Laccase-mediated synthesis of 2-methoxy-3-methyl-5-(alkylamino)- and 3-methyl-2,5-bis(alkylamino)-[1,4]-benzoquinones

Article abstract of DOI:10.1016/j.molcatb.2013.01.020

The synthesis of 5-alkylamino- and 2,5-bis(alkylamino)-[1,4]-benzoquinones, showing structural similarity to natural mitomycins, was performed through coupling of 2-methoxy-3-methylhydroquinone with primary amines such as n-octylamine, geranylamine and cy

Full text of DOI:10.1016/j.molcatb.2013.01.020

Journal of Molecular Catalysis B: Enzymatic 90 (2013) 91–97
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Laccase-mediated synthesis of 2-methoxy-3-methyl-5-(alkylamino)- and
3-methyl-2,5-bis(alkylamino)-[1,4]-benzoquinones
Susanne Hertera,c,∗, Dirk Michalikb, Annett Mikolaschc, Marlen Schmidtd, Roland Wohlgemuthe,
Uwe Bornscheuerd, Frieder Schauerc
a Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK
b Leibniz Institute for Catalysis, Albert-Einstein-Str. 29a, 18059 Rostock, Germany
c Institute of Microbiology, Department of Applied Microbiology, University of Greifswald, Friedrich-Ludwig-Jahn-Str. 15a, 17487 Greifswald, Germany
d Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, Felix-Hausdorff-Str. 4, 17487 Greifswald, Germany
e Sigma–Aldrich Chemie GmbH, Industriestr. 25, 9470 Buchs, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of 5-alkylamino- and 2,5-bis(alkylamino)-[1,4]-benzoquinones, showing structural sim-
ilarity to natural mitomycins, was performed through coupling of 2-methoxy-3-methylhydroquinone
with primary amines such as n-octylamine, geranylamine and cyclooctylamine using laccases from Myce-
liophthora thermophila (MtL) and Pycnoporus cinnabarinus SBUG-M 1044 (PcL). Product spectra of laccase
reactions differ due to reaction systems pH values (pH 7.0 for MtL and pH 5.0 for PcL) applied to assure
enzymes optimal catalytic efficiency. The MtL- and PcL-mediated formation of monoaminated products
was achieved at equimolar reactant concentrations with amine coupling at the meta-position to benzo-
quinones methyl group. Increased formation of diaminated products occurred in PcL-mediated reactions
and generally when the amine was supplied in excess. Diamination entailed elimination of the benzo-
quinone methoxy group (amination in para-position to the first amine substituent). Six products were
synthesised and characterised by NMR and HR-MS analysis. The laccase-mediated amine coupling to
2-methoxy-3-methylhydroquinone confers two of the essential pharmaceutical active motifs from mit-
omycins: (i) a stable 1,4-benzoquinoic parent structure and (ii) a biological active alkylation function
Received 12 December 2012
Received in revised form 23 January 2013
Accepted 23 January 2013
Available online 8 February 2013
Keywords:
Myceliophthora thermophila
Pycnoporus cinnabarinus
Biocatalysis
C
N coupling
Mitomycin
(
NH).
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
and also some viruses [8,9]. They are also employed as chemother-
apeutics in the treatment of several types of cancer, including
solid carcinomas [10,11]. The biological activity of mitomycins has
been related to certain biologically active structure motifs such as
a para-benzoquinoidic parent structure, a strong alkylating func-
tion, especially that of a secondary amino-group, and partially a
urethane-function (Fig. 1A) [12].
Approaches for the chemical synthesis of mitomycins are
challenging due their demanding stereochemistry, the difficult
workability of quinones, and often requiring complex reaction
steps [13–15]. With respect to the aforementioned difficulties, in
particular quinone instability, the use of the biocatalyst laccase
affords mild reaction conditions and can catalyse the transforma-
tion of dihydroxylated compounds into the corresponding stable
benzoquinones. Previously, it was shown that laccases can medi-
ate a wide range of heteromolecular reactions (e.g. C C, C O, C N,
The enzyme class of laccases (para-diphenol:dioxygen oxidore-
ductases, EC 1.10.3.2) finds increasing significance in a variety of
organic synthetic methodologies and the manufacture of synthetic
building blocks for fine chemistry due to a broad substrate scope,
the variety of reactions catalysed, and in particular the high resis-
tance towards harsh reaction conditions [1–4]. The versatility of
laccases enables a broad range of applications from the production
of polymers and food additives to the synthesis of fragrance com-
pounds, and cosmetic and pharmaceutical precursors in the field of
white biotechnology [5–7].
The family of mitomycins comprised pharmaceutical active,
antineoplastic compounds. Mitomycins provide a broad spectrum
of efficacy due to their antibiotic and cytotoxic characteristics and
therefore selected mitomycins are used in control of infectious dis-
eases caused by Gram positive as well as Gram negative bacteria,
C
S coupling) at which the enzyme substrate radical is linked
with diverse coupling agents yielding stable hybrid molecules
[16–19]. In most of the published laccase-mediated C N cou-
pling reactions, simple substituted para-hydroquinones acting as
native laccase substrates due to position of hydroxyl groups were
∗
Corresponding author. Tel.: +49 3834 864204; fax: +49 3834 864202.
E-mail address: susanne.herter@manchester.ac.uk (S. Herter).
1381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.01.020

92
S. Herter et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 91–97
phosphate-citrate buffer (pH 7.0) with M. thermophila laccase
A
(1 ␮moL mL−1) or in sodium acetate buffer (20 mM, pH 5.0) with
P. cinnabarinus SBUG-M 1044 laccase (1 ␮moL mL−1). Educts were
prepared as 50 mM methanolic stock solutions and biocatalysis
conducted either in equimolar concentration or with an excess
of the primary amines. Product formation was followed by ana-
lytical HPLC at regular intervals. Preparative scale reactions were
performed in 100-mL-Erlenmeyer flasks protected against light,
containing 50 mL of the reaction mixture using the same conditions
described above. Monoaminated products were obtained using
a 2:2 mM ratio between 2-methoxy-3-methylhdroquinone 1 and
the primary amines 3a–c from MtL-catalysed reactions. Diami-
nated products were enriched using a 2:5 mM ratio under the same
conditions as described for enrichment of monoaminated products.
