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Selectivity and mechanism in the microsomal benzylic hydroxylation
Tetr~,n~.VoUI.No.24,p~3477-3480.1990 RiitedinGreatBritain
SELECIMTY
oo40-4039/90 $3.00+.00 PqmonRc.s8pk
AND MECHANISM IN TIlE MICROSOMAL BENZYLIC HYDROXYLATION
Rachele Amodeo,
Enrico Baciocchi*,
Dipartimento
Manuela
Crescenzi
di Chimica, Universita‘
and Osvaldo
’ La Sapienza
Lanzalunga
‘I,
Piazzale A. Moro 5, 00185 Roma, Italy
Summary
: The oxidation by rat liver microsomes of 4-Z-1,2-dimethylbenzenes (1) and 4-methoxybenzyltrimethylsilane (2) has been investigated. The reaction of the former substrates leads to the expected isomeric benzyl alcohols 3 and 4, with a very low intramolecular selectivity, and only 0-demethylation is observed in the reaction of 2. These results suggest the operation of a hydrogen atom transfer mechanism.
One of the most important capacity
of carrying
alkylaromatic species,
an
transfer
iron(V)
insight
oxo-form
process between
(HAT)
are still
of the Cytochrome
SC-H
-
There of
the
object
of continuous
is attributed
a and
reaction
to the design
conversion
but debate
pathways:
of enzymes with
alkanes
on the nature
the
mechanistic
particularly
is the and
of the active aspects
of
with regard
the
to the
that involving
a hydrogen
(ET),
between
the 0x0 complex
in the Scheme).
In this
context
an electron
b, respectively
P450 family
agreement
hemoprotein,
and the one involving (paths
GC-OH
is a general
the two most likely
and the substrate relevance
the
compoundsl.
hydroxylation distinction
out
properties
transfer
and use of mechanistic
probes
atom
particular
which can provide
some
into the problem2.
(P45OFe(V)O----H----C z )* a /
\
P450Fc(V)O + H-C?
P450Fe(IV)OH + ‘Cf
b \ P45OFe(IV)O + $C-H
1+ /
Scheme
3477
-
P45OFe(III) + --)C-OH
We have recently provided,
that
with a reasonable
selectivity much
shown
degree
of the side chain
higher
in ET than
in HAT
of benzyltrimetylsilanes
observed
only in ET processes)4. of the mechanism
enzymes
and
in
4-Z-1,2-dimethylbenzenes compounds
(desilylation,
with
formation
this
note
with
specimens
).
chloroform
The microsomal of ca 20-30 %
in the Table
of alkylaromatics we
report
on
potential
where
in the photochemical
and
oxidation
with
formed
of benzylic
respect
buffer)
analyzed
and an NADPH
by GC and
of 1 leads to the isomeric to
the
starting
they are compared chlorination
(an ET process,
in the
derivatives
is
induced the
by cytochrome
microsomal
oxidation
P-450 of
(2). These
values of 0.6-0.8 V5 and thus should allow
out at 35-36 oC for 60 min. The mixture
extracted
products
Thus, it is seemed of interest to apply these criteria to the
(4.5 mM) with phenobarbital
(0.65 g in 6.5 ml of phosphate
oxidation
the reaction
(generally
of the scopes of HAT and ET mechanisms.
of the substrates
been carried
di- and tri-methylbenzenes
and
can be
the intramolecular
(1, Z = Cl, t-Bu) and 4-methoxybenzyltrimethylsilane
exploration
Incubation
concerning
reactions)3
cover a range of oxidation
a meaningful
HAT and ET mechanisms
by studies
of substituted
of the oxidation
accordingly
between
of confidence,
oxidation
oxidation
problem
a distinction
substrate.
generating
CAN = cerium(IV)
ammonium
rat liver system
has then been
GC-MS
microsomes8
(0.65 ml) have
treated
(comparison
with
with HCl, authentic
benzyl alcohols 3 and 4 with a yield The
with the corresponding
( a HAT reaction)
induced
3/4 molar ratios are reported
values
obtained
and in the CAN-promoted
side-chain
nitrate) of the same substrate@.
