Isotope-effect profiles in the oxidative N-demethylation of N,N-dimethylanilines catalysed by lignin peroxidase and a chemical model

Article abstract of DOI:10.1002/1099-0690(200106)2001:12<2305::AID-EJOC2305>3.0.CO;2-E

Lignin peroxidase catalyses the oxidative N-demethylation of ring-substituted N,N-dimethylanilines by an electron-transfer mechanism whereby an anilinium radical cation is formed which is then deprotonated by the enzyme. Information on the nature of the basic centre which deprotonates the radical cation has been obtained by determining the KDIE profile (plot of k_H/k_D vs. the pK_a of the aniline radical cations) for a number of ring-substituted N,N- bis(dideuteriomethyl)-anilines. From the bell-shaped curve it has been estimated that the pK_a of the proton-abstracting base is about 7. Interestingly, almost the same value has been obtained when the same type of study has been carried out using a water-soluble model compound: 5,10,15,20-tetraphenyl-21H,23H-porphine-p,p′,p″, p?-tetrasulfonic acid iron(III) chloride. This is a strong indication that the radical cation is deprotonated by the same species in the enzymatic and in the chemical reactions. It is suggested that this species is the reduced iron-oxo complex.

Full text of DOI:10.1002/1099-0690(200106)2001:12<2305::AID-EJOC2305>3.0.CO;2-E

FULL PAPER
Isotope-Effect Profiles in the Oxidative N-Demethylation of N,N-
Dimethylanilines Catalysed by Lignin Peroxidase and a Chemical Model
Enrico Baciocchi,*[a] M. Francesca Gerini,[b] Osvaldo Lanzalunga,[b] Andrea Lapi,[b]
Maria Grazia Lo Piparo,[b] and Simona Mancinelli[b]
Keywords: Enzyme catalysis / Oxidations / Porphyrinoids / Oxidoreductases / Anilines
Lignin peroxidase catalyses the oxidative N-demethylation
of ring-substituted N,N-dimethylanilines by an electron-
transfer mechanism whereby an anilinium radical cation is
that the pKa of the proton-abstracting base is about 7. Inter-
estingly, almost the same value has been obtained when the
same type of study has been carried out using a water-sol-
formed which is then deprotonated by the enzyme. Informa- uble model compound: 5,10,15,20-tetraphenyl-21H,23H-por-
tion on the nature of the basic centre which deprotonates the
radical cation has been obtained by determining the KDIE
profile (plot of kH/kD vs. the pKa of the aniline radical cations)
for a number of ring-substituted N,N-bis(dideuteriomethyl)-
phine-p,pЈ,pЈЈ,pЈЈЈ-tetrasulfonic acid iron(III) chloride. This is
a strong indication that the radical cation is deprotonated by
the same species in the enzymatic and in the chemical reac-
tions. It is suggested that this species is the reduced iron-
anilines. From the bell-shaped curve it has been estimated oxo complex.
