Copper-promoted overall transformation of 4-tert-butylphenol to its para-hydroxyquinonic derivative, 2-hyroxy-5-tert-butyl-1,4-benzoquinone. Biomimetic studies on the generation of Topaquinone in copper amine oxidases

Article abstract of DOI:10.1016/S0960-894X(00)00145-1

Topaquinone (TPQ) is a cofactor present at the active site of copper amine oxidases, derived from a Tyr residue inserted in the polypeptide chain through a copper-dependent but otherwise largely unknown mechanism. A simple model system was developed that permits to obtain the overall transformation of 4-tert-butylphenol, chosen as a model for Tyr, into a TPQ-like, para-hydroxyquinonic structure in the presence of Cu(II)-imidazole mononuclear complexes. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00145-1

Bioorganic & Medicinal Chemistry Letters 10 (2000) 989±992
Copper-Promoted Overall Transformation of 4-tert-Butylphenol
to its para-Hydroxyquinonic Derivative,
2-Hydroxy-5-tert-butyl-1,4-benzoquinone.
Biomimetic Studies on the Generation of Topaquinone in
Copper Amine Oxidases
Andrea C. Rinaldi, a,* Gustavo Ponticelli, b Stefania Oliva, a Antonio Di Giulio c
and Enrico Sanjust a
a
Á
Cattedra di Chimica Biologica, Dipartimento di Scienze Mediche Internistiche, Universita di Cagliari, I-09042 Monserrato (CA), Italy
b
Á
Dipartimento di Chimica Inorganica ed Analitica, Universita di Cagliari, I-09042 Monserrato (CA), Italy
Á
Dipartimento di Scienze e Tecnologie Biomediche, Universita dell'Aquila, Via Vetoio, Loc. Coppito, I-67100 L'Aquila, Italy
c
Received 20 December 1999; accepted 1 March 2000
AbstractÐTopaquinone (TPQ) is a cofactor present at the active site of copper amine oxidases, derived from a Tyr residue inserted in
the polypeptide chain through a copper-dependent but otherwise largely unknown mechanism. A simple model system was developed
that permits to obtain the overall transformation of 4-tert-butylphenol, chosen as a model for Tyr, into a TPQ-like, para-hydroxy-
quinonic structure in the presence of Cu(II)-imidazole mononuclear complexes. # 2000 Elsevier Science Ltd. All rights reserved.
Copper amine oxidases (CuAOs, EC 1.4.3.6) are ubi-
quitous enzymes that catalyze the oxidative deamina-
tion of primary amines to produce aldehydes, hydrogen
peroxide, and ammonia.1 These enzymes contain an
active-site redox cofactor, identi®ed by Klinman and co-
workers as 2,4,5-trihydroxyphenylalanine quinone 1
(topaquinone, TPQ; Fig. 1).2 Recent studies have shown
that TPQ is produced by the post-translational mod-
i®cation of a strictly conserved active-site tyrosine resi-
due by enzyme-bound copper and oxygen,1b,3 in which
appears to be a unique process among known enzymatic
systems. In addition to the several fascinating mechan-
istic implications of the reaction, the full elucidation of
TPQ biogenesis is also stimulated by the increasing
medical interest in amine oxidases.4 Indeed, among the
CuAOs, the physiologically relevant lysyl oxidase has
been recently shown to contain a modi®ed form of
TPQ, designated lysine tyrosylquinone (LTQ),5 that is
expected to be generated in a manner similar to that of
TPQ. In patients with Menkes disease, which is char-
acterized by a state of copper de®ciency, the activity of
lysyl oxidase is strongly impaired, giving rise to several
clinical manifestations.6 Very recent data indicate that
copper de®ciency does not in¯uence lysyl oxidase
expression, but results in an enzyme with reduced levels
of its active site cofactor LTQ.7
Although the detailed mechanism of TPQ and LTQ bio-
genesis has yet to be elucidated, several plausible path-
ways leading from Tyr to TPQ (or LTQ) have been
proposed that invoke a certain number of intermediates.