O
OCONH
Y a
2
X
Y
Z
X
OMe OMe
NH2
H
H
mitomycin A
mitomycin C
mitomycin F
profiromycin
OMe
N
Z
N
Me
OMe OMe Me
NH2
O
OMe Me
B
OMe
OH
H N
2
3a
H N
2
Me
3c
H N
2
OH
1
2.4. Analytical HPLC
3b
Reaction batches were sampled at regular intervals over 24 h
and analyzed via HPLC (Shimadzu, Duisburg, Germany) consisting
of a SCL-10A VP control unit, a LC-A FCV-10AL VP fluid chromato-
graph and a SPD-10A VP photodiode array detector. Grading of
analytes was accomplished with a LiChroCart 125-4 RP-18 end-
capped column of 5-␮m particle size (Merck, Darmstadt, Germany).
The mobile phase consisted of an aqueous component A (0.1% phos-
phoric acid) and a methanol component B at an initial ratio of 30%
B to 70% A, simultaneously raised up to 100% B within 14 min.
The flow rate was always adjusted to 1 mL min−1 with an injection
volume of 40 ␮L.
Fig. 1. Model structure for selected naturally occurring mitomycins (A). Laccase-
substrate 1 and primary amines 3a–c (coupling agents) used in reactions focused
on the enzyme-mediated synthesis of mitomycin-like compounds (B).
applied in assays containing aromatic amines [20–22]. It was
demonstrated that product formation in such laccase-mediated
C
N coupling results in satisfying yields and is equivalent or bet-
ter when compared to synthetic routes, as for example use of the
coupling-mediator sodium iodate [23,24].
These findings encouraged us to study the laccase-mediated
synthesis of mitomycin-like compounds. The 2-methoxy-3-
methylbenzoquinone represents in its protonated form a suitable
laccase substrate (1) and was hence investigated in the cou-
pling with aliphatic (n-octylamine 3a and geranylamine 3b) as
well as cyclic (cyclooctylamine 3c) primary amines using laccases
from Myceliophthora thermophila (MtL) and Pycnoporus cinnabar-
inus SBUG-M 1044 (PcL) (Fig. 1B). The influence of pH values of
buffered reaction systems, as well as reactant ratios, on product
yields and formation of mono- or diaminated products was studied.
2.5. Isolation of heteromolecular coupling products
Products were isolated by solid reverse phase extraction with
a Strata C18-E silicagel column (60 mL Giga Tubes, 10 g absorbent
material, Merck Darmstadt, Germany), progressively charged with
50 mL of the reaction mixture. Residual starting material and
low-molecular by-reaction products were eluted with 60 mL of a
solution composed of 60% methanol and 40% bidistilled water. The
monoaminated products were obtained with 60 mL of a mixture
consistent of 85% methanol and 15% acetic acid (0.1%, v/v in bidis-
tilled water), followed by elution of the corresponding diaminated
products due to addition of pure methanol (40 mL). The methanol
concentration of samples was initially reduced via rotary evapo-
ration at 30 ◦C. Afterwards, samples were diluted with bidistilled
water to reach a final methanol concentration of 5% and subse-
quently frozen at −20 ◦C for 24 h, followed by freezing at -70 ◦C
for further 4 h. Products were obtained as solids via lyophilisation
(20 ◦C).
2. Experimental
2.1. Chemicals
All chemicals were of reagent grade or better and purchased
from Sigma–Aldrich (Steinheim, Germany) and Merck (Darmstadt,
Germany).
2.2. Enzymes
M. thermophila laccase (MtL) was commercially obtained from
Novozymes® (Bagsvaerd, Denmark). Laccase from the white-rot
fungus P. cinnabarinus SBUG-M 1044 was produced as described by
Herter et al. [25] according to methods of Kordon et al. [26]. Laccase
activity was determined by monitoring the oxidation of 2,2ꢀ-
azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium
2.6. Structural characterisation
HR-MS measurements were conducted on an ESI-TOF/MS (Agi-
lent Technologies, Böblingen, Germany) in positive ion mode
([M+H]+, [M+Na]+) by direct injection of methanolic samples
lacking a previous chromatographic column separation using
a MeOH/0.1% acetic acid mobile phase in a 9:1 ratio (v/v).
1H, 13C, DEPT and two-dimensional NMR spectra (1H,1H COSY,
1H,1H NOESY, 13C,1H HMBC, 13C, 1H HSQC) were recorded on
Bruker Avance spectrometers AV 600, AV 500, and AV 250, Karl-
sruhe, Germany, in deuterated methanol (MeOH-d4, ı(1H) = 3.31,
ı(13C) = 49.1).
(ABTS) spectrophotometrically at 420 nm (ε = 3.6 × 104 M−1 cm−1
)
in sodium acetate buffer (100 mM, pH 5) at 25 ◦C as described ear-
lier. One unit (1 U) of activity is defined as the amount of enzyme,
which catalyzes the conversion of 1 ␮moL mL−1 min−1 substrate at
25 ◦C.
2.3. Laccase-mediated heteromolecular coupling of
2-methoxy-3-methylhydroquinone with primary amines
2.6.1. 2-Methoxy-3-methyl-5-(octylamino)-[1,4]-benzoquinone
(5a)
Analytical assays were conducted in sealed brown 6-mL-glass
flasks with a total reaction volume of 5 mL at room temperature
and shaking at 200 rpm. Reactions were performed either in
Red solid. HPLCRt 14.9 min, UV-vis (MeOH) ꢀmax 212, 309,
498 nm. HR-MS (ESI): calcd for C16H26NO3 [M+H]+ 280.19072;

S. Herter et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 91–97
93
found 280.19068 (error: −0.15 ppm); calcd for C16H25NNaO3
[M+Na]+ 302.17266; found 302.17257 (error: −0.33 ppm).