Introduction
Lignin peroxidase (LiP), a heme-containing glycoprotein
isolated from the ligninolytic cultures of the white-rot
fungus Phanerochaete chrysosporium, is one of the most im-
portant enzymes involved in the biodegradation of lignin.[1]
LiP is also able to catalyse the oxidation of phenolic and
non-phenolic electron-rich aromatic lignin model com-
pounds[1c,1d,2] as well as the oxidation of different classes of
easily oxidisable organic compounds.[3]
Recently, we have reported on the ability of LiP to cata-
lyse the oxidative N-demethylation of N,N-dimethylanil-
ines,[4] a process of great biological importance. For this
reaction it has been suggested an initial electron transfer
(ET) between the N,N-dimethylaniline and the active spe-
cies of the enzyme (compound I or LiP I),[5] formed by ox-
idation of the native enzyme with H2O2 and described as
an iron(IV)-oxo porphyrin radical cation (Scheme 1, pa-
th a). Such an ET leads to an anilinium radical cation and
to the reduced form of LiP I (compound II or LiP II).[5]
The anilinium radical cation should then undergo depro-
tonation to give an α-amino carbon radical,[6] this reaction
being promoted by LiP II or whichever base is present in
the medium. In the former case the carbon radical may be
converted into a carbinolamine by oxygen rebound (path
d). In the second case the carbinolamine may be formed by
oxidation of the carbocation followed by reaction with H2O
(path e). The carbinolamine is finally converted into the N-
demethylated product and CH2O (path f).[9]
Scheme 1. Electron transfer mechanism for the oxidative N-de-
methylation of N,N-dimethylanilines catalysed by LiP
Concerning the basic centre responsible for the anilinium
radical cation deprotonation, strong evidence in favour of
an enzyme-promoted process (Scheme 1, path b or c) was
recently provided by the complete masking [kH/kD ϭ
1.04 (Ϯ 0.06)] of the intramolecular kinetic deuterium iso-
tope effect (KDIE) observed for the LiP-catalysed N-de-
methylation of 2,4,6-trichloro-N-methyl-N-trideuteriome-
thylaniline.[4] However, the actual nature of the proton-ab-
stracting base remained uncertain.
[a]
`
Dipartimento di Chimica, Universita ‘‘La Sapienza’’,
P.le A. Moro 5, 00185, Rome, Italy,
Fax: (internat.) ϩ39-06/490-421
[b]
Dipartimento di Chimica and Centro CNR di Studio sui
Meccanismi di Reazione, Universita ‘‘La Sapienza’’,
`
P.le A. Moro 5, 00185, Rome, Italy
Eur. J. Org. Chem. 2001, 2305Ϫ2310
WILEY-VCH Verlag GmbH, 69451 Weinheim, 2001
1434Ϫ193X/01/0612Ϫ2305 $ 17.50ϩ.50/0
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E. Baciocchi et al.
FULL PAPER
In order to acquire more information in this respect an porphyrin radical cation and closely resemble the active
intramolecular KDIE study of the oxidative N-demethyl- species of LiP (LiP I) (Scheme 3).
ation of ring substituted N,N-dimethylanilines catalysed by
Results
LiP has been undertaken. The idea was that the KDIE de-
pendence on the radical cation pKa might give useful in-
formation on the strength and the nature of the deprotonat-
ing base. As substrates, the N,N-bis(dideuteriomethyl)anil-
ines reported in Scheme 2 were used. With these substrates,
masking effects should be minimised since H and D are
bonded to the same carbon atom.
N,N-dimethylanilines (10 µmol) were reacted in the pres-
ence of LiP (0.96 units, 1.16 nmol) or FeTPPSCl (0.3 µmol)
with an equimolar amount of hydrogen peroxide added,
over a period of 1 h, by an infusion pump, to an argon-
degassed 50 m sodium-tartrate-buffered solution, pH ϭ
4.0, containing 2% of CH3CN as the cosolvent, at 25 °C.
A clean N-demethylation reaction occurred with formation
of the corresponding N-methylaniline and formaldehyde,
which was detected by GC-MS after conversion into the
dimedone adduct. Reaction products were characterised by
GC-MS and 1H NMR spectroscopy. Yields were deter-
mined by GC and 1H NMR spectroscopy and were referred
to the starting material. The results are reported in Table 1
where the reduction potentials of the ArNϩ·(CH3)2/
ArN(CH3)2 couple are also displayed. Some experiments
with FeTPPSCl were also carried out in the presence of
imidazole, but the results were practically the same as in
its absence.
