process can still take place, but not the deprotonation of the
radical cation by the ferryl oxygen.8 One would therefore
predict a non-enzymatic pathway for the deprotonation of the
radical cation in the LiP-catalysed N-demethylation reactions.
However, strong evidence in favour of a deprotonation
promoted by the enzyme has been provided by the complete
masking [kH/kD = 1.04 (± 0.06)] of the intramolecular KDIE
observed for the N-demethylation of N-methyl-N-trideutero-
gica (MURST) and the Consiglio Nazionale delle Ricerche
(CNR). We also thank Professor Pietro Tagliatesta, University
of ‘Tor Vergata’, Rome, Italy, for cyclic voltammetry meas-
urements.
Notes and references
methyl-2,4,6-trichloroaniline
1 catalysed by LiP (25%
1 (a) M. Tien and T. K. Kirk, Science, 1983, 221, 661; (b) J. K. Glenn,
M. A. Morgan, M. B. Mayfield, M. Kuwahara and M. H. Gold,
Biochem. Biophys. Res. Commun., 1983, 114, 1077; (c) H. E.
Schoemaker, Recl. Trav. Chim. Pays-Bas, 1990, 109, 255; (d) G. Labat
and B. Meunier, Bull. Soc. Chim. Fr., 1990, 127, 553.
yield).9,10
2 (a) P. J. Kersten, M. Tien, B. Kalyanaraman and T. K. Kirk, J. Biol.
Chem., 1985, 260, 2609; (b) K. E. Hammel, M. D. Mozuch, P. J. Kersten
and K. A. Jensen, Biochemistry, 1994, 33, 13349 and references therein;
(c) K. Joshi and M. H. Gold, Eur. J. Biochem., 1996, 237, 45.
3 (a) S. D. Haemmerli, M. S. A. Leisola, D. Sanglard and A. Fiechter,
J. Biol. Chem., 1986, 261, 6900; (b) D. K. Joshi and M. H. Gold,
Biochemistry, 1994, 33, 10969; (c) D. C. Goodwin, S. D. Aust and T. A.
Grover, Biochemistry, 1995, 34, 5060; (d) B. Kalyanaraman, Xenobio-
tica, 1995, 25, 667; (e) A. Paszczynski, S. Goszczynski, R. L. Crawford
and D. L. Crawford, Microb. Processes Biorem., 1995, 187; (f) M.
Chivukula, J. T. Spadaro and V. Renganathan, Biochemistry, 1995, 34,
7765.
4 M. Tien and T. K. Kirk, Methods Enzymol., 1988, 161, 238.
5 (a) V. D. Parker and M. Tilset, J. Am. Chem. Soc., 1991, 113, 8778. (b)
This value has been measured in MeCN, the value in water should be
significanly lower (of ca. 0.4 V) (ref. 6).
6 M. Jonsson, D. D. M. Wayner and J. Lusztyk, J. Phys. Chem., 1996, 100,
17539.
7 (a) Peroxidases in Chemistry and Biology, ed. J. Everse, K. E. Everse
and M. B. Grisham, CRC Press, Boca Raton, 1991, vol. I and II; (b) O.
Okazaki and F. P. Guengerich, J. Biol. Chem., 1993, 268, 1546 and
references therein.
8 A. M. English and G. Tsaprailis, Adv. Inorg. Chem., 1995, 43, 79.
9 All the KDIE values reported in this work were determined by GC-MS
analysis of the formaldehyde–dimedone adduct, and they are an average
of at least three independent determinations.
