time of 2a (Rf 3.30 min; Tab. 1) was lower than that of the catechol 1a (Rf
5.50 min). Similarly, Albarran et al. [31] described the appearance of the ortho-quinone
9

before the catechol in HPLC analysis of the reaction products of a chemically-catalyzed
reaction with hexachloroiridate(IV). In addition, the UV-vis spectrum of the ortho-
benzoquinone showed an absorption maximum at 389 nm [31] which corresponds exactly to
2a. The ortho-benzoquinone has a large dipole moment and hence high polarity which may
explain the low retention time [31]. Albarran et al. [31] described for the LC/MS (API-ES)
analyses the formation of cation(s) and anion(s) with m/z 109 for the ortho-benzoquinone.
The mass m/z 109 for an anion was explained by the formation of a hydride adduct. In
contrast, our LC/MS (API-ES positive mode) analysis of 2a resulted in the detection of
fragment ions at m/z (rel. intensity) 126.1 [M+NH4]+ (100) and 131.0 [M+Na]+ (25) (Suppl.
Mat. Tab. S2). The m/z 109 [M+H]+ for a cation was detected with a low relative intensity of
6. In addition, API-ES negative mode studies resulted in m/z 109 of an anion but the
ionization was considerably less than in positive mode (data not shown). The MS analyses of
the oxidation products of 1b and 1c showed again ammonium adducts as well as the
molecular ion peak at m/z 123.0 [M+H]+ confirming the o-quinone formation (Suppl. Mat.
Tab. S3,4). The oxidation of o-hydroquinones to o-benzoquinones via laccase has been well
known [32-34] but detailed analyses about the benzoquinones formed are missing to date.
Despite the lack of an authentic sample and limited analytical data in the literature, the ortho-
benzoquinone (2a) as well as its methylated derivatives (2b,c) were clearly identified by MS
analyses.
The o-benzoquinone (2a-c) formation was a prerequisite for the heteromolecular reaction with
2-aminobenzamide (3a) resulting in heteromolecular dimers (6a-c) with yields of 40-44%.
The coupling of one molecule of the catechol and one molecule of the amino compound were
confirmed by the presence of all carbon atoms of the catechols 1a-c and of the amino partner
3a in the 13C NMR spectra of the products 4a-c. Two signals in the range of 140 - 150 ppm
indicated a hydroquinonoid character of the products (e.g. for product 4a 141.7 ppm (C-4’)
and 145.8 ppm (C-3’)). No signals were detected in the range of 180 ppm - the range of
carbonylic groups. Furthermore, all proton signals of each of the two reactants, as well as the
proton of the C-1’, were detectable in the 1H NMR spectra of the products 4a-c. The number
of CH proton signals of the dihydroxylated phenyl rings changed from two - in the reactant 1a
- to three signals in the product 4a and from three - in the reactants 1b-c - to two signals in the
products 4b and 4c. The multiplicity of the signals indicated a further substituent at the C-1’
position of 4a-c. The HMBC spectrum of 4a showed correlations between the aliphatic amine
proton (9.69 ppm) and the C-1’, C-2’ and C-6’ of the hydroquinonoid ring on the one hand
and on the other with the C-1, C-2 and C-3 of the amino partner. These facts show 4a to be
10