Scheme 2. Ring substituted N,N-bis(dideuteriomethyl)anilines used
in this work
The results of the LiP-catalysed N-demethylation reac-
tions have also been compared with those obtained using a
chemical model compound: FeTPPSCl [5,10,15,20-tetra-
phenyl-21H,23H-porphine-p,pЈ,pЈЈ,pЈЈЈ-tetrasulfonic
acid
Table 1. Yields of N-demethylated products (ArNHCH3) in the LiP
or FeTPPSCl catalysed oxidation of ring substituted N,N-dimethyl-
anilines [ArN(CH3)2] by H2O2 and reduction potentials of the
ArNϩ·(CH3)2/ArN(CH3)2 couple
iron(III) chloride]. Water-soluble metalloporphyrins like
FeTPPSCl are known to mimic LiP reactivity,[10] since the
active species, formed by reaction with a suitable oxygen
donor (e.g. H2O2), can be described as an iron(IV)-oxo
Ar
E° vs. NHE
Yields (%)[b]
LiP[c]
(V)[a]
FeTPPSCl
2,4,6-Cl3C6H2
4-NO2C6H4
3,4,5-Cl3C6H2
4-CNC6H4
4-CF3C6H4
4-BrC6H4
1.58[c]
1.46
25
20
27
7
11
5
30
23
3
12
10
1.40[c]
1.37
62
1.33[d]
1.15[d]
1.11
28[e]
16
4-ClC6H4
4-CH3C6H4
n.d.
Ϫ
0.94
[a] Values estimated in water (ref.[11]) from the corresponding values
in CH3CN (ref.[12]). Ϫ [b] Referred to the starting material, equimo-
lar to H2O2. Average of at least two determinations, the error is in
[c]
all cases less than Ϯ 1. Ϫ From ref.[4] Ϫ [d] Value estimated from
a Hammett correlation. Ϫ [e] A small amount of 4-trifluoromethyl-
aniline (5%) was also detected.
KDIE values were determined by GC-MS analysis of the
formaldehyde-dimedone adduct by the ratio of the intensity
of the molecular peaks m/z ϭ 294 and 293, corrected for a
statistical factor and the 13C contribution. The KDIE
values are reported in Table 2, together with the pKa of the
N,N-dimethylaniline radical cations, which were estimated
by the usual thermochemical cycle.[13]
Discussion
With both the enzymatic and the biomimetic systems the
yields of the N-demethylated product do not follow any
regular trend upon increasing the electron-donating proper-
ties of the ring substituent, probably due to the variable
Scheme 3. Formation of the active oxidant by reaction of FeTPPS
with H2O2
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Eur. J. Org. Chem. 2001, 2305Ϫ2310

Isotope-Effect Profiles in the Oxidative N-Demethylation of N-Dimethylanilines
FULL PAPER
extent of substrate protonation. In this respect, it should be
noted that 4-methyl-N,N-dimethylaniline reacted only with
FeTPPSCl. LiP I is probably a weaker oxidant than the act-
ive species of FeTPPSCl[17] and is not able to promote the
oxidation of this substrate, which is almost completely pro-
tonated at the low pH required for the enzymatic cata-
lysis.[18]
Examining the data reported in Table 2, a very remark-
able observation is that the KDIE values are very similar
for both the enzymatic and the biomimetic systems. More-
over, increasing the electron-donating properties of the ring
substituents (and the pKa of the N,N-dimethylaniline rad-
ical cations) the KDIE values increase up to a maximum
value and then decrease for both systems. Thus, a plot of
the KDIE values as a function of the pKa of the substituted
N,N-dimethylaniline radical cations presents the bell-shaped
profile depicted in Figure 1 for the enzymatic (Figure 1, A)
and biomimetic (Figure 1, B) systems. The solid curves re-
ported are the theoretical ones obtained from Equation (1),
[where ∆pKa ϭ pKa Ϫ pKa (max)] based on the Marcus the-
ory as applied to acid-base reactions.[19]
Table 2. KDIE values for LiP or FeTPPSCl catalysed oxidative N-
demethylation of ring substituted N,N-bis(dideuteriomethyl) anil-
ines [ArN(CHD2)2] by H2O2
Ar
KDIE[a]
FeTPPSCl
pKa radical cation[b]
LiP
2,4,6-Cl3C6H2
4-NO2C6H4
3,4,5-Cl3C6H2
4-CNC6H4
4-CF3C6H4
4-BrC6H4
3.4 (1)
5.3 (2)
5.6 (1)
5.9 (1)
5.8 (1)[c]
5.3 (1)
5.1 (2)
n.d.
