Instrumentation
References
A VAꢀIAN 3400 Star gas chromatograph, fitted with a 20 m ×
1 M. Tien and T. K. Kirk, Science, 1983, 221, 661–663.
2 M. Kuwahara, J. K. Glenn, A. Morgan and M. H. Gold, FEBS
Lett., 1984, 169, 247–250.
0
.25 mm methyl silicone gum capillary column, was employed
in the GC analyses. GC-MS analyses were performed on a HP
892 GC, equipped with a 12 m × 0.2 mm methyl silicone gum
3
A. Messerschmidt, Multi-Copper Oxidases, World Scientific,
Singapore, 1997.
5
capillary column, and coupled to a HP 5972 MSD instrument,
operating at 70 eV. The acquisition of the MS signal was in the
SIM mode for the Ar-CHO vs. Ar-CDO relative intensity
determinations.
4
(a) F. Xu, J. J. Kulys, K. Duke, K. L. K. Krikstopaitis,
H.-J. W. Deussen, E. Abbate, V. Galinyte and P. Schneider, Appl.
Environ. Microbiol., 2000, 66, 2052–2056; (b) F. Xu, W. Shin,
S. H. Brown, J. A. Wahleithner, U. M. Sundaram and E. I. Solomon,
Biochim. Biophys. Acta, 1996, 1292, 303–311; (c) K. Li, F. Xu
and K.-E. L. Eriksson, Appl. Environ. Microbiol., 1999, 65,
Enzymatic reactions
2
654–2660.
The oxidation reactions were performed at room temperature
in water solution (3 mL), buffered at pH 5 (0.1 M in sodium
5 (a) ꢀ. Bourbonnais and M. G. Paice, FEBS Lett., 1990, 267, 99–102;
(b) ꢀ. Bourbonnais and M. G. Paice, Appl. Microbiol. Biot., 1992,
3
6, 823–827; (c) ꢀ. Bourbonnais, M. G. Paice, B. Freiermuth,
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627–4632.
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161–2169.
citrate) and purged with O for 30 min prior to the addition of
2
7
the reagents. In case of sparingly soluble substrates, 10%
4
acetonitrile was added. The concentration of the reagents was:
6
[substrate], 20 mM; [mediator], 6 mM, with 10 units of laccase.
2
The incubation was carried out for 24 h under constant
stirring, keeping a latex balloon half-filled with oxygen on top
the reaction vessel. Following a conventional work-up, the
yields of oxidation were determined by GC analysis with
respect to an internal standard (acetophenone or p-methoxy-
acetophenone), suitable response factors being determined
from authentic products. The identity of the products was also
confirmed by GC-MS analyses. No other products, besides
those indicated in the Tables, were detected.
7 M. Fabbrini, C. Galli and P. Gentili, J. Mol. Catal. B: Enzym., 2002,
16, 231–240.
8
L. Banci, S. Ciofi-Baffoni and M. Tien, Biochemistry, 1999, 38,
205–3210.
F. d’Acunzo, C. Galli and B. Masci, Eur. J. Biochem., 2002, 269,
330–5335.
3
9
5
1
0 M. Fabbrini, C. Galli and P. Gentili, J. Mol. Catal. B: Enzym., 2002,
18, 169–171.
11 C. Eggert, U. Temp, J. F. D. Dean and K.-E. L. Eriksson, FEBS
Lett., 1996, 391, 144–148.
1
1
2 H.-P. Call and I. Mücke, J. Biotechnol., 1997, 53, 163–202.
3 M. Fabbrini, C. Galli, P. Gentili and D. Macchitella, Tetrahedron
Lett., 2001, 42, 7551–7553.
Competition experiments
Competition experiments of two substrates, either for the
Hammett’s correlations (k /k ), but also for the k /k and k /k
X
H
3
1
3
7
relative rate determinations, were similarly run on a 40 mmol
amount of each of the substrates, giving the following initial
concentrations: [ArCH OH], 20 mM; [PhCH OH], 20 mM,
2
2
[mediator], 6 mM, with 10 units of laccase. The yields of
products were determined by GC, after a reaction time (4–5 h)
that would ensure only a modest/moderate conversion into
products (10–25%). In order to determine the k /kD ratios,
H
determination of the relative amount of the Ar-CHO and
Ar-CDO oxidation products (Scheme 4) was done by GC-MS
analyses after a 5 h reaction time.
