Enzymatic thiol Michael addition using laccases: Multiple C-S bond formation between p-hydroquinones and aromatic thiols

Article abstract of DOI:10.1016/j.molcatb.2015.12.012

Laccases create C-C, C-O or C-N bonds and have been investigated intensively as catalysts for green chemistry and white biotechnology. However, little is known about C-S bond formation in laccase-catalyzed reactions. We have used the laccases from Pycnopo

Full text of DOI:10.1016/j.molcatb.2015.12.012

Accepted Manuscript
Title: Enzymatic thiol Michael addition using laccases:
Multiple C<ce:glyph name="sbnd"/>S bond formation
between p-hydroquinones and aromatic thiols
Author: Max Schlippert Annett Mikolasch Veronika Hahn
Frieder Schauer
PII:
S1381-1177(15)30132-6
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2015.12.012
MOLCAB 3302
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date:
Revised date:
Accepted date:
20-8-2015
30-12-2015
31-12-2015
Please cite this article as: Max Schlippert, Annett Mikolasch, Veronika Hahn, Frieder
Schauer, Enzymatic thiol Michael addition using laccases: Multiple CS bond formation
between p-hydroquinones and aromatic thiols, Journal of Molecular Catalysis B:
Enzymatic http://dx.doi.org/10.1016/j.molcatb.2015.12.012
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Enzymatic thiol Michael addition using laccases: Multiple C-S bond
formation between p-hydroquinones and aromatic thiols
Max Schlipperta, Annett Mikolascha,* Veronika Hahna and Frieder Schauera
Institut für Mikrobiologie, Ernst-Moritz-Arndt-Universität Greifswald, Friedrich-Ludwig-Jahn Straße 15, 17487
Greifswald, Germany
* corresponding author: Fax: ++49 3834 864202; Tel: ++49 3834 864229; e-mail: annett.mikolasch@uni-
greifswald.de
1
Electronic Supplementary Information (ESI) available: Experimental methods; H NMR, 13C NMR, HSQC,
HMBC data, retention time, UV-vis, and MS-data.
Graphical abstract
Enzymatic thiol Michael addition using laccases: Multiple C-S bond formation between p-hydroquinones
and aromatic thiols,
Max Schlippert, Annett Mikolasch, Veronika Hahn and Frieder Schauer
oxidation by laccase
thiol Michael addition
1

Highlights
 Laccase created C-S bonds between p-hydroquinones and aromatic thiols.
 We discovered different thiolated products, ranging from dimers to tetramers.
 Laccase created S-S bonded dimers from aromatic thiols.
 The use of laccase is a convenient way for catalysis under eco-friendly conditions.
Abstract
Laccases create C-C, C-O or C-N bonds and have been investigated intensively as catalysts for green chemistry
and white biotechnology. However, little is known about C-S bond formation in laccase-catalyzed reactions. We
have used the laccases from Pycnoporus cinnabarinus and Myceliophthora thermophila to create
heteromolecular reaction products from p-hydroquinones and aromatic thiols via C-S bond formation. We
discovered a broad assortment of different thiolated products, ranging from dimers to tetramers. During laccase-
catalysis of the aromatic thiols without p-hydroquinones homomolecular dimers were formed and these were
always linked by S-S bonds. The use of laccase is a convenient way to form heteromolecular thiolated products
and homomolecular S-S bonds under eco-friendly environments.
Abbreviations: ABTS (2,2’azino-bis(3-ethylbenzothioazoline-6-sulfonic acid)diammonuim salt), HPLC (high
performance liquid chromatography), LC-MS (liquid chromatography-mass spectrometry), API-ES (atmospheric
pressure ionization electrospray), NMR (nuclear magnetic resonance), MeOH (methanol), 2MPD (2-
mercaptopyrimidine), 4MBA (4-mercaptobenzoic acid), MPAA (4-mercaptophenylacetic acid)
Keywords: laccase, C-S linkage, aromatic thiol, SH-containing compound, p-hydroquinone
2

1 Introduction
Enzyme-catalyzed oxidations using atmospheric oxygen as a non-toxic oxidant are of considerable general
interest. Laccases (E.C. 1.10.3.2, benzendiol:dioxygen oxidoreductase) are phenol oxidases, which contain four
copper atoms in their catalytic center [1]. They are used for a wide range of organic syntheses with a broad
spectrum of reactants, including monophenols, anilines and dihydroxylated substrates [2]. They use atmospheric
oxygen to oxidize hydroxyl groups and abstract the hydrogen, thus creating radicals. These radicals can either
undergo bonding reactions with another compound or transfer the radical character onto another reactant and act
as a radical mediator [3].
Laccases are widely distributed in fungi and plants, but can also be found in insects and bacteria [4, 5]. Their
natural functions include the synthesis and degradation of lignin and aromatic compounds [6], the synthesis of
pigments in fungi [7] and the sclerotization of insects [8]. Laccases find applications in the disposal of soil
pollutants [9] such as pentachlorophenol and TNT [10]. They are also used in the paper industry for the
preparation of pulp [11], in the food industry for the removal of unwanted phenols and in the textile industry for
the treatment of waste water [12, 13]. The oxidation of suitable substrates often yields radicals, which can
undergo various reactions such as the coupling of two different reaction partners. In the case of phenolic
compounds bond formations like C–C [14, 15], C–O [16] and C=C [17] or in case of reactions with amines C–N
[18-20] or C=N [17-19] may result. Laccase-catalyzed bond formation can be used in green chemistry for the
synthesis of many different substances, e.g. the formation of benzofurans and benzofuran-1-ones [21-23],
naphthoquinones [24, 25], aminobenzoquinones[19, 20, 26-31], substituted azoles [32, 33], 3-substituted 1,2,4-
triazolo[4,3-b][4.1.2]benzothiadiazine-8-ones [34], phenazines [35], and phenoxazinone derivatives [36-38], as
well as the dimerization of salicylic esters [39] and penicillins [40]. It has been shown, that the modification of
existing antibiotics with laccases further improves their inhibiting abilities, even against MRSA-strains [20, 27].
All the above mentioned reactions create bonds by using C-C, C-O or C-N interlinkages [19, 35, 41] and have
been investigated in depth. In contrast C-S bond formation has so far not been dealt with to any great extent in
the relevant literature [42-47] although it is of great interest both for green chemistry and white biotechnology.
To broaden the range of application of laccases in fine-chemical synthesis, we exploited the laccases of
Pycnoporus cinnabarinus and Myceliophthora thermophila for reactions involving C-S bond formation. These
fungal laccases catalyzed the reactions of several p-hydroquinones with three different thiols. The products were
analyzed by MS and NMR spectroscopy to gain more insight into their properties and structures.
2 Experimental
2.1 Enyzmes. The laccases used were obtained from the white rot fungi Pycnoporus cinnabarinus and
Myceliopthora thermophila.
Pycnoporus cinnabarinus was isolated in northern Germany and is deposited at the strain collection of the
Department of Biology of the University in Greifswald, from where it can be obtained. The fungus was
3