In his simplest form (Fig. 2), the overall reaction is postu-
lated to involve the initial hydroxylation of Tyr to 3,4-
dihydroxyphenylalanine (dopa), followed by oxidation of
dopa to dopaquinone. Nucleophilic attack by water (or
hydroxide ion) on the dopaquinone ring leads to topa.
Further oxidation of topa would ®nally give TPQ. Alter-
natively, the reaction could produce directly dopaquinone
without passing through the compulsory intermediacy of
the catechol, as suggested by Sayre and Nadkarni.8
We have previously addressed the issue of TPQ generation
by means of biomimetic studies that utilized non-enzy-
matic model compounds for TPQ and putative biogenetic
intermediates.1b,9 In particular, the steps following the
formation of dopa, have been investigated. More
*Corresponding author. Fax: +39-070-675-4527; e-mail: rinaldi@
vaxcal.unica.it
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00145-1

990
A. C. Rinaldi et al. / Bioorg. Med. Chem. Lett. 10 (2000) 989±992
Table 1. Dependence on pH value of the production of HtBQ from
a
4-tert-butylphenol in the presence of CuIm4(ClO4)2
pH
HtBQ [mM]
Eciency relative to
that at pH 11 (100)
8
0.01
0.16
0.45
0.20
2.23
35.56
100
10
11
12
44.45
Figure 1. Structure of TPQ and of its depronated form. The anionic
form shows a characteristic absorbtion band in the visible region,
centered around 485±490 nm.
aExperimental conditions as reported in the Notes.10 HtBQ yield esti-
1
mated from absorbance at 485 nm (e=1800 M cm 1).
Table 2. Relative eciency in the production of HtBQ from 4-tert-
butylphenol in the presence of free or complexed Cu(II)a
Cu(II)
compound
HtBQ
[mM]
Eciency relative to that
with CuIm4(ClO4)2 (100)
CuIm4(ClO4)2
CuIm4Cl2
CuIm2Cl2
free Cu(ClO4)2
free CuCl2
0.45
0.43
0.36
0.27
0.26
100
95.56
80.00
60.00
57.78
aAll experiments were performed at pH 11. Other experimental con-
ditions as reported in the Notes.10 HtBQ yield estimated from absor-
1
bance at 485 nm (e=1800 M cm 1).
noticed. The achieved yield of conversion of 4-tert-
butylphenol into HtBQ was low (approx. 2.25%), and
most of the starting material remained unreacted (data
not shown). However, this is not surprising, given the
diculty to get the bulk production of para-hydro-
xyquinonic compounds under alkaline conditions even
when starting from catechols.11a
Figure 2. Postulated pathway for the biogenisis of TPQ cofactor from
a tyrosine residue in CuAOs. LTQ cofactor in lysyl oxidase is thought
to derive from Tyr through a similar mechanism.
recently, we started to focus on the ®rst step of the TPQ
generation, i.e., the oxidative conversion of tyrosine to
dopa, which is by far the least understood portion of the
biogenetic reaction. We herewith report some data show-
ing that Cu(II)±imidazole mononuclear complexes are
able, under experimental conditions, to induce the overall
transformation of 4-tert-butylphenol, chosen as a model
for Tyr, into a TPQ-like structure, namely 2-hydroxy-5-
tert-butyl-1,4-benzoquinone (HtBQ). This model system
might therefore o€er a frame of reference for interpreta-
tion of studies on TPQ biogenesis in CuAOs.
The identity of the ®nal product of 4-tert-butylphenol
hydroxylation and oxidation in our model system,
HtBQ, is proven by its absorption spectrum (Fig. 3),
showing a broad peak centered around 485 nm, which
closely resembles that of an authentic sample of
HtBQ,9b of other TPQ-like models,11 and of native
CuAOs. Moreover, in both cases, a very similar spec-
troscopic behaviour can be observed when the model
compound is reacted with a 10-fold molar amount of
the carbonyl reagent phenylhydrazine, with the pro-
gressive bleaching of the peak at 485 nm and the rapid
increase at shorter wavelength (ꢀ430 nm) (Fig. 3). TPQ
in native CuAOs is also well known to give stable
hydrazine adducts when reacted with phenylhydrazine
and 4-nitrophenylhydrazine, with comparable e€ects on
the absorption spectrum.
