C-N coupling of 3-methylcatechol with primary amines using native and recombinant laccases from Trametes versicolor and Pycnoporus cinnabarinus

Article abstract of DOI:10.1016/j.tet.2011.09.123

Five laccase genes from Pycnoporus cinnabarinus and Trametes versicolor encoding for different isoenzymes have been cloned, recombinantly expressed and characterized. Following C-N coupling of primary linear, branched-chained and cyclic amines to 3-methyl

Full text of DOI:10.1016/j.tet.2011.09.123

Tetrahedron 67 (2011) 9311e9321
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
CeN coupling of 3-methylcatechol with primary amines using native and
recombinant laccases from Trametes versicolor and Pycnoporus cinnabarinus
,
Susanne Herter a, Annett Mikolasch a, Dirk Michalik b, Elke Hammer a y, Frieder Schauer a,
,
Uwe Bornscheuer c, Marlen Schmidt c
*
a Department of Applied Microbiology, Institute of Microbiology, Greifswald University, Friedrich-Ludwig-Jahn-Str. 15a, D-17487 Greifswald, Germany
b Leibniz Institute for Catalysis, Albert-Einstein-Str. 29a, D-18059 Rostock, Germany
c Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Greifswald University, Felix-Hausdorff-Str. 4, D-17487 Greifswald, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 August 2011
Received in revised form 18 September 2011
Accepted 28 September 2011
Available online 6 October 2011
Five laccase genes from Pycnoporus cinnabarinus and Trametes versicolor encoding for different iso-
enzymes have been cloned, recombinantly expressed and characterized. Following CeN coupling of
primary linear, branched-chained and cyclic amines to 3-methylcatechol was mediated by native and
recombinant laccases yielding the corresponding secondary amines. Formation of C5-monoaminated
ortho-methylquinones occurred within 1e2 h; prolonged incubation led to the formation of high-
molecular mass products. No difference between the use of native or recombinant isoenzymes from P.
cinnabarinus or T. versicolor was observed. Optimization of the reaction conditions included variation of
amine donor ratios, pH, amount and type of enzyme preparations. The formation of by-products could be
suppressed at pH values corresponding to the enzymes optima (pH 4e5). A total of 10 secondary amines
were synthesized with product formations of up to 80%. Furthermore, all purified secondary amines were
characterized by NMR-, LCeMS- and HRMS-analysis and log P values were determined.
Keywords:
Laccase
Recombinant
CeN coupling
3-Methylcatechol
Secondary amines
log P
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
recombinant expression. Thus, recombinant production of laccases
is important. Indeed they could already be successfully expressed in
The synthesis of fine chemicals using biocatalysts represents an
efficient alternative to conventional chemical routes and the in-
vestigation of various enzyme classes lead to widespread applica-
tions in industry.1 Laccases (p-diphenol:dioxygen oxidoreductases,
EC 1.10.3.2) catalyze the oxidation of aromatic compounds, espe-
cially of substituted phenols and anilines, whilst molecular oxygen
is reduced and water formed without the need of a cofactor and
harsh reaction conditions.2,3 Many laccases have been studied and
they exhibit a broad temperature and pH range (usually pH 4e9),4
are stable in organic solvents5 and towards proteolytic enzymes as
well.6
In principle laccases can be easily obtained by cultivation of
wild-type organisms, but often a mixture of isoenzymes is received,
which might be problematic for a biocatalytic application as it was
described for pig liver esterase isoenzymes.7 At the same time high
amounts of enzymes are needed for application in large-scale
synthesis and therefore enzymes are commonly produced by
a range of heterologous hosts, for example, fungal laccases in Pichia
pastoris, Saccharomyces cerevisiae, Aspergillus sp. or Trichoderma sp.,
and bacterial laccases in Escherichia coli.8 However, fungal laccases
were not expressed in E. coli so far and known recombinant ex-
pression systems are not sufficient or still too expensive for in-
dustrial applications.
The substrate scope of laccases is very broad. Beside activity
towards para-dihydroxylated substrates, they also convert ortho-
dihydroxylated compounds, monophenols and nonphenolic sub-
stances with a wide range of substituents.6a,9 In the presence of
oxygen, laccases mediate an oxidation of the substrate molecule
generating reactive radicals, which usually undergo homo-
molecular coupling generating polymers.10 More interesting for
synthesis is the heteromolecular coupling, where an acceptor
molecule reacts with the radical forming new CeC-, CeO-, CeN- or
CeS-coupling products in a non-enzymatic step.11
In the present study, we investigated the laccase-mediated het-
eromolecular coupling of an ortho-dihydroxylated substrate mole-
cule, 3-methylcatechol (1), with primary linear amines with alkyl
chain lengths ranging from C4 (n-butylamine) to C9 (n-nonylamine)
(2aef), the branched-chained compounds 2-ethyl-1-hexylamine
(2g), (R)-2-aminohexane (2h), 2-amino-5-methylhexane (2i), and
the cyclic (R)-(þ)-bornylamine (2j) to obtain the pharmaceutically
* Corresponding author. Tel.: þ49 3834 86 22819; fax: þ49 3834 86 4373; e-mail
address: marlen.schmidt@uni-greifswald.de (M. Schmidt).
y
Present address: Department of Functional Genomics, Institute of Genetics and
Functional Genomics, Friedrich-Ludwig-Jahn-Str. 15a, Greifswald University,
D-17487 Greifswald, Germany
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.09.123

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S. Herter et al. / Tetrahedron 67 (2011) 9311e9321
Table 1
valuable secondary amines 3aej. These reactions were performed
using native and recombinant laccases from the white-rot fungi
Pycnoporus cinnabarinus or Trametes versicolor and various reaction
parameters were studied.
Summary of isolated laccase isoenzymes, their comparison with published se-
quences and expression levels in Pichia pastoris as secreted enzyme, using the alpha
factor of S. cerevisiae as signal sequence into the culture supernatant containing
0.3 mM CuSO4 at 20 ꢀC or 30 ꢀ
C
In a previously published example, a structural analogue com-
pound, the secondary amine 4-butylamino-5-methyl-ortho-qui-
none (MLTQ), acting as a model cofactor for lysyl oxidase12 and as
a cross-linking inhibitor on osteolathyrism induction,13 was syn-
thesized using 4-methylcatechol and n-butylamine and the
chemical coupling mediator sodium iodate.14 Furthermore Burzio
and Waite examined the synthesis and structural characterization
of mushel adhesive proteins and their cross-linking mediates by
tyrosinase and byssal catechol oxidase in mixtures containing
4-methyl- and 4-ethylcatechol with several amino acids.15 In order
to design potential biomaterials against the background of mushel
adhesive proteins, Mikolasch et al. investigated the laccase-
Laccase Alternative Analogous
Amino acid
substitutions [mU mgprotein]
Enzymeꢁa1ctivities
isoenzyme name22
GenBank
accession no.
30 ꢀC
16
20 ꢀ
423
C
TvL5
Lccb
Lcca
Lccg
CAA77015
AAW2942
Asp75Asn
Asp519Tyr
Phe350Leu
Asp360Asn
Ala155Pro
Gly249Asp
Asp520Gly
Ile71Val
TvL10
TvL13
728
n.d.
168
n.d.
Q12717a or
AAC49829
TvL20
PcL35
Lcc
d
Q12719 or
CAA59161
n.d.
15
n.d.
364
Ile186Val
Thr499Ala
Ala285Val
catalyzed cross reaction between L-lysine or lysine-containing
d
AAF13052.1
or AAG13724.1
peptides and dihydroxylated aromatics, for example, ortho-dihy-
droxylated hydrocaffeic acid.16 Besides this, novel cephalosporins,
penicillins and carbacephems, inhibiting the growth of several
Gram positive bacterial strains, as the methicillin-resistant Staph-
ylococcus aureus strains and vancomycin-resistant Enterococci,
were synthesized by amination of 3- and 4-methylcatechol with
n.d.: not detectable.
a
Protein sequence of Q12717 has 99% identity to BAA23284, which corresponds
to the described Lccg. Q12717 and BAA23284 differ additionally in three amino
acids, while Q12717 consists of 527 amino acid and BAA23284 of 526.
amino-
b
-lactams using laccase from Trametes sp.5b ortho-Quinones
expression of laccase PcL35, while in case of TvL10 expression was
lowered (Table 1). In case of TvL13 and TvL20 only marginal lac-
cases activities could be observed, which were only detectable by
activity staining with ABTS in native polyacrylamide gel electro-
phoresis. An explanation might be the assumed lower protein level
of laccases due to the constitutive expression in comparison to an
induced and controlled expression using the AOX3 promotor. An-
other reason can be seen in the three amino acid substitutions for
both isoenzymes in comparison to the known protein sequences as
shown in Table 1.