3479
Table. Intramolecular Selectivity 4-Z- 1.2-Dimethylbenzenes.
[(3/4)
3/4 Z
molar
ratio]
CAN/AcOHb
1.4
t-Bu
Side Chain
Reactions
of
molar ratio
oxidation by microsomesa
Cl
in Some
C12,hv/CC14C
13.0
2.0
6.0
1.2
1.6
(a) Average of two or more determinations. For the reaction conditions see text. (b) Ref. 9. In this case 3 and 4 are the corresponding benzyl acetates. (c) Ref. 9. After hydrolysis of the corresponding chlorides.
It
can
immediately
intramolecular
be
selectivity.
in the photochemical
Clearly,
benzylic
oxidation.
This
2.
mechanism of
a
In this
since
reaction
that attributed this conclusion
extends
promoted
for
the
mechanism
oxidation
cytochrome potentials
the former
(yield
btnzyl Only
case,
porphyrins
whereas
low
in the reactions
for the microsomal
by the
results
obtained
in the
form,
which
excludes
an ET
radical the
is
the
expected
0-demethylation
fate
product of the
with an oxidation
potential
P-45011.
investigations
induced
even lower than
It has to be noted that which have suggested
oxidations
of heteroatom
an
containing
up to 2.3 Vlo.12. been
porphyrins
preferential
shown
to hold
also
and iodosobenzene
for the benzylic
in CH2Cl213,
in an apolar mcdiumlsb,
however,
by the model
are used.
very
ca 5 %), which again is indicative
of cytochrome
P-450
A difference,
and that induced
mechanism
derivatives a
substrates
has recently
P-450.
a
Thus, since the Ep of 2 is ca 1.8 V vs NHE, the scope of the
of the use of these compounds,
of cytochrome
exhibits
from those found
supported
radical4.
is observed
iron(III)
oxidation
a HAT
is not in line with that of previous
by synthetic
the validity
produce
to include
with oxidation
A HAT
iron(II1)
to
( ca 2 V ) to the oxo-form
ET mechanism substrates
suggest
no benzylic
of a HAT mechanismlo.
HAT mechanism
different
is also
cation
4-hydroxybenzyltrimethylsilane operation
results
conclusion
benzyltrimethylsilane
microsomal
and greatly
these
desilylation
the
the 3/4 molar ratios are quite close to those observed
chlorinations
by CAN.
of
that
Moreover,
induced
oxidation
seen
has
systems:
attack
been
noted
which confirms
to mimic the properties between
with 2, 0-demethylation
at the benzylic
oxidation
hydrogens
the
microsomal
is observed takes
place
in
when
3480
It is probable oxidation
that the lack of reactivity
has to be ascribed
difficult
an appropriate
of the benzylic
to an effect
approach
of the protein
of the bulky
hydrogens
in the microsomal
structure14
CH2SiMe3
which
might
make
group to the active site of the
enzyme. Acknowledgments. Scientifica financial
Thanks
e Tecnologica
are due to the Minister0
(MURST) and to the Consiglio
support. We are also greatly indebted
S. Gallicano,
Rome) for assistance
per la Universita’
Nazionale
delle Ricerche
to Dr. S. Passi (Laboratorio
and advice in the preparation
e la Ricerca (CNR) for
Ricerca, Ospedale
of micro.somes.
References 1. (a) “Cytochrome
P-450”,
(b) F. .P. Guengerich,
P.R.Ortiz
de Montellano,
T. L. Macdonald,
ti.
Tetr.u
P. F. Hollenberg, Montellano,
(1989);
L Biol.
Cba,
3. E. Baciocchi,
and
F. P. Guengerich,
A, Dalla Cart.
L. Eberson,
Press, New York (1986); 9 (1984) 2446
G. T. Miwa,
14445 (1983); 4081 (1985);
u,
J.
L. T. Burka, P. Peyman
1l.