1H NMR (600.13 MHz, MeOH-d4) ı (ppm) = 5.26 (s, 1H, H-6); 4.04
(s, 3H, H-1ꢀꢀ); 3.14 (t, 1H, 3J = 7.3 Hz, H-1ꢀ); 1.86 (s, 3H, H-2ꢀꢀ); 1.62
(m, 2H, H-2ꢀ); 1.33 (m, 10H, H-7ꢀ, H-6ꢀ, H-5ꢀ, H-4ꢀ, H-3ꢀ); 0.90 (t, 3H,
3J = 7.2 Hz, H-8ꢀ). 13C NMR (150.9 MHz, MeOH-d4) ı (ppm) = 185.0
(C-4); 182.8 (C-1); 159.8 (C-2); 149.2 (C-5); 124.3 (C-3); 61.8 (C-1ꢀꢀ);
43.3 (C-1ꢀ); 32.7 (C-6ꢀ); 28.8 (C-2ꢀ); 28.0 (C-3ꢀ); 30.1, 30.1 (C-4ꢀ,5ꢀ);
23.4 (C-7ꢀ); 14.3 (C-8ꢀ); 8.1 (C-2ꢀꢀ).
found 278.17553 (error: 1.65 ppm); calcd for C16H23NaNO3
[M+Na]+ 300.15701; found 300.15741 (error: 1.33 ppm).
1H NMR (250.13 MHz, MeOH-d4) ı (ppm) = 5.19 (s, 1H, H-6); 4.04
(s, 3H, H-1ꢀꢀ); 3.45 (m, 1H, H-1ꢀ); 1.85–1.53 (m, 14H, H-2ꢀ, H-3ꢀ, H-4ꢀ,
H-5ꢀ); 1.85 (s, 3H, H-2ꢀꢀ). 13C NMR (62.9 MHz, MeOH-d4) ı = 185.3
(C-1); 182.8 (C-4); 160.0 (C-2); 147.8 (C-5); 124.5 (C-3); 96.1 (C-
6); 62.0 (C-1ꢀꢀ); 53.9 (C-1ꢀ); 32.6 (C-2ꢀ); 28.0, 25.1 (C-3ꢀ, C-4ꢀ); 27.0
(C-5ꢀ); 8.5 (C-2ꢀꢀ).
2.6.6. 3-Methyl-2,5-bis-cyclooctylamino-[1,4]-benzoquinone (6c)
Brown-ochre solid. HPLCRt 16.5 min, UV-vis (MeOH) ꢀmax
216, 348, 508 nm. HR-MS (ESI): calcd for C23H37N2O2 [M+H]+
373.28495; found 373.28542 (error: 1.25 ppm); calcd for
2.6.2. 3-Methyl-2,5-bis-octylamino-[1,4]-benzoquinone (6a)
Brown-ochre solid. HPLCRt 17.0 min, UV–vis (MeOH) ꢀmax
217, 346, 508 nm. HR-MS (ESI): calcd for C23H41N2O2 [M+H]+
377.31625; found 377.3165 (error: 0.66 ppm); calcd for
C
23H36N2NaO2 [M+Na]+ 395.26671; found 395.26671 (error:
C
23H40N2NaO2 [M+Na]+ 399.30014; found 399.30018 (error:
−0.48 ppm).
1H NMR (500.13 MHz, MeOH-d4) ı (ppm) = 5.20 (s, 1H, H-6);
0.54 ppm).
1H NMR (500.13 MHz, MeOH-d4) ı (ppm) = 5.23 (s, 1H, H-6); 3.60
4.19 (s, 3H, H-1ꢀꢀ); 3.52 (m, 1H, H-1ꢀ); 2.03 (s, 3H, H-1ꢀꢀꢀ); 1.98–1.53
(m, 28H, H-2ꢀ,2ꢀꢀ,3ꢀ,3ꢀꢀ,4ꢀ,4ꢀꢀ,5ꢀ,5ꢀꢀ). 13C NMR (125.8 MHz, MeOH-d4)
ı = 180.6 (C-1); 180.0 (C-4); 151.5 (C-5); 148.4 (C-2); 102.9 (C-3);
92.5 (C-6); 54.8 (C-1ꢀꢀ); 53.9 (C-1ꢀ); 34.4 (C-2ꢀꢀ); 32.6 (C-2ꢀ); 28.3,
28.2, 25.0, 24.4 (C-3ꢀ,3ꢀꢀ,4ꢀ,4ꢀꢀ); 26.9, 26.8 (C-5ꢀ,5ꢀꢀ); 10.7 (C-1ꢀꢀꢀ).
(t, 1H, 3J1 ꢀꢀ = 7.2 Hz, H-1ꢀꢀ); 3.18 (t, 1H, J1 ,2 = 7.2 Hz, H-1 ); 2.04
ꢀꢀ
3
ꢀ
ꢀ
ꢀ
,2
(s, 3H, H-1ꢀꢀꢀ); 1.63 (m, 4H, H-2ꢀ, H-2ꢀꢀ); 1.40–1.27 (m, 20H, H-3ꢀ-
H-7ꢀ, H-3ꢀꢀ-H-7ꢀꢀ); 0.90 (m, 6H, H-8ꢀ, H-8ꢀꢀ). 13C NMR (125.8 MHz,
MeOH-d4) ı (ppm) = 180.5, 179.9 (C-1, C-4); 153.3 (C-5); 150.2 (C-
2); 102.9 (C-3); 92.1 (C-6); 45.9 (C-1ꢀꢀ); 43.6 (C-1ꢀ); 33.0, 33.0 (C-6ꢀ,
C-6ꢀꢀ); 31.9, 29.3 (C-2ꢀ, C-2ꢀꢀ); 30.4, 30.4 (3) (C-4ꢀ, C-4ꢀꢀ, C-5ꢀ, C-5ꢀꢀ);
28.1, 27.8 (C-3ꢀ, C-3ꢀꢀ); 23.8, 23.8 (C-7ꢀ, C-7ꢀꢀ); 14.5 (2) (C-8ꢀ, C-8ꢀꢀ);
10.5 (C-1ꢀꢀꢀ).
3. Results and discussion
3.1. Laccase-mediated coupling reactions: product formation and
recovery
2.6.3. 5-[[(2E)]-3,7-dimethylocta-2,6-dienyl]-amino]-2-
methoxy-3-methyl-[1,4]-benzo-quinone (5b)
Prior to heteromolecular coupling reactions which targeted
the synthesis of mitomycin-like compounds, the laccase-mediated
homomolecular reaction (reaction without amines 3a–c) of the
enzyme substrate 2-methoxy-3-methylhydroquinone 1 (1 mM)
was studied. For all reactions, phosphate-citrate buffer (pH 7.0) was
used for M. thermophila laccase (MtL) and sodium acetate buffer (pH
5.0) for P. cinnabarinus laccase (PcL). The aforementioned reaction
systems and pH values were chosen to ensure the optimal catalytic
efficiency of both laccases [27–29].
