Nuclear Amination Catalyzed by Fungal Laccases
and amines has been observed before.36,37 In contrast to
the reactions described in these reports, no diamination
took place in the amination of 1j. The propionic acid
residue seems not to be displaceable as it is described
for alkyl groups,37 so that the reaction stops at the
monoaminated product.
Our results suggest that laccase-catalyzed amination
of o- or p-hydroquinones with amines always results in
the formation of the corresponding mono- or diaminated
quinonamines or quinonimines, and that laccase-cata-
lyzed reactions are not suitable for the synthesis of
aminohydroquinones. In fact, it would be highly surpris-
ing if aminohydroquinones were synthesizable with use
of oxidative enzymes under oxygen, given that amino-
hydroquinones are highly susceptible to oxidation.38
As addition of amines to quinonoid systems are well-
known reactions in organic synthesis,39 further studies
will be performed to investigate the differences and
similarities between laccase-catalyzed and nonenzymatic
nuclear amination.
described above. For production scale, laccase of M. thermo-
phila (final activity 1.0 units‚mL-1) was added to 100 mL of a
solution of the compounds (2 mM hydroquinone, 10 mM amine)
dissolved in citrate-phosphate buffer under the same condi-
tions. Products were isolated approximately 24 h after addition
of the enzyme.
Oxidation of tert-Butylhydroquinone. Laccase of M.
thermophila (final activity 1.0 units‚mL-1) was added to 100
mL of a 4 mM solution of 1g dissolved in citrate-phosphate
buffer under the conditions used for the amination of alkylated
hydroquinones. The product was isolated approximately 2 h
after starting the reaction.
Isolation of the Products. Products soluble in buffer
(3a-h, 3l, 3n,o, 4a-c, 5): All isolation steps were performed
by solid-phase extraction with an RP18 silica gel column (Strata
C18-E, 50 µm, 70 Å, 5 g/20 mL, Phenomenex, Aschaffenburg,
Germany). After activation with methanol and equilibration
with methanol/aqua dest. (10:90 v/v), the column was charged
with 50 mL of the reaction mixture. Washing steps with 25
mL of aqua dest. and 25 mL of methanol/aqua dest. (10:90
v/v) were carried out to remove laccase and polar impurities
from the column. Monoaminated products were eluted with
methanol/aqua dest. (50:50 v/v). The dark red fraction was
collected, ignoring the less dark front and tail of this fraction.
After complete elution of this fraction from the column, solvent
was switched to methanol to elute the yellow-brown fraction
(if diaminated products were formed). This procedure was
repeated. After solid-phase extraction of the products, the
corresponding fractions were combined and dried under vacuum
at 30 °C or lyophilized. In the case of 3f and 5, acetonitrile
was used instead of methanol due to fast degradation of the
products in methanol.
Conclusions
Though nuclear amination reactions of p-hydroquino-
nes with aromatic amines have been studied before, we
showed here for the first time that laccases provide a new
synthetic route to aminoquinones. Employing laccase-
catalyzed reactions, mono- and diaminated quinones can
be synthesized in good to very good yields starting from
p-dihydroxylated benzoic acid derivatives or alkylated
hydroquinones and primary aromatic amines.
Products with low solubility in buffer (3i-k, 3m, 4d):
Reaction mixtures were spun in portions of 50 mL for 20 min
at 4000 rpm with a centrifuge. The combined residues were
washed thrice with 20 mL of methanol/aqua dest. (5:95 v/v).
Products were dissolved in methanol and dried under vacuum
at 30 °C.
Experimental Section
Enzymes. Extracellular laccase C of Trametes spec. (EC
1.10.3.2) was obtained from ASA Spezialenzyme (Wolfenbu¨ttel,
Germany) and used as received (activity 1000 U/g; substrate:
syringaldazine). Boiled enzyme was held at 100 °C for 60 min
in buffer prior to use.
Laccase from Myceliophthora thermophila (expressed in
genetically modified Aspergillus sp.) was bought from
NovoNordisk (Bagsvaerd, Denmark). It was used as received
(activity 1000 U/g; substrate: syringaldazine).
Yields have not been optimized.
2-[4′-(Carboxyphenyl)amino]-3-(2′′-hydroxyethylcar-
bamoyl)-1,4-benzoquinone (3a). Synthesis and isolation as
described above. Dark red to purple solid. Yield 70% (46.2 mg);
mp 184-186 °C. 1H NMR δ 12.79 (br s, 1H), 12.42 (br s, 1H),
9.14 (t, J ) 5.3 Hz, 1H), 7.84 (d, J ) 8.5 Hz, 2H), 7.30 (d, J )
7.9 Hz, 2H), 6.81 (d, J ) 9.5 Hz, 1H), 6.76 (d, J ) 9.5 Hz, 1H),
4.77 (br s), 3.41 (t, J ) 5.6 Hz, 2H), 3.17 (q, J ) 5.7 Hz, 2H).