aminated at the C-1’ position of the hydroquinone ring. The HMBC included also correlations
between the H-2’, H-5’ and H-6’ protons of the hydroquinone ring and the carbons C-3’ and
C-4’ which are substituted by hydroxyl groups, confirming the hydroquinonoid character.
Nevertheless, the LC/MS (API-ES positive mode) analyses of 4a-c resulted in molecular ion
peaks (e.g. for 4a: m/z (rel. intensity) 243.0 [M+H]+ (100)) for monoaminated ortho-
quinonoid compounds. In addition, the UV-vis spectra (4a: 204, 273, 497 nm; 4b: 208, 300,
505 nm; 4c: 202, 293, 495 nm) resemble those of aminated para-quinonoid products with two
absorption maxima between 200-300 nm and a weak maximum around 500 nm [6].
Depending on the measurement used we can postulate hydroquinonoid or quinonoid
structures, but with all methods we could demonstrate the coupling of one molecule of
catechol and one molecule of the amino compound (Fig. 1).
Further reactions of 1a-c with 2-aminobenzoic acid (3b), 4-aminoimidazole-5-carboxamide
(3c) and 3-aminopyrazole-4-carboxamide (3d) resulted also in the formation of
heteromolecular dimers (4d-l) which were structurally characterized by LC/MS (Suppl. Mat.
Tab. S8-16). The products consist of one molecule of the oxidized catechol and one molecule
of the benzoic acid or aminated azole, respectively. It was not possible to isolate 4d-l due to
complex reaction kinetics and instability of some of these products.
The formation of heteromolecular dimers for the reaction of ortho-dihydroxylated aromatic
acids with amino-β-lactam antibiotics was reported by Mikolasch et al. [33]. The products
presented by Mikolasch et al. [33] had a quinonoid structure in contrast to our identified
products 4a-c with hydroquinonoid structure.
For 1b the regioselective formation of catechol thioethers was described by Abdel-Mohsen et
al. [35] with a nucleophilic attack at C4 or C5 (C4 favoured with approx. 66%). In contrast,
for 1a and 1c only a Michael-addition at C5 was described by Abdel-Mohsen et al. [35].
These results are in accordance with our experiments for 1a and 1c and confirm C5 as the
favoured position for a nucleophilic attack at catechols.
We note that in some reactions also trimeric products consisting of one molecule of the
oxidized catechol and two molecules of the amino compound were detected in accordance
with other laccase-mediated reactions [36,37]. However in the experiments reported here only
traces of such trimers were formed because of the use of equimolar concentrations of
reactants and the strong competing homomolecular reaction.
3.2 Second reaction type: The reactions of the catechols (1a-c) described above are not
straightforward kinetics to the o-quinones. Instead these reactions resulted not only in the
11

formation of the respective o-quinones (2a-c) but also in the production of homomolecular
dimers (5a-c) which were formed only in small amounts (according to the peak area at 254
nm; Fig. 2).
The LC/MS (API-ES positive mode) analyses of 5a-c resulted in the detection of fragment
ions at m/z (rel. intensity) 215.0 [M+H]+ (100; Suppl. Mat. Tab. S17-19) implying an
connection of two molecules 1a-c, respectively. The products may consist of two quinones
connected via one C-C bond or may be formed by one C-C and one C-O bond. According to
Hajdok et al. [19,20] the first step of the reaction process is the laccase catalyzed oxidation of
one molecule of catechol (1a-c) with O2 to o-benzoquinone (2a-c). The o-benzoquinones can
undergo an intermolecular 1,4-addition with a second molecule of 1a-c acting as a
nucleophile to yield a dihydroxy intermediate (I) that cannot be isolated, but that can be
further transformed. On one hand two laccase catalyzed oxidation steps can result in 5a1-c1.
On the other hand side one laccase catalyzed oxidation step followed by a second 1,4-addition
and a second laccase catalyzed oxidation step can result in the intramolecular formation of
5a2-b2. Due to the methyl group of 1c the formation of a 5c2 structure is not possible.
However, a comparable product to 5a-b could be analyzed for 1c, which is why this third
probable pathway must be considered. One laccase catalyzed oxidation step followed by a
1,3-addition and a second laccase catalyzed oxidation step may result in the formation of 5a3-
c3. In contrast to the homomolecular reactions of 1a-c described herein, Hajdok et al.
described the formation of products comparable to 5a2-b2 but for heteromolecular reactions of
cyclohexane-1,3-diones [19] or 1,3-dicarbonyls [20] with catechols.
In the heteromolecular reaction of 1a-c with 3a-c heteromolecular trimers (6a-l) were
detected consisting of one homomolecular dimer (5a1-c1, 5a2-c2 or 5a3-c3) and one molecule
of the respective amino compound 3a-d (LC/MS data for 6a-l: Suppl. Mat. Tab. S20-31). The
products 6a-l were formed in small amounts and thus only one product (6h) could be isolated
at a yield of 9% (Fig. 3).
The LC/MS (API-ES positive mode) analysis for 6h resulted in a molecular ion peak at m/z
(rel. intensity) = 367.1 (100). The NMR analyses revealed the formation of at least three
structurally different products. The structure 4-[(4-hydroxy-3,8-dimethyl-6,7-
dioxodibenzofuran-2-yl)amino]-1H-imidazole-5-carboxamide is proposed for one of these
products (Tab. 2). The structure of 4-[(4-hydroxy-3,8-dimethyl-6,7-dioxodibenzofuran-2-
yl)amino]-1H-imidazole-5-carboxamide was confirmed by the presence of all carbon atoms of
two catechol molecules and of one molecule of the amino partner in the 13C NMR spectrum of
the product 6h. A signal in the range of 180 ppm indicated a quinonoid character of ring A.
12