cultivated on malt agar plates for 7 days at 30 °C and then maintained at 4 °C. Three 1 cm2 agar fragments were
placed in a nitrogen-rich medium [48] and incubated without shaking at 30°C for 7 days. This preculture was
homogenized and 6 mL of homogenate was incubated with 40 mL medium. To stimulate laccase production, 10
mM veratryl alcohol was added and then agitated in a water bath for 3 days at 30 °C and 160 rpm. To purify the
laccase from the medium the culture was filtered through a glass fiber filter and stirred with DEAE-Sephacel
(Sigma-Aldrich, Steinheim, Germany) for 1h. Afterwards the enzyme was eluted with 20 mM sodium acetate
buffer (pH 5) and 70 mM NaCl. To desalt the laccase-containing buffer a PD10 column (SephadexTM; GE
Healthcare, Buckinghamshire, UK) with 20 mM sodium acetate buffer (pH 5) was used. The purified laccase
was stored at -20 °C and used at room temperature in sodium acetate buffer (pH 5) because of its pH optimum of
around pH 5.0 (ABTS) [49-51].
Laccase from Myceliophthora thermophila (expressed in a genetically modified Aspergillus sp.) was obtained
from Novozymes (Bagsvaerd, Denmark). It was used as received (activity 1000 U*g-1; substrate: syringaldazine)
in citrate phosphate buffer (CPB, 18 mM citrate, 165 mM phosphate) at its pH optimum of pH 7.0 [50, 52].
2.2 Measurement of laccase activity. The activity of laccase was determined spectrophotometrically at 420 nm
with ABTS as substrate using the method described by Jonas [51].
2.3 Experimental procedures. For analytical experiments one thiol (1-5 mM) and one p-hydroquinone (1 mM)
were incubated with laccase (activity 1 U) in a final volume of 4 ml of the respective buffer (sodium acetate
buffer, pH 5 for Pycnoporus cinnabarinus and citrate phosphate buffer, pH 7 for Myceliophthora thermophila,
both stored at 4 °C and used at room temperature for reactions). Reaction mixtures were incubated with agitation
at 200 rpm at room temperature in brown-glass-bottles. After incubation for 20 min, 2 h, or 24 h the mixture was
analyzed by HPLC, LC-10AT VP system (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. The
separation of substances was achieved on a RP18 column at a flow rate of 1 mL min-1. The solvent system
consisted of methanol (eluent A) and 0.1 % phosphoric acid (eluent B), starting from an initial ration of 10% A
and 90% B and reaching 100% methanol within 14 min.
The isolated products were characterized using a LC-MS system. The 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 ES ionization source in positive and negative
mode (dry and nebulizer gas: nitrogen; nebulizer pressure, 45 psig; drying gas flow, 10 L min-1; drying gas
temperature, 350 °C; capillary voltage, 4 kV; fragmentor voltage, 75 V). HPLC separation was performed on a
Zorbrax SB-C18 (2.1×50 mm, 1.8 μm) column (Agilent, Waldbronn, Germany), at a flow rate of 0.3 mL min-1.
A solvent system consisting of acetonitrile (eluent A) and 0.1 % aqueous ammonium formate (eluent B), starting
from an initial ratio of 10 % A and 90 % B and reaching 100 % acetonitrile within 7 min, holding this until 13
min, was used.
4

Product isolation was achieved using solid phase extraction on a RP18 silica gel column (6 mL, 40-64 µm,
LiChrolut, 2000 mg, Merck, Darmstadt). Product 3d was isolated from the reaction mixture (40 mL) after an
incubation period of 24 h (1a : 2d 2 mM : 1 mM assay). After charging the column with 6 mL reaction mixture,
6 mL of a methanol-distilled water (60 % : 40 % v/v) mixture was used to remove undesired impurities. Elution
of the red product fraction was performed with 2 mL methanol. Product 3g was isolated from the reaction
mixture (50 mL) after an incubation period of 20 min (1b : 2b 1 mM : 1 mM assay). After charging the column
with 6 mL reaction mixture, the orange product fraction was eluted with 2 mL methanol. Product 3h was isolated
from the reaction mixture (50 mL) after an incubation period of 20 min (1b : 2c 1 mM : 1 mM assay). After
charging the column with 6 mL reaction mixture, the orange product fraction was eluted with 2 mL methanol.
Product 3j was isolated from the reaction mixture (50 mL) after an incubation period of 20 min (1b : 2d 1 mM :
1 mM assay). After charging the column with 6 mL reaction mixture, the orange product fraction was eluted
with 2 mL methanol. Product 3o was isolated from the reaction mixture (60 mL) after an incubation period of 20
min (1c : 2d 1 mM : 1 mM assay). After charging the column with 6 mL reaction mixture, the orange-yellow
product fraction was eluted with 2 mL methanol. Product 4i was isolated from the reaction mixture (50 mL) after
an incubation period of 2 h (1b : 2e 5 mM : 1 mM assay). After charging the column with 6 mL reaction
mixture, 6 mL of a methanol-distilled water (60 % : 40 % v/v) mixture was used to remove undesired impurities.
Elution of the dark red product product fraction was performed with 2 mL methanol. Product 4j was isolated
from the reaction mixture (80 mL) after an incubation period of 2 h (1c : 2a 1 mM : 1 mM assay). After
charging the column with 6 mL reaction mixture, 6 mL of a methanol-distilled water (10 % : 90 % v/v) mixture
was used to remove undesired impurities. Elution of the brown product fraction was performed with 2 mL
methanol. Product 4m was isolated from the reaction mixture (80 mL) after an incubation period of 20 min (1c :
2d 5 mM : 1 mM assay). After charging the column with 6 mL reaction mixture, 4 mL of methanol-distilled
water (50 % : 50 % v/v) was used to remove undesired impurities. Elution of the red product fraction was
performed with 8 mL of a methanol-distilled water (60 % : 40 % v/v) mixture. 4n was isolated from the reaction
mixture (40 mL) after an incubation period of 2 h (1c : 2e 1 mM : 1 mM assay). After charging the column with
6 mL reaction mixture, the brown product fraction was eluted with 2 mL methanol.
1
The NMR spectra were obtained at 600 MHz (1H, H1H COSY, HSQC, HMBC) and at 150 MHz (13C) on a
Bruker Avance 600 instrument (Rheinstetten, Germany). The solvent used was deuterated methanol. Chemical
shifts expressed in δ (ppm) calibrated on the resonaces of the residual nondeuterated solvent. For NMR
spectroscopy the isolated products were dried by lyophilization.
2.4 Analytical data of products
Atom numbering for each isolated product is shown in Electronic Supplementary Information.
(3a). Rf (LC-MS) 6.9 min. UV-vis (MeOH) λmax 225, 251, 360 nm. MS m/z AP-ESI neg. mode [M-H]- 219, AP-
ESI: pos. Mode [M+H]+ 221.
(3b). Rf (LC-MS) 11.6 min. UV-vis (MeOH) λmax 254, 384 nm. MS m/z AP-ESI neg. mode [2M-H]- 465, AP-
ESI: pos. Mode [M+H]+ 235.
(3c). Rf (LC-MS) 15.3 min. UV-vis (MeOH) λmax 249, 310 nm. MS m/z AP-ESI neg. mode [M-H]- 247, AP-ESI:
pos. Mode [M+H]+ 249.
5