Further characterization of laccase isoenzymes was only per-
formed with TvL5, TvL10 and PcL35, since the activity of the other
laccases was too low. The pH optima of native (laccase isoenzyme
mixture) and recombinant laccases from T. versicolor and P. cinna-
barinus were very similar. All of them showed two optima, one at
pH 2 using phosphate buffer and one at pH 4 in acetate buffer, in
which activities of approx. 50e60% could be detected in compari-
son to activity in phosphate buffer (pH 2). The three recombinant
isoenzymes showed as well a higher relative activity in maleate
buffer at pH 3 than the native isoenzyme mixture, but lower rela-
tive activity in Tris or Bis-Tris buffer at pH 6 or 6.5.
are known for their biological activity, like acting as oxidants
generating oxidative stress and as electrophiles forming covalent
adducts with cellular macromolecules and partially intercalate
with DNA, and thus a range of pharmacokinetic effects had been
discussed.17,18 In addition, secondary amines and arylalkylamine
structures are motifs in various drugs like b
-blockers19 and hallu-
cinogens20 and were applied in medication of neuropathic pain
causing antidepressant effects.21 Therefore, a further part of this
work deals with the determination of log P values of products 3aef
to estimate their distribution in biomembranes or cytosolic areas
for potential applications in the pharmaceutical area.
2. Results and discussion
2.1. Biochemical properties of native and recombinant
laccases
First, four different laccase encoding genes from T. versicolor
SBUG-M 1050 and one laccase gene from P. cinnabarinus SBUG-M
1044 were isolated, cloned and sequenced. The four isoenzymes
from T. versicolor (TvL) showed 68e76% amino acid sequence sim-
ilarity and were identical to the isoenzymes described recently
(Table 1).22 Unfortunately, only one laccase isoenzyme gene could
be isolated from P. cinnabarinus, although several protein-, DNA-
and mRNA-sequences with 72e99% homology are known from
literature.23
All five genes were cloned into pET22b(þ) without their native
signal peptide for a recombinant expression in E. coli. Regrettably,
soluble expression of active laccase could not be achieved despite
the use of various E. coli strains, co-expression with different
chaperones (Takara), or fusion-tags (data not shown). Therefore all
laccase genes were cloned for a constitutive expression in pGAP-
Zalpha_B, using the alpha secretion factor of S. cerevisiae, instead of
their native signal peptide, for a secretion into the culture super-
natant. TvL5, TvL10 and PcL35 could be expressed in sufficient
amounts at 30 ꢀC and with an addition of 0.3 mM CuSO4 (Table 1).
When adding 0.3 mM CoSO4 to the culture media, 75% and without
any addition, still 50% of laccase activity compared to the addition
of CuSO4 (100%) could be obtained. In comparison to the group of
Urlacher22 the activity of TvL5 could not be increased by using its
native signal peptide, but by cultivation at 20 ꢀC a 25-fold increase
was reached. The same observation was made in the recombinant
All studied laccases were stable from 4 to 45ꢀC during in-
cubation for 3h and remained stable from 4 to 30ꢀC after incubation
for 16 h. After 16 h incubation at 45 ꢀC activity decreased to approx.
50%, and after 3 h incubation at 60 ꢀC all isoenzymes were com-
pletely inactive. Stability of PcL35 at 65 ꢀC was further analysed and
it could be seen that activity increased after incubation for 2 min at
65 ꢀC (due to a maturation process24), but decreased drastically
within further incubation; after 30 min the laccase was completely
inactive.
Furthermore the methanol stability of laccases was investigated
as this alcohol is often required to dissolve substrates in the
aqueous reaction system. As expected, activity decreased with in-
creasing methanol concentrations using native laccase from
P. cinnabarinus and recombinant PcL35, which was more pro-
nounced for the latter (Fig. S1 in Supplementary data). An expla-
nation might be the different glycosylation pattern of native and
recombinant enzymes. In comparison, activity of the native iso-
enzyme mixture of T. versicolor increased with increasing methanol
concentration up to 20e30% (v/v), at concentrations above 30% the
activity also started to decrease until 80% of methanol, where lac-
cases are completely inactive. The same effect was observed for the
commercial available laccase from Myceliophthora thermophila.

S. Herter et al. / Tetrahedron 67 (2011) 9311e9321
9313
The two recombinant laccases from T. versicolor showed the same
behaviour as PcL35 and displayed strong negative correlation of
activity with increasing methanol concentrations until 50%, where
both laccases were completely inactive.
addition reactions of water or CH3OH from stock solutions and/or
reaction milieu to activated aromatics. Unfortunately, a definite
evidence for the occurrence of the non-substituted o-methyl-
benzoquinone could not be achieved in HPLC analysis as it was
described for other para-dihydroxylated enzyme substrates.25
Nevertheless, in time-course analysis an evidence for a displace-
ment reaction, where the methoxy group was replaced by the
amine group, exists. Similar nucleophile displacement reactions on
2.2. Synthesis of secondary amine structures via CeN
coupling
readily eliminable groups (e.g., methoxy substituents) of activated
Coupling-reactions between 3-methylcatechol (1) and the pri-
mary amines (2aej) were studied using native and recombinant
laccases from P. cinnabarinus SBUG-M 1044 and T. versicolor SBUG-
M 1050 and only marginal differences between the enzymes were
observed. Formation of the secondary amines (3aej) (Scheme 1)
was determined via HPLC analyses, additionally successful amina-
tion could also be monitored qualitatively by formation of yellow
and later red reaction mixtures.
26
€
hydroquinones were previously described by Schafer and Aguado
for reactions focused on a sodium iodate-mediated synthesis of
substituted 2,5-diamino-1,4-benzoquinones.
During the formation of secondary amines 3aej, the generation
of the magenta-coloured by-product 4 was monitored as well.
Compound 4 was unavailable for further heteromolecular coupling
with the used primary amines 2aej and instable during preparation
3
O
O
HO
HO
O
O
-
2a j
4
Laccase, MeOH, O2
-2H2O
2
R1
5
-MeOH
1
O
NH
6
-
3a j
1a
1
High-molecular mass products
&
5
6
O
R2
O
4
H2N
2g
R1 =
2a-f
H2N
n = 2 - 6
NH2
NH2
2i
2j
2h
NH2
R =
O-species or NH2
2
Scheme 1. Reaction pathway for the laccase-mediated heteromolecular coupling of 3-methylcatechol with primary amines 2aej aiming at the synthesis of secondary amines 3aej.
In the HPLC analysis, the decrease of the concentration of 1 was
detected due to simultaneous appearance of a yellow coloured
product 1a, which was subsequently converted into the hetero-
molecular coupling products 3aej and partially to the by-product 4
(Fig. S2 in Supplementary data). Efforts in the structural charac-
terization of compound 1a via NMR-spectroscopy were not suc-
cessful, because during sample preparation by dehydration via
lyophilisation, 1a decomposed into several other products. How-
ever, LCeMS-analysis in a mixture of 50% methanol and 50% acetic
acid in water (0.1%), revealed a defined single peak with a molecular
mass of 153.1 g moLꢁ1 [(MþH)þ]. The corresponding mass
for a sodium addition (175.1 g moLꢁ1, [MþNa]þ) could be observed
as well (Table S1 in Supplementary data). Therefore, we postulate
for the structure of 1a the activated 3-methylbenzoquinone
substituted by electrophile addition with a methoxy group. Since
reactants were prepared as methanolic stock solutions, we propose
that this low-molecular mass substituent was preserved from
methanol, which was coupled non-enzymatically to the ortho-
methylbenzoquinone as already described for 2,5-dihydroxy-N-(2-
hydroxyethyl)-benzamide by Manda et al.,25 who also determined
for NMR analysis as it was already described for 1a. The by-product
resulted in identical UV-absorption spectra as the secondary
amines 3aej, suggesting a 3-methylbenzoquinone’s substitution at
the cyclic ring system and the formation of the ortho-quinoid
structure same position determined for the synthesized secondary
amine products, but with a low-molecular mass substituent caus-
ing significantly more hydrophilic characteristics (Table S1 in
Supplementary data). LCeMS-analysis of a fraction solely contain-
ing compound 4, obtained from solid phase extraction, resulted in
a peak possessing a molar mass of 138.1 g moLꢁ1 [(MþH)þ] and
a
molecular mass for the corresponding sodium adduct
(160.1 g moLꢁ1, [MþNa]þ) (Table S1 in Supplementary data), which
indicates a substitution of the ortho-methylbenzoquinone with
a low-molecular mass substituent also originating from reaction
milieu. Referring to the estimated molar mass of product 4
(137 g moLꢁ1) in comparison to the activated substrate molecule
(122 g moLꢁ1), the mass difference between both leads to the as-
sumption of an addition of either one NH2-group or a certain kind
of oxygen species. The structure of compound 4 could not be ter-
minatory identified due to it’s instability within further preparation

9314
S. Herter et al. / Tetrahedron 67 (2011) 9311e9321
steps, however a displacement reaction between this ortho-meth-
ylbenzoquinone’s low-molecular mass substituent and the amine
group of coupling agents had never took place as it was examined
for compound 1a. Therefore, generation of product 4 was nega-
tively affecting the yields of the desired secondary amines.