Red solid. HPLCRt 14.5 min, UV–vis (MeOH) ꢀmax 212, 308,
488 nm. HR-MS (ESI): calcd for C18H26NO3 [M+H]+ 304.19072;
found 304.19013 (error: −1.95 ppm); calcd for C18H25NNaO3
[M+Na]+ 326.17266; found 326.1736 (error: 2.87 ppm).
1H NMR (600.13 MHz, MeOH-d4) ı (ppm) = 5.22 (s, 1H, H-6); 5.19
(t, 1H, 3J = 6.5 Hz,H-2ꢀ); 5.08 (t, 3J = 7.0 Hz, 1H, H-6ꢀ); 4.04 (s, 3H, H-
1ꢀꢀ); 3.76 (d, 2H, 3J = 6.5 Hz,H-1ꢀ); 2.12 (m, 2H, H-5ꢀ); 2.05 (m, 2H,
H-4ꢀ); 1.86 (s, 3H, H-2ꢀꢀ); 1.72 (s, 3H, H-10ꢀ); 1.65 (s, 3H, H-9ꢀ); 1.59
(s, 3H, H-8ꢀ). 13C NMR (150.9 MHz, MeOH-d4) ı (ppm) = 184.9 (C-
4); 181.8 (C-1); 159.6 (C-2); 149.1 (C-5); 140.8 (C-3ꢀ); 132.3 (C-7ꢀ);
124.4 (C-6ꢀ); 124.2 (C-3); 119.7 (C-2ꢀ); 95.7 (C-6); 61.5 (C-1ꢀꢀ); 41.1
(C-1ꢀ); 40.1 (C-4ꢀ); 26.9 (C-5ꢀ); 25.4 (C-9ꢀ); 17.5 (C-8ꢀ); 16.1 (C-10ꢀ);
7.9 (C-2ꢀꢀ).
In a homomolecular reaction, enzyme substrate 1 was com-
pletely oxidized within 60 min using MtL and 100 min using PcL.
Laccase-mediated oxidation of 1 led to simultaneous formation of
the corresponding 2-methoxy-3-methylbenzoquinone 2a (HPLCRt
6.0 min, ꢀmax 283, 381 nm; Scheme 1), appearing in each case as the
main product with yellow colouration of the initially uncoloured
reaction mixture. In reactions with MtL, concomitant formation of
one further main product 2b (HPLCRt 5.7 min, ꢀmax 208, 294 nm)
and a minor product 2c (HPLCRt 8.4 min, ꢀmax 254, 324 nm)
appeared together with decrease of benzoquinone 2a. Products 2b
and 2c were not detected in the PcL-catalyzed reaction of 1, how-
ever, formation of two minor products 2d (HPLCRt 4.9 min, ꢀmax
221, 267 nm) and 2e (HPLCRt 13.5 min, ꢀmax 272 nm) occurred.
The structures of the homomolecular products were not examined
within this study, but additional data is available in Supplementary
Material (Table S1). The observed differences in the spectrum and
quantities of homomolecular products are reasoned with differ-
ent pH values of the reactions rather than with laccase catalysed
activity. Referring to this, the benzoquinone 2a showed a higher
reactivity in a phosphate-citrate buffer at pH 7.0 (used for MtL) and
which also resulted in an increased formation of by-products when
compared to a sodium acetate buffer system at pH 5.0 (PcL). This
is in accordance with a general characteristic of para- as well as
ortho-benzoquinones, which possess at neutral to alkaline pH val-
ues an increased affinity for side reactions due to increased redox
potentials [30,31].
2.6.4. 2,5-bis[[2E]-3,7-dimethylocta-2,6-dienyl]amino]-3-
methyl-[1,4]-benzoquinone (6b)
Brown-ochre solid. HPLCRt 16.7 min, UV–vis (MeOH) ꢀmax
217, 347, 508 nm. HR-MS (ESI): calcd for C27H41N2O2 [M+H]+
425.31625; found 425.3165 (error: 0.57 ppm); calcd for
C27H40N2NaO2 [M+Na]+ 447.2982; found 447.29853 (error:
0.74 ppm).
1H NMR (600.13 MHz, MeOH-d4) ı (ppm) = 5.29 (m, 1H, H-2ꢀꢀ);
5.21 (m, 1H, H-2ꢀ); 5.20 (s, 1H, H-6); 5.08 (m, 2H, H-6ꢀ,6ꢀꢀ); 4.20 (d,
2H, 3J = 6.3 Hz,H-1ꢀꢀ); 3.80 (d, 2H, 3J = 6.8 Hz,H-1ꢀ); 2.12 (m, 4H, H-
5ꢀ,5ꢀꢀ); 2.07 (m, 4H, H-4ꢀ,4ꢀꢀ); 2.05 (s, 3H, H-1ꢀꢀꢀ); 1.73 (m, 3H, H-10ꢀ);
1.70 (m, 3H, H-10ꢀꢀ); 1.66 (m, 3H), 1.65 (m, 3H), (H-9ꢀ,9ꢀꢀ); 1.60 (br
s, 3H), 1.59 (br s, 3H), (H-8ꢀ,8ꢀꢀ). 13C NMR (150.9 MHz, MeOH-d4)
ı (ppm) = 180.7 (C-1); 180.3 (C-4); 153.0 (C-5); 150.0 (C-2); 141.5
(C-3ꢀ); 141.1 (C-3ꢀꢀ); 132.8 (2) (C-7ꢀ,7ꢀꢀ); 125.0 (2) (C-6ꢀ,6ꢀꢀ); 122.6 (C-
2ꢀꢀ); 120.1 (C-2ꢀ); 103.3 (C-3); 92.6 (C-6); 43.8 (C-1ꢀꢀ); 41.5 (C-1ꢀ);
40.6, 40.6 (C-4ꢀ,4ꢀꢀ); 27.4, 27.4 (C-5ꢀ,5ꢀꢀ); 26.0, 26.0 (C-9ꢀ,9ꢀꢀ); 17.9 (2)
(C-8ꢀ,8ꢀꢀ); 16.6, 16.6 (C-10ꢀ,10ꢀꢀ); 10.4 (C-1ꢀꢀꢀ).