(3d). Rf (LC-MS) 11.6 min. UV-vis (MeOH) λmax 260, 295 nm. MS m/z AP-ESI neg. mode [2M-H]- 495.
(3e). Rf (LC-MS) 13.1 min. UV-vis (MeOH) λmax 234, 331 nm. MS m/z AP-ESI neg. mode [M-H]- 306, AP-ESI:
pos. Mode [M+H]+ 308.
(3f). Rf (LC-MS) 8.5 min. UV-vis (MeOH) λmax 223, 261 nm, 435 nm. MS m/z AP-ESI neg. mode [M-H]- 259,
AP-ESI: pos. Mode [M+H]+ 261.
4-(4-Methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)sulfanyl)benzoic acid (3g). Synthesis and isolation as described
above. Orange solid. Yield 66.6 % (8.89 mg). 1H NMR: δ 2.02 (s(broad), 3H, H-7’), 5.81 (s(broad), 1H, H-2’),
6.72 (s(broad), 1H, H-5’), 7.55 (d, J = 8.3 Hz, 2H, H-3/H-5), 8.09 (d, J = 8.3 Hz, 2H, H-2/H-6). 13C NMR: δ
14.3 (C-7’), 120.7( C-2’), 117.7 (C-5)’, 134.6 (C-3/C-5), 130.6 (C-2/C-6), 129.7 (C-4), 140.3 (C-1), 172.5 (C-7),
1
183.9 (C-6‘), 184.5( C-3’). 1H H COSY: H-3/H-5 (H-2/H-6); H-2/H-6 (H-3/H-5). HMBC: H-3/H-5 (C-2/C-6,
C-3/C-5, C-1), H-2/H-6 (C-4, C-2/C-6, C-3/C-5, C-7). Rf (HPLC) 12.1 min. UV-vis (MeOH) λmax 230, 280, 425
nm. MS m/z AP-ESI neg. mode [M-H]- 273, AP-ESI: pos. Mode [M+H]+ 275.
4-(4,5-Dimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)sulfanyl)benzoic acid (3h). Synthesis and isolation as
1
described above. Orange solid. Yield 72 % (10.2 mg). H NMR: δ 2.00, s, 3H, H-7’, 2.07, s, 3H, H-8’, 5.78, s,
1H, H-2’, 7.55, d, J = 8.2 Hz, 2H, H-3/H-5, 8.08, d, J = 8.2 Hz, 2H, H-2/H-6. 13C NMR: δ 10.8 C-7’, 10.9 C-8’,
125.5 C-2’, 134.6 C-3/C-5, 130.6 C-2/C-6, 129.7 C-4, 140.2 C-1/C-5’, 141.5 C-4’, 153.4 C-1’, 172.5 C-7, 183.7
1
1
C-6’, 184.2 C-3’. H H COSY: H-7’ (H-3/H-5); H-8’ (H-3/H-5); H-3/H-5 (H-7‘, H-8‘, H-2/H-6); H-2/H-6 (H-
3/H-5). HMBC: H-7’ (C-5’, C-3’); H-8’ (C-4’, C-6’); H-2’ (C-4’, C-1’, C-6’); H-3/H-5 (C-2/C-6, C-3/C-5, C-1);
H-2/H-6 (C-4, C-2/C-6, C-3/C-5, C-7). Rf (HPLC) 13,2 min. UV-vis (MeOH) λmax 219, 287, 428 nm. MS m/z
AP-ESI neg. mode [M-H]- 287, AP-ESI: pos. Mode [M+H]+ 289.
4-(4-Methoxy-3,6-dioxocyclohexa-1,4-dien-1-yl)sulfanylbenzoic acid (3i). Synthesis and isolation as
1
described above. Orange-brown solid. Yield 79.3 % (11.2 mg). H NMR: δ 3.86, s(broad), 3H, H-7’, 5.71,
s(broad), 1H, H-2’, 6.11, s(broad), 1H, H-5’, 7.56, d, J = 8.3 Hz, 2H, H-3/H-5, 8.09, d, J = 8.5 Hz, 2H, H-2/H-6.
13C NMR: δ 55.6 C-7’, 123.2 C-2’, 106.1 C-5’, 134.5 C-3/C-5, 130.7 C-2/C-6, 129.1 C-4, 140.4 C-1, 159.9 C-4’,
1
1
72.5 C-7, 178.7 C-3’, 183.8 C-6’ . H H COSY: H-3/H-5 (H-3/H-5), H-2/H-6 (H-3/H-5). HMBC: H-7’ (C-4’),
H-3/H-5 (C-2/C-6, C-3/C-5, C-1), H-2/H-6 (C-4, C-2/C-6, C-3/C-5, C-7). Rf (HPLC) 10.8 min. UV-vis (MeOH)
λmax 217, 319, 444 nm. MS m/z AP-ESI neg. mode [M-H]- 289, AP-ESI: pos. Mode [M+H]+ 291.
(3j). Rf (LC-MS) 5.9 min. UV-vis (MeOH) λmax 285 nm, 486 nm. MS m/z AP-ESI neg. mode [M-H]- 319, AP-
ESI: pos. Mode [M+H]+ 321.
(3k). Rf (LC-MS) 1.3 min. UV-vis (MeOH) λmax 226 nm, 262 nm, 343 nm. MS m/z AP-ESI neg. mode [M-H]-
362, AP-ESI: pos. Mode [M+H]+ 364.
(3l). Rf (HPLC) 10.8 min. UV-vis (MeOH) λmax 210 nm, 310 nm, 433 nm. MS m/z AP-ESI neg. mode [M-H]-
273, AP-ESI: pos. Mode [M+H]+ 275.
(3m). Rf (HPLC) 12.5 min. UV-vis (MeOH) λmax 220 nm, 270 nm, 433 nm. MS m/z AP-ESI neg. mode [M-
COOH]- 243, AP-ESI: pos. Mode [M+H]+ 289.
(3n). Rf (LC-MS) 2.9 min. UV-vis (MeOH) λmax 229 nm, 260 nm, 346 nm. MS m/z AP-ESI neg. mode [M+OH-
H]- 376, AP-ESI pos. Mode [M+OH+H]+ 378.
2-[4-(4-Methoxy-3,6-dioxo-cyclohexa-1,4-dien-1-yl)sulfanylphenyl]phenylic acid (3o). Synthesis and
1
isolation as described above. Orange solid. Yield 63.7 % (12.04 mg). H NMR: δ 3.72, s, 2H, H-7; 3.85, s, 3H,
6