3.4 (1)
5.3 (2)
5.5 (1)
5.7 (2)
5.6 (2)
5.7 (2)
5.1 (1)
4.0 (2)
2.2
4.1
5.2
5.7
6.4
9.4
10.1
13.0
4-ClC6H4
4-CH3C6H4
[a] Average of at least four determinations. The error (standard devi-
[b]
ation) in the last significant digit is given in parentheses. Ϫ
In
water. Ϫ [c] A lower amount of LiP (0.64 units, 0.77 nmol) was used
to avoid the formation of 4-trifluoromethylaniline.
(1)
This result is fully consistent with the proposed ET mech-
anism (Scheme 1), with the KDIE being determined in the
step involving the dimethylaniline radical cation depro-
tonation (Scheme 1, path b, or c). A hydrogen-atom transfer
(HAT) mechanism appears unlikely since the strength of
the NCH2ϪH bond in the neutral aniline is almost insensit-
ive to the presence of ring substituents[14] and the same
should hold for the KDIE values. Accordingly, only a small,
steady decrease of the KDIE values was observed upon de-
creasing the electron-withdrawing properties of the ring
substituents in the oxidative N-demethylation of N,N-di-
methylanilines promoted by cytochrome P450, a reaction
suggested to occur by an HAT mechanism.[20]
The pKa values corresponding to the maximum of the
profiles reported in Figure 1 are 7 and 8. According to the
theory, the maximum of the bell-shaped curve should be
reached when the pKa of the acid equals that of the depro-
tonating base.[19,21] It follows that bases of very similar
strength act in both the enzymatic and biomimetic reac-
tions. Thus, unless we are dealing with an extraordinary
coincidence, the most simple conclusion is that the same
deprotonating base, with a pKa value around 7, is operating
in the two reactions. In this situation, the most reasonable
suggestion is that the deprotonation of the intermediate
radical cation is promoted by the reduced iron-oxo complex
(LiP II). Very interestingly, a pKa greater than that of pyrid-
ine (and therefore Ͼ5, in water) has already been proposed
for the PϪFeIVϭO species on the basis of a study of the N-
demethylation of 9-tert-butyl-N-methylacridane catalysed
by an iron porphyrin.[22,23]
Figure 1. Bell-shaped curve for the LiP (A) and FeTPPSCl (B)
catalysed oxidative N-demethylation of ring substituted N,N-bis(di-
deuteriomethyl)anilines
This suggestion, if correct, would be very interesting
since it would seem to contrast the current views about the
Eur. J. Org. Chem. 2001, 2305Ϫ2310
2307

E. Baciocchi et al.
FULL PAPER
restricted accessibility of the heme in LiP,[25] thereby the
substrate should have access only to the heme edge, as very
crudely shown in Figure 2, where the electron-transfer pro-
cess can still take place, but not the deprotonation of the
radical cation by the ferryl oxygen. Accordingly, the pos-
sibility that compound II would act as a base in the N-de-
methylation reactions was suggested for CPO and
cytochrome P450, but not for HRP, whose access to the
heme is much more restricted than in the other two enzymes
and similar to that in LiP.[9] However, the structure depicted
in Figure 2 might not be rigid and it cannot be excluded
that the substrate radical cation may move to put the α-
hydrogen close enough to the oxygen of the reduced form
of the iron-oxo complex, as suggested by the present re-
sults.[26] On the other hand, it should be remembered that
LiP (and HRP) is able to effect sulfoxidation with the oxy-
gen-transfer process occurring from the oxoferryl species of
the enzyme.[27]
MS analyses were performed on an HP5890 GC (OV1 capillary
column, 12 m ϫ 0.2 mm) coupled with an HP5970 MSD. GC ana-
lyses were performed on a Varian 3400 GC (OV1 capillary column,
25 m ϫ 0.2 mm) and Varian Vista 6000 (OV1701 capillary column,
30 m ϫ 0.35 mm).