Regardless to the by-product formation, maximum concentra-
tions of secondary amines were observed after 1e2 h, depending
on the used laccase preparation, the pH of sodium acetate buffer
and the concentrations of reactants. However, prolonged in-
cubation led to the formation of the high-molecular mass coupling
products 5 and 6 (Fig. S2 in Supplementary data), exclusively oc-
curring in heteromolecular transformation reactions. Attempts to
isolate these high-molecular mass products as stable pure com-
pounds or in a mixture were not successful due to products high
reactivity and therefore structural data cannot be provided within
this study.
Generally, formation of secondary amines could be increased
with higher amounts of aliphatic coupling agents 2aej and best
results were observed in reactions with a ratio of 1 and primary
amines of 1e5 mM (Table 2, Fig. 1). For example, the product for-
mation of 3f could be increased by 30%, of 3g by 18%, of 3h by 8%
and 3j by 26% by using 5 mM instead of 1 mM amino donor. Further
increases were observed when using directly 5 mM of the amino
donor instead of the methanolic stock solutions, in case of forma-
tion of 3g a 12% enhancement and for 3i a 16% enhancement could
be monitored (Table 2). The direct use of amino partners in 10 mM
and 40 mM concentration ranges resulted in an additional increase
of product generation, as determined for 3h (þ9%) and 3i (þ34%).
Complementary to those findings, the use of the amino donor in
excess resulted in a suppressed formation of the by-product 4.
The use of larger amounts of enzyme activities gave only mar-
ginally increased product formation by 5%. Nevertheless, laccase
units above 4 U mLꢁ1 caused a decreased formation of secondary
amines due to increasing generation of by-product 4 and high-
molecular by-products 5 and 6 (data not shown). Interestingly,
when using low activities of recombinant PcL35 (0.2 U mLꢁ1), a si-
multaneous appearance of a further product (7) was detected.
Based on its UVevis-absorption characteristics (lmax 262 nm), it
appears similar to the UVevis-absorption behaviour of 1 (lmax
274 nm) rather than to the secondary amine structure (lmax 220,
298, 490 nm). However, with further incubation product 7 was
converted into the desired secondary amine. When increasing
PcL35 activity up to 0.4 U mLꢁ1 again only the targeted products
3aej were detected.
According to literature known pH optima and optima based on
this study, all biocatalyses were performed in corresponding buffer
Table 2
Influence of amine donor 2aej concentrations (methanolic stock solutions, v/v), enzyme units used and type of laccase preparation on the conversion of the laccase substrate,
formation of secondary amines 3aej in 5 mL scale
Amino
donor
Ratio of 1 to
amino donor
[mM]
Enzyme
(U mLꢁ1
Laccase
preparation
Conversion of 1 [%]
Formation of
monoaminated
productb [%]
a
)
40 min
120 min
2a
2b
2c
1:5
1:5
2
1
Native PcL
Native PcL
PcL35
74
60
100
10
16
93.5
1:5
1:5
1:5
1:5
1
1.5
2
20
37
44
56
72.2
76.3
81.5
72.6
0.5
1
1
0.5
Native PcL
2
2d
1:5
1:5
1:5
0.5
0.5
0.5
TvL5
TvL10
Native PcL
41
39
33
90.3
79.7
81.4
16
12
14
2e
2f
1:5
2
Native PcL
57
100
58
1:5
1:5
1:5
1:5
1:1
1:5
0.015
0.03
0.05
0.15
1
TvL5
TvL5
TvL5
TvL5
Native PcL
Native PcL
2
7
20
46
94
88
18.6
24.5
32.4
96.5
100
100
0
0
0
19
15
46
1
2g
2h
2i
1:1
1:5
1
1
1
1
1
2
Native PcL
Native PcL
94
100
89
93
92
89
100
100
100
100
100
100
12
30
42
35
29
30
1:5c
1:10c
1:40c
1:40c
1:1
1:5
1
1
1
2
4
1
84
98
84
92
100
93
100
100
100
100
100
100
16
23
19
18
25
29
1:5c
1:5c
1:5c
1:10c
1:1
1
1
1
1
Native PcL
Native PcL
98
100
67
100
100
100
100
37
30
47
81
1:5
1:5c
1:10c
88
2j
1:1
1:5
1:5c
1
1
1
99
100
95
100
100
100
18
45
45
a
Based on ABTS assay.
b
c
Evaluated yields of product formation [%] compared to a 100% conversion into desired compounds 3aej, estimated in HPLC analysis of 5 mL scale in reaction after 2 h.
In selected reactions instead of applying methanolic stock solutions the direct use of amine donors was tested.

S. Herter et al. / Tetrahedron 67 (2011) 9311e9321
9315
Fig. 1. Effect of concentrations of aliphatic amines on substrate consumption (1 mM amine: open square; 5 mM amine: closed square) and product formation (1 mM amine: open
circle; 5 mM amine: closed circle) in a 5 mL scale, exemplary shown for the reaction between 3-methylcatechol and n-nonylamine mediated by 2 U mLꢁ1 native P. cinnabarinus
laccase (20 mM sodium acetate, pH 5).
systems to assure catalytic efficiency and preventing acidification
by release of protons due to enzymatic substrate activation. In-
dependent from an optimal catalytically environment of laccases,
also the physicochemical nature of 1 referring to the pH of reaction
milieu resulted in a significant influence on the formation of the
desired secondary amine structures 3aej. According to this, in ex-
periments using M. thermophila laccase in 20 mM phosphate citrate
buffer at pH 7 higher amounts of by-products were observed in
comparison to reactions at lower pH ranges (data not shown).
Therefore, application of laccases preferring slightly acidic pH
values (pH 4) reduced the formation of by-products such as 4, since
a higher amount of ortho-dihydroxylated enzyme substrate is
available for synthesis of monoaminated target products.
Consequently, reaction progress and yields are also based on
reactant properties, which are positively affected by the pH optima
of the laccases from P. cinnabarinus and T. versicolor. An increased
by-product formation was also observed in the synthesis of 3d and
3f using small amounts of recombinant isoenzymes TvL5 and TvL10
at pH 4, (Fig. 2), whereas increased enzyme activity resulted in
increased product formation.
Fig. 2. Formation and decrease of desired secondary amine (filled circle) due to increasing accumulation of high-molecular weight products 5 (closed triangle) and 6 (open triangle)
in comparison to substrate consumption (filled square) in a reaction of 1 mM 3-methylcatechol and 5 mM n-heptylamine catalyzed by recombinant TvL10 (20 mM sodium acetate,
pH 4) (mAu¼milliabsorption units).

9316
S. Herter et al. / Tetrahedron 67 (2011) 9311e9321
In this study, we mainly focused on the optimization of
lipophilic biomembranes and aqueous cytosolic areas.28 Further-
more, it is accepted as an important parameter in QSAR-studies
(quantitative structure activity relationships) and in areas of de-
signing pharmaceutical active compounds focused on their char-
acteristics like absorption, biological availability, metabolism and
toxicity.29 As ortho-dihydroxylated benzenediols are commonly
described as pharmaceutical valuable compounds,30 showing ac-
tivities as analgesics31 and other interesting physiological activities,
for example, as extremely important pharmacophores in numerous
biologically active compounds,32 log P of the synthesized alkyl
aminated ortho-benzoquinones 3aef was of great interest (Table 4).
The log P of the secondary amines with increasing chain
length resulted in an increased hydrophobicity of the laccase-
mediated reaction products, and therefore each product should
elute in-between the hydrophilic and hydrophobic reference
substances. Accordingly, the use of three different hydrophobic
references was essential. A comparison of the log P data derived
from chromatographic analysis with theoretical log P values,
obtained from ACD/ChemSketch, suggested 2-(4-hydroxyphenyl)
ethylalcohol as hydrophilic standard while 3-methylcatechol
(log P¼0.75) was used as second reference. The newly synthe-
sized secondary amines with C4 and C5 alkyl substituents (3a and
3b) were determined to be more hydrophilic (log P¼ꢁ0.75 to
0.41). In contrast, amination of 1a with alkyl substituents with
longer alkyl chain length (3cef) led to rather high log P values up
to 3.64. Generally, an additional methylene group enhanced log P
about 0.89ꢂ0.21.
product formation detectable by HPLC for the synthesis of novel
secondary amines 3aej. With increased amino donor molarities
(5e40 mM) and more enzyme units, the formation of 3ee3j in
good to high amounts (up to 81%) within only 2 h (Table 2) were
possible.
2.3. Structural characterization of secondary amines
Structural characterization of the secondary amine products is
exemplary explained here for compound 3b (Table 3). The signals
appearing at 5.53 ppm and 6.74 ppm in the 1H NMR spectrum of 3b
could be assigned to H-6 and H-4, respectively. Within this, the signal
of H-4 exists as a broad signal according to its long-range coupling to
meta-positioned H-6 and further methyl protons of C-100 as also in-
dicated by coupling constants (Table 3). The coupling of n-pentyl-
amine tothe C5positionisclearlyconfirmedbythecorrelationofH-10
protons with C-5. The presence of a quinoid, hydroquinoidorquinone
imine structure was proved by 13C and HMBC data. As quinones
revealed low-field shifted resonance signals (180 ppm up to
190 ppm), 3b was identified as a 1,2-quinoid structure according to
13C NMR signals at 175.4 ppm for C-1 and 185.7 ppm for C-2 as de-
scribed by Mikolasch et al.5b for the amination of catecholsand for the
hybrid dimer quinone production from 3,4-dichloroaniline with
protocatechuic acid and syringic acid.27 Additionally, the HMBC
spectra confirmed the presence of a 1,2 quinoid structure as in 3-
methyl-5-(pentylamino)-1,2-benzoquinone.