2.6.5. 2-Methoxy-3-methyl-5-(cyclooctylamino)-[1,4]-
benzoquinone (5c)
In order to obtain mitomycin-like compounds by laccase-
mediated transformation reactions, the heteromolecular coupling
between 1 and amines n-octylamine 3a, geranylamine 3b and
Red solid. HPLCRt 13.7 min, UV–vis (MeOH) ꢀmax 212, 309,
485 nm. HR-MS (ESI): calcd for C16H24NO3 [M+H]+ 278.17507;

94
S. Herter et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 91–97
in the homomolecular reaction (Fig. 2A). A further difference to
O
OH
1"
MeO
1
1
6
6
H
H
the PcL-mediated reaction was observed in the number as well as
quantities of heteromolecular coupling products. Besides the minor
product 4a (HPLCRt 14.5 min, ꢀmax 300, 498 nm), which was not
formed in the PcL-reaction, the monoaminated 5a was observed as
the main product in the MtL-mediated reaction. Products 4a and
5a were generated in a 1:2 ratio based upon their HPLC peak areas
within the first 2 h of reaction (Fig. 2A). With regard to product
retention time and UV/VIS-spectra, the presence of monoaminated
regioisomers was highly presumed, although not examined within
this study. Proceeding reaction led to a decrease of monoaminated
product 5a together with concomitant increase of the diaminated
product 6a (Fig. 2A).
In summary, the following conclusions for heteromolecular cou-
pling reactions between 2-methoxy-3-methylhydroquinone 1 and
n-octylamine 3a at pH 5.0 and pH 7.0, can be made: (i) In the PcL-
catalyzed system (pH 5.0) the diaminated product 6a is formed as
the main product, the monoaminated regioisomer 5a occurred only
in negligible concentrations. (ii) In the MtL-catalyzed system (pH
7.0) two monoaminated regioisomeric products 4a and 5a were
simultaneously generated, whereby 5a presented the main prod-
uct and proceeding incubation led to increased formation of the
diaminated product 6a. (iii) The formation of heteromolecular cou-
pling products in MtL- and PcL-mediated systems is dependent on
assay pH.
a
b
2
5H
5 H
3
3
Me
4
4
2"
OH
O
2a
1
O
O
1"
1
1
R NH
6
H
MeO
6
H
c
2
2
2a
5
5
4
3
3
Me
4
Me
R
NH
NH R
2"
2"
O
6a-c
O
5a-c
O
M1e"O
1
6NH
R
2
5
3
Me
4
2"
O
Scheme 1. Laccase-mediated synthesis of mono- (5a–c) and diaminated (6a–c)
mitomycin-like compounds by heteromolecular coupling of 2-methoxy-3-
methylhydroquinone 1 with primary amines 3a–c at RT and 200 rpm. Reaction
and conditions: (a) Oxidation of 1 by MtL (phosphate–citrate buffer, pH 7.0) or PcL
(sodium acetate buffer, pH 5.0) yielding benzoquinone 2a. (b) Monoamination of 2a
by coupling of one molecule of amines 3a–c. (c) Diamination of 5a–c by coupling of
a second molecule of amines 3a–c. Postulated structure for products 4a–c formed
in reaction with MtL is shown in brackets.
As observed in the MtL-catalysed reaction, in each case the
products 4a–6a were formed, and their generation in relation
to concentration of the amine donor 3a was analysed. During
product isolation only the main products 5a (monoaminated)
and 6a (diaminated) could be enriched in sufficient quantities
and purities. Therefore, data given in Table 1 are restricted
to these products. At an equimolar reactant ratio, and with a
2 mM excess of 3a, formation of monoaminated product 5a was
achieved with yields up to 32.9% (Table 1). Generation of the
diaminated product 6a occurred only at marginal rates. Although
equal concentrations of 1 and amine 3a led predominantly to
formation of monoaminated product 5a (and 4a), the concen-
tration of by-product 2b was still remarkable and correlated
with comparable yields of 5a. Even though the structure of by-
product 2b was not elucidated within this study, we suggest a
competing reaction for the same position at the benzoquinone
2a between the amine and solvent molecules (e.g. water or
methanol). Therefore, at relatively low amine concentrations, an
advanced coupling of water or methanol to benzoquinone 2a
is highly presumed, and as described previously for amination
reactions of 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide or
cyclooctylamine 3c was analysed (Fig. 1B). As already described
for homomolecular reactions, a different spectrum of heteromolec-
ular products was obtained depending upon reaction pH, and
the laccase used. Generally, formation of up to three hetero-
molecular main products occurred with each of the amines used.
Heteromolecular coupling reactions are described for the reaction
between 1 (1 mM) and n-octylamine 3a (5 mM) catalysed by MtL
and PcL (Fig. 2A and B, respectively).
In both reaction systems, enzymatic oxidation of
1 was
associated with the primary generation of the corresponding ben-
zoquinone 2a as already described. In the PcL-catalysed reaction
(pH 5.0), benzoquinone 2a accumulated up to 60 min. A subse-
quent decrease of 2a was related to the formation of product 5a
(HPLCRt 14.9 min, ꢀmax 212, 309, 498 nm), generally appearing only
in low concentrations, and of the main product 6a (HPLCRt 17.0 min,
ꢀmax 217, 346, 508 nm) (Fig. 2B). Products 5a and 6a were later
identified as mono- and diaminated compounds, respectively. In
contrast to the reaction with PcL, in the MtL-catalysed reaction (pH
7.0), decreasing concentrations of benzoquinone 2a were partially
related to an increase of by-product 2b which was already detected
6000000
600
6000000
600
A
B
5000000
5000000
4000000
400
4000000
400
3000000
3000000
2000000
200
2000000
200
1000000
1000000
0
0
0
0
0
100
200
300
400
0
0
100
100
200
300
400
400
200
300
0
100
200
300
400
Reaction time [min]
Reaction time [min]
Fig. 2. Decrease of 2-methoxy-3-methylhydroquinone 1 (1 mM, ᭹) within formation of the 2-methoxy-3-methylbenzoquinone 2a (ꢁ), by-product 2b ( ) and the mitomycin-
like products 4a (ꢀ), 5a ( ), and 6a ( ) in heteromolecular coupling reactions with n-octylamine 3a (5 mM) catalysed by MtL (A) in phosphate-citrate buffer (pH 7.0) and
PcL (B) in sodium-acetate buffer (pH 5.0). Enzyme activities[ABTS] 1.0 ␮moL mL−1, 200 rpm, RT (mAu = milliabsorption unit).