H-7’; 5.73, s, 1H, H-2’; 6.11, s, 1H, H-5’; 7.50, d, J = 8.5 Hz, 2H, H-2/H-6; 7.52, d, J = 8.5 Hz, 2H, H-3/H-5. 13
C
NMR: δ 40.6 C-7; 55.8 C-7’; 123.2 C-2’; 106.3 C-5’; 131.2 C-2/C-6; 135.2 C-3/C-5; 125.6 C-4, 138.2 C-1,
156.2 C-1’, 159.9 C-4’, 173.7 C-8, 178.6 C-3’, 183.9 C-6’. 1H 1H COSY: H-7 (H-2/H-6), H-7’ (H-5’, H-3/H-5),
H-5’ (H-7’), H-2/H-6 (H-7), H-3/H-5 (H-7’). HMBC: H-7 (C-2/C-6, C-1, C-8), H-7’ (C-5’, C-4’), H-2’ (C-1’, C-
4’, C-6’), H-5’ (C-1’, C-4’, C-3’, C-6’), H-2/H-6 (C-7, C-4, C-2/C-6), H-3/H-5 (C-4, C-3/C-5, C-1). Rf (HPLC)
10,5 min. UV-vis (MeOH) λmax 250, 303, 450 nm. MS m/z AP-ESI neg. mode [M-COOH]- 259, AP-ESI: pos.
Mode [M+H]+ 305.
(3p). Rf (HPLC) 10.4 min. UV-vis (MeOH) λmax 268 nm, 294 nm, 492 nm. MS m/z AP-ESI neg. mode [M-H]-
333, AP-ESI: pos. Mode [M+H]+ 335.
(3q). Rf (HPLC) 11 min. UV-vis (MeOH) λmax 230 nm, 309 nm, 384 nm. MS m/z AP-ESI neg. mode [M+OH-
H]- 376, AP-ESI: pos. Mode [M+OH+H]+ 378.
(4a). Rf (LC-MS) 6.3 min. UV-vis (MeOH) λmax 239 nm, 304 nm. MS m/z AP-ESI neg. mode [M-H]- 329, AP-
ESI pos. Mode [M+H]+ 331.
(4b). Rf (LC-MS) 13.3 min. UV-vis (MeOH) λmax 226 nm, 269 nm, 321 nm. MS m/z AP-ESI pos. Mode [M+H]+
345.
(4c). Rf (LC-MS) 14.2 min. UV-vis (MeOH) λmax 250 nm, 287 nm. MS m/z AP-ESI neg. mode [M-H]- 357, AP-
ESI pos. Mode [M+H]+ 359.
(4d). Rf (LC-MS) 14.2 min. UV-vis (MeOH) λmax 240 nm, 340 nm. MS m/z AP-ESI pos. Mode [M+H]+ 416.
(4e). Rf (LC-MS) 11.7 min. UV-vis (MeOH) λmax 230 nm, 271 nm, 374 nm. MS m/z AP-ESI neg. mode [M-H]-
411, AP-ESI pos. Mode [M+H]+ 413.
(4f). Rf (HPLC) 14.7 min. UV-vis (MeOH) λmax 230 nm, 280 nm, 410 nm. MS m/z AP-ESI pos. Mode [M+H]+
427.
(4g). Rf (LC-MS) 11.6 min. UV-vis (MeOH) λmax 275 nm, 488 nm. MS m/z AP-ESI neg. mode [M-H]- 439, AP-
ESI pos. Mode [M+H]+ 441.
(4h). Rf (HPLC) 13.1 min. UV-vis (MeOH) λmax 282 nm, 475 nm. MS m/z AP-ESI neg. mode [M-H]- 441, AP-
ESI pos. Mode [M+H]+ 443.
(4i). Rf (HPLC) 13.2 min. UV-vis (MeOH) λmax 288 nm, 405 nm. MS m/z AP-ESI neg. mode [M-H]- 471, AP-
ESI pos. Mode [M+H]+ 473.
2-[4-[4-[4-(Carboxymethyl)phenyl]sulfanyl-2,5-dihydroxyphenyl]sulfanylphenyl]acetic acid (4j). Synthesis
1
and isolation as described above. Brown solid. Yield 29.5 % (10.4 mg). H NMR: δ 3.58, s, 4H, H-7, 5.89,
s(broad), 1H, H-2’/H-5’, 7.45, d, J = 8.1 Hz, 4H, H-3/H-5, 7.51, d, J = 8.1 Hz, 4H, H-2/H-6. 13C NMR: δ 44.7 C-
7, 124.2 C-2’/C-5’, 134.9 C-3/C-5, 131.1 C-2/C-6, 124.1 C-4, 141.4 C-1, 177.7 C-8. 1H 1H COSY: H-7 (H-2/H-
6); H-3/H-5 (H-2/H-6); H-2/H-6 (H-7, H-3/H-5). HMBC: H-7 (C-2/C-6, C-1, C-8), H-3/H-5 (C-3/C-5, C-1), H-
2/H-6 (C-7, C-4, C-2/C-6). Rf (HPLC) 12.8 min. UV-vis (MeOH) λmax 219, 370 nm. MS m/z AP-ESI neg. mode
[M-H]- 439, AP-ESI: pos. Mode [M+H]+ 441.
(4k). Rf (HPLC) 13.5 min. UV-vis (MeOH) λmax 250 nm, 292 nm. MS m/z AP-ESI neg. mode [M-H]- 453, AP-
ESI pos. Mode [M+NH4]+ 472.
(4l). Rf (HPLC) 14.1 min. UV-vis (MeOH) λmax 204 nm, 247 nm, 325 nm. MS m/z AP-ESI neg. mode [M-H]-
467, AP-ESI pos. Mode [M+H]+ 469.
7