Table 3
1H and 13C NMR assignments and HMBC/1H,1H COSY correlations for 3ba
Structure of 3b
Pos.
13C
1H
1H,13C correlations (HMBC)
1H,1H COSY
4
6
133.5
94.6
45.1
29.0
6.74, br, 1H
C-2 (185.7), C-6 (94.6), C-100 (15.6)
C-1 (175.4),b C-2 (185.7), C-4 (133.5)
C-5 (160.7), C-20 (29.0), C-30 (30.3)
C-10 (45.1), C-30 (30.3), C-40 (23.5)
H-30/H-40 (1.40)
H-4 (6.74)
5.53, d, 1H (2.5)
3.33, t, 2H (7.2)
1.71, m, 2H
10
20
H-20 (1.71)
H-10 (3.33), H-30/H-40
(1.40)
H-20 (1.70), H-50 (0.96)
30
40
30.3
23.5
14.4
15.6
1.40, m, 4H
C-10 (45.1),b C-30 (30.3), C-40 (23.5),
C-50 (14.4)b
C-30 (30.3), C-40 (23.5)
50
0.96, t, 3H (7.0)
1.98, d, 3H (1.3)
H-30/H-40 (1.40)
H-4 (6.74)
100
C-4 (133.5), C-2 (185.7), C-3 (142.7)
a
Chemical shifts are expressed in
Correlation with low intensity.
d
(ppm) referred to TMS (calibration was done on solvents signals:
d
MeOH-d4¼3.31 (1H), 49.0 (13C)). J values are in hertz in brackets.
b
Table 4
log P values determined in comparison to the theoretical log P values for the secondary amines 3aef
Compound
log Pa
Theoretical log P
log P difference (eCH2)
3,5-Dihydroxybenzoic
acid
2-(4-Hydroxyphenyl)
ethyl alcohol
ACD/ChemSketch
HPLC
Theoretical
1
3-Methylcatechol
ꢁ0.007
ꢁ2.15
ꢁ0.58
0.77
1.77
n.d.
0.75
ꢁ0.75
0.41
1.29
2.02
1.34
1.34
1.88
2.41
2.94
3.47
4.00
3a
3b
3c
3d
3e
3f
3-Methyl-5-(butylamino)-1,2-benzoquinone
3-Methyl-5-(pentylamino)-1,2-benzoquinone
3-Methyl-5-(hexylamino)-1,2-benzoquinone
3-Methyl-5-(heptylamino)-1,2-benzoquinone
3-Methyl-5-(octylamino)-1,2-benzoquinone
3-Methyl-5-(nonylamino)-1,2-benzoquinone
1.16
0.88
0.73
0.59
1.03
0.53
0.53
0.53
0.53
0.53
2.61
3.64
n.d.
n.d.: not determined.
a
log P according to Donavan and Pescatore.33
2.4. Evaluation of log P and product stability
Stability studies for 3aej showed that they remained stable as
dry chemicals at room temperature and 4 ꢀC. Furthermore, stability
in organic solvents controlled via HPLC analysis confirmed stability
in methanol and acetonitrile, while they decomposed and lost their
typical reddish colour in DMSO or THF.
Since log P is used for quantitative description of the hydro-
phobicity of compounds, it can be used as a key criterion of phar-
macokinetic properties by means of dispersive behaviour in

S. Herter et al. / Tetrahedron 67 (2011) 9311e9321
9317
FW_AF025481: ATGTCCAGATTCCAATCTCTCC
3. Conclusion
RV_AF025481: TCAGAGATCGCTGGGGTCAA
FW_AF123571: ATGATCAGCATGGGCTTCCG
RV_AF123571: CTAATGGTCAGACTCCGGGA
In conclusion, we demonstrate the use of native and several
recombinant laccase isoenzymes for the synthesis of a variety of
secondary amines, which are potential biologically active com-
pounds and thus of great interest for pharmaceutical industry.
Product formation could be increased by applying excessively con-
centration of the amino donors set either as a methanolic stock
solution, but ideally used directly in ranges from 5 to 10 mM.
However, preventing formation of a two-phase system due to
hydrophaty of alkylamines, enzyme substrate molecule 1 was al-
ways introduced as a methanolic stock solution in a 1 mM concen-
tration (v/v). Based upon reaction kinetics, maximal yields of
secondary amines were recovered after 2h using 1 U mLꢁ1 of es-
pecially native laccase preparations. Referring to this, increase of
enzyme activities were found to repress concentration levels of
aimed synthesis products due to a simultaneous elevation of low-
molecular mass by-product 4. Since the herein studied laccases
are stable in methanol to a certain extent and no alternative
chemical methods for the product synthesis exist, the introduced
laccase-catalyzed coupling reaction, progressed at enzymes optimal
pH ranges of pH 4 and 5, respectively, presents the general appli-
cability of recombinant and native enzymes in organic synthesis and
mayserve as a tool in cases where chemical methods are exercisable.
Primers used for isolation of T. versicolor isoenzymes:
FW_Y1801: ATGTCGAGGTTTCACTCTCTTCTCGCTTTCGTCG
RV_Y1801: TTACTGGTCGCTCGGGTCGCGCGCG
FW_AY693776: FTV2: ATGGGTCTGCAGCGATTCAGCTTCTTCGTCACCC
RV_AY693776: TCACTGGTTAGCCTCGCTCAGCCCG
FW_AB212734: ATGGGCAAGTTTCACTCTTTTGTGAACG
RV_AB212734: TCAGAGGTCGGACGAGTCCAAAGCACCG
FW_AB212733: ATGGGCAGGGTCTCATCTCTCTGCGCGC
RV_AB212733: TTAGAGGTCGGATGAGTCAAGAGCGTTGTACG
FW_AM422387: ATGGGCAGGTTCTCATCTCTCTGCGCGC
FW_AF414109: ATGTCGAGGTTTCACTCTCTTTTCGCTTTCGTCG
The PCR product was analysed on a 1% agarose gel and a 1.5 kb
fragment was isolated and purified via QIAquick Gel Extraktion Kit.
The pure fragment was ligated into pCRÒII_TOPOÒ vector, which
was used for transformation of E. coli DH5alpha. Transformands
were plated on agar plates containing X-gal for a blue-white
screening. White colonies were used for a colony PCR to identify
clones bearing laccase encoding fragments. Appropriate colonies
were used for plasmid isolation and sequencing. Laccase encoding
fragments were used for cloning into the vectors pET22b(þ) for
expression in E. coli and pGAPZalpha_B for expression in yeast,
where the native signal peptides were removed for expression in E.
coli and replaced by the alpha-mating factor signal peptide of S.
cerevisiae for expression in yeast. Therefore laccase encoding genes
were amplified using standard PCR protocols and the following
primers for the expression in E. coli without their native signal
peptides:
4. Experimental section
4.1. General
All chemicals were purchased from Fluka (Buchs, Switzerland),
Sigma (Steinheim, Germany) and Merck (Darmstadt, Germany)
unless stated otherwise. Restriction enzymes, ligase, polymerases,
cDNA preparation kits, reagents for DNA isolation and purification
were obtained from New England BioLabs GmbH (Frankfurt am
Main, Germany), Fermentas (St. Leon-Roth, Germany) and Qiagen
(Hilden, Germany), all primers, cloning and expression vectors, as
well as Pichia strains from Invitrogen (Darmstadt, Germany). Se-
quencing was performed at GATC (Konstanz, Germany).