S. Herter et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 91–97
95
Table 1
Influence of reactant ratios on the formation of monoaminated product 5a and diaminated product 6a. MtL-catalysed reactions between the 2-methoxy-3-
methylhydroquinone 1 and n-octylamine 3a in a 20-mL-reaction scale in phosphate-citrate buffer (pH 7.0). Enzyme activities[ABTS] 1 ␮moL mL−1, 200 rpm, RT.
Ratio of 1 to amine 3a [mM]
Concentration of products 5a and 6a [mM]
Formation of 5a and 6a [%]a
1 h
6 h
6 h
5a
6a
5a
6a
5a
6a
4.5
4.5
78.8
1:1
1:2
2:5
0.043
0.183
0.254
0.039
0.047
0.854
0.207
0.329
0.279
0.045
0.045
1.69
20.7
32.9
12.9
a
Yields of product formation [%] compared to a 100% conversion into desired compounds 5a and 6a, respectively, estimated via HPLC-analysis after 6 h.
3-methylcatechol [19,25]. Furthermore, amine 3a applied in an
excess (5:2 mM) resulted in formation of the diaminated product
6a with a HPLC-yield of 78.8%. This again supports the aforemen-
tioned hypothesis of a competing reaction between the amine and
solvent molecules as an increased amine concentration caused
decreased formation of by-product 2b. Therefore, at almost equal
reactant ratios, the reaction could be directed to the formation of
monoaminated product (together with a distinct presence of 2b),
whereby a higher amine concentration favours diamination. An
increased formation of diaminated products in nuclear amination
reactions with excessively applied amine donors was already
demonstrated [22,32].
309, 485 nm) at higher concentrations. The diaminated product
6c (HPLCRt 16.5 min, ꢀmax 216, 348, 508 nm) was also formed. In
the PcL-catalyzed heteromolecular reactions using a 1:5 mM ratio
between 1 and amines 3b and 3c respectively, the diaminated
products 6b (yield[6h HPLC] 84.7%) and 6c (yield[6h,HPLC] 89.8%) were
,
always identified as the main products. The monoaminated prod-
ucts 5b and 5c were only transiently detected, acting as precursors
for diamination reaction.
3.2. Structural characterisation of heteromolecular coupling
products
As described in detail for the reaction between 1 and n-
octylamine 3a, and with respect to the pH of the reaction systems,
the same results were obtained in experiments with geranylamine
3b and cyclooctylamine 3c as amine donors. With amine 3b in an
MtL-catalysed reaction system, formation of the minor monoam-
inated product 4b (HPLCRt 14.1 min, ꢀmax 289, 488 nm) and the
major monoaminated product 5b (HPLCRt 14.5 min, ꢀmax 212, 308,
488 nm) as well as the diaminated product 6b (HPLCRt 16.7 min,
ꢀmax 217, 347, 508 nm) occurred. MtL-catalysed coupling reac-
tions with cyclooctylamine 3c led to formation of monoaminated
product 4c (HPLCRt 13.2 min, ꢀmax 299, 485 nm) in low concentra-
tions and monoaminated product 5c (HPLCRt 13.7 min, ꢀmax 212,
The monoaminated products 5a–c were obtained from MtL-
catalysed reaction batches (6 h) in which educts were used in a
2:2 mM ratio: 16.3 mg 5a (and 1.4 mg 6a, 4 mL × 50 mL); 20.7 mg
5b (and 2.6 mg 6b, 4 mL × 50 mL); 22.4 mg 5c (and 3.1 mg 6c,
7 mL × 50 mL). Diaminated products 6a–c were obtained using a
2:5 mM ratio at the same conditions described above: 6.7 mg 6a,
9.3 mg 6b, and 6.6 mg 6c each with 6 mL × 50 mL. The solids were
stable at room temperature as well as 4 ◦C within the analytic period
of 3 months. HPLC analysis of products dissolved in methanol
revealed no decay over 24 h. Structural characterisation of the
monoaminated products 5a–c is shown for compound 5b (Table 2).
Table 2
1H and 13C NMR assignments and HMBC correlations for the monoaminated product 5b isolated from a reaction between 2-methoxy-3-methylhydroquinone 1 and gerany-
lamine 3b.a
.
O
1"
1
MeO
H
2
10`
3`
9`
7`
6
1`
3
5`
5
Me
2"
N
H
4
8`
2`
4`
6`
O
Pos.
13C
1H
1H,13C correlations (HMBC)
4
1
2
5
184.9
181.8
159.6
149.1
140.8
132.3
124.4
124.2
119.7
95.7
–
–
–
–
–
–
–
–
–
–
–
–
3ꢀ
7ꢀ
6ꢀ
3
5.08, t, 1H, (7.0)
–
5.19, m, 1H, (6.5)
5.22, s, 1H
C-8ꢀ (17.5), C-9ꢀ (25.4), C-5ꢀ (26.9), C-4ꢀ (40.1)
–
2ꢀ
6
C-10ꢀ (16.1), C-4ꢀ (40.1), C-1ꢀ (41.1)
C-5 (149.1), C-2 (159.6), C-1 (181.8), C-4 (184.9)
C-2 (159.6)
1ꢀꢀ
1ꢀ
4ꢀ
5ꢀ
9ꢀ
8ꢀ
10ꢀ
2ꢀꢀ
1ꢀ
61.5
41.1
40.1
26.9
25.4
17.5
16.1
7.9
4.04, s, 3H
3.76, d, 2H, (6.5)
2.05, m, 2H
2.12, m, 2H
1.65, s, 3H
1.59, s, 3H
1.72, s, 3H
C-10ꢀ(16.1), C-5ꢀ(26.9), (C-4ꢀ (40.1))b, (C-6 (95.7))b, C-2ꢀ (119.7), C-3ꢀ (140.8), C-5 (149.1), (C-4 (184.9))b
C-10ꢀ(16.1), C-5ꢀ(26.9), C-1ꢀ (41.1), C-2ꢀ (119.7), C-6ꢀ (124.4), C-3ꢀ (140.8)
C-4ꢀ (40.1), C-6ꢀ (124.4), C-7ꢀ (132.3), (C-3ꢀ (140.8))b
C-8ꢀ (17.5), C-6ꢀ (124.4), C-7ꢀ (132.3)
C-9ꢀ (25.4), C-6ꢀ (124.4), C-7ꢀ (132.3)
C-4ꢀ (40.1), C-1ꢀ (41.1), C-2ꢀ (119.7), C-3ꢀ (140.8)
C-1ꢀꢀ (61.5), C-3 (124.2), C-2 (159.6), C-4 (184.9)
C-10ꢀ(16.1), C-5ꢀ(26.9), (C-4ꢀ (40.1))b, (C-6 (95.7))b, C-2ꢀ (119.7), C-3ꢀ (140.8), C-5 (149.1), (C-4 (184.9))b
1.86, s, 3H
3.76, d, 2H, (6.5)
41.1
a
Chemical shifts are expressed in ı (ppm) referred to TMS = 0 (calibration was performed on solvent signals: ı (MeOH-d4) = 3.31 (1H), 49.1 (13C)). J values are in Hz in
brackets.