J.&
2. L. R. Hall, R. T. Iwamoto, R. P. Hanzlick, R. P. Hanzlich,
Ed., Plenum
Act..
(1989);
J. S. Walsh,
R. A. Stearns, P. R. Ortiz de T. L. Macdonald, K. Zirvi,
J.
SOC,~,,
L. Mandolini.
P. Riley and
G. L. Kedderis,
C. Rol,
2050
(1982).
J. Ore. Chew=
4544
(1986). 3573 (1989). 4. E. Baciocchi, T. Del Giacco, C. Rol, G. V. Sebastiani, Tetr Leu, 5. An Ep value of 1.8 V vs NHE is reported for 2 6. For 1 (Z=Cl) an Ep value of ca 2.5 V vs NHE can
be estimated7.
6. J. P. Dinnocenzo, -Am.
S. Farid,
Sot,
7. S. Fukuzumi,
m,
8973
J. K. Kochi.
8. R. P. Hanzlich. 9. E. Baciocchi,
K. Hogberg, M. Crescenzi,
10. J. R. Lindsay
J. L. Goodman,
Smith,
I. R.
Gould,
W. P. Todd,
S.
L. Mattes,
(1989).
J.
m, C. M. Judson, Tetrahedron
R. E. Piggott,
7240
vu. 44,
(1981). 3048
6525
P. R. Sleath,
(1984).
(1988). J. Chem.
Sot.
Chem.
Co-
55
(1982) 11. T. L. Macdonald, 2071
W. G. Gutheim,
R. B. Martin,
F. P. Guengerich,
Biochem.X,
(1989).
12. F. P. Guengerich. J.
R. J. Willard,
Soc._m
6446
J. P. Shea,
L. E. Richards,
T. L. Macdonald.
(1984)
13. (a)E. Baciocchi, M. Crescenzi, 0. Lanzalunga, J. Chem. Sot. Cv .. in press. (b) However, when a more polar solvent is used, radical cation formation is possible. 14. G. T. Miwa and A. Y. H. Lu, Ref. la Chapter 3; P. R. Ortiz de Montellano Act.
Chem
Res,a,
289
(Received in UK 14 March 1990)
(1987).
SELECIMTY
oo40-4039/90 $3.00+.00 PqmonRc.s8pk
AND MECHANISM IN TIlE MICROSOMAL BENZYLIC HYDROXYLATION
Rachele Amodeo,
Enrico Baciocchi*,
Dipartimento
Manuela
Crescenzi
di Chimica, Universita‘
and Osvaldo
’ La Sapienza
Lanzalunga
‘I,
Piazzale A. Moro 5, 00185 Roma, Italy
Summary
: The oxidation by rat liver microsomes of 4-Z-1,2-dimethylbenzenes (1) and 4-methoxybenzyltrimethylsilane (2) has been investigated. The reaction of the former substrates leads to the expected isomeric benzyl alcohols 3 and 4, with a very low intramolecular selectivity, and only 0-demethylation is observed in the reaction of 2. These results suggest the operation of a hydrogen atom transfer mechanism.
One of the most important capacity
of carrying
alkylaromatic species,
an
transfer
iron(V)
insight
oxo-form
process between
(HAT)
are still
of the Cytochrome
SC-H
-
There of
the
object
of continuous
is attributed
a and
reaction
to the design
conversion
but debate
pathways:
of enzymes with
alkanes
on the nature
the
mechanistic
particularly
is the and
of the active aspects
of
with regard
the
to the
that involving
a hydrogen
(ET),
between
the 0x0 complex
in the Scheme).
In this
context
an electron
b, respectively
P450 family
agreement
hemoprotein,
and the one involving (paths
GC-OH
is a general
the two most likely
and the substrate relevance
the
compoundsl.
hydroxylation distinction
out
properties
transfer
and use of mechanistic
probes
atom
particular
which can provide
some
into the problem2.
(P45OFe(V)O----H----C z )* a /
\
P450Fc(V)O + H-C?