TvL5 forward: GGCGTATACATATGGGTATCGGTCCTGTCGCCG
TvL5 reverse: GTGGTGCTCGAGTGCGGCCGCCTGGTAGCTCGGGTCGC
GCGCG
TvL10 forward: GGCGTATACATATGGCCATCGGGCCGGTGGCG
TvL10 reverse: GTGGTGCTCGAGTGCGGCCGCCTGGTTAGCCTCGCTC
AGCCCG
4.2. Isolation of native laccases
TvL13 forward: GGCGTATACATATGGCGATTGGGCCCGTCACCG
TvL13 reverse: GTGGTGCTCGAGTGCGGCCGCGAGGTCGGACGAGTCC
AAAGCACCG
TvL20 forward: GGCGTATACATATGGCTATCGGGCCTGTGACCGACC
TvL20 reverse: GTGGTGCTCGAGTGCGGCCGCGAGGTCGGATGAGTC
AAGAGCG
PcL35 forward: GGCGTATACATATGGCCATAGGGCCTGTGGCGG
PcL35 reverse: GTGGTGCTCGAGTGCGGCCGCGAGGTCGCTGGGGT
CAAGTGC
Laccases from the white-rot fungus P. cinnabarinus SBUG-M
1044 were produced with 3,4-dimethoxybenzyl alcohol (10 mM)
as an enzyme inducer, isolated and purified as recently described
by Kordon et al.34 Under these conditions P. cinnabarinus produced
laccase as a single extracellular enzyme with an activity of
0.5 U mLꢁ1. Laccases from T. versicolor SBUG-M 1050 were obtained
as described earlier by Jonas et al.35 after 9 d of cultivation in
nitrogen-rich medium with
a maximal recovered activity of
0.5 U mLꢁ1 in cell-free culture supernatants.
All PCR products were cloned into pET22b(þ) using restriction
enzymes NdeI and NotI. For cloning of laccase isoenzymes into
pGAPZalpha_B for expression in P. pastoris without their native
signal peptides and secretion via alpha factor of S. cerevisiae all
laccase encoding genes were amplified using standard PCR pro-
tocols and the following primers:
4.2.1. Cloning of recombinant laccases. During isolation of laccases
from their native white-rot fungi, whole cells from culture super-
natant were separated by filtration through a glass fibre filter in
a Buchner funnel and used for total RNA isolation by using TRI
REAGENTÔ. Afterwards polyA RNA was enriched with OligotexÒ
and cDNA preparation was performed using the ProtoscriptÒ First
Strand cDNA Synthesis Kit. The second DNA strand was then syn-
thesized via PCR and the following primer, based on literature
known laccase sequences, in all possible combinations of forward
(FW) and reverse (RV) primers.
TvL5 forward: GCTGCAGGAATTCTGGGTATCGGTCCTGTCGCCG
TvL5 reverse: GAAAGCTGGCGGCCGCCTGGTAGCTCGGGTCGCG
TvL10 forward: GCTGCAGGAATTCTGGCCATCGGGCCGGTGGCG
TvL10 reverse: CTAGAAAGCTGGCGGCCGCCTGGTTAGCCTCGCTC
AGCC
Primers used for isolation of P. cinnabarinus isoenzymes:
TvL13 forward: CTCGAGCCGCGCGGCGGCCGCTGGCGATTGGGCCC
GTCACCGAC
TvL13 reverse: CGCTTGTTCTAGAAAGAGGTCGGACGAGTCC
FW_AF170093: ATGTCGAGGTTCCAGTCCCTC
RV_AF170093: TCAGAGGTCGCTGGGGTCAA

9318
S. Herter et al. / Tetrahedron 67 (2011) 9311e9321
TvL20 forward: GCTGCAGGAATTCTGGCTATCGGGCCTGTGACCG
TvL20 reverse: CTAGAAAGCTGGCGGCCGCGAGGTCGGATGAGTCA
AGAGC
PcL35 forward: GCTGCAGGAATTCTGGCCATAGGGCCTGTGGCGG
PcL35 reverse: GAAAGCTGGCGGCCGCGAGGTCGCTGGGGTC
amines 2gei and cyclic (R)-(þ)-bornylamine 2j were performed
in sealed 6 mL brown glass flasks in a final reaction volume of 5 mL
at room temperature with agitation at 200 rpm in presence of at-
mospheric oxygen. For enrichment of the expected hetero-
molecular products, reactions were scaled up in 100 mL
Erlenmeyer-flasks, which were protected against light, containing
50 mL of reaction mixture using the same conditions as already
described. Educts were prepared as 50 mM methanolic stock so-
lutions and biocatalysis conducted either in equimolar concentra-
tions of the aromatic or aliphatic amines (1 mM) or with an excess
of the aliphatic coupling agents (5, 10 or 40 mM). Reactions were
generally performed in sodium acetate buffer (20 mM) at pH 4 or
PCR products TvL5, TvL10, TvL20 and PcL35 were cloned into
pGAPZalpha_B using restriction enzymes EcoRI and NotI, TvL13 was
cloned using XbaI and NotI. Later, also constructs containing the
native signal peptide for a secretion while expressed in yeast, were
produced by cloning into pGAPZ_B. The laccase encoding genes
were amplified using standard PCR protocols and the following
primers:
5
34,35 depending on the pH optima of the native and recombinant P.
cinnabarinus and T. versicolor laccases.
TvL5 forward: CGACGAGGAATTCAAGCATGGCATCGAGGTTTCACT
CTCTTCTC
TvL5 reverse: CCGCTGGCGGCCGCTCTGGTAGCTCGGGTCGCG
TvL10 forward: CGACGAGGAATTCAAGCATGGGTCTGCAGCG
TvL10 reverse: CCGCTGGCGGCCGCTCTGGTTAGCCTCGCTCAGCC
TvL13 forward: GCGCGTACCTCGAGGATGGGCAAGTTTCAC
TvL13 reverse: CCGCTGGCGGCCGCTGGACGAGTCC
TvL20 forward: CGACGAGGAATTCAAGCATGGGCAGGTTCTC
TvL20 reverse: CCGCTGGCGGCCGCTGAGGTCGGATGAGTCAAGAGC
PcL35 forward: CGACGAGGAATTCAAGCATGGCATCGAGGTTCCAGT
CCCTC
4.6. Analytical HPLC
For kinetic studies, reaction mixtures were sampled in regular
intervals over 24 h and analysed via HPLC. Grading of analytes was
accomplished with a LiChroCart 125-4 RP-18 endcapped column of
5-mm particle size. The mobile phase consisted of an aqueous
component A (0.1% phosphoric acid) and a methanol component B
at an initial ratio of 10% B to 90% A, simultaneously raised up to
100% B within 14 min. The flow rate was always adjusted to
1 mL minꢁ1 with an injection volume of 40
mL.
PcL35 reverse: CCGCTGGCGGCCGCTGAGGTCGCTGGGGTC
PCR products TvL5, TvL10, TvL20 and PcL35 were cloned into
pGAPZ_B using restriction enzymes EcoRI and NotI, TvL13 was
cloned using XhoI and NotI.
4.7. Determination of log P
Lyophilized secondary amines 3aef were analysed for their
n-octanol/water partition coefficient using a modified log P method
as described by Donovan and Pescatore33 with instruments and
analytical parameters as already described. Chromatographic sep-
4.3. Recombinant expression of laccases in yeast
After cloning different laccase encoding gene fragments into the
shuttle vector pGAPZalpha_B, the plasmid was used for trans-
formation of P. pastoris X33 according to the manufacturer. Cells
aration was conducted on a 20 mmꢃ4.0 mm short HPLC-column of
ꢀ
5
m
m (250 A) particle size with an octdecyl-poly(vinylalcohol) ODP
were plated out on YPDS agar plates containing 100
cin. Colonies grown after approx. 3 d were inoculated on YPDS
plates containing 500, 1000 and 2000
g mLꢁ1 Zeocin to select
m
g mLꢁ1 Zeo-
50 matrix. Samples were eluted with an aqueous phase consisting
of phosphoric acid (0.1%) and methanol. Measurements were con-
ducted at an initial ratio of 10% methanol to 90% phosphoric acid,
whereby methanol concentration raised up to 100% in 9.4 min with
6 min equilibration time. The flow rate was adjusted to 1 mL minꢁ1
m
multi-copy recombinants. These clones were used for recombinant
constitutive expression according to the manufactures protocol.
Laccases were secreted into the culture supernatant by the alpha
secretion factor from the expression vector.
and injection volumes set to 5 mL. To get evidence of change in log P
by modifying the activated ortho-benzenediol with linking to pri-
mary amines 2aef consisting of increasing chain length,
3-methylcatechol as well as the synthesized dimeric compounds
3aef were individually combined in standard mixtures containing
hydrophobic (toluene, ibuprofen, triphenylamine) and also hydro-
philic (3,5-dihydroxybenzoic acid or 2-(4-hydroxyphenyl)ethyl al-
cohol) reference substances. Standard mixtures were prepared by
adding 20 mg of the hydrophilic substance to 2 mL of hydrophobic
reference and filled up to a final volume of 200 mL with methanol.
For log P determination, 0.5 mg of the product was diluted in 1 mL
of standard mixtures and subsequently measured. For arylalkyl
products 3a,b ranging from C4 to C5 side chains, toluene was set as
hydrophobic standard. Ibuprofen was used for products 3c,d car-
rying C6 and C7-substitutents, whereas triphenylamine was applied
for 3e,f with addition of n-octylamine or n-nonylamine. Examina-
tions were conducted in standard mixtures containing either 3,5-
dihydroxybenzoic acid or 2-(4-hydroxyphenyl)ethyl alcohol as
hydrophilic reference. With known log P values (Table S2 in
Supplementary data) of reference substances, n-octanol/water
partition coefficients of the secondary amines were calculated.
Products 3aef were analysed in triplicate. Results obtained from
log P measurements were validated with a software tool of ACD/
ChemSketch, whereby theoretical log P values of the products
could be estimated.