b
Correlation with low intensity.

96
S. Herter et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 91–97
Table 3
1H and 13C NMR assignments and HMBC correlations for the diaminated product 6b isolated from a reaction between 2-methoxy-3-methylhydroquinone 1 and geranylamine
3b.a
.
O
1```
Me
6`
H
N
4`
2`
4
8`
3`
9``
7``
7`
10``
3``
5
3
5`
1`
1``
5``
6
9`
10`
1
2
H
N
H
8``
2``
4``
6``
O
Pos.
13C
1H
1H,13C correlations (HMBC)
1
4
5
180.7
180.3
153.0
150.0
141.5
141.1
132.8 (2)
125.0 (2)
122.6
–
–
–
–
–
–
–
–
–
–
–
–
–
–
2
3ꢀ
3ꢀꢀ
7ꢀ,7ꢀꢀ
6ꢀ,6ꢀꢀ
2ꢀꢀ
5.08, m, 2H
5.29, m, 1H
5.21, m, 1H
–
C-9ꢀ,9ꢀꢀ (26.9), C-8ꢀ,8ꢀꢀ (17.9), C-5ꢀ,5ꢀꢀ (27.4), C-4ꢀ,4ꢀꢀ (40.6)
C-10ꢀꢀ (16.6), C-4ꢀꢀ (40.6), C-1ꢀꢀ (43.8)
C-10ꢀ (16.6), C-4ꢀ (40.6)
2ꢀ
3
120.1
103.3
–
6
92.6
43.8
5.20, s, 1H
4.20, d, 2H,
(6.3)
3.80, d, 2H,
(6.8)
2.07, m, 4H
2.12, m, 4H
1.66, s, 3H
1.65, s, 3H
1.60, br s, 3H
1.59, br s, 3H
1.73, s, 3H
1.70, s, 3H
2.05, s, 3H
2.12, m, 4H
1.66, s, 3H
1.65, s, 3H
1.60, br s, 3H
1.59, br s, 3H
1.73, s, 3H
2.05, s, 3H
(C-3 (103.3))b, C-2 (150.0), C-4 (180.3)
C-5ꢀꢀ (27.4), C-4ꢀꢀ (40.6), C-2ꢀꢀ (122.6), C-3ꢀꢀ (141.1), C-2 (150.0)
1ꢀꢀ
1ꢀ
41.5
C-5ꢀ (27.4), (C-4ꢀ (40.6))b, C-2ꢀ (120.1), C-3ꢀ (141.5), C-5 (153.0)
4ꢀ,4ꢀꢀ
5ꢀ,5ꢀꢀ
9ꢀ,9ꢀꢀ
40.6 (2)
27.4 (2)
26.0 (2)
C-10ꢀ,10ꢀꢀ (16.6), C-5ꢀ,5ꢀꢀ (27.4), C-2ꢀ (120.1), C-2ꢀꢀ (122.6), C-3ꢀ (141.5), C-3ꢀꢀ (141.1)
C-4ꢀ,4ꢀꢀ (40.6), C-6ꢀ,6ꢀꢀ (125.0), C-7ꢀ,7ꢀꢀ (132.8), C-3ꢀꢀ (141.1), C-3ꢀ (141.5)
C-8ꢀ,8ꢀꢀ (17.9), C-4,4ꢀꢀ (40.6), C-6ꢀ,6ꢀꢀ (125.0), C-7,7ꢀꢀ (132.8)
8ꢀ,8ꢀꢀ
17.9 (2)
C-9ꢀ,9ꢀꢀ (26.0), (C-4ꢀ,4ꢀꢀ (40.6))b, C-6ꢀ,6ꢀꢀ (125.0), C-7ꢀ,7ꢀꢀ (132.8)
10ꢀ
16.6
16.6
10.4
27.4 (2)
26.0 (2)
C-4ꢀ (40.3), C-2ꢀ (120.1), C-3ꢀ (141.5)
10ꢀꢀ
1ꢀꢀꢀ
C-4ꢀꢀ (40.6), C-2ꢀꢀ (122.6), C-3ꢀꢀ (141.1)
C-3 (103.3), C-2 (150.0), C-4 (180.3)
5ꢀ,5ꢀꢀ
9ꢀ,9ꢀꢀ
C-4ꢀ,4ꢀꢀ (40.6), C-6ꢀ,6ꢀꢀ (125.0), C-7ꢀ,7ꢀꢀ (132.8), C-3ꢀꢀ (141.1), C-3ꢀ (141.5)
C-8ꢀ,8ꢀꢀ (17.9), C-4,4ꢀꢀ (40.6), C-6ꢀ,6ꢀꢀ (125.0), C-7,7ꢀꢀ (132.8)
8ꢀ,8ꢀꢀ
17.9 (2)
C-9ꢀ,9ꢀꢀ (26.0), (C-4ꢀ,4ꢀꢀ (40.6))b, C-6ꢀ,6ꢀꢀ (125.0), C-7ꢀ,7ꢀꢀ (132.8)
10ꢀ
1ꢀꢀꢀ
16.6
10.4
C-4ꢀ (40.3), C-2ꢀ (120.1), C-3ꢀ (141.5)
C-3 (103.3), C-2 (150.0), C-4 (180.3)
Chemical shifts are expressed in ı (ppm) referred to TMS = 0 (calibration was performed on solvent signals: ı (MeOH-d4) = 3.31 (1H), 49.1 (13C)). J values are in Hz in
a
brackets.
b
Correlation with low intensity.