In our model system,10 Cu(II)±imidazole mononuclear
complexes were used in order to account for the active-
site copper ion, ligated by three histidines. 4-tert-Butyl-
phenol was chosen as a model for Tyr, since the two
compounds have a similar value of pKa for the phenol
moiety, and the hydroxylated and quinonoid derivatives
of 4-tert-butylphenol have been already studied.9 The
higher yield of HtBQ was obtained when 4-tert-butyl-
phenol was incubated in the presence of CuIm4(ClO4)2,
in 20% aqueous MeOH at pH 11. Tables 1 and 2 list the
results obtained varying the pH value from 8 to 12, and
in the presence of other Cu(II)±imidazole complexes or
uncomplexed copper. Attempts to lower (10%) or to
increase (50 and 80%) the proportion of MeOH in the
reaction solution, or to substitute it for another organic
solvent (acetonitrile, dioxane, acetone) did always lead
to a decrease in the yield of HtBQ (data not shown). In
some cases, the formation of a brown precipitate, pos-
sibly due to the production of polymeric products, was
There are some precedents for the ortho-hydroxylation of
phenols by mononuclear copper systems to obtain cate-
chols without cleavage of the aromatic system.12 More
recently, Urano and co-workers reported the selective
ortho-hydroxylation of para-substituted phenols by a
Cu2+±ascorbic acid-O2 system.13 When para-sub-
stituted phenols bearing electron-donating groups (as
para-cresol) were used as substrates, only small amounts
of hydroxylated products were obtained (3%),13 com-
parable to the yield obtained in this study, con®rming
that electron-donating groups probably retard the initial
hydroxylation reaction of the phenol to the correspond-
ing catechol. Other catalytic systems lead to oxidative

A. C. Rinaldi et al. / Bioorg. Med. Chem. Lett. 10 (2000) 989±992
991
Figure 3. Absorbtion spectrum of HtBQ (0.39 mM, estimated from e485 nm), in aqueous solution at pH 11, obtained from 4-tert-butylphenol.10 (B)
Time of reaction of HtBQ, as derived from 4-tert-butylphenol, with phenylhydrazine. HtBQ, 0.39 mM in aqueous solution, pH 11, was reacted with
a 10-fold molar amount of phenylhydrazine. Spectra have been recorded at several times after addition of phenylhydrazine: 5, 10, 15, 20, 25, 30, 45,
and 60 min. A similar spectroscopic behaviour was observed when an authentic sample of HtBQ9b was reacted with phenylhydrazine under similar
experimental conditions (data not shown).
aromatic ring cleavage or to the formation of coupling
products.14 Also the direct conversion of phenols to
ortho-quinones by Cu(I) in the presence of O2, and the
copper catalyzed oxidation of phenol to hydroquinone,
have been demonstrated.8,15
that the oxidation of tyrosine to TPQ consumes two
mol of dioxygen and produces one mol of H2O2 for
every mol of TPQ generated.18
Our results indicate that, under experimental condi-
tions, it is possible to obtain a TPQ-like compound
starting from a phenol, and that this reaction is pro-
moted by Cu(II) reagents. In particular, complexation
of copper by imidazoles enhances the yield of HtBQ
with respect to uncomplexed Cu(II) species, possibly
favoring the initial hydroxylation of the phenol ring to a
catechol. Site-directed mutagenesis studies have shown
that the copper-ligating histidines in native CuAOs are
required for TPQ formation, con®rming the key role
played by coordinated copper in the biogenesis reaction.
Moreover, data reported in Table 1 suggest that proton
abstraction from the phenol moiety to give a phenolate
ion, obtained by raising the pH of the reaction solution
(pKa value of the hydroxyl group of 4-tert-butylphenol
being 10.0), it is a prerequisite for the e€ective ortho-
hydroxylation of the aromatic system. This is consistent
There is considerable agreement in the literature that
copper plays an essential role in the initial conversion of
Tyr to dopa during TPQ biogenesis in CuAOs, although
it is not yet clear whether copper is required for acti-
vating the phenol ring of tyrosine, dioxygen, or both.