Two signals in the range of 140 - 150 ppm suggested that a furanic structure is part of the
product (143.4 ppm (C-9a) and 145.2 ppm (C-9b)). The HMBC included correlations between
the H-14 and C-9a (143.4 ppm) on one hand and between H-13 and C-11 (184.2 ppm) on the
other, confirming the hydroquinonoid character of ring B and the quinonoid character of ring
A. The singulet character of the proton signal H-14 indicated a further substituent at the C-7
position. The chemical shift to lower field of the C-7 carbon (143.9 ppm) compared with the
C-8 carbon (132.7 ppm) demonstrated the presence of an electron-withdrawing group such as
the amino partner 3c.
According to the NMR analyses of 6h the homomolecular dimers 5a3-c3 could be postulated
to be dibenzofuran derivatives.
As described previously the dibenzofurans resulted from homo-intermolecular dimerizations
involving C-C and C-O bond formation. Descriptions of the laccase-mediated formation of C-
C and C-O bonds in a one-pot reaction with only one compound are very rare. Thus, Nicotra
et al. [38] described the formation of a dehydrodimer during laccase-catalyzed reaction of
resveratrol. The formation of a heteroaromatic five-membered furan ring within a
dibenzofuran system has been described for the laccase-mediated homo-intramolecular
reaction of 3,5-dichloro-2-hydroxybiphenyl by Kordon et al. [39].
In contrast to the homomolecular reactions described by Nicotra et al. [38], Kordon et al. [39]
and in our study, the formation of heteroaromatic five-membered rings resulting from
heteromolecular reactions of ortho-hydroquinones have been repeatedly studied [17-22].
In some reactions, such as with 1c, the homomolecular product (5c) was hydroxylated (data
not shown) probably as a result of an attack by water possibly after the formation of 2c or 5c.
Furthermore, this hydroxylated product can undergo a coupling reaction with 3c or 3d as
described for the heteromolecular reaction of 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide
and morpholine [12].
In general morpholines and azoles (the last are elements of products 4 and 6) are building
blocks for antimicrobial drugs [40-42]. In addition, dibenzofurans may also possess biological
activity as is the case with usnic acid derived from lichens that exerts antibacterial [43] and
anticancer [44] activity.
3.3 Third reaction type: The laccase-mediated reaction of epinephrine (1d) resulted in the
formation of one sole product (7). The HPLC analysis showed that 7 (Rf 1.7 min) had a
13

similar retention time than 1d (Rf 1.3 min). The UV-vis spectrum of 7 showed absorption
maxima at 219, 301, 487 nm which corresponds to those found in the literature for
adrenochrome (220, 305, 490 nm; [32]). The LC/MS (API-ES positive mode) analysis
resulted in fragment ions at m/z (rel. intensity) 180.0 [M+H]+ (100) and 381.1 [2M+Na]+ (19)
confirming an intramolecular cyclization to form adrenochrome.
In the heteromolecular reactions of 1d with 3c,d only one product (8a,b) was formed,
respectively. The products were detected within 20 min and their concentrations increased
over at least 24 hours. The LC/MS (API-ES positive mode) analyses for the products resulted
in molecular ion peaks at m/z (rel. intensity) 302.0 (100) for 8a and 302.1 (100) for 8b (Suppl.
Mat. Tab. S32-33). We measured various NMR sprectra – 1H NMR, 13C NMR, HSQC,
HMBC and 1H1H COSY – and from all these spectra we tentatively propose the structures of
8a and 8b as shown in Fig. 4.
The LC/MS (API-ES positive mode) analysis for the product 8c of the reaction from 1d with
3a resulted in molecular ion peaks at m/z (rel. intensity) 312.0 (100) (Suppl. Mat. Tab. S34)
confirming a similar structure as shown for 8a,b.
The first step for the formation of 8a-c is the postulated oxidation of 1d to o-quinone followed
by a nucleophilic attack of the side chain nitrogen atom to the C5-position of the ring
resulting in leukoadrenochrome which is oxidized to adrenochrome [45].
The oxidation of epinephrine (adrenaline) by enzymes such as laccase [46,47] or tyrosinase
[48] is used for the development of methods/sensors for the detection of this neurotransmitter
in blood and plasma.
The formation of 7 was the prerequisite for the heteromolecular reaction with the amino
partners 3a,c,d. Thus, the cyclization - and not the 1,4-addition forming a dimer as described
for products 4a-l - is essential for the formation of a stable product. We described previously
[26] the reaction of para-dihydroxylated benzoic acid derivatives with 3c,d resulting in
cyclization after 1,4-addition of the azole and subsequent intramolecular 1,2-addition forming
cycloheptenes (diazepines) or addition-elimination-reaction forming cyclooctenes
(diazocines).
We propose a similar scheme for the formation of 8a-c. At first, the 1,4-addition starts from
the amino group of the carboxamide of 5a-c (Fig. 4) and the resulting non-detectable
heteromolecular dimer undergoes a 1,3-addition to form the derivatives 8a-c. As expected, the
reaction of 1d with 3b leads to no heteromolecular product due to the lack of two amino
groups which are required for cyclooctene formation.
14