The signal appearing at 5.22 ppm in the 1H NMR spectrum of 5b
Consequently, monoamination reactions on
1 led to 1,4-
benzoquinoid coupling products 5a–c, revealing amine coupling in
para-position to benzoquinone’s methoxy substituent and in meta-
position to the methyl substituent, respectively (Scheme 1). With
regard to lower-concentration regioisomer products 4a–c, amine
coupling at benzoquinones C-6-atom could be assumed, however
this was not examined within this study.
could be assigned to H-6. A signal of the ortho-neighboured proton
H-5 did not occur. Therefore, initial consideration was given for
amine coupling at the C-5-atom. Signals at 4.04 ppm and 1.86 ppm
respectively could be related to methyl protons of the methoxy
substituent (H-1ꢀꢀ) and the protons of the methyl substituent
(H-2ꢀꢀ). A coupling of geranylamine 3b to C-5-position was clearly
confirmed by the correlation of the proton H-1ꢀ with C-5 in the
HMBC spectrum, whereas additional relatively weak correlations
appeared also with C-6 and C-4. All further signals in the aliphatic
range could be assigned to geranylamine 3b. Presence of either
a quinoidic, hydroquinoidic or quinone imine parent structure
was obvious in 13C and HMBC data. As the carbonyl-groups of
benzoquinones possess low-field resonance signals (180 ppm
up to 190 ppm), compound 5b was identified as a 1,4-quinoidic
structure according to 13C signals at 181.8 ppm for C-1 and
184.9 ppm for C-4. Moreover, the coupling between the H-2ꢀꢀ pro-
tons with the C-4 as well as between H-6 and C-4 and C-1 (weak)
Structural characterisation of diaminated products 6a–c is
explained as with compound 6b obtained from a reaction between
1 and geranylamine 3b (Table 3). HR-MS recorded in positive mode
gave for the pseudo-molecule ion [M+H]+ a mass of 425.3165 amu
(error: 0.57 ppm) and a molecular formula of C27H41N2O2. The cor-
responding sodium adduct [M+Na]+ (447.29853, error 0.74 ppm,
molecular formula: C27H40N2NaO2) was also detected. With ref-
erence to pseudo-molecule ions masses and molecular formulas,
a diamination by coupling of two geranylamine molecules was
clearly proven as product 6b revealed presence of two nitrogen
atoms. However, only two oxygen atoms appeared in the analyte
molecule indicating loss of one of the oxy-functionalised groups
of 2-methoxy-3-methylbenzoquinone 2a, either belonging to the
carbonyl groups or the methoxy substituent. With interpretation
of the 1H NMR spectrum of compound 6b, the signal appearing
at 5.20 ppm was dedicated to H-6. Furthermore, the signal for
methyl protons of the 2a methyl substituent at 2.05 ppm was also
detected. However, a signal for methyl protons of the methoxy
strengthened the presence of
a 1,4-benzoquinoidic structure
in 5-[[(2E)]-3,7-dimethylocta-2,6-dienyl]-amino]-2-methoxy-3-
methyl-[1,4]-benzoquinone (5b). Additionally, data obtained from
HR-MS measurement of product 5b ([M+H]+ 304.19013 (error:
−1.95 ppm); [M+Na]+ 326.1736 (error: 2.87 ppm), molecular
formula: C18H26NO3) reconfirmed presence of a monoaminated
2-methoxy-3-methylbenzoquinone.

S. Herter et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 91–97
97
substituent did not appear as determined for the monoaminated
product 5b (cf. Table 2). Therefore, loss of the methoxy substituent
due to coupling of a geranylamine molecule was indicated. More-
over, besides HMBC correlation between the H-1ꢀ protons of the
first coupled geranylamine with C-5 (monoamination), additional
correlations of the H-1ꢀꢀ protons of the second coupled amine
molecule with C-2 appeared. Presence of carbonyl-groups due to
diamination and therefore existence of a 1,4-benzoquinoidic ring
was again clearly observed by the low-field resonance signals at
180.7 ppm (C-4) and 180.3 ppm (C-4). Hence, diamination reaction
on 2-methoxy-3- methylbenzoquinone 2a entailed a replacement
reaction of benzoquinone’s methoxy group (Scheme 1) and thus
occurred in para-position to the first amine substituent. Sim-
ilar to the herein determined nucleophilic replacement of the
methoxy group of 2b due to diamination reactions, Schäfer and
Aguado [33] previously described comparable mechanisms for
the sodium-iodate catalysed diamination reaction on alkylsubsti-
tuted para-hydroquinones. In an earlier study we have already
described a comparable replacement reaction for the monoam-
ination of ortho-dihydroxylated 3-methylcatechol, in which a
methoxy-activated radical intermediate was identified as pre-
cursor for a following amination, entailing the replacement of the
methoxy substituent [25]. Furthermore, our results obtained for
structural characterisation of diaminated mitomycin-like products
6a–c stand in accordance with other works focused on diamination
of para-hydroquinones whereby coupling of the second amine to
the monoaminated compound usually appeared para to the first
amine substituent [22,34,35].
(Manchester Institute of Biotechnology, University of Manchester)
for help in preparing the manuscript.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.molcatb.2013.01.020.
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The reactions presented herein provide a convenient approach
to amino-functionalised para-benzoquinones with structural alike-
ness to known mitomycins. The laccase-mediated amine coupling
to 2-methoxy-3-methylhydroquinone 1 confers two of the essen-
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1,4-benzoquinoic parent structure and (ii) a biological active alkyl-
ation function ( NH). Reactions can be driven to the formation
of monoaminated mitomycin-like products in particular in a MtL-
catalysed reaction system at pH 7.0, and within this, by using equal
ratios of enzyme substrate and amine. Diaminated mitomycin-like
products are obtained in high yields in the PcL-catalysed reaction
(pH 5.0) and in general when amine donors are applied in excess.
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Acknowledgments
Financial support by the Deutsche Bundesstiftung Umwelt
(Osnabrück, Germany, grant no. AZ13191) is gratefully acknowl-
edged. S. H. is also grateful to the government of Mecklenburg-
Vorpommern, Germany, for financial support in the framework
of Landesgraduiertenförderung. We thank M. L. Thompson