13C NMR δ 184.2 (C-4), 182.9 (C-1), 166.7 (C-7′), 166.3 (C-7),
148.6 (C-2), 143.4 (C-1′), 138.9 (C-6), 133.8 (C-5), 129.7 (C-3′,
C-5′), 127.2 (C-4′), 123.3 (C-2′, C-6′), 105.5. (C-3), 59.3 (C-2′′),
41.2 (C-1′′). HMBC correlations see Table 3. IR (KBr) ν 3436,
2941, 1691, 1644, 1563, 1542 cm-1. UV-vis (MeOH) λmax (log
ꢀ) 201 (4.46), 261 (4.24), 501 (3.63) nm. LC/MS m/z (rel
intensity) 331 ([M + 1]+, 63), 270 (100) APCI, pos. mode; 330
([M]-, 100) APCI, neg. mode; 353 ([M + Na]+, 100), 683 ([2M
+ Na]+, 19) API-ES, pos. mode; 329 ([M - 1]-, 100) API-ES,
neg. mode. HRFT-ICRMS [M + H]+ calcd for C16H15N2O6
331.0930, found 331.0921.
2,3-Dimethyl-5-[(4′-carboxyphenyl)amino]-1,4-bezo-
quinone (3l). Synthesis and isolation as described above.
Dark violet solid. Yield 60% (32.4 mg); mp 179-181 °C. 1H
NMR δ 7.96 (d, J ) 8.4 Hz, 2H), 7.24 (d, J ) 8.4 Hz, 2H), 6.06
(s, 1H), 2.02 (s, 3H), 2.00 (s, 3H). 13C NMR δ 188.4, 184.8,
174.6, 145.3, 144.3, 141.5, 138.5, 135.8, 131.7, 122.5, 101.2,
12.6, 12.1. IR (KBr) ν 3281, 1646, 1596, 1524 cm-1. UV-vis
(MeOH) λmax (log ꢀ) 202 (4.33), 278 (4.28), 497 (3.52) nm. LC/
MS m/z (rel intensity) 272 ([M + 1]+, 100) APCI, pos. mode;
271 ([M]-, 100) APCI neg. mode; 270 ([M - 1]-, 100), 136 (31)
API-ES, neg. mode. HRFT-ICRMS [M + H]+ calcd for
C15H14NO4 272.0923, found 272.0919.
Amination of 2,5-Dihydroxybenzoic Acid Derivatives
and 1j with Aromatic Amines. For analytical scale experi-
ments, the compounds were incubated at equimolar concentra-
tions of 1, 2, 5, and 10 mM with laccase in 10 mL of 20 mM
sodium acetate buffer, pH 5 (laccase from T. spec., final activity
0.15 units‚mL-1) or 10 mL of citrate-phosphate buffer (16 mM
citrate, 164 mM phosphate), pH 7 (laccase from M. thermo-
phila, final activity 1.0 units‚mL-1). The reaction mixture was
incubated at room temperature with agitation at 300 rpm. For
production scale, laccase of T. spec. (final activity 0.1 units‚mL-1
)
was added to 100 mL of a 2 mM solution of the compounds
dissolved in sodium acetate buffer under the same conditions.
Products were isolated 60 to 90 min after starting the reaction.
For the synthesis of diaminated quinones, amine excess was
used (10:2 mM).
Amination of Alkylated Hydroquinones with Aro-
matic Amines. Analytical scale experiments were done as
(36) Horspool, W.; Smith, P.; Tedder, J. J. Chem. Soc. C 1971, 1,
138-140.
(37) Horspool, W.; Smith, P.; Tedder, J. J. Chem. Soc., Perkin Trans.
1 1972, 8, 1024-1030.
(38) Kuckla¨nder, U.; Ulmer, P.; Kuna, K.; To¨berich, H. Chem. Ber.
1989, 122, 209-212.
2,5-Bis[4′,4′′′-(carboxyphenyl)amino]-3-(2′′-hydroxy-
ethylcarbamoyl)-1,4-benzoquinone (4a). Synthesis and
isolation as described above. Yellow-brown to brown solid.
(39) Finley, K. In The Chemistry of the quinonoid compounds;
Rappoport, Z., Ed.; John Wiley and Sons Ltd.: Bath, UK, 1988; Vol.
2, pp 537-717.
J. Org. Chem, Vol. 70, No. 6, 2005 2007