4.4. Activity measurements of laccases
Generally laccase activity was determined by monitoring the
oxidation of 2,20-azino-bis-(3-ethylbenzothiazoline-6-sulfonic
acid)diammonium (ABTS) spectrophotometrically at 420 nm
(
3
¼3.6ꢃ104 Mꢁ1 cmꢁ1) in sodium acetate buffer (20 mM pH 5) at
30 ꢀC as described earlier.35 One unit (1 U) is defined as the amount
of enzyme, which converts 1
say conditions.
mmol substrate per minute under as-
For estimation of pH optima, malate, sodium acetate, sodium
phosphate, Bis-Tris or Tris buffers (all 20 mM) were used. To de-
termine the stability and activity in methanol, the enzyme activity
was monitored by adding different methanol concentrations to the
sodium acetate buffer (20 mM, pH 5) followed by activity mea-
surements using the ABTS assay.
4.5. Laccase-mediated coupling of 3-methylcatechol with
aliphatic amines
Examinations on the laccase-mediated coupling of
3-methylcatechol with various linear primary amines ranging from
C4 (n-butylamine) to C9 (n-nonylamine), the branch-chained

S. Herter et al. / Tetrahedron 67 (2011) 9311e9321
9319
3
4
0
00
0
0
00
4.8. Isolation of heteromolecular coupling products
J1 ,2 ¼7.2 Hz, 2H, H-1 ), 1.98 (d, J4,1 ¼1.5 Hz, 3H, H-1 ), 1.70 (m, 2H,
H-20), 1.43 (m, 2H, H-30), 1.39e1.33 (m, 4H, H-30, H-40, H-50), 0.93 (t,
3
J5 ,6 ¼7.0 Hz, 3H, H-6 ). 13C NMR (150 MHz, MeOH-d4)
d
(ppm)¼
0
0
0
The secondary amines were purified by solid reverse phase
extraction with a silicagel column (Strata C18-E 60 mL Giga Tubes,
10 g absorbent material), progressively charged with 50 mL of the
reaction mixture. Homomolecular by-products were eluted with
50 mL of a solution composed of 50% methanol and 50% double
destilled water. The desired alkylamine substituted hetero-
molecular coupling products were obtained with mixtures consis-
tent of 60% methanol and 40% acetic acid (0.1% v/v). After elution,
the fraction containing the desired coupling product was diluted to
a final methanol concentration of 5% with bidestilled water and
frozen at ꢁ20 ꢀC for 24 h, followed by freezing at ꢁ80 ꢀC for further
4 h. Via lyophilisation, products were obtained as dry chemicals
and further available for aimed characterization studies.
185.8 (C-2), 175.8 (C-1), 160.5 (C-5), 142.7 (C-3), 133.7 (C-4), 94.7
(C-6), 45.1 (C-10), 32.6 (C-40), 29.3 (C-20), 27.8 (C-30), 23.6 (C-50), 15.4
(C-100), 14.3 (C-60). HMBC correlations see Supplementary data.
HPLC Rf 11.6 min. UVevis (MeOH) lmax 220, 298, 490 nm. LCeMS Rf
5.6 min (MeOH); API-ES: pos. mode [MþH]þ calculated m/z 221;
found m/z 222.1. HRMS (ESI) calcd for C13H19NO2 222.14886; found
222.14856.
4.9.4. 3-Methyl-5-(heptylamino)-1,2-benzoquinone (3d). Dark-red
solid. 1H NMR (600 MHz, MeOH-d44)
d
(ppm)¼6.74 (dq, 4J4,6¼2.7 Hz,
4
00
J4,1 ¼1.5 Hz, 1H, H-4), 5.53 (d, J4,6¼2.7 Hz, 1H, H-6), 3.33 (t,
3
4
0
00
0
0
00
J1 ,2 ¼7.2 Hz, 2H, H-1 ), 1.98 (d, J4,1 ¼1.5 Hz, 3H, H-1 ), 1.70 (m, 2H,
3
H-20), 1.45e1.29 (m, 8H, H-30, H-40, H-50, H-60), 0.91 (t, J6 ,7 ¼6.9 Hz,
0
0
4.9. Structural characterization by LCeMS, HRMS and NMR
studies
3H, H-70). 13C NMR (150 MHz, MeOH-d4)
d
(ppm)¼185.6 (C-2),160.5
(C-5), 142.6 (C-3), 133.6 (C-4), 94.5 (C-6), 44.8 (C-10), 32.7 (C-50),
29.8 (C-40), 29.0 (C-20), 27.9 (C-30), 23.5 (C-60), 15.3 (C-100), 14.3 (C-
70). HMBC correlations see Supplementary data. HPLC Rf 12.7 min.
UVevis (MeOH) lmax 220, 298, 490 nm. LCeMS Rf 6.2 min (MeOH);
API-ES: pos. mode [MþH]þ calculated m/z 235; found m/z 236.1.
HRMS (ESI) calcd for C14H21NO2 236.16451; found 236.16459.
The heteromolecular coupling products were characterized by
liquid chromatography coupled with mass spectroscopy. For sam-
ple ionization, the API-ES method was used in positive ion [MþH]þ
mode. All measurements were performed on a 1200 Series 6120
€
Quadrupole mass spectrometer (Agilent Technologies, Boblingen,
Germany). Grading of analytes was accomplished on a ZORBAX SB-
4.9.5. 3-Methyl-5-(octylamino)-1,2-benzoquinone
(3e). Dark-red
C18 column (2.1ꢃ50 mm) possessing a 1.8
m
m pore size (Agilent
solid. 1H NMR (600 MHz, MeOH-d4)
d
(ppm)¼6.74 (br, 1H, H-4),
4
3
0
€
0
0
Technologies, Boblingen, Germany) with a mobile phase consistent
of acetonitrile and 0.1% ammonium formate. HRMS measurements
5.53 (d, J4,6¼2.5 Hz, 1H0,0 H-6), 3.33 (t, J1 ,2 ¼7.2 Hz, 2H, H-1 ), 1.98
(d, 4J4,1 ¼1.4 Hz, 3H, H-1 ), 1.70 (m, 2H, H-2 ), 1.45e1.29 (0m, 10H, H-
0
00
3
30, H-40, H-50, H-60, H-70), 0.90 (t, J7 ,8 ¼7.1 Hz, 3H, H-8 ). HPLC Rf
13.6 min. UVevis (MeOH) lmax 220, 298, 490 nm. LCeMS Rf 6.7 min
(MeOH); API-ES: pos. mode [MþH]þ calculated m/z 249; found m/z
250.1. HRMS (ESI) calcd for C15H23NO2 250.18016; found 250.17915.
0
0
were conducted on an ESI-TOF/MS (Agilent Technologies,
þ
€
Boblingen, Germany) using positive ion [MþH] mode within
a direct injection of methanolic samples lacking a previous chro-
matographic column separation. HRMS measurements were ac-
complished using a MeOH/0.1% acetic acid mobile phase in a 9:1
ratio. For complete structure determination of the isolated prod-
ucts, HMBC-, HSQC-, and HH-COSY were recorded on a Bruker
spectrometer (Advance 600, 600 MHz, Karlsruhe, Germany) in
deuterated methanol (MeOH-d4).
4.9.6. 3-Methyl-5-(nonylamino)-1,2-benzoquinone (3f). Red-brown
solid. 1H NMR (600 MHz, MeOH-d4)
d
3
(ppm)¼6.75 (br, 1H, H-4);
4
0
0
0
5.53 (d, J4,6¼2.5 Hz, 1H, 0H0 -6), 3.33 (t, J1 ,2 ¼7.2 Hz, 2H, H-1 ), 1.98
4
0
00
(d, J4,1 ¼1.4 Hz, 3H, H-1 ), 1.70 (m, 2H, H-2 ), 1.45e1.25 (m, 12H,
3
H-30, H-40, H-50, H-60, H-70, H-80), 0.90 (t, J8 ,9 ¼7.1 Hz, 3H, H-9 ).
0
0
0
4.9.1. 3-Methyl-5-(butylamino)-1,2-benzoquinone (3a). Light red
13C NMR (150 MHz, MeOH-d4)
d
(ppm)¼185.5 (C-2), 160.4 (C-5),
solid. 1H NMR (600 MHz, MeOH-d44)
d
(ppm)¼6.74 (dq, 4J4,6¼2.7 Hz,
142.3 (C-3), 133.2 (C-4), 94.2 (C-6), 44.6 (C-10), 32.7 (C-70), 30.2 (C-
40), 29.7, 21.5 (C‑50, C-60), 28.9 (C-20), 27.8 (C-30), 23.4 (C-80), 15.0
(C-100), 14.3 (C-90). HMBC correlations see Supplementary data.
HPLC Rf 14.3 min. UVevis (MeOH) lmax 220, 298, 490 nm. LC-MS Rf
7.3 min (MeOH); API-ES: pos. mode [MþH]þ calculated m/z 263 ;
found m/z 264.1. HRMS (ESI) calcd for C16H25NO2 264.19581; found
264.19619.