Chemical oxidations
III
Oxidations with K Co W O (viz. Co()W) (60 mmol) of
5
12 40
a substrate (30 mmol) were conducted in 2 mL citrate buffer,
at room temperature for 1 or 3 days; conventional work-up
22 (a) M. Bietti, E. Baciocchi and S. Steenken, J. Phys. Chem. A, 1998,
02, 7337–7342; (b) J. Howell, J. M. Goncalves, C. Amatore,
1
L. Klasinc, ꢀ. M. Wightman and J. K. Kochi, J. Am. Chem. Soc.,
1984, 106, 3968–3976; (c) P. J. Kersten, B. Kalyanaraman,
K. E. Hammel, B. ꢀeinhammar and T. K. Kirk, Biochem. J., 1990,
268, 475–480.
with CHCl or ethyl acetate followed. In the oxidations with
3
ϩϩ
‘
preformed ABTS ’, 20 µmol of ABTS were dissolved in 1.5
IV
mL of 2 M H SO ; 40 µmol of (NH ) Ce (NO ) (viz. CAN)
2
4
4
2
3 6
dissolved in 1.5 mL of 2 M H SO were added, and the red
23 T. Iwahama, Y. Yoshino, T. Keitoku, S. Sakaguchi and Y. Ishii,
J. Org. Chem., 2000, 65, 6502–6507.
2
4
colour of the dication developed immediately; 20 µmol of
substrate were added very quickly at this point, and the result-
ing solution stirred at room temperature for 3 min, or until
2
2
2
2
2
4 F. Minisci, C. Punta, F. ꢀecupero, F. Fontana and G. F. Pedulli,
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5 F. d’Acunzo, P. Baiocco, M. Fabbrini, C. Galli and P. Gentili,
New J. Chem., 2002, 26, 1791–1794.
6 F. Minisci, C. Punta, F. ꢀecupero, F. Fontana and G. F. Pedulli,
Chem. Commun., 2002, 688–689.
ؒ
ϩ
the red colour had turned blue (i.e., ABTS ). Conventional
work-up followed.
7 D. F. McMillen and D. M. Golden, Ann. Rev. Phys Chem., 1982, 33,
Abbreviations
4
93–532.
8 C. Walling, A. L. ꢀieger and D. D. Tanner, J. Am. Chem. Soc., 1963,
5, 3129–3134.
HBT
HPI
VLA
ABTS
1-Hydroxybenzotriazole
N-Hydroxyphthalimide
Violuric acid
2,2Ј-Azinobis(3-ethylbenzothiazoline-6-
sulfonic acid)
8
2
3
9 E. Baciocchi and O. Lanzalunga, Tetrahedron, 1993, 49, 7267–7276.
0 T. H. Lowry and K. S. ꢀichardson, Mechanism and Theory in
Organic Chemistry, 2nd ed., Harper & ꢀow, New York, 1981.
31 G. J. Gleicher, J. Org. Chem., 1968, 33, 332–336.
3
3
3
3
2 E. Baciocchi, T. Del Giacco, C. ꢀol and G. V. Sebastiani,
Tetrahedron Lett., 1989, 30, 3573–3576.
3 E. Baciocchi, S. Belvedere, M. Bietti and O. Lanzalunga, Eur. J. Org.
Chem., 1998, 299–302.
4 K. Li, ꢀ. F. Helm and K.-E. L. Eriksson, Biotechnol. Appl. Bioc.,
Acknowledgements
Thanks are due to Novo Nordisk Biotech (Denmark) for a
generous gift of laccase. We also thank the EU for financial
support (grant QLK5-CT-1999-01277) to the OXYDELIGN
project.
1
998, 27, 239–243.
5 E. Baciocchi, M. Mattioli, ꢀ. ꢀomano and ꢀ. ꢀuzziconi, J. Org.
Chem., 1991, 56, 7154–7160.
1
96
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 9 1 – 1 9 7