2-[4-[2-[4-(Carboxymethyl)phenyl]sulfanyl-4-methoxy-3,6-dioxocyclohexa-1,4-dien-1-
yl]sulfanylphenyl]acetic acid (4m). Synthesis and isolation as described above. Darkblue solid. Yield 32.9 %
1
(12.37 mg). H NMR: δ 3.61, s, 8H, C-7, 3.81, s, 3H, H-7’, 6.07, s, 1H, H-5’, 7.28, m, 12H, 7.47, m, 4H. 13C
NMR: δ 40.2 C-7, 55.9 C-7’, 107.8 C-5’, 129.9 C-, 127.9 C-, 148.1 C-1’, 159.8 C-4’, 175.7 C-3’, 180.7 C-6’. 1H
1H COSY: H-7’ (H-5’); H-5’ (H-7’); 7.28 (HC-7); 7.47 (H-7). HMBC: C-7 (C-8); H-7’ (C-4’); H-5’ (C-1’, C-4’,
C-3’). Rf (HPLC) 12 min. UV-vis (MeOH) λmax 251, 339, 491 nm. MS m/z AP-ESI neg. mode [M-H]- 469, AP-
ESI: pos. Mode [M+H]+ 471.
2-[4-[5-[4-(Carboxymethyl)phenyl]sulfanyl-2,4-dimethoxy-3,6-dioxocyclohexa-1,4-dien-1-
yl]sulfanylphenyl]acetic acid (4n). Synthesis and isolation as described above. Darkbrown solid. Yield 29.4 %
(9.6 mg). 1H NMR: δ 3.58, s, 4H, H-7, 3.89, s, 6H, H-7‘/H-8‘, 7.27, d, J = 8.3 Hz, 4H, H-2/H-6, 7.45, d, J = 8.3
Hz, 4H, H-3/H-5. 13C NMR: δ 41.1 C-7, 60.2 C-7’/C-8’, 129.8 C-2/C-6, 127.9 C-3/C-5, 157.4 C-2’/C-4’, 175.8
1
1
C-3’, 180.7 C-6’. H H COSY: H-7 (H-2/H-6, H-3/H-5), H-2/H-6 (H-7, H-3/H-5), H-3/H-5 (H-7, H-2/H-6).
HMBC: H-7 (C-2/C-6, C-1, C-8), H-7‘/H-8‘ (C-2’/C-4’), H-2/H-6 (C-7, C-3/C-5, C-2/C-6, C-1), H-3/H-5 (C-
3/C-5, C-1). Rf (HPLC) 12,5 min. UV-vis (MeOH) λmax 249, 310 nm. MS m/z AP-ESI neg. mode [M-H]- 499,
AP-ESI: pos. Mode [M+NH4]+ 518.
(4o). Rf (LC-MS) 3.5 min. UV-vis (MeOH) λmax 227 nm, 255 nm, 384 nm. MS m/z AP-ESI neg. mode [M-H]-
526, AP-ESI pos. Mode [M+H]+ 528.
(5a). Rf (LC-MS) 11.6 min. UV-vis (MeOH) λmax 247 nm, 341 nm. MS m/z AP-ESI neg. mode [M-H]- 439, AP-
ESI pos. Mode [M+H]+ 441.
(5b). Rf (LC-MS) 12.2 min. UV-vis (MeOH) λmax 251 nm, 339 nm. MS m/z AP-ESI neg. mode [M-H]- 453,
AP-ESI pos. Mode [M+H]+ 455. (5c). Rf (LC-MS) 11.3 min. UV-vis (MeOH) λmax 229 nm, 271 nm, 401 nm.
MS m/z AP-ESI neg. mode [M-H]- 563, AP-ESI pos. Mode [M+H]+ 565.
(5d). Rf (LC-MS) 5.9 min. UV-vis (MeOH) λmax 227 nm, 255 nm, 384 nm. MS m/z AP-ESI neg. mode [M-H]-
577, AP-ESI pos. Mode [M+H]+ 579.
(5e). Rf (HPLC) 13.9 min. UV-vis (MeOH) λmax 216 nm, 254 nm, 389 nm. MS m/z AP-ESI neg. mode [M-H]-
605.
Product mixture: 2-[4-[4,5-bis[[4-(carboxymethyl)phenyl]sulfanyl]-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-
yl]sulfanylphenyl]acetic acid (5f), 2-[4-[[4-(carboxymethyl)phenyl]disulfanyl]phenyl]acetic acid (6c).
Synthesis and isolation as described above. Darkbrown solid. Yield 18.8 % (9.34 mg). 1H NMR: δ 2.18, s, 3H,
H-7’, 3.59, s, 2H, H-7, 3.61, s, 2H, H-7, 3.616, s, 2H, H-7, 3.619, s, 2H, H-7, 7.13, d, J = 8.5 Hz, 2H, 7.16, d, J =
8.5 Hz, 2H, 7.18, d, J = 8.3 Hz, 2H, 7.22, d, J = 8.3 Hz, 2H, 7.27, d, J = 8.3 Hz, 4H, 7.34, d, J = 8.3 Hz, 2H, 7.47,
d, J = 8.3 Hz, 2H. 13C NMR: δ 14.26 C-7’, 40.00 C-7, 40.09 C-7, 40.12 C-7, 129.86, 130.80 129.89, 130.56,
129.91, 130.87, 127.89, 175.84 C-6’, 178.59 C-3’. 1H 1H COSY: H-7’ (3.59, 3.61, 7.13, 7.16, 7.47), H-7 (H-7’),
7.13 (H-7’, H-7, 7.16), 7.16 (H-7’, 7.13), 7.18 (3.616/3.619, 7.22), 7.22 (7.18), 7.27 (3.616/3.619, 7.34, 7.47),
7.34 (7.27), 7.47 (H-7’, 3.61, 7.27), HMBC: H-7’ (131.54 C-,143.62 C-, 147.59 C-, C-3’), H-7 (129.86, 129.89,
129.91, 129.92); H-7 (134.33, 134.45, 134.58, 134.68), H-7 (173.68, 173.70, 173.73, 173.79), 7.13 (129.86,
130.56), 7.16 (130.80, 134.45), 7.18 (129.89, 131.55), 7.22 (130.56, 134.33), 7.27 (130.87, 135.10), 7.34
(130.87, 134.58), 7.47 (127.89, 134.68), Rf (HPLC) 14.2 min. UV-vis (MeOH) λmax 250 nm, 412 nm. MS m/z
AP-ESI neg. mode [M-H]- 619, AP-ESI pos. Mode [M+NH4]+ 638.
8