4
00
J4,1 ¼1.5 Hz, 1H, H-04), 5.53 (d, J4,6¼2.7 Hz, 1H, H-6), 3.34 (t,
3
4
00
0
0
00
J1 ,2 ¼7.2 Hz, 2H, H-1 ), 1.98 (d, J4,1 ¼1.5 Hz, 3H, H-1 ), 1.69 (m, 2H,
H-20); 1.45 (m, 2H, H-30), 0.99 (t, J3 ,4 ¼7.4 Hz, 3H, H-4 ). 13C NMR
3
0
0
0
(150 MHz, MeOH-d4)
d
(ppm)¼185.6 (C-2), 175.6 (C-1), 160.4 (C-5),
142.4 (C-3), 133.4 (C-4), 94.4 (C-6), 44.2 (C-10), 31.0 (C-20), 21.0 (C-
30), 15.1 (C-100), 13.8 (C-40). HPLC Rf 8.8 min. UVevis (MeOH) lmax
220, 298, 490 nm. LCeMS Rf 4.2 min (MeOH); API-ES: pos. mode
[MþH]þ calculated m/z 193; found m/z 194.1. HRMS (ESI) calcd for
C11H15NO2 194.11756; found 194.11758.
4.9.7. 3-Methyl-5-(2-ethylhexylamino)-1,2-benzoquinone (3g). Red
solid. 1H NMR (600 MHz, MeOH-d4)
d
(ppm)¼6.78 (dq, 4J4,6¼2.7 Hz,
4
3
4
00
J4,1 ¼1.5 Hz, 1H, H-4), 5.55 (d, J4,6¼2.7 Hz, 1H, H-6), 3.25 (d,
4
0
00
0
0
00
4.9.2. 3-Methyl-5-(pentylamino)-1,2-benzoquinone (3b). Reddish-
J1 ,2 ¼6.7 Hz, 2H, H-1 ), 1.98 (d, J4,1 ¼1.5 Hz, 3H, H-1 ), 1.71 (m, 1H,
crimson solid. 1H NMR (600 MHz, MeOH-d4)
d
0
(ppm)¼6.74 (br, 1H,
H-20), 1.44 (m, 2H, H-70), 1.40e1.33 (m, 6H, H-30, H-40, H-50), 0.95 (t,
H-4), 5.54 (d, 4J4,6¼2.5 Hz, 1H, H-6), 3.33 (t, 3J1 ,2 ¼7.2 Hz, 2H, H-1 ),
J7 ,8 ¼7.5 Hz, 3H, H-8 ), 0.93 (t, J5 ,6 ¼7.0 Hz, 3H, H-6 ). 13C NMR
3
3
0
0
0
0
0
0
0
0
1.98 (d, 4J4,1 ¼1.3 Hz, 3H, H-1 ), 1.71 (m, 2H, H-2 ), 1.40 (m, 4H, H-3 ,
(150 MHz, MeOH-d4)
d
(ppm)¼185.4 (C-2), 175.2 (C-1), 160.6 (C-5),
00
0
0
00
H-40), 0.96 (t, J4 ,5 ¼7.0 Hz, 3H, H-5 ). 13C NMR (150 MHz, MeOH-d4)
142.5 (C-3), 133.2 (C-4), 94.6 (C-6), 48.3 (C-10), 39.9 (C-20), 31.8 (C-
30), 29.6 (C-40), 25.0 (C-70), 23.8 (C-50), 15.2 (C-100), 14.2 (C-60), 10.8
(C-80). HMBC correlations see Supplementary data. HPLC Rf
11.4 min. UVevis (MeOH) lmax 219, 299, 486 nm. LCeMS Rf 6.4 min
(MeOH); API-ES: pos. mode [MþH]þ calculated m/z 249; found m/z
250.1. HRMS (ESI) cald for C15H23NO2 250.18016; found 250.18086.
3
0
0
0
d
(ppm)¼185.7 (C-2), 175.4 (C-1), 160.7 (C-5), 142.7 (C-3), 133.5 (C-
4), 94.6 (C-6), 45.1 (C-10), 30.3 (C-30), 29.0 (C-20), 23.5 (C-40), 15.6 (C-
100), 14.4 (C-50). 1H,1H COSY and 1H,13C correlations (HMBC): see
Table 3 and Supplementary data. HPLC Rf 10.2 min. UVevis (MeOH)
lmax 220, 298, 490 nm. LCeMS Rf 4.9 min (MeOH); API-ES: pos.
mode [MþH]þ calculated m/z 207; found m/z 208.1. HRMS (ESI)
calcd for C12H17NO2 208.13321; found 208.1332.
4.9.8. 3-Methyl-5-(1-methylpentylamino)-1,2-benzoquinone
(3h). Red solid. 1H NMR (600 MHz, MeOH-d4)
d
(ppm)¼6.74 (dq,
4
4
4
00
4.9.3. 3-Methyl-5-(hexylamino)-1,2-benzoquinone (3c). Red solid.
J4,6¼2.7 Hz, J4,1 ¼1.5 Hz, 1H, H-4), 5.56 (d, J4,6¼2.7 Hz, 1H, H-6),
4
4
1H NMR (600 MHz, MeOH-d4)
d
4
(ppm)¼6.74 (dq, J4,6¼2.7 Hz,
3.72 (m,1H, H-10),1.98 (d, J4,1 ¼1.5 Hz, 3H, H-1 ),1.63 (m, 2H, H-2 ),
00
0
00
4
3
J4,1 ¼1.5 Hz, 1H, H-4), 5.53 (d, J4,6¼2.7 Hz, 1H, H-6), 3.33 (t,
1.37 (m, 4H, H-30, H-40), 1.28 (d, J1 ,6 ¼6.5 Hz, 3H, H-6 ), 0.93 (t,
0
00
0
0

9320
S. Herter et al. / Tetrahedron 67 (2011) 9311e9321
3
J4 ,5 ¼7.0 Hz, 3H, H-5 ). 13C NMR (150 MHz, MeOH-d4)
d
(ppm)¼
0
Pazarlıoglu, N. K.; Sariis¸ ik, M.; Telefoncu, A. Process Biochem. 2005, 40,
1673e1678.
0
0
185.7 (C-2), 175.6 (C-1), 160.0 (C-5), 142.7 (C-3), 133.3 (C-4), 94.4 (C-
6), 51.2 (C-10), 36.6 (C-20), 29.2 (C-30), 23.4 (C-40), 19.9 (C-60), 15.3 (C-
100), 14.1 (C-50). HMBC correlations see Supplementary data. HPLC Rf
10.7 min. UVevis (MeOH) lmax 220, 298, 491 nm. LCeMS Rf 5.3 min
(MeOH); API-ES: pos. mode [MþH]þ calculated m/z 221; found m/z
222.1. HRMS (ESI) calcd for C13H19NO2 222.14886; found 222.14854.
3. (a)Morozova, O. V.;Shumakovich, G. P.; Shleev, S. V.; Yaropolov, Y. I. Appl. Biochem.
Microbiol. 2007, 43, 523e535; (b) Nyanhongo, G. S.; Gubitz, G.; Sukyai, P.; Leitner,
C.; Haltrich, D.; Ludwig, R. Food Technol. Biotechnol. 2007, 45, 250e268.
€
4. (a) Ryan, S.; Schnitzhofer, W.; Tzanov, T.; Cavaco-Paulo, A.; Gubitz, G. M.
Enzyme Microb. Technol. 2003, 33, 766e774; (b) De Cavalho, M. E.; Monteiro,
M. C.; Sant Anna, G. L., Jr. Appl. Biochem. Biotechnol. 1999, 77-79, 723e733;
(c) Jordaan, J.; Mathye, S.; Simpson, C.; Brady, D. Enzyme Microb. Technol.
2009, 45, 432e435.
5. (a) Mikolasch, A.; Hessel, S.; Gesell Salazar, M.; Neumann, H.; Manda, K.;
Gordes, D.; Schmidt, E.; Thurow, K.; Hammer, E.; Lindequist, U.; Beller, M.;
Schauer, F. Chem. Pharm. Bull. 2008, 56, 781e786; (b) Mikolasch, A.; Wurster,
M.; Lalk, M.; Witt, S.; Seefeldt, S.; Hammer, E.; Schauer, F.; Julich, W. D.; Lin-
dequist, U. Chem. Pharm. Bull. 2008, 56, 902e907.
6. (a) Thurston, C. F. Microbiology 1994, 140, 19e26; (b) Yoshitake, A.; Katayama,
Y.; Nakamura, M.; Iimura, Y.; Kawai, S.; Morohoshi, N. J. Gen. Microbiol. 1993,
139, 179e185.