Bi-distilled water and CH3CN (CARLO ERBA-HPLC grade) were
used as solvents. High purity commercial samples of tartaric acid,
4-methoxyacetophenone and dimedone (Aldrich) were used as re-
ceived. LiP was prepared and purified as described in the literat-
ure.[28] The concentration of the enzyme solution was determined
spectrophotometrically (ε409nm ϭ 169 mϪ1 cmϪ1).[29] The concen-
tration of H2O2 (Carlo Erba Reagents) was determined by titration
with permanganate.[30] 4-Chloro-N,N-bis(dideuteriomethyl)aniline,
4-cyano-N,N-bis(dideuteriomethyl)aniline and 4-nitro-N,N-bis-
(dideuteriomethyl)aniline were prepared according to the
literature procedure.[20] 5,10,15,20-Tetraphenyl-21H,23H-porphine-
p,pЈ,pЈЈ,pЈЈЈ-tetrasulfonic acid tetrasodium salt dodecahydrate (Ald-
rich) was metallated according to the literature procedure.[31]
General Procedures for Substrate Preparation: The ring-substituted
N,N-bis(dideuteriomethyl)anilines were prepared by reaction of the
corresponding anilines with [D2]paraformaldehyde and sodium
borohydride according to the literature procedure.[20] A Schlenk
tube charged with [D2]paraformaldehyde (Aldrich, 99%, 7.5 mmol)
was heated at 200 °C under a stream of argon and the evolved gas
collected in sulfuric acid (3 , 12 mL). The formaldehyde/sulfuric
acid solution was cooled in an ice-water bath and a slurry of aniline
(2.5 mmol) and sodium borohydride (20 mmol) in tetrahydrofuran
(30 mL) was added dropwise over a period of 10 min. The reaction
mixture was allowed to warm to room temperature and stirred for
40 min. After recooling in an ice-water bath, the reaction mixture
was made basic by dropwise addition of a sodium hydroxide solu-
tion. The organic layer was separated and the aqueous solution
was extracted with diethyl ether. The combined organic layers were
washed with water and then brine and dried over anhydrous
Na2SO4. Each compound was purified by chromatography on silica
Figure 2. Rough visualisation of the heme-edge approach of N,N-
dimethylaniline in the LiP active site
Finally, it should be noted that the hypothesis of a base
with pKa 7Ϫ8 operating at pH ϭ 4 may be reasonable only
if this base is a transient species, as is the case with
PϪFeIVϭO, which should form in close association with gel. The purity of all these compounds (Ͼ99%) was checked by
GC, GC-MS and 1H NMR spectroscopy. The isotopic composition
of the substituted N,N-bis(dideuteriomethyl)anilines was deter-
the radical cation.
mined by GC-MS in the selected ion monitoring mode from the
D4/D0 ratios. In all cases the percentage of the deuterated product
was Ͼ98%.
Conclusion
LiP and its model compound FeTPPSCl catalyse the ox-
3,4,5-Trichloro-N,N-bis(dideuteriomethyl)aniline: This material was
prepared using the procedure described above. Purification on silica
gel with petroleum ether/diethyl ether (9:1) as the eluent gave, after
solvent removal, a pale yellow solid (70%). H NMR (CDCl3, 25
°C, TMS): δ ϭ 6.67 (s, 2 H), 2.88Ϫ2.92 (m, 2 H).