(5g). Rf (HPLC) 13.7 min. UV-vis (MeOH) λmax 251 nm, 410 nm. MS m/z AP-ESI neg. mode [M-H]- 635,
AP-ESI pos. Mode [M+NH4]+ 654.
(5h). Rf (LC-MS) 6.1 min. UV-vis (MeOH) λmax 250 nm, 420 nm. MS m/z AP-ESI neg. mode [M-H]- 692,
AP-ESI pos. Mode [M+H]+ 694.
1
2-(Pyrimidin-2-yldisulfanyl)pyrimidin (6a). White solid. Yield 22 % (12 mg). H NMR: δ 7.27, t, J = 4.9 Hz,
1H, H-5, 8.62, d, J = 4.9 Hz, 2H, H-4/H-6. 13C NMR: δ 118.5 C-5, 158.0 C-4/C-6, 169.0 C-2. 1H 1H COSY: H-5
(H-4/H-6); H-4/H-6 (H-5). HMBC: H5 C-5 (C-4/C-6, C2); H-4/H-6 C-4/C-6 (C-5, C-4/C-6, C-2). Rf (HPLC) 8,3
min. UV-vis (MeOH) λmax 237 nm. MS m/z AP-ESI neg. mode [M-H]- 221, AP-ESI: pos. Mode [M+H]+ 223.
1
4-[(4-Carboxyphenyl)disulfanyl)]benzoic acid (6b). Yellow solid. Yield 71.8 % (10.7 mg). H NMR: δ 7.51,
d, J = 8.4 Hz, 2H, H-2/H-6, 7.92, d, J = 8.4 Hz, 2H, H-3/H-5. 13C NMR: δ 126.1 C-2/C-6, 129.7 C-3/C-5, 136.9
1
1
C-4, 138.8 C-1, 172.9 C-7. H H COSY: H-2/H-6 (H-3/H-5), H-3/H-5 (H-2/H-6). HMBC: H-2/H-6 (C-2/C-6,
C-3/C-5, C-4, C-1, C-7), H-3/H-5 (C-2/C-6, C-3/C-5, C-1, C-7). Rf (HPLC) 13.7 min. UV-vis (MeOH) λmax 273
nm. MS m/z AP-ESI neg. mode [M-H]- 305, AP-ESI: pos. Mode [M+Na]+ 239.
1
2-[4-[[4-(Carboxymethyl)phenyl]-disulfanyl]phenyl]acetic acid (6c). White solid. Yield 88.2 % (8.7 mg). H
NMR: δ3.48, s, 2H, H-7, 7.29, d, J = 8.3 Hz, 2H, H-3/H-5, 7.41, d, J = 8.3 Hz, 2H, H-2/H-6. 13C NMR: δ 44.4 C-
1
1
7, 129.7 C-3/C-5, 128.1 C-2/C-6, 134.2 C-1, 137.9 C-4, 178.1 C-8. H H COSY: H-7 (H-3/H-5, H-2/H-6); H-
3/H-5 (C-7, H-3/H-5, H-2/H-6), H-2/H-6 (C-7, H-3/H-5, H-2/H-6). HMBC: H-7 (H C-3/C-5, C-4, C-8); H-3/H-5
(C-7, C-2/C-6, C-3/C-5, C-1), H-2/H-6 (C-2/C-6, C-3/C-5, C-4). Rf (HPLC) 14.1 min. UV-vis (MeOH) λmax 245
nm. MS m/z AP-ESI neg. mode [M-H]- 333, AP-ESI: pos. Mode [M+NH4]+ 352.
3 Results
3.1 General observations. The three thiols used in this study, 2MPD, 4MBA and MPAA, consist of six-
membered aromatic or hetero-aromatic rings carrying a mercapto group. They are all able to form a
homomolecular product during the laccase-catalyzed reaction with both laccases in the same way. The dimers
formed were all linked by S-S bonds (see Scheme 1). These reactions could also be analyzed without laccase, but
the reaction time was shortened by more than tenfold by adding laccase.
The p-hydroquinones used in this study can be described as alkyl- or methoxy-substituted and are themselves
laccase substrates (for structures see Table 1). All p-hydroquinones were converted to their respective quinones,
as described previously [19, 29]. Nearly every combination of thiols and p-hydroquinones resulted in at least one
heteromolecular product. Depending on the amount of thiol in the reaction, the products were dimers (see Table
1), trimers (see Table 2) or tetramers (see Table 3). The reactions of the laccase from Pycnoporus cinnabarinus
were basically the same as those of Myceliophthora thermophila.
3.2 Reactions of 2MPD. In the reaction mixtures of 2MPD (1a) and the p-hydroquinones (2a-2f) the formation
of heteromolecular products was detected by HPLC/UV-vis and LC-MS analysis. The reaction rate was slower
than for the other two thiols. Product formation took place in the first few hours, but most of the products were
highly unstable and could no longer be detected after 24 h. After 24 h there was still an excess of 2MPD,
demonstrating the slow reaction rate of the heteromolecular C-S formation. With HPLC and LC-MS analyses we
found 5 dimers (Table 1), 4 trimers (Table 2) and 2 tetramers (Table 3). Only one of these products was stable
9

enough to be isolated. The dimer 3d, consisting of one molecule 2MPD and one molecule
methoxyhydroquinone, was isolated by solid phase extraction and shown to have a yield of 28.3 % using the
laccase of Pycnoporus cinnabarinus. The reaction mixture changed color from light yellow to colorless.
Heteromolecular C-S product formation only took place in the presence of laccase.
3.3 Reactions of 4MBA. In contrast to the reactions of 2MPD, the reaction of 4MBA (1b) and p-hydroquinones
(2a-2f) were much more promising. In every reaction mixture C-S heteromolecular products were detected
(Table 1, 2, 3).
Laccase-catalyzed reactions of 4MBA with p-hydroquinones (2a-2f) generally proceeded very fast. Using
laccase from Myceliophthora thermophila, the reactants were consumed within 20 min. The products seemed to
be stable in the reaction solution (as example, see figure S1 in Electronic Supplementary Information for the
formation of 3h). If equimolar concentrations of compounds were used, the formation of dimers was favored.
Three have been isolated (3g-3i) after using the laccase of Myceliophthora thermophila. In reaction mixtures
with an excess of 4MBA (2-5 : 1 mM), the formation of trimers (Table 2) and tetramers (Table 3) was observed.
It was possible to isolate the trimer 4i by solid phase extraction after using the laccase of Pycnoporus
cinnabarinus for the transformation.
The formation of the homomolecular product of 4MBA was detected in all reactions as side product. It was
present in smaller amount in the equimolar reaction mixtures and in higher amount in the reaction mixtures with
an excess of 4MBA. The retention time of the homomolecular products only differed slightly from those of the
C-S heteromolecular products and in consequence the isolations were difficult to accomplish for heteromolecular
trimers and tetramers. The reaction mixtures changed color from clear to orange or dark red. The products
formed were stable in solution and could still be detected after weeks, when stored at 4° C.
3.4 Reactions of MPAA. As in the reactions of 4MBA, MPAA (1c) also showed promising results. In every
reaction mixture the formation of stable hetermolecular products was demonstrated. The amount of MPAA
influenced the type and the yields of the resulting products. In equimolar reaction mixtures, dimers were favored.
Excess MPAA favoured the formation of trimers, of tetramers and also of the undesired homomolecular product
of MPAA. All newly formed links between the reactants involved C-S-bonds. As shown above, the reactions
were very fast, with the reactants being consumed within 20 min for equimolar reactions and within 2h for
higher concentrations of MPAA. The formation of products was analyzed over this time scale (as example, see
figure S2 in Electronic Supplementary Information for the formation of tetramer 5f).
For all products formed, it was possible to derive the structure from the HPLC and LC-MS analysis. We detected
in total 6 C-S coupled dimers (Table 1), 6 C-S coupled trimers (Table 2) and 4 C-S coupled tetramers (Table 3)
from MPAA (1c) and p-hydroquinones (2a-2f). With MPAA it was easier to separate the transformation product
from undesired impurities and from the homomolecular product of MPAA. As a result, 1 dimer (3o), 3 trimers
(4j, 4m, 4n) and 1 tetramer (5f) were isolated by solid phase extraction. All these reactions were catalyzed by the
10