4.9.9. 5-(1,4-Dimethylpentylamino)-3-methyl-1,2-benzoquinone
(3i). Red solid. 1H NMR (600 MHz, MeOH-d4)
d
(ppm)¼6.74 (dq,
4
4
4
€
00
J4,6¼2.7 Hz, J4,1 ¼1.5 Hz, 1H, H-4), 5.56 (d, J4,6¼2.7 Hz, 1H, H-6),
4
00
3.69 (m, 1H, H-10), 1.98 (d, J4,1 ¼1.5 Hz, 3H, H-1 ), 1.63 (m, 2H, H-
00
3
20), 1.57 (m, 2H, H-30), 1.28 (d, J1 ,6 ¼6.5 Hz, 3H, H-6 ), 1.25 (m, 1H,
0
0
0
H-40); 0.92 (2d, J4 ,5 ¼ J4 ,7 ¼6.6 Hz, 6H, H-50,70). 13C NMR
3
3
0
0
0
0
7. (a) Musidlowska, A.; Lange, S.; Bornscheuer, U. T. Angew. Chem., Int. Ed. 2001,
(150 MHz, MeOH-d4)
d
(ppm)¼185.5 (C-2), 175.2 (C-1), 159.7 (C-5),
€
€
40, 2851e2853; (b) Hummel, A.; Brusehaber, E.; Bottcher, D.; Doderer, K.;
Trauthwein, H.; Bornscheuer, U. T. Angew. Chem., Int. Ed. 2007, 46,
8492e8494.
142.7 (C-3), 133.5 (C-4), 94.3 (C-6), 51.3 (C-10), 34.8 (C-20), 28.9 (C-
30), 20.0 (C-60), 36.1 (C-40), 22.6 (C-50, C-70), 15.2 (C-100). HMBC
correlations see Supplementary data. HPLC Rf 11.4 min. UVevis
(MeOH) lmax 220, 298, 492 nm. LCeMS Rf 5.8 min (MeOH); API-ES:
pos. mode [MþH]þ calculated m/z 235; found m/z 236.1. HRMS (ESI)
calcd for C14H21NO2 236.16451; found 236.16449.
8. For example: (a) Brown, M. A.; Zhao, Z.; Mauk, A. G. Inorg. Chim. Acta 2002, 331,
232e238; (b) Gelo-Pujic, M.; Kim, H.-H.; Butlin, N. G.; Palmore, G. T. R. Appl.
Environ. Microbiol. 1999, 65, 5515e5521; (c) Kojima, Y.; Tsukuda, Y.; Kawai, Y.;
Tsukamoto, A.; Sugiura, J.; Sakaino, M.; Kita, Y. J. Biol. Chem. 1990, 265,
15224e15230; (d) Record, E.; Punt, P. J.; Chamkha, M.; Labat, M.; van den
Hondel, C. A. M. J. J.; Asther, M. Eur. J. Biochem. 2002, 269, 602e609; (e) Sigoillot,
J. C.; Record, E.; Belle, V.; Robert, J. L.; Levasseur, A.; Punt, P. J.; van den Hondel,
C. A. M. J. J.; Fournel, A.; Sigoillot, J. C.; Asther, M. Appl. Microbiol. Biotechnol.
2004, 64, 346e352; (f) Baker, C. J. O.; White, T. C. ACS Symp. Ser. 2001, 785,
413e423; (g) Sanchez-Amat, A.; Lucas-Elio, P.; Fernandez, E.; Garcia-Borron, J.
C.; Solano, F. Biochim. Biophys. Acta 2001, 1547, 104e116.
4.9.10. 3-Methyl-5-[(4,7,7-trimethylnorbornan-2-yl)amino]-1,2-
benzoquinone (3j). Light red solid. 1H NMR (600 MHz, MeOH-d4)
4
4
00
d
(ppm)¼6.94 (dq, J4,6¼2.7 Hz, J4,1 ¼1.5 Hz, 1H, H-4), 5.58 (d,
4
3 3
0
0
0
0
J4,6¼2.7 Hz, 1H, H-6), 3.94 (ddd, J2 ,3 a¼10.8 Hz, J2 ,3 b¼4.4 Hz,
9. Bourbonnais, R.; Paice, M. G. FEBS Lett. 1990, 267, 99e102.
4
3
4
2
10. Jonas, U.; Hammer, E.; Haupt, E. T. K.; Schauer, F. Arch. Microbiol. 2000, 174,
393e398; (b) Intra, A.; Nicotra, S.; Riva, S.; Danieli, B. Adv. Synth. Catal. 2005,
347, 973e977.
0
0
0
0
J2 ,6 b¼2.2 Hz, 1H,
H-20),
2.45
0
(dddd,
J3 a,3 b¼13.4 Hz,
4
4
J2 ,3 a¼10.8 Hz, J3 a,5 a¼4.7 Hz, J1 ,3 a¼3.2 Hz, 1H, H-30a), 1.99 (d,
0
0
0
0
00
0
0
0
00
J4,1 ¼1.5 Hz, 3H, H-1 ), 1.85 (m, 1H, H-5 a), 1.80e1.75 (m, 2H, H-1 ,
11. Review with summarized CeC-, CeO-, CeN- or CeS-coupling reactions: Mi-
kolasch, A.; Schauer, F. Appl. Microbiol. Biotechnol. 2009, 82, 605e624.
12. Akagawa, M.; Suyama, K. Biochem. Biophys. Res. Commun. 2001, 281,
193e199.
2
3
H-60a),
1.47
(dddd,
J6 a,6 b¼13.8 Hz,
J5 a,6 b¼12.1 Hz,
0
0
0
0
3
4
H-60b),
1.37 (ddd,
0
0
0
0
J5 b,6 b¼4.5 Hz,
J2 ,6 b¼2.2 Hz,
1H,
2
3
3
J5 a,5 b¼12.3 Hz, J5 b,6 a¼9.5 Hz, J5 b,6 b¼4.5 Hz, 1H, H-50b), 1.16
0
0
0
0
0
0
€
13. Dawson, D. A.; Scott, B. D.; Ellenberger, M. J.; Poch, G.; Rinaldi, A. C. Environ.
(dd, 2J3 a,3 b¼13.4 Hz, 3J2 ,3 b¼4.4 Hz,1H, H-30b),1.03, 0.95 (2s, 6H, H-
Toxicol. Chem. 2004, 16, 13e23.
14. Tamaka, T.; Miura, A.; Kato, K.; Nakamura, N.; Komamura, T. Chem. Abstr. 1994,
0
0
0
0
80, H-90); 0.94 (s, 3H, H-100). 13C NMR (150 MHz, MeOH-d4)
121, 943.
d
(ppm)¼185.5 (C-2), 175.5 (C-1), 161.1 (C-5), 142.3 (C-3), 133.3 (C-
15. Burzio, L. A.; Waite, J. H. Biochemistry 2000, 39, 11147e11153.
4), 94.9 (C-6), 60.5 (C-20), 52.2 (C-40), 49.9 (C-70), 46.3 (C-10), 37.3 (C-
30), 28.8 (C-60), 28.8 (C-50), 19.9, 18.8 (C-80, C-90), 15.4 (C-100), 14.6 (C-
100). HMBC and HH COSY correlations see Supplementary data.
HPLC Rf 12.2 min. UVevis (MeOH) lmax 220, 306, 498 nm. LCeMS Rf
6.2 min (MeOH); API-ES: pos. mode [MþH]þ calculated m/z 273;
found m/z 274.1. HRMS (ESI) calcd for C17H23NO2 274.18016; found,
274.18003.
€
16. Mikolasch, A.; Hahn, V.; Manda, K.; Pump, J.; Illas, N.; Gordes, D.; Lalk, M.;
€
Gesell Salazar, M.; Hammer, E.; Julich, W. D.; Rawer, S.; Thurow, K.; Lindequist,
U.; Schauer, F. Amino Acids 2010, 39, 671e683.
17. Finley, K.T. Quinones: the present state of addition and substitution chemistry.
In Patai, S. (ed.) The Chemistry of Hydroxyl, Ether and Peroxide Groups. John
Wiley, Chichester, Suppl. E, 1993; pp. 1027e1134.
18. Cavalieri, E. L.; Li, K. M.; Balu, N.; Saeed, M.; Devanesan, P.; Higgibotham, S.;
Zhao, J.; Gross, M. L.; Rogan, E. G. Carcinogenesis 2002, 23, 1071e1077.
19. Park, Y. J.; Lee, D. W.; Lee, W.-Y. Anal. Chim. Acta 2002, 471, 51e59.
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2009.
Acknowledgements
21. U’Prichard, D. C.; Greenberg, D. A.; Sheehan, P. P.; Snyder, S. H. Science 1978, 199,
197e198.
22. Koschorreck, K.; Richter, S. M.; Swierczek, A.; Beifuss, U.; Schmid, R. D.;
Financial support by the Deutsche Bundesstiftung Umwelt
€
(Osnabruck, Germany, grant no. AZ13191) is gratefully acknowl-
Urlacher, V. B. Arch. Biochem. Biophys. 2008, 474, 213e219.
edged. S.H. is also grateful to the government of Mecklenburg-
Vorpommern, Germany, for financial support in the framework of
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Longhi, S.; Asther, M.; Moukha, S. Eur. J. Biochem. 2000, 267, 1619e1625; (b)
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€
Landesgraduiertenforderung. The authors wish also to thank
Dr. M. Lalk and Prof. Dr. K. Weisz (both University of Greifswald) for
conducting NMR analysis of products.
Supplementary data
Supplementary data associated with this article can be found in
online version, at doi:10.1016/j.tet.2011.09.123.
24. Heinze, B.; Hoven, N.; O’Connell, T.; Maurer, K. H.; Bartsch, S.; Bornscheuer, U. T.
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