idative N-demethylation of ring substituted N,N-dimethyl-
anilines by an ET mechanism leading to the formation of
N-methylanilines and formaldehyde. The intermediate anili-
nium radical cation thus formed is then deprotonated by
the enzyme. Bell-shaped curves are obtained in the KDIE
1
profiles (plot of kH/kD vs. the pKa of the radical cations) 2,4,6-Trichloro-N,N-bis(dideuteriomethyl)aniline: This material was
prepared using the procedure described above. Purification on silica
gel with petroleum ether as the eluent gave, after solvent removal,
a colourless liquid (54%). 1H NMR (CDCl3, 25 °C, TMS): δ ϭ
7.26 (s, 2 H), 2.81Ϫ2.85 (m, 2 H).
both for the enzymatic and biomimetic oxidations. From
these curves, pKa values of 7 (enzymatic reaction) and 8
(biomimetic reaction) have been estimated for the proton-
abstracting base. The close similarity of the two cases is a
strong indication that the radical cation is deprotonated by
the same species in the enzymatic and in the chemical reac-
tions. It is therefore most likely that this species is the re-
duced iron-oxo complex.
N,N-Bis(dideuteriomethyl)-4-methylaniline: This material was pre-
pared using the procedure described above. Purification on silica
gel using n-pentane/chloroform (5:1) as the eluent gave, after solv-
ent removal, a colourless liquid (70%). 1H NMR (CDCl3, 25 °C,
TMS): δ ϭ 7.07 (d, J ϭ 8.2 Hz, 2 H), 6.74 (d, J ϭ 8.3 Hz, 2 H),
2.87 (m, 2 H), 2.26 (s, 3 H).
Experimental Section
General: 1H NMR spectra were recorded on a Bruker AC300P
N,N-Bis(dideuteriomethyl)-4-trifluoromethylaniline: This material
was prepared using the procedure described above. Purification on
silica gel using petroleum ether as the eluent gave, after solvent
spectrometer in CDCl3 using TMS as the internal standard. GC-
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Eur. J. Org. Chem. 2001, 2305Ϫ2310

Isotope-Effect Profiles in the Oxidative N-Demethylation of N-Dimethylanilines
FULL PAPER
[4]
removal, a white solid (75%). 1H NMR (CDCl3, 25 °C, TMS): δ ϭ
7.45 (d, J ϭ 8.6 Hz, 2 H), 6.70 (d, J ϭ 8.6 Hz, 2 H), 2.97 (m, 2 H).
E. Baciocchi, M. F. Gerini, O. Lanzalunga, A. Lapi, S. Manci-
nelli, P. Mencarelli, Chem. Commun. 2000, 393Ϫ394.
V. Renganathan, M. H. Gold, Biochemistry 1986, 25,
1626Ϫ1631.
The possibility that the radical cation reacts with PϪFeIVϭO
by a HAT mechanism has also been considered at least for the
biomimetic process.[7] However, the results reported by Lind-
say-Smith seem clearly in favour of a deprotonation step.[8]
Moreover, the HAT mechanism is unlikely also on energetic
[5]
[6]
4-Bromo-N,N-bis(dideuteriomethyl)aniline: This material was pre-
pared using the procedure described above. Purification on silica
gel using petroleum ether/diethyl ether (4:1) as the eluent gave, after
solvent removal, a white solid (72%). 1H NMR (CDCl3, 25 °C,
TMS): δ ϭ 7.30 (d, J ϭ 9.0 Hz, 2 H), 6.58 (d, J ϭ 9.0 Hz, 2 H),
2.88 (m, 2 H).
grounds, the homolytic CϪH BDE free energy (180 kJ molϪ1
,
in MeCN) being much higher than the heterolytic one
Enzymatic or Biomimetic Oxidation: H2O2 (10 µmol) was added,
over a period of 1 h by an infusion pump, to a magnetically stirred
argon-degassed solution of the substrate (10 µmol), LiP (0.96 units,
1.16 nmol) or FeTPPSCl (0.3 µmol) in 3 mL of 50 m sodium-tart-
rate-buffered solution with 2% acetonitrile as cosolvent, pH ϭ 4,
at 25 °C. At the end of the reaction the mixture was made basic,
the products of the reaction were extracted with CH2Cl2 and dried
over Na2SO4. In the LiP-catalysed oxidation of 4-trifluoromethyl-
N,N-bis(dideuteriomethyl)aniline a lower amount of LiP (0.64
units, 0.77 nmol) was used to avoid the formation of 4-trifluoro-
methylaniline.