Mechanisms proposed so far involve an oxidation of the
precursor tyrosine to a tyrosyl radical, concomitant
with the conversion of active site Cu(II) to Cu(I), with a
copper-dependent activation of both the phenol ring
and dioxygen,16 or the formation of a charge-transfer
complex of Cu(II) and a tyrosinate ion,17 with dioxygen
reacting directly with the activated phenol ring, without
the necessity to generate Cu(I) and tyrosyl radical as
discrete precursor species. A recent study on a native
CuAO from Arthrobacter globiformis has ascertained

992
A. C. Rinaldi et al. / Bioorg. Med. Chem. Lett. 10 (2000) 989±992
with the expectation that the phenolate ring would be
more susceptible to direct, or indirect (through copper),
attack by dioxygen.
metals. Cu(II)±Imidazole complexes CuIm2Cl2, CuIm4Cl2,
and CuIm4(ClO4)2 were prepared according to the methods
reported in Goodgame, D. M. L.; Goodgame, M.; Rayner-
Canham, G. W. Inorg. Chim. Acta 1969, 3, 406. and in
Krushna, C.; Mohapatra, C.; Dash, K. C. J. Inorg. Nucl.
Chem. 1977, 39, 1253. Analytical results (C, H, and N) for all
the compounds were good. Characterization of the complexes
involved measurement of infrared spectra, re¯ectance spectra,
and electric conductance (data not shown). Hydroxylation of
4-tert-butylphenol was attempted as follows. In a typical
experiment, a mixture of 20 mM 4-tert-butylphenol and 2 mM
Cu(II)±imidazole complex (or uncomplexed copper) in 10 mL
of 20% aqueous MeOH was adjusted to pH 11 with NaOH.
The pH value was corrected for the mixed solvent system, and
remained at the initial set value over the course of the reaction,
or fell slightly. The solution was magnetically stirred vigor-
ously at 40 ꢁC in the dark. Several experiments at di€erent pH
values (in the range 8±12), or with organic solvents other than
MeOH (dioxane, acetonitrile, acetone), were also performed.
In another set of experiments, the MeOH concentration in the
reaction solution was varied progressively from 10 to 80%. In
all cases, after 48 h, the reaction mixture was acidi®ed with
HCl and then extracted with dichloromethane for three times,
and the combined organic layer was dried over anhydrous
Na2SO4 and then evaporated under reduced pressure. The
concentrate was then resuspended in 2 mL of 0.1 M NaOH, to
ensure a rapid autoxidation of all the species present, and the
absorbance spectra recorded at 10 and 60 min after addition of
NaOH. Authentic samples of HtBQ and its deprotonated ana-
logue were prepared from 4-tert-butylcatechol,9b and the con-
centration estimated using an E484 nm of 1800 M 1 cm 1 in H2O,
as reported by Moenne-Loccoz, P.; Nakamura, N.; Steinebach,
V.; Duine, J. A.; Mure, M.; Klinman, J. P.; Sanders-Loehr, J.
Biochemistry 1995, 34, 7020. The reaction of HtBQ, as derived
from 4-tert-butylphenol, with phenylhydrazine is described in
the caption of Figure 3.
Obviously, the results obtained from model studies must
be in any case carefully evaluated and interpreted before
exporting to the relevant enzymatic system, and is
therefore not possible to translate in a straightforward
manner our results to the mechanism of TPQ biogenesis in
the protein active site. However, present data tend to sup-
port the hypothesis that a tyrosinate ion may be involved
in the ®rst step of cofactor generation in native CuAOs.17
References and Notes
1. For a recent review see (a) Hartmann, C.; McIntire, W. S.
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10. Prior to use, 4-tert-butylphenol was analyzed by means of
HPLC, to ensure it was free from contaminating 4-tert-butyl-
catechol. Solutions were made using deionized, distilled water
but not puri®ed further, so contained usual levels of trace