P450Fe(IV)OH + ‘Cf
b \ P45OFe(IV)O + $C-H
1+ /
Scheme
3477
-
P45OFe(III) + --)C-OH
We have recently provided,
that
with a reasonable
selectivity much
shown
degree
of the side chain
higher
in ET than
in HAT
of benzyltrimetylsilanes
observed
only in ET processes)4. of the mechanism
enzymes
and
in
4-Z-1,2-dimethylbenzenes compounds
(desilylation,
with
formation
this
note
with
specimens
).
chloroform
The microsomal of ca 20-30 %
in the Table
of alkylaromatics we
report
on
potential
where
in the photochemical
and
oxidation
with
formed
of benzylic
respect
buffer)
analyzed
and an NADPH
by GC and
of 1 leads to the isomeric to
the
starting
they are compared chlorination
(an ET process,
in the
derivatives
is
induced the
by cytochrome
microsomal
oxidation
P-450 of
(2). These
values of 0.6-0.8 V5 and thus should allow
out at 35-36 oC for 60 min. The mixture
extracted
products
Thus, it is seemed of interest to apply these criteria to the
(4.5 mM) with phenobarbital
(0.65 g in 6.5 ml of phosphate
oxidation
the reaction
(generally
of the scopes of HAT and ET mechanisms.
of the substrates
been carried
di- and tri-methylbenzenes
and
can be
the intramolecular
(1, Z = Cl, t-Bu) and 4-methoxybenzyltrimethylsilane
exploration
Incubation
concerning
reactions)3
cover a range of oxidation
a meaningful
HAT and ET mechanisms
by studies
of substituted
of the oxidation
accordingly
between
of confidence,
oxidation
oxidation
problem
a distinction
substrate.
generating
CAN = cerium(IV)
ammonium
rat liver system
has then been
GC-MS
microsomes8
(0.65 ml) have
treated
(comparison
with
with HCl, authentic
benzyl alcohols 3 and 4 with a yield The
with the corresponding
( a HAT reaction)
induced
3/4 molar ratios are reported
values
obtained
and in the CAN-promoted
side-chain
nitrate) of the same substrate@.
3479
Table. Intramolecular Selectivity 4-Z- 1.2-Dimethylbenzenes.
[(3/4)
3/4 Z
molar
ratio]
CAN/AcOHb
1.4
t-Bu
Side Chain
Reactions
of
molar ratio
oxidation by microsomesa
Cl
in Some
C12,hv/CC14C
13.0
2.0
6.0
1.2
1.6
(a) Average of two or more determinations. For the reaction conditions see text. (b) Ref. 9. In this case 3 and 4 are the corresponding benzyl acetates. (c) Ref. 9. After hydrolysis of the corresponding chlorides.
It
can
immediately
intramolecular
be
selectivity.
in the photochemical
Clearly,
benzylic
oxidation.
This
2.
mechanism of
a
In this
since
reaction
that attributed this conclusion
extends
promoted
for
the
mechanism
oxidation
cytochrome potentials
the former
(yield
btnzyl Only
case,
porphyrins
whereas
low
in the reactions
for the microsomal
by the
results
obtained
in the
form,
which
excludes
an ET
radical the
is
the
expected
0-demethylation
fate
product of the
with an oxidation
potential
P-45011.
investigations
induced
even lower than
It has to be noted that which have suggested
oxidations
of heteroatom
an
containing
up to 2.3 Vlo.12. been
porphyrins
preferential
shown
to hold
also
and iodosobenzene
for the benzylic
in CH2Cl213,
in an apolar mcdiumlsb,
however,
by the model
are used.
very
ca 5 %), which again is indicative
of cytochrome
P-450
A difference,
and that induced
mechanism
derivatives a
substrates
has recently
P-450.
a
Thus, since the Ep of 2 is ca 1.8 V vs NHE, the scope of the
of the use of these compounds,
of cytochrome
exhibits
from those found
supported
radical4.
is observed
iron(III)
oxidation
a HAT
is not in line with that of previous
by synthetic
the validity
produce
to include
with oxidation
A HAT
iron(II1)
to
( ca 2 V ) to the oxo-form
ET mechanism substrates
suggest
no benzylic
of a HAT mechanismlo.