In this system, due to the steric effect of the two ortho-
chlorine atoms, the two N-methyl groups are forced to stay one
above and the other below the plane of aromatic ring as
confirmed by theoretical calculations based on the Density
Functional approach (DFT), carried out on the radical cation of
N,N-dimethyl-2,6-dichloroaniline, a model compound for 1.11
These calculations moreover show that there is a quite large
barrier (33.5 kJ mol21) to the rotation around the C(aromatic)–
N bond.12 Thus, the absence of a deuterium isotope effect for
this substrate strongly suggests that deprotonation has to take
place in the enzyme pocket, being significantly faster than
rotation of CH3 and CD3 groups around the C(aromatic)–N
bond.13 Under these conditions, the loss of hydrogen or
deuterium will only depend on which one of the two methyl
groups in the radical cation is oriented towards the proton
abstracting center and reasonably there is the same probability
that this group is CH3 or CD3. Thus, the intramolecular KDIE
with this substrate should be completely masked, as is actually
observed. This interpretation is confirmed by the significant
value of intramolecular KDIE (3.36 ± 0.07) found instead in the
oxidation of N,N-bis(dideuteromethyl)-2,4,6-trichloroaniline 2
(27 % yield), where deuterium and hydrogen are bonded to the
same carbon.14 In 2, the KDIE is no longer influenced by the
hindered rotation mentioned before. Further support also comes
from the high intramolecular KDIE value (7.0 ± 0.8) measured
in the oxidation of N-methyl-N-trideuteromethyl-3,4,5-tri-
chloroaniline, where the absence of ortho-substituents allows
the two methyl groups to freely interchange within the enzyme
pocket.
In conclusion, our data clearly show that LiP can catalyse the
oxidative N-demethylation of aromatic tertiary amines with
fairly good efficiency. Moreover, the intramolecular KDIEs
measured with 1 and 2 indicate that the aminium radical cation
is deprotonated by the enzyme. Concerning the basic center,
Compound II seems unlikely, in the light of the already
mentioned current views about the accessibility of the heme in
this enzyme.8 Another hypothesis is that the deprotonation of
the radical cation is promoted by some specific amino acid
residue located in or very close to the active site. Histidine 82
might be a suitable candidate in this respect. An additional
possibility might be a medium induced deprotonation of an
enzyme complexed radical cation.15
10 Compound 1 was prepared by reacting 2,4,6-trichloroaniline with MeI
and then with CD3I.
11 (a) The calculations were carried out, with the GAUSSIAN 94 package
[ref. 11(b)], by using the DFT approach at the B3LYP/6-311+G(d,p)
level of theory. Spin contamination due to states of multiplicity higher
than the doublet state was negligible, in that the < S2 > parameter was,
in all cases, well within 10% of the expectation value for a doublet
(0.75). (b) M. J. Frisch, G. W. Trucks, H. B. Schlegel, P. M. W. Gill,
B. G. Johnson, M. A. Robb, J. R. Cheeseman, T. Keith, G. A. Petersson,
J. A. Montgomery, K. Raghavachari, M. A. Al-Laham, V. G.
Zakrzewski, J. V. Ortiz, J. B. Foresman, J. Cioslowski, B. B. Stefanov,
A. Nanayakkara, M. Challacombe, C. Y. Peng, P. Y. Ayala, W. Chen,
M. W. Wong, J. L. Andres, E. S. Replogle, R. Gomperts, R. L. Martin,
D. J. Fox, J. S. Binkley, D. J. Defrees, J. Baker, J. P. Stewart, M. Head-
Gordon, C. Gonzalez and J. A. Pople, GAUSSIAN 94, Revision D.2,
Gaussian, Inc., Pittsburgh PA, 1995.
12 This can also be argued by the oxidation peak potential (Ep) measured
for 1 (1.34 V vs. SCE) [ref. 5(b)] which is significantly higher than that
measured for its isomer N,N-dimethyl-3,4,5-trichloroaniline (Ep = 1.16
V vs. SCE) [ref. 5(b)].
13 Even assuming that the barrier to rotation does not increase in the
enzyme pocket, a deprotonation faster than rotation around the
C(aromatic)–N bond is plausible, as an activation enthalpy less than
33.5 kJ mol21 is possible for the former reaction [ref. 5(a)].
14 Compound 2 was prepared by reacting 2,4,6-trichloroaniline with CD2O
and NaBH4 (S. B. Karki, J. P. Dinnocenzo, J. P. Jones and K. R.
Korzekwa, J. Am. Chem. Soc., 1995, 117, 3657).
15 We thank one of the referees for this suggestion.
This work was carried out with the financial support of the
Ministero dell’Università e della Ricerca Scientifica Tecnolo-
Communication a908394d
394
Chem. Commun., 2000, 393–394