laccase of Pycnoporus cinnabarinus. The reaction mixtures changed color from clear to red or brown-red. The
products formed were stable in solution and could still be detected after weeks, when stored at 4° C.
3.5 Detailed Structural Characterization of 3h, 4n, 5f.
In the UV-vis spectrum 3h showed similar absorption maxima to monoaminated aminoquinones[20, 26], such as
the weak maximum between 400 and 500 nm and two maxima under 300 nm, indicating the formation of a
monothiolated quinone.
LC-MS analyses with AP-ESI in both positive and negative modes showed the molecular mass of 3h to be 288.
1
This mass could be attributed to the thiolation of 2c with 1b under loss of four hydrogen atoms. H NMR
spectral data of 3h showed characteristic signals for both compounds (Table 4). Multiplicity of H-2’ suggests the
thiolation having taken place at C-1’. 13C NMR showed typical signals for quinones in the range of 180 ppm, an
indication for the quinoid character of 3h. The HMBC spectrum showed correlations between the proton H-
2’and the quinone carbonyl carbon C-6’ and additional between the protons H-7’and H-8’ and the quinone
carbonyl carbons, unambiguously proving 3h to be a thiolquinone and being substituted at C-1’. The bond
formation between 1b and 2c was also confirmed by cross-signals from the methyl protons H-7’and H-8’ with
H-3 and H-5 in the 1H1H COSY. All results led to the identification of 3h as 4-(4,5-dimethyl-3,6-
dioxocyclohexa-1,4-dien-1-yl)sulfanyl)benzoic acid (Figure 1A).
The UV-vis spectrum of 4n showed maxima only under 400 nm in accordance with those of known diaminated
quinines [26] indicating the formation of a dithiolated quinone.
LC-MS with API-ESI in both positive and negative modes showed the molecular mass of 4n to be 500. This
mass could be attributed to the thiolation of 2e with two molecules of 1c accompanied by the loss of six
hydrogen atoms. 1H NMR spectral data of 4n showed the presence of two MPAA residues (Table 4). No protons
of the aromatic system of 2e remained. 13C NMR showed again typical signals for quinones. This showed 4n to
be a dithiolated quinone substituted at C-1’ and C-5’. All results led to the identification of 4n as 2-[4-[5-[4-
(carboxymethyl)phenyl]sulfanyl-2,4-dimethoxy-3,6-dioxocyclohexa-1,4-dien-1-yl]sulfanylphenyl]acetic
(Figure 1B).
acid
The UV-vis spectrum of 5f showed maxima only under 400 nm indicating the formation of a trithiolated
quinone. LC-MS analyses with AP-ESI in both positive and negative modes showed the molecular mass of 5f to
be 620. This mass could be attributed to the thiolation of 2b with three molecules of 1c accompanied by the loss
of eight hydrogen atoms. 1H NMR spectral data of 5f showed the presence of three MPAA residues. No protons
of the aromatic system of 2b remained. 13C NMR showed again typical signals for quinones. The bond formation
between 1c and 2b was also confirmed by cross-signals from the methyl protons H-7’ with H-3 and H-5 in the
1H1H COSY. All together this showed 5f to be a trithiolated quinone substituted at C-1’, C-4’ and C-5’ and led
to the identification of 5f as 2-[4-[4,5-bis[[4-(carboxymethyl)-phenyl]sulfanyl]-2-methyl-3,6-dioxocyclohexa-
1,4-dien-1-yl]sulfanylphenyl]acetic acid (Figure 1C).
4 Discussion
11

Structural characterization of three homomolecular reaction products showed that in the course of laccase-
catalyzed reactions, the three selected thiols act as laccase substrates. The homomolecular reaction products were
formed as dimers with S-S links in all three experiments (Scheme 1). No higher oligomers or other monomers
have been detected. The reaction rate was high for 4MBA and MPAA with the reactants being consumed within
20 min. 2MPD reacted slower in all reactions. Even after 24 h, we could detect a moderate amount of 2MPD.
Still, the data show that thiols can be laccase substrates, and that they have a reaction rate similar to that of the
classic phenolic laccase substrates.
In heteromolecular reactions of three selected thiols p-hydroquinones can be nuclear thiolated, because thiols can
also be used as heteroatomic donors in the Michael addition [53]. Depending on the substituents of the p-
hydroquinone, the properties of the aromatic thiol, and the concentration of the compounds in solution, the
course of the laccase-catalyzed reaction and the properties of the resulting products differed to a considerable
extent. The thiolation of p-hydroquinones proceeded in one step without the detection of the corresponding
quinones and much faster than aminations of p-hydroquinones, where in a first step a quantitative formation of
the corresponding quinone took place [19, 29]. This observation can be explained with a general higher
nucleophilicity of thiols compared with amines. This may also be the reason for the fast formation of trimers and
tetramers. The thiolate anion is often the active species in Michael additions [53]. The mechanism underlying the
reaction is likely to be an intermediate laccase-catalyzed formation of the corresponding quinones (7) and
subsequent thiolation with aromatic thiols by Michael addition (Scheme 2). After a second oxidation the
monothiolated products 3, which were isolated for 3d, g, h, i and o, are formed. Products 3 undergo a second
Michael addition, resulting in the formation of intermediates 9, which are comparable to the intermediates 8.
After a third oxidation the dithiolated products 4, which were isolated for 4i, j, m and n, are produced. The
position of the thiolations at the aromatic ring is determined by the substituents of the aromatic or heteroaromatic
thiol partner and by the substitution of the laccase substrate used. The chemical synthesis of trimers like 4 has
also been described for aminoquinones [19, 30]. Depending on the number of substituents at the laccase
substrates (2a-f) a third Michael addition could be shown resulting in the formation of intermediates 10, which
are comparable to the intermediates 8 and 9 and undergo a fourth oxidation to the trithiolated quinones 5.
The use of laccase for fine chemistry has been well studied. The coupling of p-hydroquinones and amines have
been described several times [19, 26, 30, 41, 54] forming C-N linked heteromolecular compounds. However, to
date little has been reported about laccase-catalyzed reactions with thiols generating C-S linkages.
The heteromolecular reaction approaches resulted in a multitude of transformation products. All of them were
characterized by HPLC and LC-MS and some were isolated in higher yields and analyzed by NMR. All
compounds are linked by C-S bonds. Oligomers consisted of one molecule p-hydroquinone and one to three
molecules of the thiol used. 4MBA and MPAA had rapid reaction rates and formed stable C-S heteromolecular
products with p-hydroquinones. Overall, we detected 39 heteromolecular transformation products with C-S
bonds. As stated above, we used two different laccases in these experiments. The laccase of Pycnoporus
cinnabarinus has a pH optimum of 5 [49-51] and the laccase of Myceliophthora thermophila a pH optimum of 7
[50, 52]. We did not detect any differences in the spectrum of products formed though there were differences in
the amount and stability of the products. This is most likely caused by the different pH values. The thiol Michael
addition reaction rates increase with pH due to the increased concentration of the thiolate anion [53].
12