3.4 Additional remarks
Some products were detected in small quantities also in the respective controls without
addition of laccase. We described such autocatalytic reactions previously for the dimeric
products of para-dihydroxylated substances and amino acids [36]. The concentration of the
products (4b, 4c, 4e, 4f) was, however, very small. Thus, within 24 h only 3% of 4c was
formed autocatalytically in comparison to the amount formed in 20 minutes in the laccase-
catalyzed reaction. Consequently, the autocatalytic reaction is negligible.
3.5 Summary
In summary, for the reactions of 1a-c with 3a-d the formation of dimers (4a-l) takes place in
the order (from large to low quantities; Fig. 5): 1a-c with 3a (products 4a-c) = 1a-c with 3b
(products 4d-f) > 1a-c with 3c (products 4g-i) > 1a-c with 3d (products 4j-l). In contrast, for
6a-l the amino compound 3c (with products 6g-i) gave the highest yield. Thus, 3a and 3b
were the most effective nucleophiles resulting in fast and efficient Michael-addition forming
dimeric products (4a-f) whereas for the reaction involving the homomolecular products 5a-c
the azole 3c was most efficient. In the reactions of 1a-c with 3a or 3b (for products 6a-f) the
formation of homomolecular products, beyond the o-benzoquinone, is strongly suppressed
due to a fast reaction of the amino compound with the catechols enabling an almost
completely consumption of the two reaction partners to one heteromolecular product (4a-f). In
contrast, the reactions with the azoles 3c or 3d and 1a-c are not as fast as with 3a,b allowing
both reaction types simple amination via Michael-addition resulting in products 4g-l and a
reaction including homomolecular formation of potential dibenzofuran derivatives and only
then reaction with 3c,d forming 6g-l.
Beyond this, for the products 8a-c the reaction of 1d to 7 is the only possibility for the
reaction with 3a,c,d resulting in cyclooctene derivatives.
The yield of the products for the non-optimized reactions was between 9 and 44%. For
reactions of para-dihydroxylated aromatic substances and amino compounds yields of 20-
80% are achievable [12] and for optimized reactions a yield of 96% is possible [6].
15

4. Conclusions
The three identified reaction types between ortho-dihydroxylated aromatic substances and
amino compounds result in Michael-addition forming heteromolecular dimers or included
homomolecular dimerization with amination and ring closure mechanisms.
The catalytic action of the enzyme laccase allows multi-step or domino reactions and thereby
enlarges the application possibilities. Furthermore, the reactions demonstrated here enable the
use of laccase for the production of new substances not only for fine chemical synthesis but
also for novel antimicrobial or anticancer pharmaceuticals.
Acknowledgements
We thank M. Lalk (Institute of Pharmacy, University of Greifswald) for providing NMR data.
R. Jack is gratefully acknowledged for help in preparing the manuscript.
16

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19

Figure Captions
Fig. 1 Laccase-mediated reactions of catechol (1a), 3-methylcatechol (1b) or 4-
methylcatechol (1c) with 2-aminobenzamide (3a)
Fig. 2 Laccase-catalyzed reaction of catechols (1a-c) resulting in the tentatively proposed
products 5a-c
Fig. 3 Laccase-mediated reaction of 3-methylcatechol (1b) with 4-aminoimidazole-5-
carboxamide (3c)
Fig. 4 Laccase-mediated reaction of epinephrine (1d) with 4-aminoimidazole-5-carboxamide
(3c) or 3-aminopyrazole-4-carboxamide (3d)
Fig. 5 Concentration of products 4a-l (white columns) and 6a-l (black columns) formed in
reactions of 1a-c with 3a-d (2:2 mM)
20