(70 kJ molϪ1).
[7]
[8]
[9]
Y. Goto, Y. Watanabe, S. Fukuzumi, J. P. Jones, J. P. Dinno-
cenzo, J. Am. Chem. Soc. 1998, 120, 10762Ϫ10763.
J. R. Lindsay-Smith, D. N. Mortimer, J. Chem. Soc., Perkin
Trans. 2 1986, 1743Ϫ1749.
A similar mechanism was proposed for other hemoproteins. O.
Okazaki, F. P. Guengerich, J. Biol. Chem. 1993, 268,
1546Ϫ1552 and references therein.
B. Meunier, in Metalloporphyrins in Catalytic Oxidations (Ed.:
R. A. Sheldon), Marcel Dekker, Inc, New York, 1994, Chap-
ter 5.
[10]
[11]
[12]
[13]
Product Analysis: Yields were determined by GC and 1H NMR
spectroscopy (with 4-methoxyacetophenone as the internal stand-
ard) and referred to the starting material. A good material balance
(Ͼ90%) was observed in all the experiments.
M. Jonsson, D. D. M. Wayner, J. Lusztyk, J. Phys. Chem. 1996,
100, 17539Ϫ17543.
V. D. Parker, M. Tilset, J. Am. Chem. Soc. 1991, 113,
8778Ϫ8781.
The pKa of the N,N-dimethylaniline radical cations were estim-
ated by the following equation: pKa ϭ (2.3 RT)Ϫ1 ϫ [∆G°hom
Ϫ 96.48 E°ArNϩ·(CH3)2/ArN(CH3)2 ϩ 96.48 E°Hϩ/H·]where ∆G°hom
is the standard free energy associated with the homolytic cleav-
age of the NCH2ϪH bond, taken as 346 kJ molϪ1 (at 25 °C),
resulting from the difference between ∆H°hom (380 kJ molϪ1),
an average value for the NCH2ϪH homolytic bond dissoci-
ation enthalpy for a number of N,N-dimethylanilines,[14] and
T∆S°hom, where ∆S°ϭ 115 J KϪ1 is the standard entropy of the
hydrogen atom.[15] E°ArNϩ·(CH3)2/ArN(CH3)2 is the standard
redox potential (V vs. NHE in H2O) of the couple
KDIEs Measurement: At the end of the enzymatic or biomimetic
oxidation the reaction mixture was treated with 400 µL of a basic
solution of dimedone (0.2 ) in order to allow the formation of the
dimedone-formaldehyde adduct. After 30 minutes the mixture was
extracted with CH2Cl2. All the KDIE values, averaged over at least
four independent determinations, were determined by GC-MS ana-
lysis of the formaldehyde-dimedone adduct by the ratio of the in-
tensity of the molecular peaks m/z ϭ 294 and 293, corrected for
the statistical factor and for the 13C contribution, in the oxidation
of N,N-bis(dideuteriomethyl)anilines.
ArNϩ·(CH3)2/ArN(CH3)2 (Table 1). E°Hϩ/H·
, the standard
redox potential of the couple Hϩ/H·, is taken as Ϫ2.29 V vs.
NHE in H2O.[16]
[14]
G. W. Dombrowski, J. P. Dinnocenzo, S. Farid, J. L. Goodman,
I. R. Gould, J. Org. Chem. 1999, 64, 427Ϫ431.
Acknowledgments
This work was carried out with the financial support of Consiglio
[15] [15a]
A. M. de P. Nicholas, D. R. Arnold, Can. J. Chem. 1982,
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Received February 9, 2001
[O01066]
2310
Eur. J. Org. Chem. 2001, 2305Ϫ2310