C-S-linkage in a traditional chemical pathway is only possible under extreme reaction conditions. For the
bonding process of thiols with alkyl halides, it is necessary to maintain a reaction temperature of 200 °C and the
polar solvent hexamethylphosphoramide [55]. Another coupling of thiols and alkyls described requires a
temperature of 90 °C and a solvent like ethanol or dimethyl sulfoxide [56]. Other copper-catalyzed reactions also
have the disadvantage of requiring high temperatures and large amounts of copper salts [57]. Therefore, the use
of laccase to form C-S-linkages is economic and the reactions take place under mild conditions, room
temperature, fast reaction rate, low resource input and an aqueous environment, making them a prime example
for green chemistry.
5 Conclusion
In this study we present new transformation products of thiols and p-hydroquinones. The formation of laccase
induced S-S and C-S bonds is described. We demonstrate the use of thiols as laccase substrates, thus increasing
the potential of laccases in organic synthesis and green chemistry. By using laccase we can synthesize products
in environmentally friendly conditions.
6 Acknowledgments
The authors thank Michael Lalk for providing NMR data and Robert Jack for reviewing the manuscript.
13

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15

Figure captions
1
1
Figure 1: Atom numbering and important H-13C (HMBC) (H ► C) and H-1H (COSY) correlations (H ---
► C) of (A) 3h, (B) 4n, (C) 5f
16

Scheme captions
Scheme 1: Homomolecular laccase-catalyzed reactions of 1a (2MPD), 1b (4MBA) and 1c (MPAA)
17

Scheme 2: Possible reaction mechanism. Describtion of R1 to R5 see table 1 to 3.
18

Tables
Table 1: p-Hydroquinones and thiols used in laccase-catalyzed biotransformation and synthesized dimeric C-S coupling
products (yield in parentheses, n.d. = not determined; Pcl = Pycnoporus cinnabarinus-laccase, Mtl = Myceliophthora
theromphila-laccase)
Sulfur compound
p-Hydroquinone
Dimeric product
1a: R1 = H; R2 = N
2a: R3=R4=R5=H
3a: (n.d.)
2b: R3= R5=H; R4= CH3
3b: (n.d.)
1a
1a
1a
1a
2c: R3= H; R4=R5= CH3
3c: (n.d.)
2d: R3= R5=H; R4= OCH3
3d: (28.3% Pcl)
3e: (n.d.)
2f: R3= R5=H;
R4= CONHCH2CH2OH
1b: R1 = COOH; R2 = CH
3f: (n.d.)
2a
3g: (66.6% Mtl)
3h: (72.0% Mtl)
3i: (79.3% Mtl)
3j: (n.d.)
1b
2b
1b
2c
1b
2d
2e: R5= H; R3=R4= OCH3
1b
3k: (n.d.)
1b
2f
1c: R1 = CH2COOH; R2 = CH
3l: (n.d.)
2a
2b
2c
2d
2e
2f
3m: (n.d.)
1c
1c
1c
1c
1c
3n: (n.d.)
3o: (63.7% Pcl)
3p: (n.d.)
3q: (n.d.)
19

Table 2: p-Hydroquinones and thiols used in laccase-catalyzed biotransformation and synthesized trimeric C-S coupling
products (yield in parentheses, n.d. = not determined; Pcl = Pycnoporus cinnabarinus-laccase, Mtl = Myceliophthora
theromphila-laccase)
Sulfur compound
p-Hydroquinone
Trimeric product
1a: R1 = H; R2 = N
2a: R3=R4=R5=H
4a: (n.d.)
4b: (n.d.)
4c: (n.d.)
4d: (n.d.)
1a
1a
1a
2b: R3= R5=H; R4= CH3
2c: R3= H; R4=R5= CH3
2f: R3= R5=H;
R4= CONHCH2CH2OH
1b: R1 = COOH; R2 = CH
2a
2b
2c
4e: (n.d.)
4f: (n.d.)
4g: (n.d.)
1b
1b
1b
2d: R3= R5=H; R4= 4h: (n.d.)
OCH3
1b
2e: R5= H; R3=R4= OCH3 4i: (26.2% Pcl)
1c: R1 = CH2COOH; R2 = CH 2a
4j: (29.5% Pcl)
4k: (n.d.)
1c
1c
1c
1c
1c
2b
2c
2d
2e
2f
4l: (n.d.)
4m: (32.9% Pcl)
4n: (29.4% Pcl)
4o: (n.d.)
20

Table 3: p-Hydroquinones and thiols used in laccase-catalyzed biotransformation and synthesized tetrameric C-S coupling
products (yield in parentheses, n.d. = not determined; , Pcl = Pycnoporus cinnabarinus-laccase, Mtl = Myceliophthora
theromphila-laccase)
Sulfur compound
p-Hydroquinone
Tetrameric product
1a: R1 = H; R2 = N
2a: R3=R4=R5=H
5a: (n.d.)
1a
2b: R4= R5=H; R3= 5b: (n.d.)
CH3
1b: R1 = COOH; R2 = CH
2a
2b
5c: (n.d.)
1b
5d: (n.d.)
1c: R1 = CH2COOH; R2 = CH 2a
5e: (n.d.)
1c
1c
2b
5f: (18.8% Pcl)
2d: R4= R5=H; R3= 5g: (n.d.)
OCH3
1c
2f: R4= R5=H;
R3=
5h: (n.d.)
CONHCH2CH2OH
21