HAT mechanism
different
is also
cation
4-hydroxybenzyltrimethylsilane operation
results
conclusion
benzyltrimethylsilane
microsomal
and greatly
these
desilylation
the
the 3/4 molar ratios are quite close to those observed
chlorinations
by CAN.
of
that
Moreover,
induced
oxidation
seen
has
systems:
attack
been
noted
which confirms
to mimic the properties between
with 2, 0-demethylation
at the benzylic
oxidation
hydrogens
the
microsomal
is observed takes
place
in
when
3480
It is probable oxidation
that the lack of reactivity
has to be ascribed
difficult
an appropriate
of the benzylic
to an effect
approach
of the protein
of the bulky
hydrogens
in the microsomal
structure14
CH2SiMe3
which
might
make
group to the active site of the
enzyme. Acknowledgments. Scientifica financial
Thanks
e Tecnologica
are due to the Minister0
(MURST) and to the Consiglio
support. We are also greatly indebted
S. Gallicano,
Rome) for assistance
per la Universita’
Nazionale
delle Ricerche
to Dr. S. Passi (Laboratorio
and advice in the preparation
e la Ricerca (CNR) for
Ricerca, Ospedale
of micro.somes.
References 1. (a) “Cytochrome
P-450”,
(b) F. .P. Guengerich,
P.R.Ortiz
de Montellano,
T. L. Macdonald,
ti.
Tetr.u
P. F. Hollenberg, Montellano,
(1989);
L Biol.
Cba,
3. E. Baciocchi,
and
F. P. Guengerich,
A, Dalla Cart.
L. Eberson,
Press, New York (1986); 9 (1984) 2446
G. T. Miwa,
14445 (1983); 4081 (1985);
u,
J.
L. T. Burka, P. Peyman
1l.
J.&
2. L. R. Hall, R. T. Iwamoto, R. P. Hanzlick, R. P. Hanzlich,
Ed., Plenum
Act..
(1989);
J. S. Walsh,
R. A. Stearns, P. R. Ortiz de T. L. Macdonald, K. Zirvi,
J.
SOC,~,,
L. Mandolini.
P. Riley and
G. L. Kedderis,
C. Rol,
2050
(1982).
J. Ore. Chew=
4544
(1986). 3573 (1989). 4. E. Baciocchi, T. Del Giacco, C. Rol, G. V. Sebastiani, Tetr Leu, 5. An Ep value of 1.8 V vs NHE is reported for 2 6. For 1 (Z=Cl) an Ep value of ca 2.5 V vs NHE can
be estimated7.
6. J. P. Dinnocenzo, -Am.
S. Farid,
Sot,
7. S. Fukuzumi,
m,
8973
J. K. Kochi.
8. R. P. Hanzlich. 9. E. Baciocchi,
K. Hogberg, M. Crescenzi,
10. J. R. Lindsay
J. L. Goodman,
Smith,
I. R.
Gould,
W. P. Todd,
S.
L. Mattes,
(1989).
J.
m, C. M. Judson, Tetrahedron
R. E. Piggott,
7240
vu. 44,
(1981). 3048
6525
P. R. Sleath,
(1984).
(1988). J. Chem.
Sot.
Chem.
Co-
55
(1982) 11. T. L. Macdonald, 2071
W. G. Gutheim,
R. B. Martin,
F. P. Guengerich,
Biochem.X,
(1989).
12. F. P. Guengerich. J.
R. J. Willard,
Soc._m
6446
J. P. Shea,
L. E. Richards,
T. L. Macdonald.
(1984)
13. (a)E. Baciocchi, M. Crescenzi, 0. Lanzalunga, J. Chem. Sot. Cv .. in press. (b) However, when a more polar solvent is used, radical cation formation is possible. 14. G. T. Miwa and A. Y. H. Lu, Ref. la Chapter 3; P. R. Ortiz de Montellano Act.
Chem
Res,a,
289
(Received in UK 14 March 1990)
(1987).