Tables
Table 1 Structural data of homomolecular products (2a-c) formed during laccase-catalyzed
reaction of 1a-c
Reaction of 1a
2a
1b
2b
1c
2c
o-Benzo-
quinone
R1
R 2
O
O
2a R1=H, R2=H
2b R1=H,
R2=CH3
2c R1=CH3, R2=H
Rf (HPLC)
3.30 min
5.34 min
5.10 min
UV-vis λmax 205, 389 nm
203, 414 nm
208, 399 nm
2a
MS (AP-
[M+H]+ 109.1 (6),
(100), [M+Na]+
131.0 (25)
[M+H]+ 123.1 (27),
[M+NH4]+ 140.1
(100)
[M+H]+ 123.1 (100),
[M+NH4]+ 140.1
(68)
[M] 108.10 ESI pos. ion [M+NH4]+ 126.1
2b,c
[M] 122.12 (%)
mode): m/z
1
Table 2 H and 13C assignments and HMBC correlations for 6ha
13C
1H
1H-13C correlations
Rings
9.4 (C-16)
1.66 (s, H-16) 108.5, 132.7 (C-8), 143.9 (C-7)
B
A
B
15.9 (C-15)
102.7 (C-14)
1.74 (s, H-15) 128.4 (C-13), 135.1 (C-12), 184.2 (C-11)
7.23 (s, H-14) 112.8 (C-13b), 132.5 (C-8), 143.4 (C-9a)
6.64 (s, H-13) (15.9 (C-15))*, (113.1 (C-13ª)), (145.2 (C-9b)),
184.2 (C-11)
A
128.4 (C-13)
137.6 (C-2)
7.74 (s, H-2)
123.4 (C-5), 147.5 (C-4)
C
a Chemical shifts are expressed in δ (ppm) calibrated on the resonances of the residual
nondeuterated solvent DMSO. *() Signals with low intensity
21

Figure 1
O
NH2
R 1
6'
7
H
1
N
R 2
O
NH
NH2
6
5
7'
1'
2'
2
3
5'
4'
OH
3'
4
+
OH
laccase
(Pycnoporus
cinnabarinus)
R 1
R 1
4a R1 = H, R2 = H, yield = 44%*
4b R1 = H, R2 = CH3, yield = 41%
R 2
R 2
O
3a
air, pH5, rt
O
NH2
OH
7'
7
H
OH
O
2'
1
N
6
5
3'
1a R1 = H, R2 = H
1b R1 = H, R2 = CH3
1c R1 = CH3, R2 = H
2a-c
1'
6'
2
3
4'
OH
5'
4
OH
4c yield = 40%
* Yields refer to isolated products.

Figure 2
laccase
(Pycnoporus
cinnabarinus)
R
R
OH
OH
O
O
air, pH5, rt
-2 e- - 2 H+
1a-c
2a-c
R
R
R
O
O
O
O
OH
OH
OH
R
R
R
HO
-2 e- - 2 H+
O
1,4-addition
+
O
OH
OH
OH
R
R
OH
OH
OH
(I)
laccase
(Pycnoporus
cinnabarinus)
1,3-addition
-4 e- - 4 H+
1,4-addition
air, pH5, rt
HO
R
R
R
O
R
R
OH
O
OH
OH
R
OH
O
O
O
OH
O
5a1-c1
laccase
laccase
(Pycnoporus
cinnabarinus)
(Pycnoporus
cinnabarinus)
-2 e- - 2 H+
-2 e- - 2 H+
air, pH5, rt
air, pH5, rt
R
HO
R
R
O
O
O
O
O
R
OH
O
5a2-b2
5a3-c3

Figure 3
HN
N
H
2
O
NH2
N
1C
15
O
NH2
3c
3
N
5
12
13
6
4
laccase
(Pycnoporus
cinnabarinus)
11
14
H2N
13a
O
O
HN
13b
A
A
5
1,4-addition
7
10
B
B
2
9b
air, pH5, rt
O
O
O
8
9a
O
16
9
OH
OH
6h (NMR), yield = 9 %
OH
OH
1b
5b3

Figure 4
NH2
z
NH2
+
Y
X
O
O
OH1
HO
2
6
H
3c X = NH, Y = CH, Z = N
3d X = CH, Y = NH, Z = N
3
N
H
laccase
(Pycnoporus
cinnabarinus)
10
N
OH
9
8
z
4
O
11
O
O
12 Y
N
7
14
X
air, pH5, rt
15
NH16
5
13
N
OH
O
OH
8a X = NH, Y = CH, Z = N (NMR), yield = 15 %
8b X = CH, Y = NH, Z = N (NMR), yield = 11 %
1d
7

Figure 5
0,50
0,45
0,40
0,35
0,30
0,25
0,20
0,15
0,10
0,05
0,00
a
b
c
d
e
f
g
h
i
j
k
l